WO2025221860A1

WO2025221860A1 - Recombinant phosphatases

Info

Publication number: WO2025221860A1
Application number: PCT/US2025/024919
Authority: WO
Inventors: Marina BAKHTINA; Stephanie FORGET; Amani SHOUBBER; Ljubica Vojcic; Jonathan VROOM; David Watts; Nikki D. Kruse
Original assignee: Codexis Inc
Current assignee: Codexis Inc
Priority date: 2024-04-16
Filing date: 2025-04-16
Publication date: 2025-10-23
Anticipated expiration: 2026-10-16

Abstract

The present disclosure relates to recombinant phosphatases and compositions thereof, as well as polynucleotides encoding the recombinant phosphatases. The present disclosure also provides methods of using the recombinant phosphatases or compositions thereof for molecular biological, diagnostic, and other purposes.

Description

RECOMBINANT PHOSPHATASES

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application 63/634,868, filed April 16, 2024, the contents of which is incorporated by reference herein.

REFERENCE TO SEQUENCE LISTING, TABLE OR COMPUTER PROGRAM

[0002] The Sequence Listing concurrently submitted herewith as file name CX10- 266WO2_ST26.xml, created on March 3, 2025, with a file size of 2,193,000 bytes, is part of the specification and is incorporated by reference herein.

TECHNICAL FIELD

[0003] The present disclosure provides recombinant phosphatases and compositions thereof, and polynucleotides encoding the recombinant phosphatases. The disclosure further provides methods of using the recombinant phosphatases or compositions thereof for cleaving substrates with phosphate monoester groups and analogs thereof.

BACKGROUND

[0004] Alkaline phosphatases are widely distributed enzymes found in both prokaryotes and eukaryotes that catalyze the hydrolysis of phosphate monoesters, with an optimal activity at alkaline pH. In mammals, alkaline phosphatases are present in the intestine (i.e., intestinal alkaline phosphatase) and placenta (i.e., placental alkaline phosphatase). Phosphatases are widely used in molecular biological applications, for example for the removal of 5 ’-phosphate from polynucleotides or oligonucleotides for subsequent labeling with labeled ATP, reducing or preventing ligation of polynucleotides or oligonucleotides, and reducing susceptibility of polynucleotides or oligonucleotides to certain nucleases, e.g., X exonuclease.

[0005] Alkaline phosphatases are also found in many different types of bacteria, and some are sold commercially for molecular biological applications. Three classes of prokaryotic alkaline phosphatases are known and include PhoA, PhoD, and PhoX. These phosphatases differ in their structure, substrate specificity, and dependence on different metal ions for activity. PhoD and Pho X phosphatases are commonly found in marine and soil bacteria, while the PhoX phosphatases are also found in cyanobacteria. PhoA represents the classical phosphatase present in E. coli. Bacteria typically express at least one of the phosphatase of the three phosphatase classes.

[0006] While various phosphatases are commercially available, desirable are phosphatases that can be expressed in active soluble form, thermally stable for high temperature applications, and amenable to immobilization on support medium. SUMMARY

[0007] The present disclosure provides recombinant phosphatases and compositions thereof, as well as polynucleotides encoding the recombinant phosphatases. The present disclosure also provides methods of using the recombinant phosphatase for cleaving a substrate comprising a phosphomonoester or analogs thereof.

[0008] In one aspect, the present disclosure provides a recombinant phosphatase, or a functional fragment thereof, comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%. 85%. 86%, 87%, 88%. 89%. 90%, 91%, 92%. 93%. 94%, 95%, 96%. 97%. 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of an even-numbered SEQ ID NO. of SEQ ID NOs: 2- 18 and 24-1052, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 2-18 and 24- 1052.

[0009] In some embodiments, the recombinant phosphatase, or a functional fragment thereof, comprises an amino acid sequence having at least 70%, 75%, 80%. 81%. 82%, 83%, 84%. 85%. 86%, 87%. 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residue 12 to the carboxyl terminal of SEQ ID NO: 16 or 18, or to a reference sequence corresponding to SEQ ID NO: 16 or 18.

[0010] In some embodiments, the recombinant phosphatase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%. 82%, 83%, 84%, 85%. 86%, 87%, 88%, 89%, 90%. 91%, 92%, 93%, 94%, 95%, 96%. 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of an even-numbered SEQ ID NO. of SEQ ID NOs: 2-14 or SEQ ID NO: 18. 84, 214, 232, 562. 656, or 774. or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 2-14, or SEQ ID NO: 18. 84. 214, 232, 562, 656, or 774.

[0011] In some embodiments, the recombinant phosphatase comprises an alkaline phosphatase of Thermoflexibacter, Pyrococcus, Thennotoga, Pseudothermotoga, or Bacillus.

[0012] In some embodiments, the recombinant phosphatase further comprises an N-tenninal deletion of up to 5, 10, 25, 30, 40, 45, or 50 amino acids of the naturally occurring amino acid sequence.

[0013] In some embodiments, the N-terminal deletion removes or inactivates signaling sequences at the N-terminal sequence of the phosphatase. In some embodiments, the N-terminal deletion enhances or increases expression of the recombinant phosphatase in the cytoplasm of a bacterial host cell. In some embodiments, the N-terminal deletion enhances or increases expression of soluble form of the recombinant phosphatase. [0014] In some embodiments, the N-terminal deletion preserves the capability of the phosphatase to form the biologically active dimer of the recombinant phosphatase.

[0015] In some embodiments, the recombinant phosphatase comprises an amino acid sequence having at least 70%, 75%, 80%. 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%. 96%, 97%, 98%, 99%. or more sequence identity to a reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of an even-numbered SEQ ID NO. of SEQ ID NOs: 12 and 24-1052, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 12 and 24-1052, wherein the amino acid comprises one or more amino acid differences relative to the reference sequence corresponding to amino acid residues 12 to the carboxy l terminus of an even-numbered SEQ ID NOs: 12 and 24-1052, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 12 and 24-1052.

[0016] In some embodiments, the recombinant phosphatase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%. 96%, 97%, 98%, 99%. or more sequence identity to a reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of SEQ ID NO: 12, 18, 84. 214, 232, 562, 656, or 774, or to the reference sequence corresponding to SEQ ID NO: 12, 18, 84, 214, 232, 562, 656, or 774, wherein the amino acid comprises one or more amino acid differences relative to the reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of SEQ ID NO: 12, 18, 84, 214, 232, 562, 656, or 774, or to the reference sequence corresponding to SEQ ID NO: 12, 18, 84, 214, 232, 562, 656, or 774.

[0017] In some embodiments, the recombinant phosphatase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%. 96%, 97%, 98%, 99%. or more sequence identity to a reference sequence corresponding to amino acid residues 12 to the carboxy l terminus of an even-numbered SEQ ID NO. of SEQ ID NOs: 24-80, or to the reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 24-80, wherein the amino acid sequence comprises one or more amino acid differences relative to the reference sequence corresponding to amino acid residues 12 to the carboxy l terminus of SEQ ID NO: 18, or to the reference sequence corresponding to SEQ ID NO: 18.

[0018] In some embodiments, the recombinant phosphatase comprises an amino acid sequence having at least 70%, 75%, 80%. 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%. 96%, 97%, 98%, 99%. or more sequence identity to a reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of SEQ ID NO: 18, or to the reference sequence corresponding to SEQ ID NO: 18, wherein the amino acid sequence comprises one or more amino acid differences relative to the reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of SEQ ID NO: 18, or to the reference sequence corresponding to SEQ ID NO: 18.

[0019] In some embodiments, the amino acid sequence of the recombinant phosphatase comprises at least an amino acid difference at amino acid position 23, 25, 50, 56. 63, 99, 102. 136, 193, 195. 198, 224, 234, 235, 267, 306, or 345, or combinations thereof, wherein the amino acid difference is relative to the reference sequence corresponding to residues 12 to the carboxyl terminus of SEQ ID NO: 18, or to the reference sequence corresponding to SEQ ID NO: 18.

[0020] In some embodiments, the recombinant phosphatase comprises an amino acid sequence having at least 70%, 75%, 80%. 81%, 82%, 83%, 84%. 85%, 86%, 87%, 88%. 89%, 90%, 91%, 92%. 93%, 94%, 95%. 96%, 97%, 98%, 99%. or more sequence identity to a reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of SEQ ID NO: 12, 84, 214, 232. 562. 656, or 774. or to a reference sequence corresponding to SEQ ID NO: 12, 84. 214, 232, 562. 656, or 774, wherein the amino acid sequence comprises one or more amino acid differences relative to the reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of SEQ ID NO: 12, 84, 214, 232. 562, 656, or 774, or to tire reference sequence corresponding to SEQ ID NO: 12, 84, 214, 232, 562, 656, or 774.

[0021] In some embodiments, the recombinant phosphatase comprises an amino acid sequence having at least 70%, 75%, 80%. 81%, 82%, 83%, 84%. 85%, 86%, 87%, 88%. 89%, 90%, 91%, 92%. 93%, 94%, 95%. 96%, 97%, 98%, 99%. or more sequence identity to a reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of an even-numbered SEQ ID NO. of SEQ ID NOs: 24-1052, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 24-1052, wherein the amino acid sequence comprises one or more amino acid differences relative to the reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of SEQ ID NO: 12, or to the reference sequence corresponding to SEQ ID NO: 12.

[0022] In some embodiments, the recombinant phosphatase comprises an amino acid sequence having at least 70%, 75%, 80%. 81%, 82%, 83%, 84%. 85%, 86%, 87%, 88%. 89%, 90%, 91%, 92%. 93%, 94%. 95%. 96%, 97%, 98%, 99%. or more sequence identity to the reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of SEQ ID NO: 84, 214, 232, 562, 656. or 774, or to the reference sequence corresponding to SEQ ID NO: 84, 214. 232, 562, 656, or 774. wherein the amino acid sequence comprises one or more amino acid differences relative to the reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of SEQ ID NO: 12, or to the reference sequence corresponding to SEQ ID NO: 12.

[0023] In some embodiments, the recombinant phosphatase comprises an amino acid sequence having at least 70%, 75%, 80%. 81%, 82%, 83%, 84%. 85%, 86%, 87%, 88%. 89%, 90%, 91%, 92%. 93%, 94%. 95%. 96%, 97%, 98%, 99%. or more sequence identity to the reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of SEQ ID NO: 12, or to the reference sequence corresponding to SEQ ID NO: 12, wherein the amino acid sequence comprises one or more amino acid differences relative to the reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of SEQ ID NO: 12, or to the reference sequence corresponding to SEQ ID NO: 12.

[0024] In some embodiments, the amino acid sequence of the recombinant phosphatase comprises at least an amino acid difference at amino acid position 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44. 45, 46, 47, 48, 49, 50, 51, 52, 56, 57, 60, 61, 63, 81, 82, 83, 89, 93, 95, 96, 98, 100, 101, 105, 108, 128, 138, 139, 141, 148, 150, 158, 169, 174, 175, 181, 202, 205, 206, 207, 211, 214, 215, 223, 224, 225, 226, 227, 228, 230, 231, 234, 235, 240, 241, 244, 247, 260, 261, 263, 266, 271, 291, 299, 301, 302, 306, 307, 336, 340, 341, 343, 345, 346, 347, 353. 357, 363, 367, 368, 371, 389, 390, 391, 392, or 393, or combinations thereof, wherein the amino acid difference is relative to the reference sequence corresponding to residues 12 to the carboxyl terminus of SEQ ID NO: 12, or to the reference sequence corresponding to SEQ ID NO: 12.

[0025] In some embodiments, the amino acid sequence of the recombinant phosphatase comprises at least an amino acid difference at amino acid position 19, 22, 50, 138, 139, 141, 158, 169, 244, 260, 306, 307, 340, 357, 390, 392, or 393, or combinations thereof, wherein the amino acid difference is relative to die reference sequence corresponding to residues 12 to the carboxyl terminus of SEQ ID NO: 12, or to the reference sequence corresponding to SEQ ID NO: 12.

[0026] In some embodiments, the amino acid sequence of the recombinant phosphatase comprises at least an amino acid difference at amino acid position 11, 13, 14, 16. 17, 18, 19, 20, 21, 28, 29, 31, 35. 42, 44, or 61, or combinations thereof, wherein the amino acid difference is relative to the reference sequence corresponding to residues 12 to the carboxy l terminus of SEQ ID NO: 12, or to the reference sequence corresponding to SEQ ID NO: 12.

[0027] In some embodiments, the recombinant phosphatase comprises an amino acid sequence having at least 70%, 75%, 80%. 81 %, 82%, 83%, 84%. 85%, 86%, 87%, 88%. 89%, 90%, 91%, 92%, 93%, 94%, 95%. 96%, 97%, 98%, 99%. or more sequence identity to a reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of an even-numbered SEQ ID NO. of SEQ ID NOs: 24-1052, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 24-1052, wherein the amino acid sequence comprises one or more amino acid differences relative to the reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of SEQ ID NO: 84, 214. 232, 562, 656, or 774, or to the reference sequence corresponding to SEQ ID NO: 84, 214, 232, 562, 656, or 774. [0028] In some embodiments, the recombinant phosphatase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of SEQ ID NO: 84, 214, 232, 562, 656, or 774, or to a reference sequence corresponding to SEQ ID NO: 84, 214, 232, 562, 656, or 774, wherein the amino acid sequence comprises one or more amino acid difference relative to the reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of SEQ ID NO: 84, 214. 232, 562, 656, or 774, or to the reference sequence corresponding to SEQ ID NO: 84, 214, 232, 562, 656. or 774.

[0029] In some embodiments, the amino acid sequence of the recombinant phosphatase comprises at least an amino acid difference at amino acid position 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31. 32, 33, 34, 35, 36, 37, 38, 39, 40. 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 56, 57, 60, 61, 63, 81. 82, 83, 89, 93, 95, 96, 98, 100, 101, 105, 108, 128, 138, 139. 141, 148, 150, 158, 169, 174, 175, 181. 202, 205, 206, 207, 211, 214, 215, 223. 224, 225, 226, 227, 228, 230, 231, 234, 235. 240, 241, 244. 247, 260, 261, 263, 266, 271, 291, 299. 301, 302, 306, 307, 336, 340, 341, 343, 345. 346, 347, 353. 357, 363, 367, 368, 371, 389, 390, 391. 392, or 393. or combinations thereof, wherein the amino acid difference is relative to the reference sequence corresponding to residues 12 to the carboxyl terminus of SEQ ID NO: 84, 214, 232, 562. 656, or 774. or to the reference sequence corresponding to SEQ ID NO: 84, 214. 232, 562, 656, or 774.

[0030] In some embodiments, the amino acid sequence of the recombinant phosphatase comprises at least an amino acid difference at amino acid position 19, 22, 50, 138, 139, 141, 158, 169, 244, 260, 306, 307, 340, 357, 390, 392. or 393, or combinations thereof, wherein the amino acid difference is relative to the reference sequence corresponding to residues 12 to the carboxyl tenninus of SEQ ID NO: 84. 214, 232, 562, 656, or 774, or to the reference sequence corresponding to SEQ ID NO: 84, 214, 232, 562. 656, or 774.

[0031] In some embodiments, the recombinant phosphatase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to the carboxyl tenninus of an even-numbered SEQ ID NO. of SEQ ID NOs: 144-224, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 144-224, wherein the amino acid sequence comprises one or more amino acid differences relative to the reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of SEQ ID NO: 84, or to the reference sequence corresponding to SEQ ID NO: 84.

[0032] In some embodiments, the recombinant phosphatase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of SEQ ID NO: 84, or to the reference sequence corresponding to SEQ ID NO: 84, wherein the amino acid sequence comprises one or more ammo acid differences relative to the reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of SEQ ID NO: 84, or to the reference sequence corresponding to SEQ ID NO: 84.

[0033] In some embodiments, the amino acid sequence of the recombinant phosphatase comprises at least an amino acid difference at amino acid position 11, 15, 16, 18, 19, 22, 27, 38, 44, 48, 50, 81, 82, 83, 95, 96, 100, 101, 150, 158, 174, 175, 202, 226, 230, 235, 241, 247, 261, 266, 299, 301, 302, 307, 336, 341, 346, 357, 368, or 389, or combinations thereof, wherein the amino acid difference is relative to the reference sequence corresponding to residues 12 to the carboxyl terminus of SEQ ID NO: 84, or to the reference sequence corresponding to SEQ ID NO: 84.

[0034] In some embodiments, the recombinant phosphatase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%. 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of an even-numbered SEQ ID NO. of SEQ ID NOs: 226-348, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 226-348, wherein the amino acid sequence comprises one or more amino acid differences relative to the reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of SEQ ID NO: 214, or to the reference sequence corresponding to SEQ ID NO: 214.

[0035] In some embodiments, the recombinant phosphatase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%. 96%, 97%, 98%, 99%. or more sequence identity to the reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of SEQ ID NO: 214, or to the reference sequence corresponding to SEQ ID NO: 214, wherein the amino acid sequence comprises one or more amino acid differences relative to the reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of SEQ ID NO: 214, or to the reference sequence corresponding to SEQ ID NO: 214.

[0036] In some embodiments, the amino acid sequence of the recombinant phosphatase comprises at least an amino acid difference at amino acid position(s) 15. 15/50/150/174/175, 15/50/158/174/226/247/346, 15/50/158/226/247/357, 15/50/158/247/357, 15/50/226, 15/96, 15/96/175/247, 15/96/175/341, 15/96/226/357, 15/96/357, 15/158/174/175/357, 15/158/174/247, 15/158/174/301/346, 15/158/175/247. 15/158/175/247/307/341/346, 15/174, 15/174/175, 15/174/175/247, 15/174/175/247/346, 15/174/226/301, 15/174/341, 15/175/247, 15/175/247/301, 15/175/247/341/346, 15/175/247/346/357, 15/175/357, 15/247. 15/346, 50/174/175/247/357, 96/158/175/301, 96/174/175, 96/174/175/226, 96/174/175/301/341/346, 96/174/247/341, 96/174/247/346, 96/175, 158, 158/174/226/247, 158/174/247/307/357, 158/175, 158/175/226/247/301/341/346/363, 158/175/247/346, 158/175/247/346/357, 158/175/301, 158/247, 158/307/346, 174, 174/175/247, 174/175/357, 174/247, 174/301/341, 174/346, 174/357, 175, 226, 226/247, 226/247/346, 247/301/346, 247/341/346, 307/357, or 357, wherein the amino acid difference is relative to the reference sequence corresponding to residues 12 to the carboxyl terminus of SEQ ID NO: 214, or to the reference sequence corresponding to SEQ ID NO: 214.

[0037] In some embodiments, the recombinant phosphatase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of an even-numbered SEQ ID NO. of SEQ ID NOs: 350-650, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 350-650, wherein the amino acid sequence comprises one or more amino acid differences relative to the reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of SEQ ID NO: 232, or to the reference sequence corresponding to SEQ ID NO: 232.

[0038] In some embodiments, the recombinant phosphatase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of SEQ ID NO: 232, or to the reference sequence corresponding to SEQ ID NO: 232, wherein tire ammo acid sequence comprises one or more amino acid differences relative to the reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of SEQ ID NO: 232, or to the reference sequence corresponding to SEQ ID NO: 232.

[0039] In some embodiments, the amino acid sequence of the recombinant phosphatase comprises at least an amino acid difference at amino acid position(s) 12, 14, 15, 16, 17, 18, 19, 21, 22, 37. 43, 50, 57, 61, 89/95/141/169/234/306, 89/95/234/306/367, 89/96/367/371, 89/138/141/234/306/367/371, 89/138/234, 89/138/367, 89/141/169/234/306/371, 89/141/169/234/371, 89/141/169/306/367/371, 89/141/234/306/367/371, 89/141/306/367/371, 89/141/367, 89/141/371, 89/169, 89/169/234, 89/169/234/306/367/371, 89/169/234/367, 89/169/234/371, 89/169/306/367, 89/169/367, 89/169/371, 89/234, 89/234/306/371, 89/234/371, 89/306, 89/367/371, 89/371, 95/169/234/306/371, 96/141/234/306/371. 96/169/306. 138, 138/141, 138/141/169/234/367, 138/141/169/306, 138/141/234/306, 138/141/234/367, 138/141/234/371, 138/141/306/367, 138/141/306/371, 138/169/367/371, 138/234/367, 138/234/367/371, 138/306, 138/371, 139, 141, 141/169. 141/234, 141/234/306, 141/234/306/371, 141/234/371, 141/306/367, 141/306/371, 141/367, 169, 169/306, 205, 214, 224, 234. 234/306, 234/306/367. 234/306/367/371, 234/371, 244, 260. 271, 306, 306/367, 306/367/371, 306/371. 340, 341, 343. 367, 390, 391, 392, or 393, or combinations thereof, wherein die amino acid difference is relative to the reference sequence corresponding to residues 12 to the carboxyl terminus of SEQ ID NO: 232, or to the reference sequence corresponding to SEQ ID NO: 232.

[0040] In some embodiments, the recombinant phosphatase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%. 96%, 97%, 98%, 99%. or more sequence identity to a reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of an even-numbered SEQ ID NO. of SEQ ID NOs: 652-740, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 652-740, wherein the amino acid sequence comprises one or more amino acid differences relative to the reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of SEQ ID NO: 562, or to the reference sequence corresponding to SEQ ID NO: 562.

[0041] In some embodiments, the recombinant phosphatase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%. 96%, 97%, 98%, 99%. or more sequence identity to the reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of SEQ ID NO: 562, or to the reference sequence corresponding to SEQ ID NO: 562, wherein the amino acid sequence comprises one or more amino acid differences relative to the reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of SEQ ID NO: 562, or to the reference sequence corresponding to SEQ ID NO: 562.

[0042] In some embodiments, the amino acid sequence of the recombinant phosphatase comprises at least an amino acid difference at amino acid position(s) 12. 12/37/50/139/244/260/393, 12/37/57/139/244/260, 12/37/139/244/390/392/393, 12/37/139/393, 12/37/244/260/393, 12/37/244/392, 12/50/57/244/390/393, 12/50/139/244/260/392/393, 12/57/139/244/392, 12/139/244/260, 12/139/244/260/390/393, 12/139/244/260/392/393, 12/139/244/260/393, 12/139/244/390/392/393, 12/139/260/393, 12/260. 12/260/392. 12/390/392/393, 12/392, 37/50/57/139/244/260/392, 37/50/139/244/390/393, 37/57/139/244/390/393, 37/57/139/244/392/393, 37/57/139/390/392/393, 37/57/244/392, 37/139/223/244/393, 37/139/244/260/393, 37/139/244/390/392, 37/139/260/393, 50/139/244/260/390/392/393, 50/139/244/390/393, 50/244, 50/260, 57/244, 139/244/390/392/393, 139/244/392/393. 139/244/393, 139/260/393, 139/392, 244. 244/392, 260/390, or 392/393, wherein the amino acid difference is relative to the reference sequence corresponding to residues 12 to the carboxyl terminus of SEQ ID NO: 562, or to the reference sequence corresponding to SEQ ID NO: 562.

[0043] In some embodiments, the recombinant phosphatase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of an even-numbered SEQ ID NO. of SEQ ID NOs: 742-894, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 742-894, wherein the amino acid sequence comprises one or more amino acid differences relative to the reference sequence corresponding to amino acid residue 12 to the carboxyl terminus of SEQ ID NO: 656, or to the reference sequence corresponding to SEQ ID NO: 656.

[0044] In some embodiments, the recombinant phosphatase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity’ to the reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of SEQ ID NO: 656, or to the reference sequence corresponding to SEQ ID NO: 656, wherein the amino acid sequence comprises one or more amino acid differences relative to the reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of SEQ ID NO: 656, or to the reference sequence corresponding to SEQ ID NO: 656.

[0045] In some embodiments, the amino acid sequence of the recombinant phosphatase comprises at least an amino acid difference at amino acid position(s) 13. 22/141, 22/141/158, 22/158/340, 22/158/340/390/392, 23, 24, 25, 29. 30, 31, 32, 35, 36, 38, 44/50, 45/50, 47/50, 50, 50/51, 50/52, 50/56, 60, 63, 83, 98, 105, 128, 141/158/340, 141/340, 148, 150, 158, 158/340/371, 158/340/371/390/392, 181, 206, 207, 211, 215, 225, 227, 228, 231, 260, 291, 345, 347, or 353 wherein the amino acid difference is relative to the reference sequence corresponding to residues 12 to the carboxyl terminus of SEQ ID NO: 656, or to the reference sequence corresponding to SEQ ID NO: 656.

[0046] In some embodiments, the recombinant phosphatase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%. 96%, 97%, 98%, 99%, or more sequence identity’ to a reference sequence corresponding to amino acid residues 12 to the carboxy l terminus of an even-numbered SEQ ID NO. of SEQ ID NOs: 896-1052, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 896-1052, wherein the amino acid sequence comprises one or more amino acid differences relative to the reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of SEQ ID NO: 774, or to the reference sequence corresponding to SEQ ID NO: 774.

[0047] In some embodiments, the recombinant phosphatase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%. 96%, 97%, 98%, 99%. or more sequence identity’ to the reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of SEQ ID NO: 774, or to the reference sequence corresponding to SEQ ID NO: 774, wherein the amino acid sequence comprises one or more amino acid differences relative to the reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of SEQ ID NO: 774, or to the reference sequence corresponding to SEQ ID NO: 774.

[0048] In some embodiments, the amino acid sequence of the recombinant phosphatase comprises at least an amino acid difference at amino acid position(s) 13. 13/25/31/105, 13/25/47, 13/25/47/56/105/260, 13/25/47/207/260/353, 13/25/47/207/353, 13/25/56/207/260/353, 13/25/260/353, 13/25/353, 13/32, 13/32/47/56/353, 13/32/93/105/260/353, 13/47, 13/47/56/105/207/353, 13/47/56/353, 13/47/93/207/353, 13/47/105/207/240/260/353, 13/47/207/260, 13/56/263/353, 13/56/353, 13/105, 13/207, 13/207/260/353, 13/207/353, 13/353, 25/31/32, 25/31/32/56/105/260/353, 25/32/207/353, 25/47, 25/47/105, 25/47/105/207, 25/47/353, 25/56, 25/56/105/260/353, 25/93/207/260/353, 25/105/207/260, 25/105/207/260/353, 25/207, 25/353, 31/47/49/105/207/260/353, 32/47/105/353, 32/56/105, 47, 47/56/105/139/260, 47/105, 47/105/207/353, 47/105/260/353, 47/105/353, 47/108/207/260/353, 47/139, 47/207/260/353, 47/207/353, 47/260, 47/260/353, 47/353, 56, 56/93/105/139/207, 56/105/207/260.

56/105/207/260/353. 56/105/207/353, 56/207. 56/207/260. 56/207/353, 56/214/353, 105, 105/139/207/353, 105/139/260/353, 105/207/353, 105/260/353, 105/353, 207, 207/260/353, 207/353, or 353, wherein the amino acid difference is relative to die reference sequence corresponding to residues 12 to the carboxyl terminus of SEQ ID NO: 774, or to the reference sequence corresponding to SEQ ID NO: 774.

[0049] In some embodiments, the recombinant phosphatase comprises an amino acid sequence comprising residues 12 to die carboxyl terminus of an even-numbered SEQ ID NO. of SEQ ID NOs: 16-18 and 24-1052, or an amino acid sequence comprising an even-numbered SEQ ID NO. of SEQ ID NOs: 16-18 and 24-1052.

[0050] In some embodiments, the recombinant phosphatase has an improved property selected from i) increased phosphatase activity on an oligonucleotide having a 3 ’-phosphate group, ii) increased phosphatase activity' on an 3’-phosphate-NTP, iii) increased solubility, iv) increased expression or protein yield, v) increased thermal stability, vi) increased resistance to proteolysis, and vii) increased resistance to inhibition by immobilization on epoxide activated polyacrylate resin, or any combinations thereof, as compared to a reference phosphatase having the amino acid sequence corresponding to SEQ ID NO: 12, 18, 84, 214, 232, 562, 656, or 774.

[0051] In another aspect, the present disclosure provides a recombinant polynucleotide comprising a polynucleotide sequence encoding a recombinant phosphatase described herein.

[0052] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence having at least 70%, 75%. 80%. 81%, 82%, 83%, 84%. 85%, 90%, 91%, 92%. 93%, 94%, 95%, 96%. 97%, 98%. 99%. or more sequence identity to a reference polynucleotide sequence corresponding to a sequence from nucleotide residue 34 to the 3 ’-terminal nucleotide of an odd numbered SEQ ID NO. of SEQ ID NOs: 1-13, or to a reference polynucleotide sequence corresponding to an odd numbered SEQ ID NO. of SEQ ID NOs: 1-13, wherein the polynucleotide sequence encodes a phosphatase.

[0053] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence having at least 70%, 75%, 80%. 81%, 82%, 83%, 84%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%. or more sequence identity to a reference polynucleotide sequence corresponding to nucleotide residues 34 to the 3 ’-terminal nucleotide of an even-numbered SEQ ID NO. of SEQ ID NOs: 15-17 and 23-1051, or to a reference polynucleotide sequence corresponding to an even- numbered SEQ ID NO. of SEQ ID NOs: 15-17 and 23-1051, wherein the polynucleotide sequence encodes a phosphatase.

[0054] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence having at least 70%, 75%, 80%. 81%, 82%, 83%, 84%. 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%. or more sequence identity to a reference polynucleotide sequence corresponding to nucleotide residues 34 to the 3 ’-terminal nucleotide of SEQ ID NO: 11, 83, 213. 231, 561, 655. or 773. or to a reference polynucleotide sequence corresponding to SEQ ID NO: 11, 83, 213, 231. 561, 655, or 773, wherein the polynucleotide sequence encodes a phosphatase.

[0055] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence codon-optimized for expression of the encoded recombinant phosphatase. In some embodiments, the polynucleotide sequence is codon optimized for expression in a bacteria cell, fungal cell, insect cell, or mammalian cell.

[0056] In a further aspect, the present disclosure provides an expression vector comprising a recombinant polynucleotide encoding a recombinant phosphatase described herein. In some embodiments, the polynucleotide encoding the recombinant phosphatase is operably linked to a control sequence, such as a promoter.

[0057] In a further aspect, the present disclosure provides a host cell comprising an expression vector for expression of the encoded recombinant phosphatase. In some embodiments, the host cell comprises a prokaryotic cell or eukaryotic cell. In some embodiments, the host cell comprises a bacterial cell, fungal cell, insect cell, or mammalian cell.

[0058] In a further aspect, the host cell is used in a method of producing a recombinant phosphatase. In some embodiments, a method of producing a recombinant phosphatase in a host cell comprises culturing a host cell comprising an expression vector encoding a recombinant phosphatase under suitable culture conditions such that the recombinant phosphatase is produced.

[0059] In some embodiments, the method of producing a recombinant phosphatase also comprises recovering the recombinant phosphatase from the culture media and/or host cells. In some embodiments, the method further comprises purifying the recombinant phosphatase. [0060] In another aspect, the recombinant phosphatase is provided as a composition. In some embodiments, the composition comprises a recombinant phosphatase immobilized on a support medium. In some embodiments, the recombinant phosphatase is immobilized on the support medium through adsorption, covalent attachment, entrapment, or affinity interactions. In some embodiments, the support medium is a solid support, porous support, or membrane support.

[0061] In some embodiments, the composition comprises a recombinant phosphatase and one or more phosphatase substrates with a phosphate monoester or analog thereof. In some embodiments, the substrate phosphate monoester or analog thereof in the composition comprises NTP, NDP, NMP, 3’-P-NTP, 3’-P-NDP, 3’-P-NMP, Np, NTP-a-S, NDP-a-S, NMP-a-S, 3‘-P-NTP-a-S, 3’-P-NDP-a-S, 3 ’-P -NMP-a-S, NpS, or any combination thereof. In some embodiments, substrate phosphate monoester or analog thereof in the composition comprises a polynucleotide or oligonucleotide with a 5’-P, 3’-P, 5’-P(S), 3’-P(S), or any suitable combinations thereof.

[0062] In some embodiments, the composition comprises a polynucleotide or oligonucleotide substrate, wherein the polynucleotide or oligonucleotide is single stranded or double stranded. In some embodiments, the polynucleotide or oligonucleotide substrate comprises at least a modified nucleoside or internucleoside linkage.

[0063] In a further aspect, the present disclosure provides a method of cleaving a phosphate monoester moiety or analog thereof, comprising contacting a substrate with a phosphate monoester or analog thereof with a recombinant phosphatase described herein under suitable conditions for cleaving of the phosphate monocstcr or analog thereof. In some embodiments, the method is carried out in vitro.

[0064] In some embodiments of the method, the phosphatase substrate comprises NTP, NDP, NMP, 3’-P-NTP. 3’-P-NDP, 3’-P-NMP, Np. NTP-a-S. NDP-a-S, NMP-a-S, 3’-P-NTP-a-S, 3’-P-NDP-a-S, 3 ’-P -NMP-a-S, NpS, or any combinations thereof. In some embodiments of the method, the phosphatase substrate comprises a polynucleotide or oligonucleotide with a 5'-P, 3’-P, 5’-P(S), 3’- P(S), or any suitable combinations thereof.

[0065] In some embodiments, the reaction with the recombinant phosphatase and substrate is carried under suitable reaction conditions for cleaving of the phosphate monoester or analog thereof. In some embodiments, a suitable reaction temperature of about 4 °C-70 °C. In some embodiments, the suitable reaction conditions comprise a pH of about > 7, particularly about pH 7-10.

[0066] In some embodiments of the method, the recombinant phosphatase is immobilized on a support medium. In some embodiments, the substrate is contacted with the recombinant phosphatase immobilized on a support medium, and separated from the recombinant phosphatase immobilized on a support medium following cleaving reaction. [0067] In some embodiments of the method, a polynucleotide or oligonucleotide substrate is bound to a support medium, and the polynucleotide or oligonucleotide bound to a support medium is contacted widi the recombinant phosphatase. In some embodiments, the recombinant phosphatase is in solution, and is separated from the polynucleotide or oligonucleotide bound to the support medium following cleaving of tire phosphate monoester or analog thereof.

DETAILED DESCRIPTION

[0068] The present disclosure provides recombinant phosphatase polypeptides and compositions thereof, as well as polynucleotides encoding the recombinant phosphatase polypeptides. The disclosure also provides methods of using of the recombinant phosphatase polypeptides and compositions thereof for molecular biological, diagnostic, and other purposes. In some embodiments, the recombinant phosphatase polypeptides display, among others, increased expression in the cytoplasm of bacterial host cells, and increased production of soluble enzyme.

Abbreviations and Definitions

[0069] In reference to the present disclosure, the technical and scientific terms used in the descriptions herein will have the meanings commonly understood by one of ordinary' skill in the art, unless specifically defined otherwise. Accordingly, the following tenns are intended to have the following meanings.

[0070] As used herein, the singular forms “a”, “an” and “the” include plural referents unless the context clearly indicates otherwise. Thus, for example, reference to “a polypeptide” includes more dian one polypeptide.

[0071] Similarly, “comprise.” “comprises,” “comprising” “include,” “includes,” and “including” are interchangeable and not intended to be limiting. Thus, as used herein, the term “comprising” and its cognates are used in their inclusive sense (i.e., equivalent to the term “including” and its corresponding cognates).

[0072] It is to be further understood that where descriptions of various embodiments use the term “comprising.” those skilled in the art would understand that in some specific instances, an embodiment can be alternatively described using language “consisting essentially of’ or “consisting of.”

[0073] “About” means an acceptable error for a particular value. In some instances, “about” means within 0.05%. 0.5%. 1.0%. or 2.0%, of a given value range. In some instances, “about” means within 1. 2, 3, or 4 standard deviations of a given value.

[0074] “ EC” number refers to die Enzy me Nomenclature of the Nomenclature Committee of the International Union of Biochemistry' and Molecular Biology (NC-IUBMB). The IUBMB biochemical classification is a numerical classification system for enzymes based on the chemical reactions they catalyze.

[0075] “ ATCC” refers to the American Type Culture Collection whose biorepository collection includes genes and strains.

[0076] "NCBI” refers to National Center for Biological Information and the sequence databases provided therein.

[0077] “ Protein,” “polypeptide,” and “peptide” are used interchangeably to denote a polymer of at least two amino acids covalently linked by an amide bond, regardless of length or post-translational modification (e.g., glycosylation or phosphorylation).

[0078] “ Amino acids” and “amino acid” are referred to herein by either their commonly known thrcc-lcttcr symbols or by the one-letter symbols recommended by IUPAC-IUB Biochemical Nomenclature Commission. The abbreviations used for the genetically encoded amino acids are conventional and are as follows: alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartate (Asp or D). cysteine (Cys or C), glutamate (Glu or E), glycine (Gly or G), glutamine (Gin or Q), histidine (His or H), isoleucine (He or I), leucine (Leu or L), lysine (Lys or K), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y), and valine (Vai or V). When the three-letter abbreviations are used, unless specifically preceded by an “L” or a “D” or clear from the context in which the abbreviation is used, the amino acid may be in either the L- or D-configuration about a-carbon (C«). For example, whereas “Ala” designates alanine without specifying the configuration about the a-carbon. “D-Ala” and “L-Ala” designate D-alanine and L-alanine, respectively. When the one-letter abbreviations are used, upper case letters designate amino acids in the L-configuration about the a-carbon and lower case letters designate amino acids in the D-configuration about the a-carbon. For example, “A” designates L-alanine and “a” designates D-alanine. When polypeptide sequences are presented as a string of one-letter or three-letter abbreviations (or mixtures thereof), the sequences are presented in the amino (N) to carboxy (C) direction in accordance with common convention.

[0079] “Phosphatase” as used herein refers to enzymes that preferentially cleave phosphomonoester bonds, or analogs thereof, such as phosph orothioate. “Alkaline phosphatase” refers to a phosphatase that has activity at optimal pH of >7.

[0080] “Fusion protein,” and “chimeric protein” and “chimera” refer to hybrid proteins created through the joining of tw o or more polynucleotides that originally encode separate proteins. In some embodiments, fusion proteins are created by recombinant technology.

[0081] “Polynucleotide.” “nucleic acid,” or "oligonucleotide” is used herein to denote a polymer comprising at least two nucleotides where the nucleotides are either deoxyribonucleotides or ribonucleotides or mixtures of deoxyribonucleotides and ribonucleotides. In some embodiments, the abbreviations used for genetically encoding nucleosides are conventional and are as follow: adenosine (A); guanosine (G); cytidine (C); thymidine (T); and uridine (U). Unless specifically delineated, the abbreviated nucleosides may be either ribonucleosides or 2 ’-deoxy ribonucleosides. The nucleosides may be specified as being either ribonucleosides or 2’-deoxyribonucleosides on an individual basis or on an aggregate basis. When a polynucleotide, nucleic acid, or oligonucleotide sequences are presented as a string of one-letter abbreviations, the sequences are presented in the 5’ to 3’ direction in accordance with common convention, and the phosphates are not indicated. The term “DNA” refers to deoxyribonucleic acid. The term “RNA” refers to ribonucleic acid. The polynucleotide or nucleic acid may be single-stranded or double-stranded, or may include both singlestranded regions and double-stranded regions.

[0082] In some embodiments, the terms “polynucleotide," “nucleic acid" and “oligonucleotide" encompass polynucleotide or nucleic acid or oligonucleotide analogs or modified polynucleotide or nucleic acid or oligonucleotide, which include, among others, nucleosides linked together via internucleoside linkages other than standard phosphodiester linkages, such as non-standard linkages of phosphorothioates, amide linkages, etc.; nucleosides with modified and/or synthetic nucleobases, for example inosine, xanthine, hypoxanthine, etc.; nucleosides with modified sugar residues, such as 2’-O-alkyl, 2’-halo, 2,3-dideoxy. 2 ’-halo-2 ’-deoxy, [3-D-ribo LNA. oc-L-ribo-LNA (e.g.. locked nucleic acids), etc.; and/or 5 ’-phosphate analogs, including, among others, phosphorothioate. phosphoacetate, phosphoramidate. monomethylphosphate, methylphosphonate, or phosphonocarboxylate.

[0083] “Duplex" and “ds" refer to a double-stranded nucleic acid (e.g., DNA or RNA) molecule comprised of two single-stranded polynucleotides that are complementary in their sequence (e.g., A pairs to T or U, C pairs to G), arranged in an antiparallel 5’ to 3’ orientation, and held together by hydrogen bonds between the nucleobases (e.g., adenine [A], guanine [G] . cytosine [C], thymine [T] , uridine [U]).

[0084] “Complementary" is used herein to describe the structural relationship between nucleotide bases that are capable of forming base pairs with one another. For example, a purine nucleotide base present on a polynucleotide that is complementary to a pyrimidine nucleotide base on a polynucleotide may base pair by forming hydrogen bonds with one another. Complementary nucleotide bases can base pair via Watson/Crick base pairing or in any other manner than forms stable duplexes or other nucleic acid structures.

[0085] “Watson/Crick Base-Pairing" refers to a pattern of specific pairs of nucleobases and analogs that bind together through sequence-specific hydrogen-bonds, e.g. A pairs with T or U, and G pairs with C. [0086] “Engineered,” “recombinant,” “non-naturally occurring,” and “variant,” when used with reference to a cell, a polynucleotide or a polypeptide refer to a material or a material corresponding to the natural or native form of the material that has been modified in a manner that would not otherwise exist in nature or is identical thereto but produced or derived from synthetic materials and/or by manipulation using recombinant techniques.

[0087] “Wild-type” and “naturally-occurring” refer to the form found in nature. For example, a wild-type polypeptide or polynucleotide sequence is a sequence present in an organism that can be isolated from a source in nature and which has not been intentionally modified by human manipulation.

[0088] “Coding sequence” and synonymously “encoding” refers to that part of a nucleic acid (e.g.. a gene) that encodes an amino acid sequence of a protein.

[0089] “Percent (%) sequence identity” refers to comparisons among polynucleotides and polypeptides, and are determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence for optimal alignment of the two sequences. The percentage may be calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. Alternatively, the percentage may be calculated by determining the number of positions at which either the identical nucleic acid base or amino acid residue occurs in both sequences or a nucleic acid base or amino acid residue is aligned with a gap to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. Those of skill in the art appreciate that there are many established algorithms available to align two sequences. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman (Smith and Waterman, Adv. Appl. Math., 1981, 2:482), by the homology alignment algorithm of Needleman and Wunsch (Needleman and Wunsch, J. Mol. Biol., 1970, 48:443), by the search for similarity method of Pearson and Lipman (Pearson and Lipman, Proc. Natl. Acad. Sci. USA, 1988, 85:2444), by computerized implementations of these algorithms (e.g., GAP, BESTFIT, FAST A, and TFASTA in the GCG Wisconsin Software Package), or by visual inspection, as known in the art. Examples of algorithms that are suitable for determining percent sequence identity and sequence similarity include, but are not limited to the BLAST and BLAST 2.0 algorithms (see, e.g., Altschul et al.. J. Mol. Biol., 1990, 215: 403-410; and Altschul et al., Nucleic Acids Res., 1977, 3389-3402). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information website. This algorithm involves first identify ing high scoring sequence pairs (HSPs) by identifying short words of length “W” in the query' sequence, which either match or satisfy some positive-valued threshold score “T,” when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (see Altschul et al, supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters “M” (reward score for a pair of matching residues; always >0) and “N” (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when; the cumulative alignment score falls off by the quantity "X" from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T. and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, M=5, N=-4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see, e.g., Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA, 1989, 89:10915). Exemplary determination of sequence alignment and % sequence identity can employ the BESTFIT or GAP programs in the GCG Wisconsin Software package (Accelrys, Madison WI), using default parameters provided.

[0090] “Reference sequence” refers to a defined sequence used as a basis for a sequence comparison. A reference sequence may be a subset of a larger sequence, for example, a segment of a full-length gene or polypeptide sequence. Generally, a reference sequence is at least 20 nucleotide or amino acid residues in length, at least 25 residues in length, at least 50 residues in length, at least 100 residues in length or the full length of the nucleic acid or polypeptide. Since two polynucleotides or polypeptides may each (1) comprise a sequence (i.e., a portion of the complete sequence) that is similar between the two sequences, and (2) may further comprise a sequence that is divergent between the two sequences, sequence comparisons between tw o (or more) polynucleotides or polypeptide are ty pically performed by comparing sequences of the two polynucleotides or polypeptides over a “comparison window” to identify and compare local regions of sequence similarity'.

[0091] “Comparison window” refers to a conceptual segment of contiguous nucleotide positions or amino acids residues wherein a sequence may be compared to a reference sequence. In some embodiments, the comparison window is at least 15 to 20 contiguous nucleotides or amino acids and wherein the portion of the sequence in the comparison window may comprise additions or deletions (i.e.. gaps) of 20 percent or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the tw o sequences. In some embodiments, the comparison window can be longer than 15-20 contiguous residues, and includes, optionally 30, 40, 50, 100, or longer windows.

[0092] “Corresponding to”, “reference to,” and “relative to” when used in the context of the numbering of a given amino acid or polynucleotide sequence refer to the numbering of the residues of a specified reference sequence when the given amino acid or polynucleotide sequence is compared to the reference sequence. In other words, the residue number or residue position of a given polymer is designated with respect to the reference sequence rather than by the actual numerical position of the residue within the given ammo acid or polynucleotide sequence. For example, a given amino acid sequence, such as that of a recombinant phosphatase, can be aligned to a reference sequence by introducing gaps to optimize residue matches betw een the two sequences. In these cases, although the gaps are present, the numbering of the residue in the given amino acid or polynucleotide sequence is made with respect to the reference sequence to which it has been aligned.

[0093] “ Mutation” refers to the alteration of a nucleic acid sequence. In some embodiments, mutations result in changes to the encoded polypeptide sequence (i.e., as compared to the original sequence without the mutation). In some embodiments, the mutation comprises a substitution, such tiiat a different amino acid is produced. In some alternative embodiments, the mutation comprises an addition, such that an amino acid is added (e.g., insertion) to the original polypeptide sequence. In some further embodiments, the mutation comprises a deletion, such that an amino acid is deleted from the original polypeptide sequence. Any number of mutations may be present in a given sequence.

[0094] “ Amino acid difference” and “residue difference” refer to a difference in the amino acid residue at a position of a polypeptide sequence relative to the amino acid residue at a corresponding position in a reference sequence. The amino acid positions of amino acid differences generally are referred to herein as “Xn,” where n refers to tire corresponding position in the reference sequence upon which the residue difference is based. In some instances herein, the specific amino acid residue difference at a position is indicated as “XnY” where “Xn” specified the corresponding residue and position of the reference polypeptide (as described above), and “Y” is the single letter identifier of the amino acid found in the engineered polypeptide (i.e., the different residue than in the reference polypeptide). In some instances (e.g., in the Tables in the Examples), the present disclosure also provides specific amino acid differences denoted by the conventional notation “AnB”, where A is the single letter identifier of the residue in the reference sequence, “n” is the number of the residue position in the reference sequence, and B is the single letter identifier of the residue substitution in the sequence of the engineered polypeptide. In some embodiments, the amino acid difference, e.g., a substitution, is denoted by the abbreviation “nB.” without the identifier for the residue in the reference sequence. In some instances, an amino acid residue difference or substitution may be a deletion and may be denoted by a where appropriate. [0095] In some instances, a polypeptide of the present disclosure can include one or more amino acid residue differences relative to a reference sequence, which is indicated by a list of the specified positions where residue differences are present relative to the reference sequence. In some embodiments, where more than one amino acid can be used in a specific residue position of a polypeptide, the various amino acid residues that can be used are separated by a “/” (e.g. , XI 1 A/Xl ID, XI 1 A/D, or 11 A/D). The present disclosure includes engineered polypeptide sequences comprising one or more amino acid differences that include either/or both conservative and non-conservative amino acid substitutions, as well as insertions and deletions of amino acids in the sequence.

[0096] “Amino acid substitution set” and “substitution set” refers to a group of amino acid substitutions within a polypeptide sequence. In some embodiments, substitution sets comprise 2, 3. 4, 5. 6, 7, 8, 9, 10, 11, 12, 13. 14, 15, or more amino acid substitutions. In some embodiments, a substitution set refers to the set of amino acid substitutions that is present in any of the variant RNA ligase polypeptides listed in any of the Tables in the Examples. In some embodiments, the amino acid sequence comprises at least each of the amino acid substitutions in the referenced substitution set. In the substitution sets, the individual substitutions are separated by a semicolon (“;”; e.g., T15E;S96P) or slash (“/”; e.g.. T15E/S96P, or 15E/96P).

[0097] “Conservative amino acid substitution” refers to a substitution of a residue with a different residue having a similar side chain, and thus typically involves substitution of the amino acid in the polypeptide with amino acids within the same or similar defined class of amino acids. By way of example and not limitation, an amino acid with an aliphatic side chain may be substituted with another aliphatic amino acid (e.g., alanine, valine, leucine, and isoleucine); an amino acid with hydroxyl side chain is substituted with another amino acid with a hydroxyl side chain (e.g., serine and threonine); an amino acids having aromatic side chains is substituted with another amino acid having an aromatic side chain (e.g.. phenylalanine, tyrosine, tryptophan, and histidine); air amino acid with a basic side chain is substituted with another amino acid with a basis side chain (e.g., lysine and arginine); an amino acid with an acidic side chain is substituted with another amino acid with an acidic side chain (e.g., aspartic acid or glutamic acid); and a hydrophobic or hydrophilic amino acid is replaced with another hydrophobic or hydrophilic amino acid, respectively.

[0098] “Non-conservative substitution” refers to substitution of an amino acid in the polypeptide with an amino acid with significantly differing side chain properties. Non-conservative substitutions may use amino acids between, rather than within, the defined groups and affect: (a) the structure of the peptide backbone in the area of the substitution (e.g., proline for glycine); (b) the charge or hydrophobicity; and/or (c) the bulk of the side chain. By way of example and not limitation, exemplary' non-conservative substitutions include an acidic amino acid substituted with a basic or aliphatic amino acid; an aromatic amino acid substituted with a small amino acid; and a hydrophilic amino acid substituted with a hydrophobic amino acid. [0099] “ Deletion” refers to modification to the polypeptide by removal of one or more amino acids from the reference polypeptide. Deletions can comprise removal of 1 or more amino acids, 2 or more amino acids, 5 or more amino acids, 10 or more amino acids, 15 or more amino acids, or 20 or more amino acids, up to 10% of the total number of amino acids, or up to 20% of the total number of amino acids making up the reference polypeptide while retaining enzymatic activity and/or retaining the improved properties of an engineered RNA ligase. Deletions can be directed to the internal portions and/or terminal portions of the polypeptide. In various embodiments, the deletion can comprise a continuous segment or can be discontinuous. As noted above, deletions are indicated by and may be present in substitution sets.

[0100] “ Insertion” refers to modification to the polypeptide by addition of one or more amino acids from the reference polypeptide. Insertions can be in the internal portions of the polypeptide, or to the carboxy or amino terminus. Insertions as used herein include fusion proteins as is known in the art. The insertion can be a contiguous segment of amino acids or separated by one or more of the amino acids in the naturally occurring polypeptide.

[0101] “ Functional fragment” and “biologically active fragment” are used interchangeably herein, to refer to a polypeptide that has an amino-tenninal and/or carboxy -terminal deletion(s) and/or internal deletions, but where the remaining amino acid sequence is identical to the corresponding positions in die sequence to which it is being compared (e.g., a full length recombinant phosphatase of the present invention) and that retains substantially all of the activity of the full-length polypeptide.

[0102] “ Isolated polypeptide” refers to a polypeptide which is substantially separated from other contaminants that naturally accompany it (e.g., protein, lipids, and polynucleotides). The term embraces polypeptides which have been removed or purified from their naturally -occurring environment or expression system (e.g., host cell or in vitro synthesis). The recombinant phosphatase polypeptides may be present within a cell, present in tire cellular medium, or prepared in various forms, such as lysates or isolated preparations. As such, in some embodiments, the recombinant phosphatase polypeptides provided herein are isolated polypeptides.

[0103] “Substantially pure polypeptide” or “purified” refers to a composition in which the polypeptide species is the predominant species present (i.e., on a molar or weight basis it is more abundant than any other individual macromolecular species in the composition), and is generally a substantially purified composition when the object species comprises at least about 50 percent of the macromolecular species present by mole or % weight. Generally, a substantially pure phosphatase composition will comprise about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 95% or more, and about 98% or more of all macromolecular species by mole or % weight present in the composition. In some embodiments, the object species is purified to essential homogeneity (i.e., contaminant species camrot be detected in the composition by conventional detection methods) wherein the composition consists essentially of a single macromolecular species. Solvent species, small molecules (<500 Daltons), and elemental ion species are not considered macromolecular species. In some embodiments, the isolated recombinant phosphatase polypeptides are substantially pure polypeptide compositions.

[0104] “Improved enzyme property” refers to a recombinant phosphatase polypeptide that exhibits an improvement in any enzyme property as compared to a reference phosphatase polypeptide, such as a wild-type phosphatase polypeptide or another recombinant phosphatase polypeptide. Improved properties include but are not limited to such properties as increased enzymatic activity, increased product yield, increased protein expression, increased thcnnoactivity, increased thermostability, increased stability, increased substrate specificity' and/or affinity, increased substrate range, increased specific activity, increased resistance to substrate and/or end-product inhibition, increased chemical stability, improved solvent stability, increased solubility, and increased inhibitor resistance or tolerance. Exemplary improved properties are provided in the Examples.

[0105] “ Codon optimized” refers to changes in the codons of the polynucleotide encoding a protein to those preferentially used in a particular organism such that the encoded protein is more efficiently expressed in that organism. Although the genetic code is degenerate, in that most amino acids are represented by several codons, called “synony ms” or “synonymous” codons, it is well known that codon usage by particular organisms is nonrandom and biased towards particular codon triplets. This codon usage bias may be higher in reference to a given gene, genes of common function or ancestral origin, highly expressed proteins versus low copy number proteins, and the aggregate protein coding regions of an organism's genome. In some embodiments, the polynucleotides encoding the phosphatase enzymes are codon optimized for optimal production from the host organism selected for expression.

[0106] “ Control sequence” refers herein to include all components that are necessary’ or advantageous for the expression of a polynucleotide and/or polypeptide of the present disclosure. Each control sequence may be native or foreign (e.g., heterologous) to the nucleic acid sequence encoding the polypeptide. Such control sequences include, but are not limited to, leaders, polyadeny lation sequences, propeptide sequences, promoter sequences, signal peptide sequences, initiation sequences, and transcription terminators. In some embodiments, the control sequences include a promoter, and transcriptional and translational stop signals.

[0107] “Operably linked” or “operatively linked” refers to a configuration in which a control sequence is appropriately placed (i.e., in a functional relationship) at a position relative to a polynucleotide of interest such that the control sequence directs or regulates the expression of the polynucleotide of interest, and where appropriate, expression of the encoded polypeptide of interest. [0108] “ Promoter” or “promoter sequence” refers to a nucleic acid sequence that is recognized by a host cell for expression of a polynucleotide of interest, such as a coding sequence. The promoter sequence contains transcriptional control sequences that mediate the expression of a polynucleotide of interest. The promoter may be any nucleic acid sequence which shows transcriptional activity in the host cell of choice including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the host cell.

[0109] “ Suitable reaction conditions” or “suitable conditions” refers to those conditions in the enzy matic conversion reaction solution (e.g., ranges of enzyme loading, substrate loading, temperature, pH, buffers, co-solvents, etc.) under which a phosphatase polypeptide of the present disclosure is capable of cleaving a phosphomonoester bond. Exemplary' “suitable reaction conditions” are provided herein (see, the Examples).

[0110] “ Product” in the context of an enzymatic conversion process refers to the compound or molecule resulting from the action of the phosphatase polypeptide on the substrate.

[0111] “Culturing” refers to the growing of a population of cells under suitable conditions using any suitable medium (e.g., liquid, gel. or solid).

[0112] “Vector” is a recombinant construct for introducing a polynucleotide of interest into a cell. In some embodiments, the vector is an expression vector that is operably linked to a suitable control sequence capable of effecting the expression in a suitable host of the polynucleotide or a polypeptide encoded in the polynucleotide. In some embodiments, an “expression vector” has a promoter sequence operably linked to the polynucleotide (e.g., transgene) to drive expression in a host cell, and in some embodiments, also comprises a transcription tenninator sequence.

[0113] “Expression” includes any step involved in the production of the polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, and post-translational modification. In some embodiments, the tenn also encompasses secretion of the polypeptide from a cell.

[0114] “ Produces” refers to the production of proteins and/or other compounds by cells. It is intended that the term encompass any step involved in the production of polypeptides including, but not limited to, transcription, post-transcriptional modification, translation, and post-translational modification. In some embodiments, the term also encompasses secretion of the polypeptide from a cell.

[0115] “Heterologous” or “recombinant” refers to the relationship between two or more nucleic acid or polypeptide sequences (e.g., a promoter sequence, signal peptide, terminator sequence, etc.) that are derived from different sources and are not associated in nature. [0116] “ Host cell” and ‘’host strain” refer to suitable hosts for expression vectors comprising a polynucleotide provided herein (e.g., a polynucleotide sequences encoding at least one recombinant phosphatase). In some embodiments, the host cells are prokaryotic or eukaryotic cells that have been transformed or transfected with vectors constructed using recombinant DNA techniques, and progeny thereof, as known in the art.

Recombinant Phosphatases

[0117] In one aspect, the present disclosure provides recombinant phosphatases, polynucleotides encoding the recombinant phosphatases, and use of the recombinant phosphatases for cleaving phosphate monoesters or analogs thereof. In some embodiments, the recombinant phosphatases display increased expression into the cytoplasm, increased solubility. In some embodiments, the recombinant phosphatases display increased phosphatase activity on an oligonucleotide having a 3’- phosphate group or a 3’-phosphate-NTP, increased thermal stability, increased resistance to proteolysis, and/or increased resistance to inhibition or deactivation by immobilization on a support resin.

[0118] In some embodiments, the recombinant phosphatase, or a functional fragment thereof, comprises an amino acid sequence having at least 70%. 75%. 80%, 81%, 82%. 83%. 84%, 85%, 86%. 87%, 88%. 89%. 90%, 91%, 92%. 93%. 94%, 95%, 96%. 97%. 98%, 99%, or more sequence identity to a reference sequence correspoding to amino acid residues 12 to the carboxyl terminal of an even- numbered SEQ ID NO. of SEQ ID NOs: 2-14. or to a reference sequence corresponding to an even- numbered SEQ ID NO. of SEQ ID NOs: 2-14.

[0119] In some embodiments, the recombinant phosphatase, or a functional fragment thereof, comprises an amino acid sequence having at least 70%, 75%, 80%. 81%, 82%, 83%, 84%. 85%, 86%, 87%, 88%, 89%, 90%. 91%. 92%, 93%, 94%. 95%. 96%, 97%, 98%. 99%. or more sequence identity to a reference sequence corresponding to amino acid residues 12 to the carboxyl terminal of the sequence of SEQ ID NO: 2.

[0120] In some embodiments, the recombinant phosphatase, or a functional fragment thereof, comprises an amino acid sequence having at least 70%, 75%, 80%. 81%, 82%, 83%, 84%. 85%, 86%, 87%, 88%, 89%, 90%. 91%, 92%, 93%, 94%. 95%, 96%, 97%, 98%. 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to the carboxyl terminal of the sequence of SEQ ID NO: 4.

[0121] In some embodiments, the recombinant phosphatase, or a functional fragment thereof, comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%. 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to the carboxyl terminal of the sequence of SEQ ID NO: 6. [0122] In some embodiments, the recombinant phosphatase, or a functional fragment thereof, comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to the carboxyl terminal of the sequence of SEQ ID NO: 8.

[0123] In some embodiments, the recombinant phosphatase, or a functional fragment thereof, comprises an amino acid sequence having at least 70%, 75%. 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to the carboxyl terminal of the sequence of SEQ ID NO: 10.

[0124] In some embodiments, the recombinant phosphatase, or a functional fragment thereof, comprises an amino acid sequence having at least 70%, 75%. 80%, 81%, 82%, 83%. 84%, 85%, 86%, 87%, 88%, 89%. 90%, 91%, 92%, 93%. 94%, 95%, 96%, 97%. 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to the carboxyl terminal of the sequence of SEQ ID NO: 12.

[0125] In some embodiments, the recombinant phosphatase, or a functional fragment thereof, comprises an amino acid sequence having at least 70%. 75%. 80%, 81%, 82%. 83%. 84%, 85%, 86%, 87%, 88%. 89%. 90%, 91%, 92%, 93%. 94%, 95%, 96%, 97%. 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to the carboxyl terminal of the sequence of SEQ ID NO: 14.

[0126] In some embodiments, th recombinant phosphatase, or a functional fragment thereof, comprises an amino acid sequence having at least 70%, 75%, 80%. 81%, 82%, 83%, 84%. 85%, 86%, 87%, 88%, 89%, 90%. 91%. 92%, 93%, 94%. 95%. 96%, 97%, 98%. 99%. or more sequence identity to a reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of an even- numbered SEQ ID NO. of SEQ ID NOs: 2-18 and 24-1052. or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 2-18 and 24-1052.

[0127] In some embodiments, the recombinant phosphatase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%. 82%, 83%, 84%, 85%. 86%, 87%, 88%, 89%, 90%. 91%, 92%, 93%, 94%, 95%, 96%. 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of an even-numbered SEQ ID NO. of SEQ ID NOs: 2-14 or SEQ ID NO: 18. 84. 214, 232, 562. 656, or 774. or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 2-14, or SEQ ID NO: 18, 84, 214, 232. 562, 656, or 774.

[0128] In some embodiments, the recombinant phosphatase comprises an alkaline phosphatase of Thermoflexibacter, Pyrococcus, Thennotoga, Pseudothermotoga, or Bacillus. [0129] In some embodiments, the recombinant phosphatase comprises an alkaline phosphatase of Thennoflexibacter. In some embodiments, the recombinant phosphatase comprises an alkaline phosphatase of Thermoflexibacter ruber.

[0130] In some embodiments, the recombinant phosphatase comprises an alkaline phosphatase of Pyrococcus. In some embodiments, the recombinant phosphatase comprises an alkaline phosphatase of Pyrococcus abyssi, Pyrococcus chitonophagus, Pyrococcus furiosus, Pyrococcus glycovorans, Pyrococcus horikoshii, Pyrococcus kodakaraensis, Pyrococcus kukulkanii, Pyrococcus woesei, or Pyrococcus yayanosii.

[0131] In some embodiments, the recombinant phosphatase comprises an alkaline phosphatase of Thermotoga. In some embodiments, the recombinant phosphatase comprises an alkaline phosphatase of Thermotoga caldifontis, Thermotoga elfii, Thermotoga hypogea, Thermotoga lettingae, Thermotoga maritima, Thermotoga naphthophila, Thermotoga neapolitana, Thermotoga petrophila, Thermotoga profunda, Thermotoga subterranea, Thermotoga thermarum, P seudothermotoga caldifontis, P seudothermotoga elfii, P seudothermotoga hypogea, P seudothermotoga hypogea, P seudothermotoga lettingae, Pseudothermotoga profunda, P seudothermotoga subterranea, or P seudothermotoga therm arum.

[0132] In some embodiments, the recombinant phosphatase comprises an alkaline phosphatase of bacillus. In some embodiments, the recombinant phosphatase comprises an alkaline phosphatase of Bacillus subtilis, Bacillus licheniformis, Bacillus siamensis, Bacillus mycoides, Bacillus thuringiensis, Bacillus pseudomycoides. Bacillus spizizenii, Bacillus anthracis, Bacillus paranthracis, Bacillus cereus, Bacillus mobilis, Bacillus toyonensis, Bacillus amyloliquefaciens, Bacillus pacificus, Bacillus pumilus, Bacillus velezensis, Bacillus paramycoides, Bacillus amyloliquefaciens, Bacillus albus, Bacillus sonorensis, Bacillus badius, or Bacillus thuringiensis.

[0133] In some embodiments, the recombinant phosphatase is the alkaline phosphatase of Thermoflexibacter ruber, Pyrococcus furiosus, Thermotoga maritima, Thermotoga sp. 50 64, Pseudothermotoga lettingae, Thermotoga neapolitana, or Bacillus licheniformis.

[0134] In some embodiments, the recombinant phosphatase comprises an N-terminal deletion of up to 5, 10. 25, 30, 40, 45, or 50 amino acids of the naturally occurring amino acid sequence of the phosphatase. For the exemplary phosphatases of SEQ ID NO: 2. 4, 6, 8, 10, 12. and 14, the N- terminal deletion begins from amino acid residue 12 to account for the His-tag fused to the N- terminus of the naturally occurring phosphatase.

[0135] In some embodiments, the length of the deletion is sufficient for preferential expression of the phosphatase in the cytoplasm of a bacterial host cell. In some embodiments, the length of the deletion is sufficient to increase expression of soluble form of the phosphatase. [0136] In some embodiments, the N-terminal deletion removes or inactivates signaling sequences at die N-terminal sequence of die phosphatase. Without being bound by any theory of operation, bacterial signaling sequences for transport of proteins across the inner membrane typically involves the general secretion (Sec) pathway, the Twin arginine translocation system, or the membrane protein invertase. The targeting of pre-proteins to these pathways are dependent on the selectivity for the respective signal peptide, which include Sec signal peptide, lipoprotein signal peptide. Tat signal peptide, and the prelin signal peptide. The structure and sequences of the signal peptides are known, and the N-terminal deletions can be designed to inactivate or delete the relevant signal peptides (see, e.g., Kaushik et al., Front. Physiol.. 2022. Volume 13, Article 933153).

[0137] In some embodiments, the N-terminal deletion preserves the capability of the phosphatase to form an active dimer subunit quaternary structure, i.e., a multimer formed of two phosphatase monomers. In addition to activity', the presence of a dimer can be ascertained by known techniques, for example, molecular sieve chromatography.

[0138] In some embodiments, the recombinant phosphatase, or a functional fragment thereof, comprises an amino acid sequence hay ing at least 70%, 75%. 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence from amino acid residue 12 to the carboxy l terminal of SEQ ID NO: 16 or 18, or to a reference sequence corresponding to SEQ ID NO: 16 or 18.

[0139] In some embodiments, the recombinant phosphatase comprises one or more amino acid differences relative to the reference sequence corresponding to amino acid residue 12 to the carboxyl terminal of an even-numbered SEQ ID NO. of SEQ ID NOs: 2-14. In some embodiments, the amino acid differences are based on alignment of the amino acid sequence of the naturally occurring phosphatase, and changing the amino acid residue of one sequence to the different amino acid residue present in the amino acid sequence of another naturally occurring phosphatase sequence, thereby generating an amino acid difference relative to the parent amino acid sequence.

[0140] In some embodiments, the recombinant phosphatase comprises an amino acid sequence having at least 70%, 75%, 80%. 81 %, 82%, 83%, 84%. 85%, 86%, 87%, 88%. 89%, 90%, 91%, 92%, 93%, 94%, 95%. 96%, 97%, 98%, 99%. or more sequence identity to a reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of an even-numbered SEQ ID NO. of SEQ ID NOs: 12 and 24-1052, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 12 and 24-1052, wherein the amino acid sequence comprises one or more amino acid differences relative to the reference sequence corresponding to amino acid residues 12 to the carboxy l terminus of an even-numbered SEQ ID NOs: 12 and 24-1052, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 12 and 24-1052. [0141] In some embodiments, the recombinant phosphatase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of SEQ ID NO: 12, 18, 84, 214, 232, 562, 656, or 774, or to the reference sequence corresponding to SEQ ID NO: 12, 18, 84, 214, 232, 562, 656, or 774, wherein the amino acid comprises one or more amino acid differences relative to the reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of SEQ ID NO: 12, 18, 84. 214, 232, 562, 656, or 774, or to the reference sequence corresponding to SEQ ID NO: 12. 18, 84, 214. 232, 562, 656. or 774.

[0142] In some embodiments, the recombinant phosphatase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of an even-numbered SEQ ID NO. of SEQ ID NOs: 24-80, or to the reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 24-80, wherein the amino acid sequence comprises one or more amino acid differences relative to the reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of SEQ ID NO: 18, or to the reference sequence corresponding to SEQ ID NO: 18.

[0143] In some embodiments, the recombinant phosphatase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of SEQ ID NO: 18, or to the reference sequence corresponding to SEQ ID NO: 18, wherein the amino acid sequence comprises at one or more amino acid differences relative to the reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of SEQ ID NO: 18, or to the reference sequence corresponding to SEQ ID NO: 18.

[0144] In some embodiments, the amino acid sequence of the recombinant phosphatase comprises at least an amino acid difference at amino acid position 23, 25, 50, 56, 63, 99, 102, 136, 193, 195, 198, 224, 234, 235, 267, 306, or 345, or combinations thereof, wherein the amino acid difference is relative to the reference sequence corresponding to residues 12 to the carboxyl terminus of SEQ ID NO: 18, or to the reference sequence corresponding to SEQ ID NO: 18.

[0145] In some embodiments, the amino acid sequence of the recombinant phosphatase comprises at least an amino acid difference 23Q/R, 25L/R, 50H, 56A, 63T, 99N. 102P. 1361, 193A/G, 195D/E/S/T, 198R, 224E. 234E, 235D, 267Q, 306E, or 345R, or combinations thereof, wherein the amino acid difference is relative to the reference sequence corresponding to residues 12 to the carboxyl terminus of SEQ ID NO: 18, or to die reference sequence corresponding to SEQ ID NO: 18. [0146] In some embodiments, the amino acid sequence of the recombinant phosphatase comprises at least an amino acid difference K23Q/R, K25L/R, K50H, C56A, K63T, K99N, K102P, K136I, K193A/G/D/E/S/T, K198R, K224E, K234E, K235D, K267Q, K306E, or K345R, or combinations thereof, wherein the amino acid difference is relative to the reference sequence corresponding to residues 12 to the carboxyl terminus of SEQ ID NO: 18, or to the reference sequence corresponding to SEQ ID NO: 18.

[0147] It is to be understood in the foregoing that equivalent amino acid positions relative to SEQ ID NO: 18 can be determined for other reference phosphatase sequences by sequence aligmnent. For example, the equivalent amino acid position can be determined for the reference sequence of SEQ ID NO: 12 for the amino acid positions relative to SEQ ID NO: 18, particularly given that SEQ ID NO: 18 contains a defined deletion of the amino terminal sequence of SEQ ID NO: 12.

[0148] In some embodiments, recombinant phosphatase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%. 83%, 84%, 85%, 86%. 87%, 88%, 89%, 90%. 91%, 92%, 93%, 94%, 95%, 96%. 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of SEQ ID NO: 12, 84. 214, 232, 562, 656, or 774, or to a reference sequence corresponding to SEQ ID NO: 12, 84, 214, 232, 562, 656, or 774, wherein the amino acid sequence comprises one or more amino acid differences relative to the reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of SEQ ID NO: 12, 84, 214, 232, 562, 656, or 774, or to the reference sequence corresponding to SEQ ID NO: 12, 84, 214, 232, 562, 656, or 774.

[0149] In some embodiments, the recombinant phosphatase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%. 96%, 97%, 98%, 99%. or more sequence identity to a reference sequence corresponding to amino acid residues 12 to the carboxy l terminus of an even-numbered SEQ ID NO. of SEQ ID NOs: 24-1052, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 24-1052, wherein the amino acid sequence comprises one or more amino acid differences relative to the reference sequence corresponding to amino acid residues 12 to the carboxy l terminus of SEQ ID NO: 12, or to the reference sequence corresponding to SEQ ID NO: 12.

[0150] In some embodiments, he recombinant phosphatase comprises an amino acid sequence having at least 70%. 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%. 92%, 93%, 94%, 95%, 96%. 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of SEQ ID NO: 84, 214, 232, 562, 656, or 774, or to the reference sequence corresponding to SEQ ID NO: 84, 214, 232, 562, 656, or 774, wherein the amino acid sequence comprises one or more amino acid differences relative to the reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of SEQ ID NO: 12, or to the reference sequence corresponding to SEQ ID NO: 12.

[0151] In some embodiments, the recombinant phosphatase comprises an amino acid sequence having at least 70%, 75%, 80%. 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%. 96%, 97%, 98%, 99%. or more sequence identity to the reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of SEQ ID NO: 12, or to the reference sequence corresponding to SEQ ID NO: 12, wherein the amino acid sequence comprises one or more amino acid differences relative to the reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of SEQ ID NO: 12, or to the reference sequence corresponding to SEQ ID NO: 12.

[0152] In some embodiments, the amino acid sequence of the recombinant phosphatase comprises at least an amino acid difference at amino acid position 11, 12, 13, 14. 15, 16, 17, 18, 19, 20, 21, 22, 23. 24, 25, 26. 27, 28, 29, 30, 31, 32, 33, 34, 35. 36, 37, 38, 39, 40, 41, 42, 43, 44. 45, 46, 47, 48. 49, 50, 51, 52, 56. 57, 60, 61, 63, 81, 82, 83, 89, 93. 95, 96, 98, 100, 101, 105, 108, 128, 138, 139, 141, 148, 150, 158, 169, 174, 175, 181, 202, 205, 206. 207, 211, 214, 215, 223, 224, 225, 226, 227, 228, 230, 231, 234, 235, 240, 241, 244, 247, 260, 261, 263, 266, 271, 291, 299, 301, 302, 306, 307, 336, 340, 341, 343, 345, 346, 347, 353, 357, 363, 367, 368, 371, 389, 390, 391, 392, or 393, or combinations thereof, wherein the amino acid difference is relative to the reference sequence corresponding to residues 12 to the carboxyl terminus of SEQ ID NO: 12, or to the reference sequence corresponding to SEQ ID NO: 12.

[0153] In some embodiments, the amino acid sequence of the recombinant phosphatase comprises at least an amino acid difference or amino acid residue 11A/D, 12-/D/E/G/Q/R/V/W. 13-/A/G/P/R/V/Y. 14-/E/F/I, 15-/E/Q/Y, 16-/D/L/P/V, 17-/A/D/S/T/V, 18-/D/G/I/L/P/Q/S/T, 19-/D/E/S/T/Y, 20- /A/G/N/Q, 21-/E/K/M/V. 22-/A/E/G/I/M/R, 23-/C/S/V, 24-/K/V, 25-/E/S/V, 26-, 27-/I, 28-/S, 29- /G/M, 30-/P/R/S, 31-/G/Q/R/T/V, 32-/K/T/V, 33-, 34-, 35-/F/P/R, 36-/C/G, 37-/D, 38-/D/R/S, 39-, 40- , 41-, 42-/C, 43-/G, 44-/K/V, 45-/V, 46-, 47-/E/K/P/T, 48-/N. 49-/H, 50-/A/N/P/R/T/V, 51R, 52S, 56P, 57F/R, 60A/G, 61C/S/V, 63V, 81S, 82G, 83L/W, 89H, 93D, 95A/F/L, 96P, 98V, 100F, 101V, 105S, 108S, 128L, 138N, 139A/E/I/K/V, 141P/Q/R, 148A/W, 150K/S, 158V, 169A, 174V, 175P, 181G, 202Y, 205G, 206P, 207V, 21 IS, 214S/W, 215L, 2231, 2241, 225E, 226S, 227W, 228R, 230L, 231C/R, 234E/T, 235N, 240A, 241L, 244A/E/W, 247M, 260V, 261 A, 2631, 2661, 271A/V/W, 291A/L, 299S, 301M, 302L, 306H, 307A, 3361, 340D/H, 341L, 343G/K/M/Q/R, 345L, 3461, 347A, 353A/Q/T, 357A, 363H, 367A. 368G, 371L, 389L, 390D/H/M/R/W/Y, 391W, or 392G/L/N/R, or combinations thereof, wherein the amino acid difference is relative to the reference sequence corresponding to residues 12 to the carboxyl terminus of SEQ ID NO: 12, or to the reference sequence corresponding to SEQ ID NO: 12. [0154] In some embodiments, the amino acid sequence of the recombinant phosphatase comprises at least an amino acid difference or amino acid residue G11 A/D, C12-/D/E/G/Q/R/V/W, S13- /A/G/P/R/V/Y, P14-/E/F/I, T15-/E/Q/Y, E16-/D/L/P/V, K17-/A/D/S/T/V, K18-/D/G/I/L/P/Q/S/T, A19-/D/E/S/T/Y, K20-/A/G/N/Q, S21-/E/K/M/V, L22-/A/E/G/I/M/R, P23-/C/S/V, N24-/K/V, T25- /E/S/V, P26-, F27-/I. T28-/S, P29-/G/M, T30-/P/R/S, L31-/G/Q/R/T/V, A32-/K/T/V, E33-, Q34-, N35-/F/P/R. P36-/C/G, L37-/D, K38-/D/R/S, R39-, Q40-, W41-, A42-/C, E43-/G, P44-/K/V, I45-/V, E46-, S47-/E/K/P/T, Y48-/N. Q49-/H, I50-/A/N/P/R/T/V, K51R. N52S, A56P, K57F/R. N60A/G, G61C/S/V, P63V, A81S. A82G, M83L/W, K89H. E93D, C95A/F/L, S96P. I98V. L100F, I101V, A105S, A108S, N128L, K138N, S139A/E/I/K/V, K141P/Q/R, E148A/W, N150K/S, A158V, C169A, Q174V. K175P. E181G, F202Y, R205G, S206P, A207V, N211S, P214S/W, E215L, V223I. L224I, D225E. N226S, M227W, N228R, I230L, A231C/R. K234E/T. Q235N. G240A. F241L, Q244A/E/W, N247M, Q260V, S261A, K263I, L2661, Q271A/V/W. D291A/L. Q299S. T301M. 1302L, K306H, V307A. L336I, N340D/H. I341L, E343G/K/M/Q/R. K345L. V346I, E347A, G353A/Q/T. S357A. Y363H, C367A, Q368G, K371L, F389L, G390D/H/M/R/W/Y, F391W, T392G/L/N/R, or S393A/E/G/I/K/L/M/Q/R, or combinations thereof, wherein the amino acid difference is relative to the reference sequence corresponding to residues 12 to the carboxyl terminus of SEQ ID NO: 12. or to the reference sequence corresponding to SEQ ID NO: 12.

[0155] In some embodiments, the amino acid sequence of the recombinant phosphatase comprises at least an amino acid difference at amino acid position 19. 22, 50, 138. 139, 141, 158. 169, 244, 260. 306, 307, 340. 357, 390, 392. or 393, or combinations thereof, wherein the amino acid difference is relative to the reference sequence corresponding to residues 12 to the carboxyl terminus of SEQ ID NO: 12, or to the reference sequence corresponding to SEQ ID NO: 12.

[0156] In some embodiments, the amino acid sequence of the recombinant phosphatase comprises at least an amino acid difference or amino acid residue 19-/D/E/S/T/Y, 22-/A/E/G/I/M/R, 50H/I/R, 138N, 139A/E/I/K/V, 141K/P/Q/R. 158V, 169A, 244A/E/W, 260Q/V, 306E/H, 307A. 340D/H, 357A, 390D/H/M/R/W/Y, 392G/L/N/R. or 393A/E/G/I/K/L/M/Q/R, or combinations thereof, wherein the amino acid difference is relative to the reference sequence corresponding to residues 12 to the carboxyl terminus of SEQ ID NO: 12, or to the reference sequence corresponding to SEQ ID NO: 12.

[0157] In some embodiments, the amino acid sequence of the recombinant phosphatase comprises at least an amino acid difference or amino acid residue A19D/E, L22G, I50R, K138N, S139A, K141P, A158V, C169A, Q244W, Q260V, K306H, V307A, N340D, S357A, G390H, T392L, or S393G, or combinations thereof, wherein the amino acid difference is relative to the reference sequence corresponding to residues 12 to the carboxyl terminus of SEQ ID NO: 12, or to the reference sequence corresponding to SEQ ID NO: 12. [0158] In some embodiments, the amino acid sequence of the recombinant phosphatase comprises at least an amino acid difference at amino acid position 11, 13, 14, 16, 17, 18, 19, 20, 21. 28, 29, 31, 35, 42, 44, or 61, or combinations thereof, wherein the amino acid difference is relative to the reference sequence corresponding to residues 12 to the carboxyl terminus of SEQ ID NO: 12, or to the reference sequence corresponding to SEQ ID NO: 12.

[0159] In some embodiments, the amino acid sequence of the recombinant phosphatase comprises at least an amino acid difference or amino acid residue 1 ID, 13G. 141, 16D, 17A/D/S/T/V, 18D/G/L/P/Q/S/T, 19D/S/T, 20A/G/N/Q, 21E, 28S, 29M, 3 IT, 35P, 42C, 44V, or 61 S, or combinations thereof, wherein the amino acid difference is relative to the reference sequence corresponding to residues 12 to the carboxyl terminus of SEQ ID NO: 12, or to the reference sequence corresponding to SEQ ID NO: 12.

[0160] In some embodiments, the amino acid sequence of the recombinant phosphatase comprises at least an amino acid difference or amino acid residue G11D, S13G, P14I, E16D, K17A/D/S/T/V, K18D/G/L/P/Q/S/T, A19D/S/T, K20A/G/N/Q, S21E, T28S, P29M. L31T, N35P, A42C, P44V, or G61S. or combinations thereof, wherein the amino acid difference is relative to the reference sequence corresponding to residues 12 to the carboxy l terminus of SEQ ID NO: 12, or to the reference sequence corresponding to SEQ ID NO: 12.

[0161] In some embodiments, the recombinant phosphatase comprises an amino acid sequence having at least 70%, 75%, 80%. 81%, 82%, 83%, 84%. 85%, 86%, 87%, 88%. 89%, 90%, 91%, 92%, 93%, 94%, 95%. 96%, 97%, 98%, 99%. or more sequence identity to a reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of an even-numbered SEQ ID NO. of SEQ ID NOs: 24-1052, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 24-1052, wherein the amino acid sequence comprises one or more amino acid differences relative to the reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of SEQ ID NO: 84, 214. 232, 562, 656, or 774, or to the reference sequence corresponding to SEQ ID NO: 84, 214, 232, 562, 656, or 774.

[0162] In some embodiments, the recombinant phosphatase comprises an amino acid sequence having at least 70%, 75%, 80%. 81%, 82%, 83%, 84%. 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%. 96%, 97%, 98%, 99%. or more sequence identity to a reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of SEQ ID NO: 84, 214, 232, 562, 656, or 774, or to a reference sequence corresponding to SEQ ID NO: 84, 214, 232, 562, 656, or 774. wherein the amino acid sequence comprises one or more amino acid difference relative to the reference sequence corresponding to amino acid residues 12 to the carboxy l terminus of SEQ ID NO: 84, 214, 232, 562, 656, or 774, or to the reference sequence corresponding to SEQ ID NO: 84, 214, 232, 562, 656, or 774. [0163] In some embodiments, the amino acid sequence of the recombinant phosphatase comprises at least an amino acid difference at amino acid position 11, 12, 13, 14, 15, 16, 17, 18, 19. 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 56, 57, 60, 61, 63, 81. 82, 83, 89, 93, 95, 96, 98, 100, 101, 105, 108, 128, 138, 139. 141, 148, 150, 158, 169, 174, 175, 181. 202, 205, 206, 207, 211, 214, 215, 223, 224, 225, 226, 227, 228, 230,

231, 234, 235. 240, 241, 244. 247, 260, 261, 263, 266, 271, 291, 299. 301, 302, 306, 307, 336, 340, 341, 343, 345. 346, 347, 353. 357, 363, 367, 368, 371, 389, 390, 391. 392, or 393. or combinations thereof, wherein the amino acid difference is relative to the reference sequence corresponding to residues 12 to the carboxyl terminus of SEQ ID NO: 84, 214, 232, 562. 656, or 774. or to the reference sequence corresponding to SEQ ID NO: 84, 214. 232, 562, 656, or 774.

[0164] In some embodiments, the amino acid sequence of the recombinant phosphatase comprises at least an amino acid difference or amino acid residue 11A/D, 12-/D/E/G/Q/R/V/W, 13-/A/G/P/R/V/Y, 14-/E/F/I, 15-/E/Q/Y, 16-/D/L/P/V, 17-/A/D/S/T/V, 18-/D/G/I/L/P/Q/S/T, 19-/D/E/S/T/Y, 20- /A/G/N/Q, 21-/E/K/M/V, 22-/A/E/G/I/M/R, 23-/C/S/V, 24-/K/V, 25-/E/S/V, 26-, 27-/I, 28-/S, 29- /G/M. 30-/P/R/S, 31-/G/Q/R/T/V, 32-/K/T/V, 33-, 34-, 35-/F/P/R, 36-/C/G, 37-/D, 38-/D/R/S, 39-, 40- , 41-, 42-/C, 43-/G, 44-/K/V, 45-/V, 46-, 47-/E/K/P/T, 48-/N, 49-/H, 50-/A/I/N/P/R/T/V. 51R, 52S. 56P, 57F/R. 60A/G, 61C/S/V, 63V. 81S, 82G, 83L/W. 89H, 93D, 95A/F/L. 96P, 98V. 100F. 101V, 105S. 108S. 128L. 138N, 139A/E/I/K/V, 141K/P/Q/R, 148A/W, 150K/S. 158V, 169A, 174V, 175P, 181G, 202Y, 205G, 206P, 207V. 21 IS. 214S/W, 215E, 2231, 2241. 225E. 226S. 227W, 228R, 230L, 231C/R, 234E/T, 235N, 240A, 241L. 244A/E/W, 247M. 260Q/V, 261 A, 2631, 2661, 271A/V/W. 291A/L, 299S, 301M, 302L, 306H. 307A, 3361. 340D/H, 341L, 343G/K/M/Q/R, 345L, 3461, 347A, 353A/Q/T, 357A, 363H, 367A, 368G, 371L, 389L, 390D/G/H/M/R/W/Y, 391W, 392G/L/N/R/T, or 393A/E/G/I/K/L/M/Q/R, or combinations thereof, wherein the amino acid difference is relative to the reference sequence corresponding to residues 12 to the carboxyl terminus of SEQ ID NO: 84. 214,

232. 562, 656, or 774, or to the reference sequence corresponding to SEQ ID NO: 84, 214, 232. 562, 656. or 774.

[0165] In some embodiments, the amino acid sequence of the recombinant phosphatase comprises at least an amino acid difference at amino acid position 19. 22, 50, 138. 139, 141, 158. 169, 244, 260. 306, 307, 340. 357, 390, 392. or 393. or combinations thereof, wherein the amino acid difference is relative to the reference sequence corresponding to residues 12 to the carboxyl terminus of SEQ ID NO: 84, 214, 232, 562, 656, or 774, or to the reference sequence corresponding to SEQ ID NO: 84, 214. 232, 562, 656. or 774.

[0166] In some embodiments, the amino acid sequence of the recombinant phosphatase comprises at least an amino acid difference or amino acid residue 19-/D/E/S/T7Y, 22-/A/E/G/I/M/R, 50H/I/R, 138N, 139A/E/I/K/V, 141K/P/Q/R. 158V, 169A, 244A/E/W, 260Q/V, 306E/H, 307A. 340D/H, 357A, 390D/G/H/M/R/W/Y, 392G/L/N/R/T, 393A/E/G/I/K/E/M/Q/R, or combinations thereof, wherein the amino acid difference is relative to the reference sequence corresponding to residues 12 to the carboxyl terminus of SEQ ID NO: 84, 214, 232, 562, 656, or 774, or to the reference sequence corresponding to SEQ ID NO: 84, 214, 232, 562, 656, or 774.

[0167] In some embodiments, the recombinant phosphatase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%. 96%, 97%, 98%, 99%. or more sequence identity to a reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of an even-numbered SEQ ID NO. of SEQ ID NOs: 144-224, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 144-224, wherein the amino acid sequence comprises one or more amino acid differences relative to the reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of SEQ ID NO: 84, or to the reference sequence corresponding to SEQ ID NO: 84.

[0168] In some embodiments, the recombinant phosphatase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%. 96%, 97%, 98%, 99%. or more sequence identity to the reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of SEQ ID NO: 84, or to the reference sequence corresponding to SEQ ID NO: 84, wherein the amino acid sequence comprises one or more amino acid differences relative to the reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of SEQ ID NO: 84, or to the reference sequence corresponding to SEQ ID NO: 84.

[0169] In some embodiments, the amino acid sequence of the recombinant phosphatase comprises at least an amino acid difference at amino acid position 11, 15, 16, 18. 19, 22, Tl, 38, 44, 48, 50, 81, 82. 83, 95, 96. 100, 101, 150, 158, 174, 175, 202, 226. 230, 235, 241. 247, 261, 266, 299, 301, 302, 307, 336, 341, 346, 357, 368, or 389, or combinations thereof, wherein the amino acid difference is relative to the reference sequence corresponding to residues 12 to the carboxyl terminus of SEQ ID NO: 84, or to the reference sequence corresponding to SEQ ID NO: 84.

[0170] In some embodiments, the amino acid sequence of the recombinant phosphatase comprises at least an amino acid difference or amino acid residue 1 1 A, 15E, 16P, 181, 19E, 221. 271, 38D, 44K, 48N, 50A. 81S, 82G. 83L, 95F/L. 96P, 100F, 101V, 150K, 158V. 174V, 175P, 202Y, 226S, 230L, 235N, 241L. 247M, 261 A, 2661, 299S, 301M, 302L. 307A, 3361, 341L. 3461, 357A. 368G, or 389L, or combinations thereof, wherein the amino acid difference is relative to the reference sequence corresponding to residues 12 to the carboxyl terminus of SEQ ID NO: 84, or to the reference sequence corresponding to SEQ ID NO: 84.

[0171] In some embodiments, the amino acid sequence of the recombinant phosphatase comprises at least an amino acid difference or amino acid residue G11 A. T15E, E16P, KI 81, D19E. L22I, F27I, K38D. P44K, Y48N, I50A. A81 S, A82G, M83L, C95F/L, S96P, L I OOF, 1101 V. N150K. Al 58V. Q174V, K175P, F202Y, N226S, I230L, Q235N, F241L, N247M, S261A, L266I, Q299S, T301M, I302L, V307A, L336I, I341L, V346I, S357A, Q368G, or F389L, or combinations thereof, wherein die amino acid difference is relative to the reference sequence corresponding to residues 12 to the carboxyl terminus of SEQ ID NO: 84, or to the reference sequence corresponding to SEQ ID NO: 84.

[0172] In some embodiments, the recombinant phosphatase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%. 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to the carboxy l terminus of an even-numbered SEQ ID NO. of SEQ ID NOs: 226-348, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 226-348, wherein the amino acid sequence comprises one or more amino acid differences relative to the reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of SEQ ID NO: 214, or to the reference sequence corresponding to SEQ ID NO: 214.

[0173] In some embodiments, the recombinant phosphatase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%. 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of SEQ ID NO: 214, or to the reference sequence corresponding to SEQ ID NO: 214, wherein the amino acid sequence comprises one or more amino acid differences relative to the reference sequence corresponding to ammo acid residues 12 to the carboxyl terminus of SEQ ID NO: 214, or to the reference sequence corresponding to SEQ ID NO: 214.

[0174] In some embodiments, the amino acid sequence of the recombinant phosphatase comprises at least an amino acid difference(s) at amino acid position(s) 15, 15/50/150/174/175, 15/50/158/174/226/247/346, 15/50/158/226/247/357, 15/50/158/247/357, 15/50/226, 15/96, 15/96/175/247, 15/96/175/341, 15/96/226/357, 15/96/357, 15/158/174/175/357, 15/158/174/247, 15/158/174/301/346, 15/158/175/247. 15/158/175/247/307/341/346, 15/174, 15/174/175, 15/174/175/247, 15/174/175/247/346, 15/174/226/301, 15/174/341, 15/175/247, 15/175/247/301, 15/175/247/341/346, 15/175/247/346/357, 15/175/357, 15/247, 15/346, 50/174/175/247/357, 96/158/175/301, 96/174/175, 96/174/175/226, 96/174/175/301/341/346, 96/174/247/341, 96/174/247/346, 96/175, 158, 158/174/226/247, 158/174/247/307/357, 158/175, 158/175/226/247/301/341/346/363, 158/175/247/346, 158/175/247/346/357, 158/175/301, 158/247, 158/307/346, 174, 174/175/247, 174/175/357, 174/247, 174/301/341. 174/346, 174/357, 175, 226, 226/247, 226/247/346, 247/301/346, 247/341/346. 307/357, or 357, wherein the amino acid difference is relative to the reference sequence corresponding to residues 12 to the carboxyl terminus of SEQ ID NO: 214, or to the reference sequence corresponding to SEQ ID NO: 214. [0175] In some embodiments, the amino acid sequence of the recombinant phosphatase comprises at least an amino acid difference(s) or amino acid residue(s) 15E, 15E/50A/150S/174V/175P, 15E/50A/158V/174V/226S/247M/346I. 15E/50A/158V/226S/247M/357A, 15E/50A/158V/247M/357A, 15E/50A/226S, 15E/96P, 15E/96P/175P/247M, 15E/96P/175P/341L, 15E/96P/226S/357A, 15E/96P/357A, 15E/158V/174V/175P/357A. 15E/158V/174V/247M, 15E/158V/174V/301M/346I, 15E/158V/175P/247M, 15E/158V/175P/247M/307A/341L/346I, 15E/174V, 15E/174V/175P, 15E/174V/175P/247M, 15E/174V/175P/247M/346I, 15E/174V/226S/301M, 15E/174V/341L, 15E/175P/247M. 15E/175P/247M/301M, 15E/175P/247M/341L/346I, 15E/175P/247M/346I/357A, 15E/175P/357A, 15E/247M, 15E/346I, 50A/174V/175P/247M/357A, 96P/158V/175P/301M, 96P/174V/175P, 96P/174V/175P/226S, 96P/174V/175P/301M/341L/346I, 96P/174V/247M/341L. 96P/174V/247M/346I. 96P/175P, 158V, 158V/174V/226S/247M. 158V/174V/247M/307A/357A, 158V/175P, 158V/175P/226S/247M/301M/341L/346I/363H, 158V/175P/247M/346I, 158V/175P/247M/346I/357A, 158V/175P/301M, 158V/247M, 158V/307A/346I, 174V, 174V/175P/247M, 174V/175P/357A, 174V/247M. 174V/301M/341L, 174V/346I. 174V/357A. 175P. 226S, 226S/247M, 226S/247M/346I, 247M/301 M/3461, 247M/341L/346I, 307A/357A, or 357A. wherein the amino acid difference is relative to the reference sequence corresponding to residues 12 to the carboxyl terminus of SEQ ID NO: 214, or to the reference sequence corresponding to SEQ ID NO: 214.

[0176] In some embodiments, the amino acid sequence of the recombinant phosphatase comprises at least an amino acid difference(s) or amino acid residue(s) T15E, T15E/I50A/N150S/Q174V/K175P, T15E/I50A/A158V/Q174V/N226S/N247M/V346I, T15E/I50A/A158V/N226S/N247M/S357A, T15E/I50A/A158V/N247M/S357A, T15E/I50A/N226S, T15E/S96P, T15E/S96P/K175P/N247M, T15E/S96P/K175P/I341L, T15E/S96P/N226S/S357A, T15E/S96P/S357A, T 1 EZ A 158 V/Q 174 V/K 175 P/S357A, T 15E/ A 158 V/Q 174 V/N247M, T15E/A158V/Q174V/T301M/V346I, T15E/A158V/K175P/N247M, T15E/A158V/K175P/N247M/V307A/I341L/V346I, T15E/Q174V, T15E/Q174V/K175P, T15E/Q174V/K175P/N247M, T15E/Q174V/K175P/N247M/V346I, T15E/Q174V/N226S/T301M, T15E/Q174V/I341L. T15E/K175P/N247M, T15E/K175P/N247M/T301M, T15E/K175P/N247M/I341L/V346I, T15E/K175P/N247M/V346I/S357A, T15E/K175P/S357A, T15E/N247M. T15E/V346I, I50A/Q174V/K175P/N247M/S357A, S96P/A158V/K175P/T301M. S96P/Q174V/K175P, S96P/Q174V/K175P/N226S, S96P/Q174V/K175P/T301M/I341L/V346I, S96P/Q174V/N247M/I341L, S96P/Q174V/N247M/V346I, S96P/K175P, A158V, A158V/Q174V/N226S/N247M, A158V/Q174V/N247M/V307A/S357A, A158V/K175P, A158V/K175P/N226S/N247M/T301M/I341L/V346I/Y363H, A158V/K175P/N247M/V346I, A158V/K175P/N247M/V346I/S357A, A158V/K175P/T301M, A158V/N247M, A158V7V307A/V346I, Q174V, Q174V/K175P/N247M, Q174V/K175P/S357A, Q174V/N247M, Q174V/T301M/I341L, Q174V/V346I, Q174V/S357A, K175P, N226S, N226S/N247M, N226S/N247M/V346I, N247M/T301M/V346I, N247M/I341L/V346I, V307A/S357A, or S357A, wherein the amino acid difference is relative to the reference sequence corresponding to residues 12 to the carboxyl terminus of SEQ ID NO: 214, or to the reference sequence corresponding to SEQ ID NO: 214.

[0177] In some embodiments, the recombinant phosphatase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of an even-numbered SEQ ID NO. of SEQ ID NOs: 350-650, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 350-650, wherein the amino acid sequence comprises one or more amino acid differences relative to the reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of SEQ ID NO: 232, or to the reference sequence corresponding to SEQ ID NO: 232.

[0178] In some embodiments, the recombinant phosphatase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of SEQ ID NO: 232, or to the reference sequence corresponding to SEQ ID NO: 232, wherein the amino acid sequence comprises one or more amino acid differences relative to the reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of SEQ ID NO: 232, or to the reference sequence corresponding to SEQ ID NO: 232.

[0179] In some embodiments, the amino acid sequence of the recombinant phosphatase comprises at least an amino acid difference at amino acid position(s) 12. 14, 15, 16, 17, 18, 19, 21, 22, 37. 43, 50, 57, 61, 89/95/141/169/234/306. 89/95/234/306/367, 89/96/367/371, 89/138/141/234/306/367/371, 89/138/234, 89/138/367, 89/141/169/234/306/371, 89/141/169/234/371, 89/141/169/306/367/371, 89/141/234/306/367/371, 89/141/306/367/371, 89/141/367, 89/141/371, 89/169, 89/169/234, 89/169/234/306/367/371, 89/169/234/367, 89/169/234/371, 89/169/306/367, 89/169/367, 89/169/371, 89/234, 89/234/306/371, 89/234/371, 89/306, 89/367/371, 89/371, 95/169/234/306/371, 96/141/234/306/371. 96/169/306. 138, 138/141, 138/141/169/234/367, 138/141/169/306, 138/141/234/306, 138/141/234/367, 138/141/234/371, 138/141/306/367, 138/141/306/371, 138/169/367/371, 138/234/367, 138/234/367/371, 138/306, 138/371, 139, 141, 141/169, 141/234, 141/234/306, 141/234/306/371, 141/234/371, 141/306/367, 141/306/371, 141/367, 169, 169/306, 205, 214, 224, 234. 234/306, 234/306/367. 234/306/367/371, 234/371, 244, 260. 271, 306, 306/367, 306/367/371, 306/371. 340, 341, 343. 367, 390, 391, 392, or 393, or combinations thereof, wherein the amino acid difference is relative to the reference sequence corresponding to residues 12 to the carboxyl terminus of SEQ ID NO: 232, or to the reference sequence corresponding to SEQ ID NO: 232.

[0180] In some embodiments, the amino acid sequence of the recombinant phosphatase comprises at least an amino acid difference(s) or amino acid residue(s) 12D/E/G/Q/R/V/W. 14E/F, 15E/Q/Y, 16L/V, 17S, 18G/L, 19Y, 21K/M/V, 22A/E/G/M/R, 37D, 43G. 50N/P/R/T/V, 57F/R, 61C/V, 89H/95A/I4I P/I69A/234T/306H. 89H/95A/234T/306H/367A. 89H/96P/367A/371L, 89H/138N/141P/234T/306H/367A/371L, 89H/138N/234E, 89H/138N/367A, 89H/141P/169A/234E/371L, 89H/141P/169A/234T/306H/371L, 89H/141P/169A/306H/367A/371L, 89H/141P/234E/306H/367A/371L, 89H/141P/306H/367A/371L, 89H/141P/367A, 89H/141P/371L, 89H/169A, 89H/169A/234E, 89H/169A/234E/306H/367A/371L, 89H/169A/234E/367A, 89H/169A/234T, 89H/169A/234T/367A, 89H/169A/234T/371L, 89H/169A/306H/367A, 89H/169A/367A, 89H/169A/371L, 89H/234E/306H/371L, 89H/234T, 89H/234T/306H/371L, 89H/234T/371L, 89H/306H, 89H/367A/371L, 89H/371L, 95A/169A/234T/306H/371L, 96P/141P/234T/306H/371L, 96P/169A/306H. 138N, 138N/141P, 138N/141P/169A/234T/367A, 138N/141P/169A/306H, 138N/141P/234E/306H, 138N/141P/234E/367A, 138N/141P/234E/371L, 138N/141P/306H/367A, 138N/141P/306H/371L, 138N/169A/367A/371L, 138N/234T/367A, 138N/234T/367A/371L, 138N/306H, 138N/371L. 139A, 139E, 1391. 139K, 139V, 141P/169A, 141P/234E/306H, 141P/234E/306H/371L, 141P/234E/371L. 141P/234T. 141P/234T/371L, 141P/306H/367A. 141P/306H/371L, 141P/367A, 141Q/R. 169A, 169A/306H, 205G, 214W. 2241. 234E. 234E/306H/367A. 234T. 234T/306H, 234T/306H/367A/371L. 234T/371L. 244A/E/W, 260V. 271A/V/W. 306H. 306H/367A, 306H/367A/371L, 306H/371L, 340D/H, 341L, 343G/K/M/Q/R. 367A, 390D/H/M/R/W/Y, 391W, 392G/L/N/R, or 393A/E/G/I/K/L/M/Q/R, wherein the amino acid difference is relative to the reference sequence corresponding to residues 12 to the carboxyl terminus of SEQ ID NO: 232, or to the reference sequence corresponding to SEQ ID NO: 232.

[0181] In some embodiments, the amino acid sequence of the recombinant phosphatase comprises at least an amino acid difference(s) or amino acid residue(s) C12D/E/G/Q/R/V/W. P14E/F, TI5E/Q/Y. E16L/V, K17S, K.18G/L, E19Y, S21K/M/V/A, L22E/G/M/R, L37D, E43G, I50N/P/R/T/V, K57F/R. G61C/V, K89H/C95A/K141P/C169A/K234T/K306H. K89H/C95A/K234T/K306H/C367A. K89H/S96P/C367A/K371L, K89H/K138N/K141P/K234T/K306H/C367A/K371L, K89H/K138N/K234E. K89H/K138N/C367A. K89H/K141P/C169A/K234E/K371L, K89H/K141 P/C 169A/K234T/K306H/K371 L, K89H/K141 P/C 169 A/K306H/C367 A/K371 L, K89H/K141 P/K234E/K306H/C367A/K371 L, K89H/K141 P/K306H/C367A/K371 L. K89H/K141P/C367A, K89H/K141P/K371L, K89H/C169A, K89H/C169A/K234E,

K89H/C169A/K234E/K306H/C367A/K371 L, K89H/C1 9A/K234E/C367A, K89H/C169A/K234T, K89H/C169A/K234T/C367A, K89H/C169A/K234T/K371L. K89H/C169A/K306H/C367A, K89H/C1 9A/C367A, K89H/C1 9A/K371L, K89H/K234E/K306H/K371L, K89H/K234T, K89H/K234T/K306H/K371L, K89H/K234T/K371L, K89H/K306H, K89H/C367A/K371L, K89H/K371L, C95A/C169A/K234T/K306H/K371L, S96P/K141P/K234T/K306H/K371L, S96P/C169A/K306H, K138N, K138N/K141P, K138N/K141P/C169A/K234T/C367A, K138N/K141P/C169A/K306H, K138N/K141P/K234E/K306H, K138N/K141P/K234E/C367A, K138N/K141P/K234E/K371L, K138N/K141P/K306H/C367A, K138N/K141P/K306H/K371L, K138N/C169A/C367A/K371L, K138N/K234T/C367A, K138N/K234T/C367A/K371L, K138N/K306H, K138N/K371L, S139A/E/I/K/V, K141P/C169A, K141P/K234E/K306H, K141P/K234E/K306H/K371L. K141P/K234E/K371L, K141P/K234T, K141P/K234T/K371L. K141P/K306H/C367A, K141P/K306H/K371L, K141P/C367A, K141Q/R, C169A, C169A/K306H, R205G. P214W. L224I, K234E/T, K234E/K306H/C367A. K234T/K306H, K234T/K306H/C367A/K371L. K234T/K371L, Q244A/E/W, Q260V, Q271A/V/W. K306H, K306H/C367A, K306H/C367A/K371L, K306H/K371L. N340D/H, 1341L, E343G/K/M/Q/R, C367A, G390D/H/M/R/W/Y. F391W, T392G/L/N/R. or S393A/E/G/I/K/L/M/Q/R. wherein the amino acid difference is relative to the reference sequence corresponding to residues 12 to the carboxyl terminus of SEQ ID NO: 232, or to the reference sequence corresponding to SEQ ID NO: 232.

[0182] In some embodiments, the recombinant phosphatase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%. 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%. 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of an even-numbered SEQ ID NO. of SEQ ID NOs: 652-740, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 652-740. wherein the amino acid sequence comprises one or more amino acid differences relative to the reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of SEQ ID NO: 562, or to the reference sequence corresponding to SEQ ID NO: 562.

[0183] In some embodiments, the recombinant phosphatase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%. 82%, 83%, 84%, 85%. 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%. 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of SEQ ID NO: 562, or to the reference sequence corresponding to SEQ ID NO: 562, wherein the amino acid sequence comprises one or more amino acid differences relative to the reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of SEQ ID NO: 562, or to the reference sequence corresponding to SEQ ID NO: 562.

[0184] In some embodiments, the amino acid sequence of the recombinant phosphatase comprises at least an amino acid difference(s) at amino acid position 12, 12/37/50/139/244/260/393, 12/37/57/139/244/260. 12/37/139/244/390/392/393, 12/37/139/393, 12/37/244/260/393, 12/37/244/392, 12/50/57/244/390/393, 12/50/139/244/260/392/393, 12/57/139/244/392.

12/139/244/260. 12/139/244/260/390/393, 12/139/244/260/392/393, 12/139/244/260/393, 12/139/244/390/392/393, 12/139/260/393, 12/260, 12/260/392, 12/390/392/393, 12/392, 37/50/57/139/244/260/392, 37/50/139/244/390/393, 37/57/139/244/390/393, 37/57/139/244/392/393, 37/57/139/390/392/393, 37/57/244/392, 37/139/223/244/393, 37/139/244/260/393, 37/139/244/390/392. 37/139/260/393, 50/139/244/260/390/392/393, 50/139/244/390/393, 50/244, 50/260, 57/244, 139/244/390/392/393, 139/244/392/393, 139/244/393, 139/260/393, 139/392, 244, 244/392, 260/390, or 392/393, wherein the amino acid difference is relative to the reference sequence corresponding to residues 12 to the carboxyl terminus of SEQ ID NO: 562, or to tire reference sequence corresponding to SEQ ID NO: 562.

[0185] In some embodiments, the amino acid sequence of the recombinant phosphatase comprises at least an amino acid difference(s) or amino acid residue(s) 12D/37D/139V/393G, 12D/37D/244W/392L, 12D/50R/57R/244W/390H/393L, 12D/57R/139V/244W/392L, 12D/139A/244W/260V/390H/393L, 12D/139A/260V/393L, 12D/139V/244W/260V/393L, 12D/390H/392L/393G, 12D/392L, 12E, 12E/37D/50R/139V/244W/260V/393G, 12E/37D/57R/139A/244W/260V, 12E/37D/139V/244W/390H/392L/393G, 12E/37D/244W/260V/393L, 12E/50R/139V/244W/260V/392L/393G, 12E/139A/244W/390H/392L/393G, 12E/139V/244W/260V, 12E/139V/244W/260V/392L/393G, 12E/139V/260V/393L, 12E/260V, 12E/260V/392L, 37D/50R/57R/139A/244W/260V/392L.

37D/50R/139V/244W/390H/393G. 37D/57R/139A/244W/392L/393G, 37D/57R/139A/390H/392L/393G, 37D/57R/139V/244W/390H/393L, 37D/57R/244W/392L, 37D/139A/244W/260V/393L, 37D/139A/260V/393G. 37D/139V/2231/244W/393G, 37D/139V/244W/390H/392L, 50R/139A/244W/260V/390H/392L/393G.

50R/139V/244W/390H/393L, 50R/244W, 50R/260V, 57R/244W, 139A/260V/393L, 139V/244W/390H/392L/393G, 139V/244W/392L/393G, 139V/244W/393L, 139V/392L, 244W, 244W/392L. 260V/390H. or 392L/393G, wherein the amino acid difference is relative to the reference sequence corresponding to residues 12 to the carboxyl terminus of SEQ ID NO: 562, or to the reference sequence corresponding to SEQ ID NO: 562.

[0186] In some embodiments, the amino acid sequence of the recombinant phosphatase comprises at least an amino acid difference(s) or amino acid residue(s) C12D/L37D/S139V/S393G, C12D/L37D/Q244W/T392L. C12D/I50R/K57R/Q244W/G390H/S393L, C12D/K57R/S139V/Q244W/T392L. C12D/S139A/Q244W/Q260V/G390H/S393L.

C12D/S139A/Q260V/S393L, C12D/S139V/Q244W/Q260V/S393L, C12D/G390H/T392L/S393G, C12D/T392L, C12E. C12E/L37D/I50R/S139V/Q244W/Q260V/S393G.

C12E/L37D/K57R/S139A/Q244W/Q260V, C12E/L37D/S139V/Q244W/G390H/T392L/S393G, C12E/L37D/Q244W/Q260V/S393L, C12E/I50R/S139V/Q244W/Q260V/T392L/S393G.

C12E/S139A/Q244W/G390H/T392L/S393G, C12E/S139V/Q244W/Q260V, C 12E/S 139 V/Q244W/Q260 V/T392L/S393 G, C 12E/S 139 V/Q260 V/S393L, C 12E/Q260V. C12E/Q260V/T392L, L37D/I50R/K57R/S139A/Q244W/Q260V/T392L,

L37D/I50R/S 139 V/Q244W/G390H/S393 G, L37D/K57R/S 139 A/Q244W/T392L/S393 G, L37D/K57R/S139A/G390H/T392L/S393G, L37D/K57R/S139V/Q244W/G390H/S393L, L37D/K57R/Q244W/T392L, L37D/S139A/Q244W/Q260V/S393L, L37D/S139A/Q260V/S393G, L37D/S139V/V223I/Q244W/S393G, L37D/S139V/Q244W/G390H/T392L,

I50R/S139A/Q244W/Q260V/G390H/T392L/S393G, I50R/S139V/Q244W/G390H/S393L,

I50R/Q244W, I50R/Q260V, K57R/Q244W, S139A/Q260V/S393L, S139V/Q244W/G390H/T392L/S393G, S139V/Q244W/T392L/S393G. S139V/Q244W/S393L, S139V/T392L, Q244W, Q244W/T392L, Q260V/G390H, or T392L/S393G, wherein the amino acid difference is relative to the reference sequence corresponding to residues 12 to the carboxyl terminus of SEQ ID NO: 562. or to the reference sequence corresponding to SEQ ID NO: 562.

[0187] In some embodiments, the recombinant phosphatase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%. 82%, 83%, 84%, 85%. 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of an even-numbered SEQ ID NO. of SEQ ID NOs: 742-894, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 742-894, wherein the amino acid sequence comprises one or more amino acid differences relative to the reference sequence corresponding to amino acid residue 12 to the carboxyl terminus of SEQ ID NO: 656, or to the reference sequence corresponding to SEQ ID NO: 656.

[0188] In some embodiments, the recombinant phosphatase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%. 82%, 83%, 84%, 85%. 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of SEQ ID NO: 656, or to the reference sequence corresponding to SEQ ID NO: 656, wherein the amino acid sequence comprises one or more amino acid differences relative to the reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of SEQ ID NO: 656, or to the reference sequence corresponding to SEQ ID NO: 656.

[0189] In some embodiments, the amino acid sequence of the recombinant phosphatase comprises at least an amino acid difference at amino acid position(s) 13, 22/141, 22/141/158, 22/158/340, 22/158/340/390/392. 23, 24, 25, 29, 30, 31, 32, 35, 36. 38, 44/50, 45/50, 47/50, 50, 50/51, 50/52, 50/56, 60, 63, 83, 98, 105, 128. 141/158/340, 141/340. 148, 150, 158, 158/340/371, 158/340/371/390/392, 181. 206, 207, 211, 215, 225, 227, 228, 231. 260, 291, 345. 347, or 353 wherein the amino acid difference is relative to the reference sequence corresponding to residues 12 to the carboxyl terminus of SEQ ID NO: 656, or to the reference sequence corresponding to SEQ ID NO: 656. [0190] In some embodiments, the amino acid sequence of the recombinant phosphatase comprises at least an amino acid difference(s) or amino acid residue(s) 13A/P/R/V/Y, 22G/141K, 22G/141K/158V, 22G/158V/340D, 22G/158V/340D/390G/392T, 23C/S/V, 24K/V, 25E/S/V, 29G, 30P/R/S, 31G/Q/R/V, 32K/T/V, 35F/R, 36C/G, 38R/S, 44V/50I, 45V/50I, 47E/50I, 47K/50I, 47P/50I. 47T/50I, 501. 50I/51R, 50I/52S, 50I/56P, 60A/G. 63V, 83W, 98V, 105S, 128L, 141K/158V/340D, 141K/340D, 148A/W, 150S, 158V, 158V/340D/371L, 158V/340D/371L/390W/392N, 181G, 206P, 207V, 21 IS, 215L. 225E. 227W, 228R, 231C/R. 260Q, 291 AZL, 345L, 347A, or 353A/Q/T, wherein the amino acid difference is relative to the reference sequence corresponding to residues 12 to the carboxyl terminus of SEQ ID NO: 656, or to the reference sequence corresponding to SEQ ID NO: 656.

[0191] In some embodiments, the amino acid sequence of the recombinant phosphatase comprises at least an amino acid difference(s) or amino acid residue(s) S13A/P/R/V/Y, L22G/P141K, L22G/P141K/A158V, L22G/A158V/N340D, L22G/A158V/N340D/H390G/L392T, P23C, P23S/V, N24K/V, T25E/S/V, P29G, T30P/R/S, L31G, L31Q/R/V, A32K/T/V, N35F/R, P36C/G, K38R/S, P44V/R50I, I45V/R50I, S47E/R50I, S47K/R50I, S47P/R50I, S47T/R50I. R50I. R50I/K51R, R50I/N52S, R50I/A56P, N60A/G, P63V, M83W, I98V, A105S, N128L, P141K/A158V/N340D. P141K/N340D, E148A/W. N150S, A158V, A158V/N340D/K371L, A158V/N340D/K371L/H390W/L392N, E181G, S206P, A207V. N211S, E215L, D225E, M227W. N228R. A231C/R. V260Q. D291A/L, K345L, E347A. or G353A/Q/T, wherein the amino acid difference is relative to the reference sequence corresponding to residues 12 to the carboxyl terminus of SEQ ID NO: 656. or to the reference sequence corresponding to SEQ ID NO: 656.

[0192] In some embodiments, the recombinant phosphatase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%. 82%, 83%, 84%, 85%. 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of an even-numbered SEQ ID NO. of SEQ ID NOs: 896-1052, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 896-1052, wherein the amino acid sequence comprises one or more amino acid differences relative to the reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of SEQ ID NO: 774, or to the reference sequence corresponding to SEQ ID NO: 774.

[0193] In some embodiments, the recombinant phosphatase comprises an amino acid sequence having at least 70%, 75%, 80%, 81%. 82%, 83%, 84%, 85%. 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of SEQ ID NO: 774, or to the reference sequence corresponding to SEQ ID NO: 774, wherein the amino acid sequence comprises one or more amino acid differences relative to the reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of SEQ ID NO: 774, or to the reference sequence corresponding to SEQ ID NO: 774. [0194] In some embodiments, the amino acid sequence of the recombinant phosphatase comprises at least an amino acid difference(s) at amino acid position(s) 13, 13/25/31/105, 13/25/47, 13/25/47/56/105/260, 13/25/47/207/260/353, 13/25/47/207/353, 13/25/56/207/260/353, 13/25/260/353, 13/25/353, 13/32. 13/32/47/56/353, 13/32/93/105/260/353, 13/47, 13/47/56/105/207/353. 13/47/56/353, 13/47/93/207/353, 13/47/105/207/240/260/353, 13/47/207/260, 13/56/263/353, 13/56/353, 13/105, 13/207, 13/207/260/353, 13/207/353, 13/353, 25/31/32, 25/31/32/56/105/260/353, 25/32/207/353. 25/47, 25/47/105, 25/47/105/207, 25/47/353, 25/56, 25/56/105/260/353, 25/93/207/260/353. 25/105/207/260, 25/105/207/260/353, 25/207, 25/353, 31/47/49/105/207/260/353. 32/47/105/353, 32/56/105. 47, 47/56/105/139/260, 47/105, 47/105/207/353. 47/105/260/353. 47/105/353. 47/108/207/260/353, 47/139, 47/207/260/353, 47/207/353. 47/260, 47/260/353, 47/353, 56, 56/93/105/139/207. 56/105/207/260. 56/105/207/260/353. 56/105/207/353, 56/207. 56/207/260. 56/207/353. 56/214/353. 105, 105/139/207/353, 105/139/260/353, 105/207/353. 105/260/353, 105/353. 207, 207/260/353. 207/353, or 353, wherein the amino acid difference is relative to tire reference sequence corresponding to residues 12 to the carboxyl terminus of SEQ ID NO: 774. or to the reference sequence corresponding to SEQ ID NO: 774.

[0195] In some embodiments, the amino acid sequence of the recombinant phosphatase comprises at least an amino acid difference(s) or amino acid residue(s) 13P, 13P/25E/31R/105S, 13P/25E/47P/56P/105S/260Q, 13P/25E/47P/207V/260Q/353Q, 13P/25E/47P/207V/353Q. 13P/25E/47T. 13P/25E/56P/207V/260Q/353Q, 13P/25E/260Q/353Q. 13P/25E/353Q, 13P/32T, 13P/32T/47T/56P/353Q. 13P/32T/93D/105S/260Q/353Q, 13P/47P, 13P/47P/56P/105S/207V/353Q, 13P/47P/93D/207V/353Q, 13P/47P/207V/260Q, 13P/47T, 13P/47T/56P/353Q, 13P/47T/105S/207V/240A/260Q/353Q. 13P/56P/263I/353Q. 13P/56P/353Q, 13P/105S, 13P/207V. 13P/207V/260Q/353Q. 13P/207V/353Q, 13P/353Q, 25E/31R/32T, 25E/31R/32T/56P/105S/260Q/353Q, 25E/32T/207V/353Q. 25E/47P/105S/207V, 25E/47P/353Q. 25E/47T, 25E/47T/105S. 25E/47T/353Q, 25E/56P, 25E/56P/105S/260Q/353Q, 25E/93D/207V/260Q/353Q, 25E/105S/207V/260Q, 25E/105S/207V/260Q/353Q, 25E/207V, 25E/353Q, 31R/47T/49H/105S/207V/260Q/353Q. 32T/47P/105S/353Q, 32T/56P/105S, 47P/56P/105S/139V/260Q. 47P/105S/207V/353Q, 47P/105S/353Q, 47P/108S/207V/260Q/353Q, 47P/207V/260Q/353Q. 47P/207V/353Q, 47P/260Q, 47P/260Q/353Q, 47P/353Q, 47T. 47T/105S, 47T/105S/207V/353Q, 47T/105S/260Q/353Q, 47T/139V, 47T/207V/260Q/353Q, 47T/353Q, 56P, 56P/93D/105S/139V/207V, 56P/105S/207V/260Q, 56P/105S/207V/260Q/353Q, 56P/105S/207V/353Q, 56P/207V, 56P/207V/260Q, 56P/207V/353Q, 56P/214S/353Q, 105S, 105S/139V/207V/353Q, 105S/139V/260Q/353Q, 105S/207V/353Q, 105S/260Q/353Q, 105S/353Q, 207V, 207V/260Q/353Q, 207V/353Q, or 353Q, wherein the amino acid difference is relative to the reference sequence corresponding to residues 12 to the carboxyl terminus of SEQ ID NO: 774, or to die reference sequence corresponding to SEQ ID NO: 774.

[0196] In some embodiments, the amino acid sequence of the recombinant phosphatase comprises at least an amino acid difference(s) or amino acid residue(s) S13P, S13P/T25E/L31R/A105S.

S13P/T25E/S47P/A56P/A105S/V260Q. S13P/T25E/S47P/A207V/V260Q/G353Q, S13P/T25E/S47P/A207V/G353Q, S13P/T25E/S47T, S13P/T25E/A56P/A207V/V260Q/G353Q, S13P/T25E/V260Q/G353Q, S13P/T25E/G353Q, S13P/A32T, S13P/A32T/S47T/A56P/G353Q, S13P/A32T/E93D/A105S/V260Q/G353Q, S13P/S47P, S13P/S47P/A56P/A105S/A207V/G353Q, S13P/S47P/E93D/A207V/G353Q, S13P/S47P/A207V/V260Q, S13P/S47T, S13P/S47T/A56P/G353Q, S13P/S47T/A105S/A207V/G240A/V260Q/G353Q, S13P/A56P/K263I/G353Q, S13P/A56P/G353Q, S13P/A105S, S13P/A207V, S13P/A207V/V260Q/G353Q, S13P/A207V/G353Q, S13P/G353Q, T25E/L31R/A32T, T25E/L31R/A32T/A56P/A105S/V260Q/G353Q, T25E/A32T/A207V/G353Q, T25E/S47P/A105S/A207V, T25E/S47P/G353Q, T25E/S47T, T25E/S47T/A105S.

T25E/S47T/G353Q, T25E/A56P. T25E/A56P/A105S/V260Q/G353Q, T25E/E93D/A207V/V260Q/G353Q, T25E/A105S/A207V/V260Q, T25E/A105S/A207V/V260Q/G353Q, T25E/A207V, T25E/G353Q, L31R/S47T/Q49H/A105S/A207V/V260Q/G353Q, A32T/S47P/A105S/G353Q. A32T/A56P/A105S. S47P/A56P/A105S/A139V/V260Q, S47P/A105S/A207V/G353Q, S47P/A105S/G353Q, S47P/A108S/A207V/V260Q/G353Q. S47P/A207V/V260Q/G353Q, S47P/A207V/G353Q, S47P/V260Q. S47P/V260Q/G353Q, S47P/G353Q, S47T, S47T/A105S, S47T/A105S/A207V/G353Q, S47T/A105S/V260Q/G353Q, S47T/A139V,

S47T/A207V/V260Q/G353Q, S47T/G353Q, A56P, A56P/E93D/A105S/A139V/A207V, A56P/A105S/A207V/V260Q. A56P/A105S/A207V/V260Q/G353Q, A56P/A105S/A207V/G353Q, A56P/A207V, A56P/A207V/V260Q, A56P/A207V/G353Q, A56P/P214S/G353Q. A105S.

A105S/A139V/A207V/G353Q. A105S/A139V/V260Q/G353Q, A105S/A207V/G353Q,

A105S/V260Q/G353Q, A105S/G353Q, A207V. A207V/V260Q/G353Q, A207V/G353Q, or G353Q, wherein the amino acid difference is relative to the reference sequence corresponding to residues 12 to the carboxyl terminus of SEQ ID NO: 774, or to the reference sequence corresponding to SEQ ID NO: 774.

[0197] In some embodiments, the recombinant phosphatase comprises an amino acid sequence comprising residues 12 to the carboxyl terminus of an even-numbered SEQ ID NO. of SEQ ID NOs: 16-18 and 24-1052, or an amino acid sequence comprising an even-numbered SEQ ID NO. of SEQ ID NOs: 16-18 and 24-1052. In some embodiments, the recombinant phosphatase comprises an amino acid sequence comprising SEQ ID NO: 16 or 18. In some embodiments, the recombinant phosphatase comprises an amino acid sequence comprising residues 12 to tire carboxyl terminus of SEQ ID NO: 12, 18, 84, 214, 232, 562, 656, or 774, or an amino acid sequence comprising SEQ ID NO: 12, 18, 84, 214, 232, 562, 656, or 774. In some embodiments, the amino acid sequence of the recombinant phosphatase optionally includes 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 substitutions, insertions, and/or deletions. In some embodiments, the amino acid sequence of the recombinant phosphatase optionally includes 1, 2, 3, 4. 5, 6, 7, 8, 9, or up to 10 substitutions. In some embodiments, the amino acid sequence of the recombinant phosphatase optionally includes 1, 2, 3, 4, or 5 substitutions, insertions, and/or deletions. In some embodiments, the amino acid sequence of the recombinant phosphatase optionally includes 1, 2, 3. 4, or 5 substitutions.

[0198] In some embodiments, the recombinant phosphatase exhibits one or more improved properties as compared to a reference phosphatase having the amino acid sequence corresponding to SEQ ID NO: 12, 18, 84, 214, 232, 562, 656, or 774.

[0199] In some embodiments, the improved property of the recombinant phosphatase is selected from: i) increased phosphatase activity on an oligonucleotide having a 3’-phosphate group, ii) increased phosphatase activity on an 3’-phosphate-NTP, iii) increased solubility, iv) increased expression or protein yield, v) increased thermal stability', vi) increased resistance to proteolysis, and vii) increased resistance to inhibition by immobilization on epoxide activated polyacrylate resin, or any combinations thereof, as compared to the reference phosphatase having the amino acid sequence corresponding to SEQ ID NO: 12, 18, 84, 214, 232, 562, 656, or 774. In some embodiments, the levels of improvement in provided in the Examples.

[0200] In some embodiments, the improved property is increased phosphatase activity on a substrate oligonucleotide having a 3 ’-phosphate at the 3 ’-terminus of the oligonucleotide. In some embodiments, the increased phosphatase activity is with a substrate oligonucleotide 5'-6-FAM- T26mCmCmU*mUinA-3'P (SEQ ID NO: 19) and/or 5'-T26inCmCmU*mUmA-3'P (SEQ ID NO: 20). In some embodiments, the increased activity’ on substrate oligonucleotide having a 3 ’-phosphate at the 3‘-tenninal nucleotide is at least 1.2, 1.3, 1.4, 1.5, 1.6, 1.7. 1.8. 19. 2.5, or 3 fold, or greater as compared to the reference phosphatase. In some embodiments, the reference phosphatase comprises an amino acid sequence corresponding to SEQ ID NO: 12, 18, 84, 214, 232, 562, 656, or 774.

[0201] In some embodiments, the improved property is increased phosphatase activity on an 3’- phosphate-NTP. In some embodiments, the 3’-phosphate-NTP comprises a modified nucleoside, e.g., modified nucleobase and/dor sugar moiety. In some embodiments, the phosphatase substrate is a 2’- modified nucleotide. In some embodiments, the improved property’ is increased phosphatase activity on 2 ’-fluoro-adenosine 3 ’-phosphate -5’ -triphosphate (2’-fluoro-3’-phosphate-ATP), 2’-fluoro- guanosine-3 ’ -phosphate-5 ’-triphosphate (2’ -fluoro-3 ’ -phosphate-GTP), 2 ’ -fluoro-cy tosine-3 ’ - phosphate-5’ -triphosphate (2’ -fluoro-3 ’-phosphate-CTP), 2’ -fluoro-uridine-3 ‘-phosphate-5 ’- triphosphate (2’ -fluoro-3 ’-phosphatc-UTP), 2’-O-mcthyl-adcnosinc 3 ’-phosphatc-5 ‘-triphosphate (2‘- O-methy 1-3 ' -phosphate- ATP), 2 ’ -O-methy l-guanosine-3 ’ -phosphate-5 ’-triphosphate (2’ -O-methy 1-3 ’ - phosphate-GTP), 2 ’ -O-methy 1-cy tosine-3 ’ -phosphate-5 ’ -triphosphate (2 ’ -O-methy 1-3 ‘-phosphate- CTP), or 2 ’ -O-methy l-uridine-3’ -phosphate-5 ’-triphosphate (2’-O-methyl-3’-phosphate-UTP). In some embodiments, the increased activity on a 3’-phosphate-NTP or a 2’-modified-nucleoside-3’- phosphate-5’ -phosphate is at least 1.2, 1.3, 1.4, 1.5, 1.6, 1.7. 1.8. 19. 2.5, , 3.5, 4, 4.5 or 5 fold, or greater as compared to the reference phosphatase. In some embodiments, the reference phosphatase comprises an amino acid sequence corresponding to SEQ ID NO: 12. 18, 84, 214, 232, 562, 656. or 774.

[0202] In some embodiments, the improved property is increased resistance to proteolysis and/or increased yield of phosphatase protein. In some embodiments, the recombinant phosphate displays at least 1.2, 1.3, 1.4, or 1.5 fold, or greater yield of phosphatase protein as compared the reference phosphatase. In some embodiments, the reference phosphatase comprises an amino acid sequence corresponding to SEQ ID NO: 12, 18, 84, 214, 232, 562, 656, or 774.

[0203] In some embodiments, the improved property is increased thermal stability of the recombinant phosphatase. In some embodiments, the increased thermal stability is assessed at about 72 to 76 °C (e.g., see Table 21.3) and a defined time of treatment (e.g., 1 hr). In some embodiments, die reference phosphatase comprises an amino acid sequence corresponding to SEQ ID NO: 12, 18, 84, 214, 232, 562, 656, or 774.

[0204] In some embodiments, the recombinant phosphatase is provided as a fusion protein. In some embodiments, the recombinant phosphatase described herein can be fused to a variety of polypeptide sequences, such as, by way of example and not limitation, polypeptide tags that can be used for detection, purification, immobilization on a support medium, or fusion to another protein. In some embodiments, the fusion protein of the recombinant phosphatase comprises a glycine-histidine or histidine-tag (His-tag). In some embodiments, the fusion protein of the recombinant phosphatase comprises an epitope tag, such as c-myc, FLAG, V5, or hemagglutinin (HA). In some embodiments, die fusion protein of the recombinant phosphatase comprises a GST, SUMO, Strep, MBP, or GFP tag. In some embodiments, the recombinant phosphatase is fused to a polylysine, for example, for conjugation to a support medium via the amino group of the polylysine. In some embodiments, the polylysine is from 2-10 lysine units in length. In some embodiments, the fusion is to the amino (N-) terminus of recombinant phosphatase polypeptide. In some embodiments, the fusion is to the carboxy (C-) terminus of the recombinant phosphatase polypeptide. In some embodiments, the fusion is selected or designed to preserve the activity of the phosphatase.

[0205] In some embodiments, the recombinant phosphatase described herein is an isolated composition. In some embodiments, the recombinant phosphatase polypeptide is purified. In some embodiments, the recombinant phosphatase is provided in solution, as a lyophilizate, or immobilized on a substrate, as further discussed herein.

[0206] In some embodiments, the present disclosure further provides functional fragments or biologically active fragments of recombinant phosphatase described herein. Thus, for each and every embodiment herein of a recombinant phosphatase described herein, a functional fragment or biologically active fragment of the recombinant phosphatase is provided herewith. In some embodiments, a functional fragment or biologically active fragments of a recombinant phosphatase comprises at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the activity of the phosphatase polypeptide from which it was derived (i.c., the parent phosphatase). In some embodiments, functional fragments or biologically active fragments comprise at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the parent sequence of the phosphatase. In some embodiments, the functional fragment will be truncated by less than 5, less than 10, less than 15, less than 10, less than 25, less than 30, less than 35, less than 40, less than 45, less than 50 amino acids, less than 55 amino acids, less than 60 amino acids, less than 65 amino acids, or less than 70 amino acids.

[0207] In some embodiments, a functional fragment of a recombinant phosphatase herein comprises at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the parent sequence of the recombinant phosphatase. In some embodiments, the functional fragment will be truncated by less than 5, less than 10, less than 15, less than 10, less than 25, less than 30, less than 35, less than 40, less than 45, less than 50, less than 55, less than 60, less than 65, or less than 70 amino acids.

Polynucleotides Encoding Recombinant Phosphatases, Expression Vectors and Host Cells

[0208] In another aspect, the present disclosure provides recombinant polynucleotides encoding the recombinant phosphatases described herein. In some embodiments, the recombinant polynucleotides are operably linked to one or more heterologous regulatory sequences that control gene expression to create a recombinant polynucleotide construct capable of expressing the recombinant phosphatase.

[0209] As will be apparent to the skilled artisan, availability of a protein sequence and the knowledge of the codons corresponding to the various amino acids provide a description of all the polynucleotides capable of encoding the subject polypeptides. The degeneracy of the genetic code, where the same amino acids are encoded by alternative or synonymous codons, allows an extremely large number of nucleic acids to be made, all of which encode a recombinant phosphatase of the present disclosure. Thus, the present disclosure provides methods and compositions for the production of each and every possible variation of polynucleotides that could be made that encode the recombinant phosphatases described herein by selecting combinations based on the possible codon choices, and all such variations of polynucleotides are to be considered specifically disclosed for any polypeptide described herein, including the amino acid sequences presented in the Examples and in die accompanying Sequence Listing.

[0210] In some embodiments, the codons are preferably optimized for utilization by the chosen host cell for protein production. In some embodiments, preferred codons in bacterial cells are used for expression in bacterial cells. In some embodiments, preferred codons in fungal cells are used for expression in fungal cells. In some embodiments, preferred codons in insect cells are used for expression in insect cells. In some embodiments, preferred codons in mammalian cells are used for expression in mammalian cells. In some embodiments, codon optimized polynucleotides encoding an recombinant phosphatase polypeptide described herein contain preferred codons at about 40%, 50%, 60%, 70%, 80%, 90%, or greater than 90% of the codon positions in the full length coding region.

[0211] Accordingly, in some embodiments, a recombinant polynucleotide of the present disclosure encodes a recombinant phosphatase described herein. In some embodiments, the polynucleotide sequence of the recombinant polynucleotide is codon optimized, as discussed above.

[0212] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant phosphatase, or a functional fragment thereof, comprising an amino acid sequence having at least 70%. 75%. 80%, 81%, 82%. 83%. 84%, 85%, 86%. 87%. 88%, 89%, 90%. 91%, 92%. 93%. 94%, 95%, 96%. 97%. 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of an even-numbered SEQ ID NO. of SEQ ID NOs: 2-18 and 24-1052, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 2-18 and 24-1052.

[0213] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant phosphatase, or a functional fragment thereof, comprising an amino acid sequence having at least 70%, 75%, 80%. 81%. 82%, 83%, 84%. 85%. 86%, 87%, 88%. 89%. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of an even-numbered SEQ ID NO. of SEQ ID NOs: 2-14 or SEQ ID NO: 18, 84, 214, 232, 562, 656. or 774, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 2-14, or SEQ ID NO: 18, 84, 214, 232. 562, 656, or 774.

[0214] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant phosphatase, or a functional fragment thereof, comprising an amino acid sequence having at least 70%, 75%, 80%. 81%, 82%, 83%, 84%. 85%. 86%, 87%, 88%. 89%. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of an even-numbered SEQ ID NO. of SEQ ID NOs: 12 and 24-1052, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 12 and 24-1052, wherein the amino acid comprises one or more amino acid differences relative to the reference sequence corresponding to amino acid residues 12 to the carboxy l terminus of an even-numbered SEQ ID NOs: 12 and 24-1052, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 12 and 24-1052.

[0215] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant phosphatase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%. 83%, 84%, 85%, 86%. 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%. or more sequence identity⁷ to a reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of SEQ ID NO: 12, 18, 84, 214, 232, 562, 656, or 774, or to the reference sequence corresponding to SEQ ID NO: 12, 18, 84, 214, 232, 562, 656, or 774, wherein the amino acid comprises one or more amino acid differences relative to the reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of SEQ ID NO: 12, 18, 84, 214, 232, 562, 656, or 774, or to the reference sequence corresponding to SEQ ID NO: 12, 18, 84, 214, 232, 562, 656, or 774.

[0216] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant phosphatase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%. 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of an even-numbered SEQ ID NO. of SEQ ID NOs: 24-80, or to die reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 24-80, wherein the amino acid sequence comprises one or more amino acid differences relative to the reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of SEQ ID NO: 18, or to the reference sequence corresponding to SEQ ID NO: 18.

[0217] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant phosphatase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of SEQ ID NO: 18, or to the reference sequence corresponding to SEQ ID NO: 18, wherein the amino acid sequence comprises one or more amino acid differences relative to the reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of SEQ ID NO: 18. or to the reference sequence corresponding to SEQ ID NO: 18.

[0218] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant phosphatase comprising an amino acid sequence comprising at least an amino acid difference at amino acid position 23, 25, 50, 56. 63, 99, 102, 136, 193, 195. 198, 224, 234, 235, 267, 306, or 345, or combinations thereof, wherein the amino acid difference is relative to the reference sequence corresponding to residues 12 to the carboxyl terminus of SEQ ID NO: 18, or to the reference sequence corresponding to SEQ ID NO: 18.

[0219] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant phosphatase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%. 83%, 84%, 85%, 86%. 87%, 88%, 89%, 90%. 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%. or more sequence identity to a reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of SEQ ID NO: 12, 84, 214, 232, 562, 656, or 774, or to a reference sequence corresponding to SEQ ID NO: 12, 84, 214, 232, 562, 656, or 774, wherein the amino acid sequence comprises one or more amino acid differences relative to the reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of SEQ ID NO: 12, 84, 214, 232, 562, 656, or 774, or to the reference sequence corresponding to SEQ ID NO: 12, 84. 214, 232, 562, 656, or 774.

[0220] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant phosphatase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%. 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%. or more sequence identity’ to a reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of an even-numbered SEQ ID NO. of SEQ ID NOs: 24-1052, or to a reference sequence corresponding to an cvcn-numbcrcd SEQ ID NO. of SEQ ID NOs: 24-1052, wherein the amino acid sequence comprises one or more amino acid differences relative to the reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of SEQ ID NO: 12, or to the reference sequence corresponding to SEQ ID NO: 12.

[0221] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant phosphatase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%. 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity’ to the reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of SEQ ID NO: 84, 214, 232, 562, 656, or 774, or to the reference sequence corresponding to SEQ ID NO: 84, 214, 232, 562, 656, or 774, wherein the amino acid sequence comprises one or more amino acid differences relative to tire reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of SEQ ID NO: 12, or to the reference sequence corresponding to SEQ ID NO: 12.

[0222] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant phosphatase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of SEQ ID NO: 12, or to the reference sequence corresponding to SEQ ID NO: 12, wherein the amino acid sequence comprises one or more amino acid differences relative to the reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of SEQ ID NO: 12, or to the reference sequence corresponding to SEQ ID NO: 12.

[0223] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant phosphatase comprising an amino acid sequence comprising at least an amino acid difference at amino acid position 11, 12, 13, 14. 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27. 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39. 40, 41, 42, 43, 44, 45, 46, 47, 48. 49, 50, 51, 52, 56, 57, 60, 61, 63, 81, 82, 83, 89, 93, 95, 96, 98, 100, 101. 105, 108, 128, 138, 139, 141, 148, 150, 158, 169, 174, 175, 181, 202, 205, 206, 207, 211, 214, 215, 223, 224, 225, 226, 227, 228, 230, 231, 234, 235,

240, 241, 244, 247, 260, 261, 263, 266, 271, 291, 299, 301, 302, 306, 307, 336, 340, 341, 343, 345,

346, 347, 353, 357, 363, 367, 368, 371, 389, 390, 391, 392, or 393, or combinations thereof, wherein die amino acid difference is relative to the reference sequence corresponding to residues 12 to the carboxyl terminus of SEQ ID NO: 12, or to the reference sequence corresponding to SEQ ID NO: 12.

[0224] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant phosphatase comprising an amino acid sequence comprising at least an amino acid difference at amino acid position 19, 22, 50, 138, 139, 141, 158, 169. 244, 260, 306, 307, 340, 357, 390, 392, or 393, or combinations thereof, wherein the amino acid difference is relative to the reference sequence corresponding to residues 12 to the carboxyl terminus of SEQ ID NO: 12, or to die reference sequence corresponding to SEQ ID NO: 12.

[0225] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant phosphatase comprising an amino acid sequence comprising at least an amino acid difference at amino acid position 11, 13, 14, 16. 17, 18, 19, 20. 21, 28, 29, 31, 35, 42, 44, or 61, or combinations thereof, wherein the amino acid difference is relative to the reference sequence corresponding to residues 12 to the carboxyl terminus of SEQ ID NO: 12, or to the reference sequence corresponding to SEQ ID NO: 12.

[0226] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant phosphatase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%. 83%, 84%, 85%, 86%. 87%, 88%, 89%, 90%. 91%, 92%, 93%, 94%. 95%, 96%, 97%, 98%, 99%. or more sequence identity to a reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of an even-numbered SEQ ID NO. of SEQ ID NOs: 24-1052, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 24-1052, wherein the amino acid sequence comprises one or more amino acid differences relative to the reference sequence corresponding to amino acid residues 12 to the carboxy l terminus of SEQ ID NO: 84, 214, 232, 562, 656, or 774, or to the reference sequence corresponding to SEQ ID NO: 84, 214, 232, 562, 656, or 774. [0227] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant phosphatase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of SEQ ID NO: 84, 214, 232, 562, 656, or 774, or to a reference sequence corresponding to SEQ ID NO: 84, 214, 232, 562. 656, or 774. wherein the amino acid sequence comprises one or more amino acid difference relative to the reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of SEQ ID NO: 84, 214. 232, 562, 656, or 774. or to the reference sequence corresponding to SEQ ID NO: 84. 214, 232, 562. 656, or 774.

[0228] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant phosphatase comprising an amino acid sequence comprising at least an amino acid difference at amino acid position 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35. 36, 37, 38, 39, 40, 41, 42, 43, 44. 45, 46, 47, 48, 49, 50, 51, 52, 56. 57, 60, 61, 63, 81. 82, 83, 89, 93. 95, 96, 98, 100, 101, 105, 108, 128, 138, 139, 141, 148, 150. 158, 169, 174, 175, 181. 202, 205, 206. 207, 211, 214, 215, 223, 224, 225, 226. 227, 228, 230, 231, 234, 235,

240, 241, 244. 247, 260, 261. 263, 266, 271, 291, 299. 301, 302, 306. 307, 336, 340, 341, 343, 345,

346, 347, 353. 357, 363, 367. 368, 371, 389, 390, 391. 392, or 393. or combinations thereof, wherein the amino acid difference is relative to the reference sequence corresponding to residues 12 to the carboxyl terminus of SEQ ID NO: 84, 214, 232. 562, 656, or 774, or to the reference sequence corresponding to SEQ ID NO: 84, 214, 232, 562, 656. or 774.

[0229] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant phosphatase comprising an amino acid sequence comprising at least an amino acid difference at amino acid position 19, 22, 50, 138, 139. 141, 158, 169, 244, 260, 306, 307, 340. 357, 390, 392. or 393, or combinations thereof, wherein the amino acid difference is relative to the reference sequence corresponding to residues 12 to the carboxyl terminus of SEQ ID NO: 84, 214, 232, 562, 656. or 774, or to the reference sequence corresponding to SEQ ID NO: 84, 214. 232, 562, 656, or 774.

[0230] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant phosphatase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%. 92%, 93%, 94%, 95%. 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of an even-numbered SEQ ID NO. of SEQ ID NOs: 144-224, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 144-224, wherein the amino acid sequence comprises one or more amino acid differences relative to the reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of SEQ ID NO: 84, or to the reference sequence corresponding to SEQ ID NO: 84.

[0231] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant phosphatase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%. 83%, 84%, 85%, 86%. 87%, 88%, 89%, 90%. 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%. or more sequence identity to the reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of SEQ ID NO: 84, or to the reference sequence corresponding to SEQ ID NO: 84, wherein the amino acid sequence comprises one or more amino acid differences relative to the reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of SEQ ID NO: 84, or to the reference sequence corresponding to SEQ ID NO: 84.

[0232] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant phosphatase comprising an amino acid sequence comprising at least an amino acid difference at amino acid positionl 1, 15, 16, 18, 19, 22, 27. 38, 44, 48, 50. 81, 82, 83, 95, 96, 100, 101. 150, 158, 174, 175, 202, 226, 230, 235. 241, 247, 261, 266, 299, 301, 302, 307, 336, 341, 346. 357, 368, or 389, or combinations thereof, wherein the amino acid difference is relative to the reference sequence corresponding to residues 12 to the carboxyl terminus of SEQ ID NO: 84, or to the reference sequence corresponding to SEQ ID NO: 84.

[0233] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant phosphatase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%. 83%, 84%, 85%, 86%. 87%, 88%, 89%, 90%. 91%, 92%, 93%, 94%. 95%, 96%, 97%, 98%, 99%. or more sequence identity to a reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of an even-numbered SEQ ID NO. of SEQ ID NOs: 226-348, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 226-348, wherein the amino acid sequence comprises one or more amino acid differences relative to the reference sequence corresponding to amino acid residues 12 to the carboxy l terminus of SEQ ID NO: 214, or to the reference sequence corresponding to SEQ ID NO: 214.

[0234] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant phosphatase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%. 83%, 84%, 85%, 86%. 87%, 88%, 89%, 90%. 91%, 92%, 93%, 94%. 95%, 96%, 97%, 98%, 99%. or more sequence identity to the reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of SEQ ID NO: 214, or to the reference sequence corresponding to SEQ ID NO: 214, wherein the amino acid sequence comprises one or more amino acid differences relative to the reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of SEQ ID NO: 214, or to the reference sequence corresponding to SEQ ID NO: 214. [0235] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant phosphatase comprising an amino acid sequence comprising at least an amino acid difference(s) at amino acid position(s) 15, 15/50/150/174/175, 15/50/158/174/226/247/346, 15/50/158/226/247/357, 15/50/158/247/357, 15/50/226, 15/96, 15/96/175/247, 15/96/175/341, 15/96/226/357, 15/96/357, 15/158/174/175/357, 15/158/174/247. 15/158/174/301/346, 15/158/175/247, 15/158/175/247/307/341/346, 15/174, 15/174/175, 15/174/175/247, 15/174/175/247/346. 15/174/226/301, 15/174/341, 15/175/247, 15/175/247/301, 15/175/247/341/346, 15/175/247/346/357. 15/175/357. 15/247. 15/346, 50/174/175/247/357. 96/158/175/301. 96/174/175. 96/174/175/226. 96/174/175/301/341/346, 96/174/247/341, 96/174/247/346, 96/175, 158, 158/174/226/247, 158/174/247/307/357, 158/175, 158/175/226/247/301/341/346/363. 158/175/247/346, 158/175/247/346/357, 158/175/301. 158/247, 158/307/346. 174, 174/175/247. 174/175/357, 174/247. 174/301/341, 174/346. 174/357, 175. 226, 226/247, 226/247/346, 247/301/346, 247/341/346. 307/357, or 357, wherein the amino acid difference is relative to the reference sequence corresponding to residues 12 to the carboxyl terminus of SEQ ID NO: 214, or to the reference sequence corresponding to SEQ ID NO: 214.

[0236] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant phosphatase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%. 84%, 85%, 86%, 87%. 88%, 89%, 90%, 91%. 92%, 93%, 94%, 95%. 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of an even-numbered SEQ ID NO. of SEQ ID NOs: 350-650. or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 350-650, wherein the amino acid sequence comprises one or more amino acid differences relative to the reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of SEQ ID NO: 232. or to the reference sequence corresponding to SEQ ID NO: 232.

[0237] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant phosphatase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%. 84%, 85%, 86%, 87%. 88%, 89%, 90%, 91%. 92%, 93%, 94%, 95%. 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of SEQ ID NO: 232, or to the reference sequence corresponding to SEQ ID NO: 232. wherein the amino acid sequence comprises one or more amino acid differences relative to the reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of SEQ ID NO: 232, or to the reference sequence corresponding to SEQ ID NO: 232.

[0238] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant phosphatase comprising an amino acid sequence comprising at least an amino acid difference(s) at amino acid position(s) 12, 14. 15. 16, 17, 18, 19. 21, 22, 37, 43. 50, 57, 61, 89/95/141/169/234/306, 89/95/234/306/367, 89/96/367/371, 89/138/141/234/306/367/371. 89/138/234, 89/138/367, 89/141/169/234/306/371, 89/141/169/234/371, 89/141/169/306/367/371, 89/141/234/306/367/371, 89/141/306/367/371, 89/141/367, 89/141/371, 89/169, 89/169/234, 89/169/234/306/367/371, 89/169/234/367, 89/169/234/371, 89/169/306/367, 89/169/367, 89/169/371, 89/234, 89/234/306/371, 89/234/371, 89/306, 89/367/371, 89/371, 95/169/234/306/371, 96/141/234/306/371. 96/169/306. 138, 138/141, 138/141/169/234/367, 138/141/169/306, 138/141/234/306, 138/141/234/367, 138/141/234/371, 138/141/306/367, 138/141/306/371, 138/169/367/371, 138/234/367, 138/234/367/371, 138/306, 138/371, 139, 141, 141/169. 141/234, 141/234/306, 141/234/306/371, 141/234/371, 141/306/367, 141/306/371, 141/367, 169, 169/306, 205, 214, 224, 234. 234/306, 234/306/367. 234/306/367/371, 234/371, 244, 260. 271, 306, 306/367, 306/367/371, 306/371. 340, 341, 343. 367, 390, 391, 392, or 393, or combinations thereof, wherein the amino acid difference is relative to the reference sequence corresponding to residues 12 to the carboxyl terminus of SEQ ID NO: 232. or to the reference sequence corresponding to SEQ ID NO: 232.

[0239] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant phosphatase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%. 88%, 89%, 90%, 91%. 92%, 93%, 94%, 95%. 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of an even-numbered SEQ ID NO. of SEQ ID NOs: 652-740, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 652-740, wherein the amino acid sequence comprises one or more amino acid differences relative to the reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of SEQ ID NO: 562, or to the reference sequence corresponding to SEQ ID NO: 562.

[0240] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant phosphatase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%. 88%, 89%, 90%, 91%. 92%, 93%, 94%, 95%. 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of SEQ ID NO: 562, or to the reference sequence corresponding to SEQ ID NO: 562. wherein the amino acid sequence comprises one or more amino acid differences relative to the reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of SEQ ID NO: 562, or to the reference sequence corresponding to SEQ ID NO: 562.

[0241] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant phosphatase comprising an amino acid sequence comprising at least an amino acid difference(s) at amino acid position 12. 12/37/50/139/244/260/393, 12/37/57/139/244/260, 12/37/139/244/390/392/393, 12/37/139/393, 12/37/244/260/393, 12/37/244/392, 12/50/57/244/390/393. 12/50/139/244/260/392/393, 12/57/139/244/392, 12/139/244/260, 12/139/244/260/390/393, 12/139/244/260/392/393, 12/139/244/260/393, 12/139/244/390/392/393. 12/139/260/393, 12/260, 12/260/392, 12/390/392/393, 12/392, 37/50/57/139/244/260/392, 37/50/139/244/390/393, 37/57/139/244/390/393, 37/57/139/244/392/393, 37/57/139/390/392/393, 37/57/244/392, 37/139/223/244/393, 37/139/244/260/393, 37/139/244/390/392, 37/139/260/393, 50/139/244/260/390/392/393, 50/139/244/390/393, 50/244, 50/260, 57/244, 139/244/390/392/393, 139/244/392/393, 139/244/393, 139/260/393, 139/392, 244, 244/392, 260/390, or 392/393, wherein the amino acid difference is relative to the reference sequence corresponding to residues 12 to the carboxyl terminus of SEQ ID NO: 562, or to the reference sequence corresponding to SEQ ID NO: 562.

[0242] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant phosphatase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of an even-numbered SEQ ID NO. of SEQ ID NOs: 742-894, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 742-894, wherein the amino acid sequence comprises one or more amino acid differences relative to the reference sequence corresponding to amino acid residue 12 to the carboxyl terminus of SEQ ID NO: 656, or to the reference sequence corresponding to SEQ ID NO: 656.

[0243] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant phosphatase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%. 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of SEQ ID NO: 656, or to the reference sequence corresponding to SEQ ID NO: 656, wherein the amino acid sequence comprises one or more amino acid differences relative to the reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of SEQ ID NO: 656, or to the reference sequence corresponding to SEQ ID NO: 656.

[0244] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant phosphatase comprising an amino acid sequence comprising at least an amino acid difference at amino acid position(s) 13, 22/141, 22/141/158, 22/158/340, 22/158/340/390/392, 23, 24, 25, 29, 30, 31, 32, 35, 36, 38, 44/50, 45/50, 47/50, 50, 50/51, 50/52, 50/56, 60, 63, 83, 98, 105, 128, 141/158/340, 141/340, 148, 150, 158, 158/340/371, 158/340/371/390/392, 181, 206, 207, 211, 215, 225, 227, 228, 231, 260. 291, 345, 347, or 353 wherein the amino acid difference is relative to the reference sequence corresponding to residues 12 to the carboxyl terminus of SEQ ID NO: 656, or to the reference sequence corresponding to SEQ ID NO: 656.

[0245] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant phosphatase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of an even-numbered SEQ ID NO. of SEQ ID NOs: 896-1052, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 896-1052, wherein the amino acid sequence comprises one or more amino acid differences relative to the reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of SEQ ID NO: 774, or to the reference sequence corresponding to SEQ ID NO: 774.

[0246] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant phosphatase comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of SEQ ID NO: 774, or to the reference sequence corresponding to SEQ ID NO: 774, wherein the amino acid sequence comprises one or more amino acid differences relative to the reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of SEQ ID NO: 774, or to the reference sequence corresponding to SEQ ID NO: 774.

[0247] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant phosphatase comprising an amino acid sequence comprising at least an amino acid diffcrcncc(s) at amino acid position(s) 13, 13/25/31/105, 13/25/47, 13/25/47/56/105/260, 13/25/47/207/260/353, 13/25/47/207/353, 13/25/56/207/260/353, 13/25/260/353, 13/25/353, 13/32, 13/32/47/56/353, 13/32/93/105/260/353, 13/47, 13/47/56/105/207/353, 13/47/56/353, 13/47/93/207/353, 13/47/105/207/240/260/353, 13/47/207/260, 13/56/263/353, 13/56/353, 13/105, 13/207, 13/207/260/353, 13/207/353, 13/353, 25/31/32, 25/31/32/56/105/260/353, 25/32/207/353, 25/47, 25/47/105, 25/47/105/207. 25/47/353, 25/56, 25/56/105/260/353, 25/93/207/260/353, 25/105/207/260. 25/105/207/260/353, 25/207. 25/353, 31/47/49/105/207/260/353, 32/47/105/353, 32/56/105, 47. 47/56/105/139/260, 47/105, 47/105/207/353, 47/105/260/353, 47/105/353, 47/108/207/260/353. 47/139. 47/207/260/353. 47/207/353. 47/260. 47/260/353. 47/353, 56, 56/93/105/139/207, 56/105/207/260, 56/105/207/260/353, 56/105/207/353. 56/207, 56/207/260, 56/207/353. 56/214/353. 105, 105/139/207/353. 105/139/260/353, 105/207/353, 105/260/353, 105/353, 207, 207/260/353, 207/353. or 353, wherein the amino acid difference is relative to the reference sequence corresponding to residues 12 to the carboxyl terminus of SEQ ID NO: 774. or to the reference sequence corresponding to SEQ ID NO: 774.

[0248] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant phosphatase comprising an amino acid sequence comprising residues 12 to the carboxyl terminus of an even-numbered SEQ ID NO. of SEQ ID NOs: 16-18 and 24-1052. or an amino acid sequence comprising an even-numbered SEQ ID NO. of SEQ ID NOs: 16-18 and 24- 1052. [0249] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant phosphatase comprising an amino acid sequence comprising residues 12 to die carboxyl terminus of SEQ ID NO: 12, 18, 84, 214, 232, 562, 656, or 774, or an amino acid sequence comprising SEQ ID NO: 12, 18, 84, 214, 232, 562, 656, or 774.

[0250] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%. or more sequence identity to a reference polynucleotide sequence corresponding to a sequence from nucleotide residue 34 to the 3 ’-terminal nucleotide of an odd numbered SEQ ID NO. of SEQ ID NOs: 1-13, or to a reference sequence corresponding to an odd numbered SEQ ID NO. of SEQ ID NOs: 1-13, wherein the polynucleotide sequence encodes a recombinant phosphatase.

[0251] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence having at least 70%, 75%, 80%. 81%, 82%, 83%, 84%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%. or more sequence identity to a reference polynucleotide sequence corresponding to nucleotide residues 34 to the 3 ’-terminal nucleotide of an even-numbered SEQ ID NO. of SEQ ID NOs: 15-17 and 23-1051, or to a reference polynucleotide sequence corresponding to an even- numbered SEQ ID NO. of SEQ ID NOs: 15-17 and 23-1051, wherein the polynucleotide sequence encodes a phosphatase.

[0252] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence having at least 70%, 75%, 80%. 81%, 82%, 83%, 84%. 85%, 90%, 91%, 92%. 93%, 94%, 95%, 96%, 97%, 98%, 99%. or more sequence identity to a reference polynucleotide sequence corresponding to nucleotide residues 34 to the 3 ’-terminal nucleotide of SEQ ID NO: 11, 83, 213. 231, 561, 655. or 773. or to a reference polynucleotide sequence corresponding to SEQ ID NO: 11, 83, 213, 231. 561, 655, or 773, wherein the polynucleotide sequence encodes a phosphatase.

[0253] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence comprising nucleotide residues 34 to the 3 ’-terminal nucleotide of an odd-numbered SEQ ID NO. of SEQ ID NOs: 15-17 and 23-1051. or a polynucleotide sequence comprising an odd-numbered SEQ ID NO. of SEQ ID NOs: 15-17 and 23-1051 .

[0254] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence comprising an amino acid sequence comprising nucleotide residues 34 to the 3 ’-terminal nucleotide of SEQ ID NO: 11. 17. 83, 213. 231, 561, 655, or 773, or a polynucleotide sequence comprising SEQ ID NO: 11. 17. 83, 213. 231, 561, 655. or 773.

[0255] In some embodiments, a recombinant polynucleotide encoding any of the recombinant phosphatases herein is manipulated in a variety of ways to facilitate expression of the phosphatase polypeptide. In some embodiments, the recombinant polynucleotide encoding the phosphatase comprises expression vectors where one or more control sequences is present to regulate the expression of the phosphatase encoding polynucleotides and/or encoded polypeptides. In some embodiments, the control sequences include among others, promoters, leader sequences, polyadenylation sequences, propeptide sequences, signal peptide sequences, and transcription terminators.

[0256] In some embodiments, suitable promoters are selected based on the host cell selection. For bacterial host cells, suitable promoters for directing transcription of the nucleic acid constructs of the present disclosure, include, but are not limited to promoters obtained from the E. coli lac operon, Streptomyces coelicolor agarase gene (dagA), Bacillus subtilis levansucrase gene (sacB), Bacillus lichcniformis alpha-amylase gene (amyL), Bacillus stcarothcnnophilus maltogcnic amylase gene (amyM), Bacillus amyloliquefaciens alpha-amylase gene (amyQ), Bacillus lichenifonnis penicillinase gene (penP), Bacillus subtilis xylA and x lB genes, and prokaryotic beta-lactamase gene (see, e.g., Villa-Kamaroff et al., Proc. Natl Acad. Sci. USA, 1978, 75:3727-3731), as well as tire tac promoter (see, e.g., DeBoer et al., Proc. Natl Acad. Sci. USA, 1983, 80:21-25). Exemplary promoters for filamentous fungal host cells, include, but are not limited to promoters obtained from the genes for Aspergillus oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus niger neutral alpha-amylase, Aspergillus niger acid stable alpha-amylase, Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Rhizomucor miehei lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triose phosphate isomerase, Aspergillus nidulans acetamidase, and Fusarium oxysporum trypsin-like protease (see, e.g., WO 96/00787), as well as the NA2-tpi promoter (a hybrid of the promoters from the genes for Aspergillus niger neutral alpha-amylase and Aspergillus oryzae triose phosphate isomerase), and mutant, truncated, and hybrid promoters thereof. Exemplary yeast cell promoters can be from the genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae galactokinase (GALI), Saccharomyces cerevisiae alcohol dehydrogenase/glyceraldehyde- 3-phosphate dehydrogenase (ADH2/GAP), and Saccharomyces cerevisiae 3-phosphoglycerate kinase. Other useful promoters for yeast host cells are known in the art (see, e.g., Romanos et al., Yeast, 1992, 8:423-488). Exemplary promoters for use in insect cells include, but are not limited to, polyhedrin, plO, ELT, OpIE2, and hr5/iel promoters. Exemplary promoters for use in mammalian cells include, but are not limited to, those from cytomegalovirus (CMV), chicken P -actin promoter fused with the CMV enhancer, Simian vacuolating virus 40 (SV40), from Homo sapiens phosphoglycerate kinase, beta actin, elongation factor- la or glyceraldehyde-3-phosphate dehydrogenase, and from Gallus -actin.

[0257] In some embodiments, the control sequence is a suitable transcription terminator sequence (i.e.. a sequence recognized by a host cell to terminate transcription). In some embodiments, the terminator sequence is operably linked to the 3' terminus of the nucleic acid sequence encoding the phosphatase polypeptide. Any suitable terminator which is functional in the host cell of choice finds use in the present invention. For bacterial expression, the transcription terminators can be a Rho- dependent terminators that rely on a Rho transcription factor, or a Rho-independent, or intrinsic terminators, which do not require a transcription factor. Exemplary' bacterial transcription terminators are described in Peters et al., J Mol Biol., 2011, 412(5):793-813. Exemplary' transcription terminators for filamentous fungal host cells can be obtained from the genes for Aspergillus oiyzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, Aspergillus niger alpha-glucosidase, and Fusarium oxysporum trypsin-like protease. Exemplary terminators for yeast host cells can be obtained from the genes for Saccharomyces cerevisiae enolase, Saccharomyces cerevisiae cytochrome C (CYC1), and Saccharomyces cerevisiae glyceraldehyde-3 -phosphate dehydrogenase. Other useful terminators for yeast host cells are known in the art (see, e.g., Romanos et al., Yeast. 1992. 8(6):423-88). Exemplary terminators for insect cells and mammalian cells include, but are not limited to, those from cytomegalovirus (CMV), Simian virus 40 (SV40). from Homo sapiens growth hormone hGH. from bovine growth hormone BGH, and from human or rabbit beta globulin.

[0258] In some embodiments, the control sequence is a suitable leader sequence, a non-translated region of an mRNA that is used for translation by the host cell. In some embodiments, the leader sequence is operably linked to the 5' terminus of the nucleic acid sequence encoding the phosphatase polypeptide. Any suitable leader sequence that is functional in the host cell of choice find use in the present invention. Exemplary leaders for filamentous fungal host cells are obtained from the genes for Aspergillus oryzae TAKA amylase, and Aspergillus nidulans triose phosphate isomerase. Suitable leaders for yeast host cells are obtained from the genes for Saccharomyces cerevisiae enolase (ENO- 1), Saccharomyces cerevisiae 3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, and Saccharomyces cerevisiae alcohol dehydrogenase/glyceraldehyde-3 -phosphate dehydrogenase (ADH2/GAP). Suitable leaders for mammalian host cells include but are not limited to the 5'-UTR element present in orthopoxvirus mRNA.

[0259] In some embodiments, the control sequence is a polyadenylation sequence (i.e. , a sequence operably linked to the 3' terminus of the nucleic acid sequence and which, when transcribed, is recognized by the host cell as a signal to add polyadenosine residues to transcribed mRNA). Any suitable polyadenylation sequence which is functional in the host cell of choice finds use in the present invention. Exemplary polyadenylation sequences for filamentous fungal host cells include, but are not limited to the genes for Aspergillus oiy zae TAKA amylase. Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase. Fusarium oxysporum trypsin-like protease, and Aspergillus niger alpha-glucosidase. Useful polyadenylation sequences for yeast host cells are known (see, e.g.. Guo and Sherman. Mol. Cell. Biol.. 1995. 15:5983-5990). Useful polyadenylation and 3’ UTR sequences for insect and mammalian host cells include, but are not limited to, OpIE2 polyA sequence, D. melanogaster metallothionein (Mt) polyA signal sequence, D. melanogaster alcohol dehydrogenase (adh), SV40 poly A signal sequence, and the 3'-UTRs of a- and P-globin mRNAs harboring sequence elements that increase the stability and translation of mRNA.

[0260] In some embodiments, the control sequence, where appropriate, comprises a signal peptide (i.e., a coding region that codes for an amino acid sequence linked to the amino terminus of a polypeptide and directs the encoded polypeptide into the cell’s secretory pathway). In some embodiments, the 5 ’ end of the coding sequence of the nucleic acid sequence inherently contains a signal peptide coding region naturally linked in translation reading frame with the segment of the coding region that encodes the secreted polypeptide. Alternatively, in some embodiments, the 5‘ end of the coding sequence contains a signal peptide coding region drat is foreign to the coding sequence. Any suitable signal peptide coding region which directs the expressed polypeptide into the secretory pathway of a host cell of choice finds use for expression of the recombinant polypeptide(s). Effective signal peptide coding regions for bacterial host cells are the signal peptide coding regions include, but are not limited to those obtained from the genes for Bacillus NOB 11837 maltogenic amylase, Bacillus stearothermophilus alpha-amylase, Bacillus lichenifonnis subtilisin, Bacillus licheniformis beta-lactamase, Bacillus stearothermophilus neutral proteases (nprT, nprS, nprM). and Bacillus subtilis prsA. Further signal peptides are known in the art (see, e.g., Simonen and Palva, Microbiol. Rev., 1993, 57:109-137). In some embodiments, effective signal peptide coding regions for filamentous fungal host cells include, but are not limited to the signal peptide coding regions obtained from the genes for Aspergillus oryzae TAKA amylase. Aspergillus niger neutral amylase, Aspergillus niger glucoamylase. Rhizomucor miehei aspartic proteinase, Humicola insolens cellulase, and Humicola lanuginosa lipase. Useful signal peptides for yeast host cells include, but are not limited to those from the genes for Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiae invertase. Useful signal peptides for insect and mammalian host cells include but are not limited to. those from the genes for immunoglobulin gamma (IgG) and the signal peptide in a human secreted protein, such as human beta-galactosidase polypeptide.

[0261] In some embodiments, the control sequence is a propeptide coding region that codes for an amino acid sequence positioned at the amino terminus of a polypeptide. The resultant polypeptide is referred to as a “proenzyme,” “propolypeptide,” or “zy mogen.” A propolypeptide can be converted to a mature active polypeptide by catalytic or autocatalytic cleavage of the propeptide from the propolypeptide. The propeptide coding region may be obtained from any suitable source, including, but not limited to the genes for Bacillus subtilis alkaline protease (aprE). Bacillus subtilis neutral protease (nprT), Saccharomyces cerevisiae alpha-factor, Rhizomucor miehei aspartic proteinase, and Myceliophthora thermophila lactase (see, e.g., WO 95/33836). Where both signal peptide and propeptide regions are present at the amino terminus of a polypeptide, the propeptide region is positioned next to the amino terminus of a polypeptide and the signal peptide region is positioned next to the amino terminus of the propeptide region. [0262] In some embodiments, regulatory sequences are also utilized. These sequences facilitate the regulation of the expression of the polypeptide relative to the growth of the host cell. Examples of regulatory systems are those that cause the expression of the gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound. In prokaryotic host cells, suitable regulatory sequences include, but are not limited to the lac, tac, and trp operator systems. In yeast host cells, suitable regulatory systems include, but are not limited to the ADH2 system or GALI system. In filamentous fungi, suitable regulatory sequences include, but are not limited to the TAKA alpha-amylase promoter, Aspergillus niger glucoamylase promoter, and Aspergillus oryzae glucoamylase promoter.

[0263] In another aspect, the present disclosure provides a recombinant expression vector comprising a recombinant polynucleotide encoding a recombinant phosphatase polypeptide, and one or more expression regulating regions such as a promoter and a terminator, a replication origin, etc., depending on the type of hosts into which they are to be introduced. In some embodiments, the various nucleic acid and control sequences described herein are joined together (i.e.. operably linked) to produce recombinant expression vectors capable of expressing the encoded recombinant phosphatase.

[0264] The recombinant expression vector may be any suitable vector (e.g., a plasmid or virus), that can be conveniently subjected to recombinant DNA procedures and bring about the expression of the phosphatase encoding polynucleotide. The choice of the vector ty pically depends on the compatibility of the vector with the host cell into which the vector is to be introduced. The vectors may be linear or closed circular plasmids.

[0265] In some embodiments, the expression vector is an autonomously replicating vector (i.e., a vector that exists as an extra-chromosomal entity, the replication of which is independent of chromosomal replication, such as a plasmid, an extra-chromosomal element, a minichromosome, or an artificial chromosome). The vector may contain any means for assuring self -replication. In some alternative embodiments, the vector is one in which, when introduced into the host cell, it is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated. Furthermore, in some embodiments, a single vector or plasmid, or two or more vectors or plasmids which together contain the total DNA to be introduced into the genome of the host cell, and/or a transposon is utilized.

[0266] In some embodiment, the recombinant polynucleotides may be provided on a non-replicating expression vector or plasmid. In some embodiments, the non-replicating expression vector or plasmid can be based on viral vectors defective in replication (see, e.g., Travieso et al., npj Vaccines, 2022, Vol. 7, Article 75). [0267] In some embodiments, the expression vector contains one or more selectable markers, which permit selection of transformed cells. A “selectable marker’’ is a gene, the product of which provides for biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, and the like. Examples of bacterial selectable markers include, but are not limited to the dal genes from Bacillus subtilis or Bacillus licheniformis, or markers, which confer antibiotic resistance such as ampicillin, kanamycin, chloramphenicol or tetracycline resistance. Suitable markers for yeast host cells include, but are not limited to ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Selectable markers for use in filamentous fungal host cells include, but are not limited to, amdS (acetamidase; e.g., from A. nidulans or A. orzyae). argB (ornithine carbamoyltransferases), bar (phosphinothricin acetyltransferase; e.g., from S', hygroscopi cits'), hph (hygromycin phosphotransferase). niaD (nitrate reductase), pyrG (orotidine-5'-phosphate decarboxylase; e.g., from A. nidulans or A. orzyae), sC (sulfate adenyltransferase), and trpC (anthranilate synthase), as well as equivalents thereof.

[0268] In another aspect, the present disclosure provides a host cell comprising a recombinant polynucleotide encoding a recombinant phosphatase polypeptide described herein, the polynucleotide(s) being operably linked to one or more control sequences for expression of the recombinant phosphatase enzyme(s) in the host cell. Host cells suitable for use in expressing the polypeptides encoded by the expression vectors of the present invention are known in the art and include but are not limited to, bacterial cells, such as E. coli, B. subtilis, Vibrio fluvialis, Streptomyces and Salmonella typhimurium cells; fungal cells, such as yeast cells (e.g., Saccharomyces cerevisiae or Pichia pastoris (ATCC Accession No. 201178)); insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS, BHK, 293. and Bowes melanoma cells; and plant cells. Exemplary host cells also include various Escherichia coli strains (e.g., W3110 (AfhuA) and BL21).

[0269] In another aspect, the present disclosure provides a method of producing the recombinant phosphatase, the method comprising culturing a host cell capable of expressing a polynucleotide encoding the recombinant phosphatase under conditions suitable for expression of the polypeptide such that the recombinant phosphatase is produced. In some embodiments, the method further comprises isolating the phosphatase polypeptide from the culture and/or host cells. In some embodiments, the method further comprises purifying the expressed phosphatase polypeptide, as described herein.

[0270] In some embodiments, the recombinant phosphatase expressed in a host cell is recovered from the cells and/or the culture medium using any one or more of the known techniques for protein purification, including, among others, lysozyme or detergent treatment, sonication, filtration, saltingout. ultra-centrifugation, and chromatography, such as described herein.

[0271] Chromatographic techniques for isolation/purification of the phosphatase poly peptides include, among others, reverse phase chromatography, high-pcrfonnancc liquid chromatography , ion- exchange chromatography, hydrophobic -interaction chromatography , size-exclusion chromatography, gel electrophoresis, and affinity chromatography. Conditions for purifying the phosphatase depends, in part, on factors such as net charge, hydrophobicity, hydrophilicity, molecular weight, molecular shape, etc., and will be apparent to those having skill in the art. In some embodiments, affinity techniques may be used to isolate the phosphatase. For affinity chromatography purification, an antibody that specifically binds the recombinant phosphatase polypeptide may be used. In some embodiments, an affinity tag. e.g., His-tag, can be introduced into the phosphatase for purposes of isolation/purification.

Compositions

[0272] In a further aspect, the present disclosure provides compositions of the recombinant phosphatases disclosed herein. In some embodiments, the recombinant phosphatase is combined with other components and compounds to provide compositions and formulations comprising the recombinant phosphatase as appropriate for different applications and uses.

10273| In some embodiments, the composition comprises a recombinant phosphatase described herein provided in solution or as a lyophilizate. In some embodiments, the solution or lyophilizate comprises a protective agent, such as protein stabilizer, cryoprotective agent, and/or buffer. In some embodiments, the protein stabilizer or cryoprotective agent comprises polyalcohol, such as glycerol or polyethylene glycol; dimethylsulfoxide (DMSO); reducing agent, e.g., mercaptoethanol or dithiothreitol; metal chelator, e.g.. EDTA; anti-microbial agents, e.g., sodium azide or thimerisol; and surfactants, e g., polysorbate 20 or 80.

[0274] In some embodiments, the composition comprises the phosphatase immobilized on a support medium. In some embodiments, the phosphatase is immobilized on the support medium by adsorption, covalent attachment, ionic interactions, entrapment, or affinity. In some embodiments, the substrate is a solid substrate, porous substrate, membrane, or particles. In some embodiments, the enzyme can be entrapped in matrixes or membranes.

[0275] In some embodiments, the support medium include polymeric materials such as calciumalginate, agar, k-carrageenin. polyacrylamide, agarose or derivatives thereof (e.g., cross-linked agarose), and collagen, or solid matrices, such as activated carbon, porous ceramic, and diatomaceous earth. In some embodiments, the matrix is a particle, a membrane, or a fiber. Types of membranes include, among others, nylon, cellulose, polysulfone, or polyacrylate.

[0276] In some embodiments, the enzyme is immobilized on the surface of a support material. In some embodiments, the enzyme is adsorbed on the support material. In some embodiments, the enzyme is immobilized on the support material by covalent attachment. Support materials include, among others, inorganic materials, such as alumina, silica, porous glass, ceramics, diatomaceous earth, clay, and bentonite, or organic materials, such as cellulose (CMC, DEAE-cellulose), starch, activated carbon, polyacrylamide, polyacrylate, polymcthacrylatc. polyethy lene glycol, dextran, polystyrene, agarose (e.g., cross-linked agarose, and ion-exchange resins, such as Amberlite, Sephadex, and Dowex.

[0277] In a preferred embodiment, the composition comprises a recombinant phosphatase immobilized on a polyacry late substrate, particularly a hydrophilic polyacrylate support where the phosphatase is covalently attached using epoxide groups on the polyacrylate.

[0278] In some embodiments, the composition comprises the phosphatase and a phosphatase substrate having a phosphomonoester group, or an analog thereof, e.g., phosphorothioate. In some embodiments, the substrate comprises NTP, NDP, NMP. 3’-P-NTP. 3’-P-NDP, 3’-P-NMP. Np. NTP- oc-S. NDP-a-S, NMP-a-S, 3’-P-NTP-a-S. 3’-P-NDP-a-S, 3’-P-NMP-a-S, NpS. or any combination thereof. In the foregoing, a 3’-P or Np refers to the presence of a phosphate at the 3 ’-OH of the furanosyl sugar moiety of the nucleotide substrate.

[0279] In some embodiments, the composition comprises the phosphatase and a polynucleotide or oligonucleotide having a 5’-phosphate (5 ’-P) and/or a 3’-phosphate (3 ’-P). In some embodiments, the polynucleotide or oligonucleotide substrate comprises a 5’-P and a 3’-P. In some embodiments, the 5’-P and/or 3’-P is a phosphomonoester analog, for example, 5 ’-phosphorothioate (5 ’-S). or 3’- phosphorothioate (3’-S).

[0280] In some embodiments, the polynucleotide or oligonucleotide substrate is single stranded or double stranded. In some embodiments, the polynucleotide or oligonucleotide substrate is RNA. DNA, or a mixture of RNA and DNA. In some embodiments, the polynucleotide or oligonucleotide substrate comprises at least one modified nucleoside and/or internucleoside linkage. In some embodiments, the modified nucleoside comprises a modified nucleobase, modified sugar moiety, or a combination of modified nucleobase and modified sugar moiety.

[0281] In some embodiments, the modified polynucleotide or oligonucleotide comprises at least one modified, non-naturally occurring internucleoside linkage. In some embodiments, the modified oligonucleotide has 1%, 2%, 5%. 10% 20%, 30%. 40%, 50%, or 60% or more modified internucleoside linkages. In some embodiments, all of the intemucleoside linkages are modified internucleoside linkages.

[0282] In some embodiments, the modified intemucleoside linkages are present in the 5 ’-terminal region of the polynucleotide or oligonucleotide substrate. In some embodiments, at least 1, 2, 3, 4, or 5 modified intemucleoside linkages are present at the 5 -terminal region of die polynucleotide or oligonucleotide substrate, as limited by the length of the oligonucleotide acceptor. In some embodiments at least 1 or 2 phosphorothioate intemucleoside linkages are present at the 5 ’-terminal region of the oligonucleotide acceptor. [0283] In some embodiments, the modified intemucleoside linkages are present in the 3 ’-terminal region of a polynucleotide or oligonucleotide substrate. In some embodiments, at least 1, 2, 3, 4, or 5 modified intemucleoside linkages are present at the 3 ’-terminal region of the polynucleotide or oligonucleotide, as limited by the length of the polynucleotide or oligonucleotide. In some embodiments, at last 1 or 2 phosphorothioate internucleoside linkages are present at the 3 ‘-terminal region of a polynucleotide or oligonucleotide. In some embodiments, the modified intemucleoside linkages are present in the internal portions of the oligonucleotide acceptor and/or oligonucleotide donor.

[0284] In some embodiments, the modified intemucleoside linkage is a phosphorous containing modified intemucleoside linkage. Exemplary phosphorous-containing intemucleoside linkages include, among others, phosphotriester, alkylphosphonate (e.g., methyl phosphonate, ethyl phosphonate, etc.), phosphoramidate, phosphorothioate, and phosphorodithioate.

[0285] In some embodiments, the modified intemucleoside linkage is a non-phosphorous containing intemucleoside linkage. Exemplary non-phosphorous containing intemucleoside linkages include, among others, methylenemethylimino (-CH₂-N(CH₃)-O-CH₂), thiodiestcr, thionocarbamate (-0- C(=O)(NH)-S-); siloxane (-O-SiH₂-O-); N,N’ -dimethylhydrazine (-CH₂-N((CH₃)-N((CH₃)-); MMI (3'-CH₂-N(CH₃)-O-5'), amide-3 (3'-CH₂-C(=O)-N(H)-5'), amide-4 (3'-CH₂-N(H)-C(=O)-5'), fonnacctal (3'-O-CH₂-O-5'), methoxy propyl, and thioformacctal (3’-S-CH₂-O-5').

[0286] In some embodiments, the modified intemucleoside linkages provides for a chiral center. For example, a phosphorothioate or alkylphosphonate intemucleoside linkage can be in the Rp or Sp stereomeric configmation. In some embodiments, the polynucleotide or oligonucleotide substrate has a mixture of stereoisomers in the intemucleoside linkage. In some embodiments, the polynucleotide or oligonucleotide substrate has greater than 50% of the intemucleoside linkages as Rp or Sp configuration. In some embodiments, the polynucleotide or oligonucleotide substrate have at least 60%, 70%, 80%, 90%, or greater of Rp or Sp stereomeric configuration.

[0287] In some embodiments, the polynucleotide or oligonucleotide substrate comprises at least a phosphorothioate intemucleoside linkage, where the phosphorothioate linkage is in the Sp configuration, the Rp configuration, or a mixture of Sp and Rp configuration in the population of polynucleotide or oligonucleotide substrate.

[0288] In some embodiments, the polynucleotide or oligonucleotide substrate comprises one or more modified nucleotide residues having a modified nucleobase. In some embodiments, the modified nucleobase comprises, among others, xanthine, hypoxanthine, inosine, 6-methyladenine, 7- methylguanine. 2,6-diaminopurine, 5 -methylcytosine, 5-hydroxycytosine, 5-bromocytosine, 5- iodocytosine, 2-thiothymine, 5-fluorouracil, 5 -bromouracil. 8-bromoguanine, 8 -amino guanine, or 8- aza-7-deazaguanine. In some embodiments, the nucleobase is modified with a conjugate, such as a targeting moiety, for example, GalNAc and lipid moieties.

[0289] In some embodiments of the method, the polynucleotide or oligonucleotide substrate comprises one or more modified nucleoside sugar residues. In some embodiments, the modified nucleoside sugar residue is a 2’-O-alkyl, a 2’-halo. a P-D-ribo LNA, or a a-L-ribo-LNA (e.g., locked nucleic acids). In some embodiments, the modified nucleoside sugar residue is, among others, a 2 -0- methyl, 2‘-0-ethyl, 2’-O-propyl group, 2‘-O-methoxyethyl, 2’-ally 1, or 2'-propargyl. In some embodiments, the modified nucleoside sugar residue is 2’-fluoro, 2’-bromo, or 2’-chloro, preferably 2’ -fluoro. In some embodiments, the sugar residue is modified with a conjugate, such as a targeting moiety, for example. GalNAc or lipid moieties.

Uses of Recombinant Phosphatases

[0290] In another aspect, the present disclosure provides uses of the recombinant phosphatases for cleaving (i.e. , hydrolysis) of a substrate having a phosphate monoester or analog thereof.

[0291] In some embodiments, a method of cleaving a phosphate monoester or analog thereof, comprises contacting a substrate comprising a phosphate monoester or analog thereof with a recombinant phosphatase described herein under suitable conditions for cleaving of the phosphate monoester or analog thereof. In some embodiments, the contacting the phosphatase substrate with the recombinant phosphatase is an in vitro reaction.

[0292] In some embodiments, the phosphatase substrate comprising a phosphate monoester or analog thereof is a phosphatase substrate used to measure phosphatase activity. Exemplary phosphatase substrates used to measure activity include, among others, dinitrophenyl phosphate (pNPP), 2,6- dichloro-4-nitrophenyl phosphate, 5-bromo-4-chloro-3-indolyl phosphate, dimethylacridinone (DDAO) phosphate, 4-methylumbelliferyl phosphate, and ELF 97 phosphate.

[0293] In some embodiments, the phosphatase substrate comprises NTP, NDP, NMP, 3’-P-NTP, 3’- P-NDP, 3’-P-NMP, Np, NTP-a-S, NDP-a-S, NMP-a-S, 3’-P-NTP-a-S, 3’-P-NDP-a-S. 3’-P-NMP- a-S, NpS. or any combinations thereof.

[0294] In some embodiments, the phosphatase substrate comprises a polynucleotide or oligonucleotide with a 5‘-P, 3’-P, 5’-P(S), 3’-P(S), or any suitable combinations thereof.

[0295] In some embodiments, the polynucleotide or oligonucleotide substrate is single stranded or double stranded. In some embodiments, the polynucleotide or oligonucleotide substrate is DNA, RNA, or a mixture of DNA and RNA. In some embodiments, the polynucleotide or oligonucleotide substrate comprises at least a modified nucleoside or intemucleoside linkage, as described herein.

[0296] In some embodiments, phosphatase reaction is carried out at a suitable temperature and reaction time period. In some embodiments, the reaction temperature is from about 2° C to about 80° C. In some embodiments, the phosphatase reaction temperature is from 4 °C to 80 °C, 4 °C to 75 °C, 4 °C to 70 °C, 4 °C to 60 °C, 4 °C to 50 °C, or 10 °C to 40 °C. In some embodiments, the phosphatase reaction temperature is 2 °C, 5 °C, 10 °C, 15 °C, 20 °C, 25 °C, 30 °C, 37 °C, 40 °C, 45 °C, 50 °C. 55 °C, 60 °C, 65 °C. 70 °C, 75 °C, or 80 °C. In some embodiments, the phosphatase reaction temperature can use different temperatures, for example a temperature at which double stranded polynucleotide or oligonucleotide substrates are single stranded and a lower temperature at which the phosphatase has greater stability.

[0297] In some embodiments, the suitable reaction conditions comprise a pH of > 7. In some embodiments, the suitable reaction conditions comprise a pH of about 7-10. In some embodiments, die suitable reaction conditions comprises a pH of about 7.5-9, particularly pH 7.5-8. In some embodiments, the suitable reaction conditions comprises about pH 7. 7.5, 8, 8.5, 9, 9.5 or 10.

[0298] In some embodiments, the phosphatase reaction time can be a sufficient or suitable time for cleavage of phosphomonoester moiety or analog thereof. In some embodiments, the phosphatase reaction time is from 0.1-72 hr or longer. In some embodiments, the phosphatase reaction time is 0.1- 72 hr, 0.5-65 hr, 1-50 hr, 2-48 hr, or 2-24 hr. In some embodiments, the phosphatase reaction time is 0.5, 1. 2, 4, 5, 12, 24, 48, or 72 hr or longer. In some embodiments, the reaction time is adjusted based on, among others, whether the phosphatase is in solution or immobilized on a support medium, die nature of the substrate (c.g., nucleotide, oligonucleotide, phosphomonocstcr, phosphate monocstcr analog, etc.)

[0299] In some embodiments of the method, the recombinant phosphatase is immobilized on a support medium and the phosphatase substrate reacted with the immobilized recombinant phosphatase. In some embodiments, the immobilized recombinant phosphatase is retained in a chamber, for example a column or vessel, and the phosphatase substrate fed into the chamber, e.g., column or vessel, for reaction with the recombinant phosphatase.

[0300] In some embodiments, the phosphatase substrate is contacted with the recombinant phosphatase immobilized on a support medium, and separated from the recombinant phosphatase immobilized on a support medium following cleavage reaction. In some embodiments, the solution containing the phosphatase substrate and cleavage products can be recirculated through the immobilized recombinant phosphatase in the chamber to increase or complete the dephosphorylation reaction.

[0301] In some alternative embodiments, the polynucleotide or oligonucleotide substrate is bound to a support medium, and the polynucleotide or oligonucleotide bound to a support medium contacted with the recombinant phosphatase. In some embodiments, the recombinant phosphatase is in solution. In some embodiments, the polynucleotide or oligonucleotide substrate bound to the support medium is retained in a chamber, for example a column or a reaction vessel, and the recombinant phosphatase fed into the chamber, e.g., column or vessel, for reaction with the polynucleotide or oligonucleotide substrate bound to the support medium.

[0302] In some embodiments, the recombinant phosphatase in solution is separated from the polynucleotide or oligonucleotide bound to the support medium following depho sphorylation reaction. In some embodiments, the solution containing the recombinant phosphatase following separation from the polynucleotide or oligonucleotide is recirculated through the chamber containing the polynucleotide or oligonucleotide substrate bound to the support medium to increase or complete die dephosphorylation reaction.

EXAMPLES

[0303] The following Examples, including experiments and results achieved, are provided for illustrative purposes only and are not to be construed as limiting the present invention.

[0304] In the experimental disclosure below, the following abbreviations where relevant apply: ppm (parts per million); M (molar); mM (millimolar), uM and pM (micromolar); nM (nanomolar); mol (moles); gm and g (gram); mg (milligrams); ug and pg (micrograms); L and 1 (liter); ml and mL (milliliter); ul. uL, pl, and pL (microliter); cm (centimeters); mm (millimeters); um and pm (micrometers); sec. (seconds); min(s) (minute(s)); h(s) and hr(s) (hour(s)); U (units); OD (optical density); MW (molecular weight); rpm (rotations per minute); ref (relative centrifugal force); psi and PSI (pounds per square inch); °C (degree Celsius); RT and rt (room temperature); ds (double stranded); ss (single stranded); CDS (coding sequence); DNA (deoxyribonucleic acid); RNA (ribonucleic acid); E. coli W3110 (commonly used laboratory E. coli strain, available from the Coli Genetic Stock Center [CGSC], New Haven, CT); HPLC (high pressure liquid chromatograph} ): FPLC (fast protein liquid chromatography); PBS (phosphate buffered saline); BSA (bovine serum albumin); DTT (dithiothreitol); CAM (chloramphenicol); CAT (chloramphenicol acetyltransferase); IPTG (isopropyl [3-D-l -thiogalactopyranoside); FIOPC or FIOP (fold improvements over positive control or parent); LB (Luria-Bertani); TB (Terrific -Broth); MWCO (molecular weight cut-off).

[0305] Below is the list of oligonucleotides SEQ ID NOs. 19 and 20 with its sequence used in the phosphatase reaction, along with the products SEQ ID NOs: 21 and 22. The oligonucleotide SEQ ID 22 is part of the phosphatase activity assays but was not monitored directly.

Example 1

E. coli Expression Hosts Containing Recombinant Alkaline Phosphatase Genes

[0306] Synthetic genes (SEQ ID NO: 2, 4, 6, 8, 10, 12, 14) encoding an N-terminal 6-histidine tagged version of multiple wild-type (WT) alkaline phosphatases were designed with codon optimization for E. coli expression, synthesized, and subcloned into the E. coli expression vector pCK100900i (See e.g., US Pat. No. 7,629.157 and US Pat. Appln. Publn. 2016/0244787. both of which are hereby incorporated by reference). The resulting plasmids were transformed into E. coli W3110, using standard methods known in the art. The transformants were isolated by subjecting the cells to CAM selection, as known in the art (See e.g., US Pat. No. 8,383,346 and W02010/144103).

Example 2 Preparation of HTP Alkaline Phosphatase Containing Wet Cell Pellets

[0307] Transformed E. coli cells were selected by plating onto LB agar plates containing 1% glucose and 30 pg/mL chloramphenicol. After overnight incubation at 37 °C, colonies were placed into the wells of 96-well shallow flat bottom NUNC™ (Thermo-Scientific, US) plates filled with 180 pl/well LB medium supplemented with 1% glucose and 30 pg/mL chloramphenicol. The cultures were allowed to grow overnight for 18-20 hours in a shaker (200 rpm, 30 °C. and 85% relative humidity; Kuhner). Overnight growth samples (20 pL) were transferred into Costar 96-well deep plates filled with 380 pL of Terrific Broth supplemented with 30 pg/mL chloramphenicol. The plates were incubated for 120 minutes in a shaker (250 rpm, 30 C, and 85% relative humidity; Kuhner) until the ODeoo reached between 0.4-0.8. The cells were then induced with 40 pL of 10 mM IPTG in sterile water and incubated overnight for 18-20 hours in a shaker (250 rpm, 30 C, and 85% relative humidity; Kuhner). The cells were pelleted (4,000 rpm for 20 min), the supernatants were discarded, and the cells were frozen at -80 °C prior to analysis.

Example 3 Shake Flask Expression and Purification of Alkaline Phosphatase

Shake Flask Expression

[0308] Selected HTP cultures grown as described above were plated onto LB agar plates with 1% glucose and 30 pg/mL chloramphenicol and grown overnight at 37 °C. A single colony from each culture was transferred to 5 mL of LB broth with 1% glucose and 30 pg/mL chloramphenicol. The cultures were grown for 20 h at 30 °C, 250 rpm, and subculturcd at a dilution of approximately 1:50 into 250 mL of Terrific Broth with 30 pg/mL of chloramphenicol, to a final OD₆oo of about 0.05. The cultures were incubated for approximately 195 min at 30 °C, 250 rpm, to an ODgoo of about 0.6, and then induced with the addition of IPTG at a final concentration of 1 mM. The induced cultures were incubated for 20 h at 30 °C, 250 rpm. Following this incubation period, the cultures were centrifuged at 4,000 rpm for 10 min. The culture supernatant was discarded, and the pellets were resuspended in 35 mL of 20 mM triethanolamine, pH 7.5. This cell suspension was chilled in an ice bath and lysed using a Microfluidizer cell disruptor (Microfluidics M-l 10L). The crude lysate was pelleted by centrifugation (11.000 rpm for 60 min at 4 °C), and the supernatant was then filtered through a 0.2 pm PES membrane to further clarify the lysate.

Purification of Alkaline Phosphatase from Shake Flask Lysates

[0309] Alkaline phosphatase lysates were supplemented with 1/10* volume of SF elution buffer (50 mM Tris-HCl, 500 mM NaCl, 250 mM imidazole, 0.02% v/v Triton X-100 reagent) per well. Lysates were then purified using an AKTA Start purification system and a 5 mL HisTrap FF column (GE Healthcare) using the AC Step HiF setting (the run parameters are provided below). The SF wash buffer comprised 50 mM Tris-HCl, 300 mM NaCl, 20 mM imidazole, 0.02% v/v Triton X-100 reagent.

[0310] Elution fractions containing protein were identified by UV absorption (A280) and pooled, then dialyzed overnight in dialysis buffer (20 mM Tris-HCl, pH 7.4, 100 mM KC1, 0.1 mM EDTA. and 50% glycerol) in a 3.5K Slide-A-Lyzer™ dialysis cassette (ThennoFisher) for buffer exchange. Alkaline phosphatase concentrations in the preparations were measured by absorption at 280 nm.

Example 4

Thermal Lysis of HTP Cell Pellets with Lysozyme

[0311] For lysis, 300 uL lysis buffer containing 20 mM triethanolamine buffer, pH 7.8, and 0.1 g/L lysozyme were added to the cell pellet in each well. The cells were shaken vigorously at room temperature for 5 minutes on a bench top shaker. A 100-uL aliquot of the re-suspended cells was transferred to a 96-well fonnat 200 pL BioRad PCR plate, then briefly spun-down prior to 1 h heat treatment at 55 °C. Following heat-treatment, the cell debris was pelleted by centrifugation (4,000 rpm at 4 °C for 10 min), and clear supernatants were then used in biocatalytic reactions to determine their activity levels. The truncation library was screened and improved variants were produced in shake flask and characterized.

Example 5 Capillary Electrophoresis (CE) Analysis of Oligonucleotides

Sample preparation for reaction analysis using CE

[0312] For analysis of the reaction samples, capillary electrophoresis was performed using an ABI 3500x1 Genetic Analyzer (ThermoFisher™). Reactions (1 pL) were quenched by the addition of 99 pL of 1 mM aqueous EDTA. Quenched reactions were diluted in water to 1.25 nM oligonucleotide, and a 2-pL aliquot of this solution was transferred to a new 96-well MicroAmp Optical PCR plate or 384- well MicroAmp Optical PCR plate containing 18 pL Hi-Di™ Formamide (ThermoFisher™) containing an appropriate size standard (LIZ or Alexa633). The ABI3500xl was configured with POP6 polymer, 50 cm capillaries, and a 55 °C oven temperature. Pre-run settings were 18KV for 50 sec. Injection was 10KV for 2 sec, and the run settings were 19KV for 620 sec. FAM-labeled oligo substrates and products were identified by their mobility relative to the sizing ladder.

Example 6

Expression of Wild-type Alkaline Phosphatases [0313] Cells transformed with the alkaline phosphatase expression constructs were grown at shakeflask scale using IPTG induction as described in Example 3. Cells were then lysed, purified, and dialyzed into storage buffer (20 mM Tris-HCl, pH 7.4, 100 mM KC1, 0.1 mM EDTA, and 50% glycerol). After overnight dialysis, protein samples were removed, and enzyme concentrations were measured by absorption at 280 nm using a NanoDrop™ 1000 spectrophotometer. Soluble protein concentrations are summarized in Table 2 below, showing a fold improvement in soluble protein production following shake-flask purification relative to the alkaline phosphatase from Thermotoga neapolitana (SEQ ID NO: 2).

Example 8

Improvements Over SEQ ID NO: 12

[0314] The phosphatase of SEQ ID NO: 12 was selected as the parent enzyme after screening for expression as described in Example 7. Directed evolution techniques generally known by those skilled in the art were used to generate N-terminal truncation library of the subcloned gene variant (See e g., US Pat. No. 8,383,346 and WO 2010/144103, both of which are hereby incorporated by reference). The polypeptides encoded by each gene were produced in HTP as described in Example 1. and tire cell paste was generated as described in Example 2. Cell lysis was performed as described in Example 4.

[0315] To screen the enzyme variants, reactions included 10 pM 3’-blocked oligonucleotide, 2500 pM of unlabeled 3’-blocked oligonucleotide pair, 25 % lysate (v/v), 20 mM triethanolamine (pH 7.8), and 250 pM cobalt (II) chloride. The reactions were set up as follows: (i) all reaction components, except for AP, were pre-mixed in a single solution and were aliquoted into each well of the 96-well plates (ii) AP solution was then added into the wells to initiate the reaction. The reaction plate was heat-sealed with a peelable aluminum seal and incubated in a thermocycler at the indicated temperature and reaction time, then held at 4 °C until the reaction was quenched. Reactions were quenched and processed for CE analysis as described in Example 5.

Example 9

Isolated Protein Yield Improvements Over SEQ ID NO: 12

Relative isolated protein yield of shake-flask purified alkaline phosphatase variants

[0316] Alkaline phosphatase variants of SEQ ID NO: 16 and 18 with improved activity in HTP were produced in shake flask and purified as described in Example 3. Alkaline phosphatase concentrations were measured by absorption at 280 nm.

[0317] Protein recover}’ relative to SEQ ID NO: 12 was calculated as the ratio of mg/mL protein recovered after purification of the variant compared with SEQ ID NO: 12. The results are shown in Table 4

Example 10

3'-Phosphate Deblocking Activity of Shake-Flask Purified Alkaline Phosphatases

[0318] Alkaline phosphatases (APs) SEQ ID NO: 12. 16, and 18 were assayed for 3'- dephosphorylation activity of oligonucleotides SEQ ID NOs: 19 and 20. [0319] Reactions were performed in 96-well format 200 pL BioRad PCR plates. Reactions included 10 pM 3’-blocked oligonucleotide, 2500 pM of unlabeled 3 ’-blocked oligonucleotide pair, 5 pM - 0.04 pM AP, 20 mM triethanolamine (pH 7.8), and 250 pM cobalt (II) chloride. The reactions were set up as follows: (i) all reaction components, except for AP, were pre-mixed in a single solution and were aliquoted into each well of the 96-well plates (ii) AP solution was then added into the wells to initiate the reaction. The reaction plate was heat-sealed with a peelable aluminum seal and incubated in a thermocycler at the indicated temperature and reaction time, then held at 4 °C until the reaction was quenched. Reactions were quenched and processed for CE analysis as described in Example 5.

[0320] Activity relative to SEQ ID NO: 12 (Activity FIOP) was calculated at AP concentration of 0.08 pM as the percent product of the variant compared with the percent product observed by the reaction with SEQ ID NO: 12 (where the percent product may be set as the average of replicates or else the highest single sample as appropriate). The results are shown in Table 5.2.

Example 11

Thermal Stability Improvements Over SEQ ID NO: 12 [0321] Alkaline phosphatases (APs) SEQ ID NO: 12, 16, and 18 were assayed for 3’- dephosphorylation activity of oligonucleotides SEQ ID NOs: 19 and 20 after 15 minutes of incubation at 75.8 °C.

[0322] Reactions were performed in 96-well format 200 pL BioRad PCR plates. Reactions included 10 pM 3’-blocked oligonucleotide, 2500 pM of unlabeled 3 ’-blocked oligonucleotide pair, 1 pM AP, 20 rnM triethanolamine (pH 7.8), and 250 pM cobalt (II) chloride. The reactions were set up as follows: (i) alkaline phosphatase was pre-mixed with 250 pM cobalt (II) chloride and 20 mM triethanolamine (pH 7.8) and 20 pL of this solution was aliquoted into each well of the 96-well plate; (ii) the plate was heated for 15 minutes at 75.8 °C; (iii) 250 nL of the heat-treated solution was transferred into a 96-well plate containing 750 nL reaction mix (10 pM 3 '-blocked oligonucleotide, 2500 pM of unlabeled 3‘-blocked oligonucleotide pair, 20 mM triethanolamine (pH 7.8), and 250 pM cobalt (II) chloride); (iv) Reaction plates were heat-sealed with a peelable aluminum seal and incubated in a thennocycler at the indicated temperature, then held at 4 °C until the reaction was quenched. Reactions were quenched and diluted for analytical analysis by CE as described in Example 5.

[0323] Activity⁷ relative to SEQ ID NO: 12 (Activity FIOP) was calculated as the percent product of the variant as measured after a 75.8 °C pre-incubation compared with the percent product observed by the reaction with SEQ ID NO: 12. The results are shown in Table 6.2. Example 12

Size Exclusion Chromatography with Multi-Angle Light Scattering Analysis (SEC-MALS)

[0324] Alkaline phosphatases (APs) SEQ ID NO: 12, 16, and 18 were analyzed using size exclusion chromatography (SEC) with in line UV, multi-angle light scattering and differential refractive index (UV-MALS-dRI) detectors to determine the molecular weight and the oligomeric state of the proteins.

[0325] Alkaline phosphatase solutions were diluted at 1.0 mg/mL in the mobile phase (300 mM potassium phosphate buffer, pH 7.0) for analysis. The samples were subject to HPLC analysis mrder tire following conditions:

[0326] The peak area%, molecular weight, extinction coefficient results comparing with the theoretical values are shown in Table 7.2.

Example 13

Dephosphorylation Activity with 3’-phosphate-mATP [0327] Alkaline phosphatases (APs) SEQ ID NO: 12, 16, and 18 were assayed for 3’- dephosphorylation activity of 3’-phosphate-mATP and species decomposition was analyzed by HPLC (see Example 14).

[0328] Reactions were performed in 96-well format 200 pL BioRad PCR plates. Reactions included 1 mM 3’-phos-mATP, 5 pM - 0.04 pM AP, 50 mM triethanolamine (pH 7.8), and 500 pM cobalt (II) chloride. The reactions were set up as follows: (i) all reaction components, except for AP, were premixed in a single solution, and was aliquoted into each well of the 96-well plates (ii) AP solution was then added into the wells to initiate the reaction. The reaction plate was heat-sealed with a peelable aluminum seal and incubated in a thcrmocyclcr at 50 °C for 15 minutes then held at 4 °C until the reaction was quenched. The reactions were quenched by the addition of 75 pL of 50 % MeOH, vortexed and centrifuged for 10 minutes at 4000 rpm. The reactions were further diluted 1:1 with milli-Q water, to a substrate concentration of 50 p. The samples were then analyzed on HPLC, as described in Example 14.

[0329] Activity relative to SEQ ID NO: 12 (Activity FIOP) was calculated at AP concentration of 0.625 pM as the percent mA of the variant compared with the percent mA observed by the reaction with SEQ ID NO: 12. The results are shown in Table 8.2.

Example 14

High Performance Liquid Chromatography (HPLC) Analysis of Nucleotides [0330] Samples reactions are quenched and diluted as described above in example 10. 100 pL is transferred to a 96 well round bottom plate and then 5 pL is injected onto an Ultimate 3000 HPLC system using a PAL autosampler according to die method outlined in Table 9.

Example 15

Immobilization of Alkaline Phosphatase Variants on Polyacrylate Resin

[0331] Alkaline phosphatases (APs) SEQ ID NO: 12, 16, and 18 were immobilized onto polyacrylate resin (ChiralVision, Netherlands) using the development scale (6.6 x 50 mm) BioRad columns.

[0332] Prior mixing with the resin the buffer exchange was performed for all three alkaline phosphatase variants from storage buffer (20 mM Tris-HCl, pH 7.4, 100 mM KC1. 0.1 mM EDTA, and 50% glycerol) into 20 mM MOPS (pH 7). The enzyme was diluted 10-fold in the 20 mM MOPS (pH 7) and concentrated using the 10,000 mwco spin fdter. 3 mg of each enzyme variant was mixed with 150 mg of the polyacrylate resin and incubated overnight at 4 °C. After overnight incubation, the resin was washed 3 times with 1 mL of 50 mM triethanolamine (pH 7.8). The washed resin was loaded onto BioRad columns and heated at 50 °C. The loaded resin was washed with 50 mM triethanolamine (pH 7.8) at 0.7 ml/min. Wash fractions were collected in volume of 420 pl and the p- nitrophenol phosphate assay activity test was performed as in the Example 13 described below. The % enzyme detected in wash fractions was calculated as ratio between enzyme detected in wash fraction and total bound enzyme and multiplied with 100 shown in Table 10.

Example 16

Activity of Alkaline Phosphatase with p-Nitrophenol Phosphate

[0333] Alkaline phosphatases (APs) SEQ ID NO: 12. 16, and 18 were assayed for activity with p- nitrophenol phosphate to determine the amount of unbound enzyme to the resin as described in the Example 15.

[0334] Reactions were performed in 96-well format 200 pL BioRad PCR plates. Standard curves were prepared for each enzyme variant by mixing 35 pL alkaline phosphatase of a known concentration (0.8 pM- 0.8 nM) and 35 pL of 2 mM p-nitrophenol phosphate in 50 rnM triethanolamine buffer (pH 7.8) with 500 pM Cobalt (II) Chloride The reactions were set up as follows: (i) p-nitrophenol phosphate was aliquoted into each well of the 96-well plates (ii) AP solution was then added into the wells to initiate the reaction. The reaction plate was heat-sealed w ith a peelable aluminum seal and incubated in a thermocycler at 50 °C for 15 minutes then held at 4 °C until the reaction was quenched. The reactions were quenched by the addition of 35 pL of 2N NaOH, vortexed and centrifuged. Quenched reaction (50 pL) was transferred to half area UV star plates and the amount of produced p-nitrophenol was measured by absorption at 405 nm. For the w ash fractions in the Example 15 above, the reactions included 35 pL of the wash fraction and 35 pL of p- nitrophenol phosphate. The amount of enzyme (nmol) in w ash fractions was calculated based on a standard curve for each variant.

Example 17 Dephosphorylation Activity of Immobilized AP with 3’-phos-mATP

[0335] Immobilized alkaline phosphatases (APs) SEQ ID NO: 12, 16, and 18 were assayed for activity with 3’-phosphate-mATP and species decomposition was analyzed by HPLC (see Example 14).

[0336] Reactions were performed in flow' using the immobilized enzyme from Example 12. 10 in of solution containing 1 mM 3’-phos-mATP, 50 mM triethanolamine (pH 7.8), and 500 pM cobalt (II) chloride was run through tire column at the rate of 0.07 mL/min for 2 hours at 50 °C. 1 mL fractions were collected and the HPLC analysis from Example 12 was performed.

[0337] Activity' relative to SEQ ID NO: 12 (Activity FIOP) was calculated as the 1/ percent 3’ -phos- mATP of the variant compared with the 1 /percent 3’-phos-mATP observed by the reaction with SEQ ID NO: 12. The results are shown in Table 11.

Example 18

Oligonucleotide Deblocking Activity Improvements Over SEQ ID NO: 18.

[0338] The phosphatase of SEQ ID NO: 18 was selected as the parent enzyme to design a homology' library of surface lysine and cysteine residues. Directed evolution techniques generally known by those skilled in the art were used to produce the library . The polypeptides encoded by each gene were produced in HTP as described in Example 1 and the cell paste was generated as described in Example 2. Cell lysis was performed as described in Example 4. Alkaline phosphatase activity was evaluated in the oligonucleotides 3 ’-dephosphorylation assay as described in Example 5. The HTP screening conditions are listed in Table 12.1. [0339] Activity relative to SEQ ID NO: 18 (FIOP) was calculated as the percent dephosphorylated product formed by the variant over the percent product of SEQ ID NO: 18 (see Table 12.2).

Example 19

Improvement of Resistance to Polyacrylate Resin Inhibition Over SEQ ID NO: 18

|0340| Inhibitory effect of hydrophilic, polyacrylic resin activated with epoxide groups (ChiralVision. Netherlands) on AP activity was evaluated with HTP lysates described in Example 18. About 14 mg of the resin was aliquoted to each well on 96-well Axygen round bottom plate. Lysates were diluted 16-fold in 20 mM MOPS (pH 8.0) and 200 ul of these dilutions was added into each well with the resin. A control plate was prepared similarly except no resin was added to wells. Both plates were incubated in a multitone at 25 °C and 600 rpm for 20 hours. The alkaline phosphatase activity was evaluated in the PNPP dephosphorylation assay as described in Example 6. The reactions were initiated by addition of 200 ul solution composed of 3 mM PNPP, 0.5 mM CoCL. and 50 mM MOPS (pH 8.0), incubated a multitone at 50 °C and 600 rpm for 15 minutes. To quench the reaction, 40 pL aliquots were mixed with 20 pL 2M NaOH. The quenched samples were transferred to UV-star plates and absorbance at 405 mn was measured. The ratio of the absorbance values for a sample with and without resin was used as the measure of retained phosphatase activity in the presence of resin. Improvement in resistance to resin relative to SEQ ID NO: 18 (FIOP) was calculated as the retained phosphatase activity over the retained phosphatase activity of SEQ ID NO: 18.

Example 20

Improvement of Resistance to Proteolysis Over SEQ ID NO: 12

[0341] The phosphatase of SEQ ID NO: 12 was selected as the parent enzy me to design a library’ targeting N-terminus of the polypeptide. Directed evolution techniques generally known by those skilled in the art were used to produce the library’. The polypeptides encoded by each gene were produced in HTP as described in Example 1 and the cell paste was generated as described in Example 2. To prepare cells for lysis, 600 pL lysis buffer containing 20 mM triethanolamine buffer. pH 7.8, and 0.5 g/L lysozyme were added to the cell pellet in each well. The cells were shaken vigorously at room temperature for 4 hours on a bench top shaker. After 4 hours, the plates were centrifuged at 4000 rpm. in 4 °C. for 15 minutes. The clarified lysate was purified using HisPur™ Ni-NTA plates, using the manufacturer’s protocol. The plates were pre-equilibrated with the Wash Buffer wash buffer comprised 50 mM Tris-HCl, 300 mM NaCl, 20 mM imidazole, 0.02% v/v Triton X-100. The clarified lysate was applied to the HisPur Ni-NTA plates, 400 pL/well and the plates were incubated for 30 min at RT. After the binding step, the resin was washed 3 times, each wash with 400 pL the Wash Buffer. The enzyme was eluted with 70 pL of 50mM Tris-HCl, 500 mM NaCl. 250 mM imidazole. 0.02% v/v Triton X-100 reagent. The purified protein was desalted using Zeba (7k MWCO) spin desalting plate and following the manufacturer’s protocol. The desalting plates were pre-equilibrated with the buffer comprised 40 mM Tris-HCl, pH 7.4, 200 mM KC1, 0.2 mM EDTA. The desalted samples were mixed with equal volume of 80% glycerol and stored at -20 °C prior analysis.

[0342] Purified protein yield was determined using Pierce Protein 660 nm assay according to the manufacturer’s protocol. Standard curves were prepared using alkaline phosphatase of a known concentration (0.1 - 1 mg/mL). Protein recovery relative to SEQ ID NO: 12 was calculated as the ratio of mg/mL protein recovered after purification of the variant compared with SEQ ID NO: 12.

The results are shown in Table 14.

Example 21

Isolated Protein Yield and Resistance to Proteolysis Improvements Over SEQ ID NO: 12

[0343] Alkaline phosphatase variants of SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 90. SEQ ID NO: 92, SEQ ID NO: 112. and SEQ ID NO: 124 with improved yield in HTP were produced in shake flasks and purified as described in Example 3. Improvement of resistance to proteolysis was evaluated by SDS-PAGE analysis of the cell lysates.

[0344] Protein recovery relative to SEQ ID NO: 12 was calculated as the ratio of mg protein recovered after purification of tire variant compared with SEQ ID NO: 12. The results are shown in Table 15. Improvement of resistance to proteolysis was evaluated by SDS-PAGE analysis of the cell lysates. The results are shown in Table 15.

Example 22

Oligonucleotide Deblocking Activity Improvements Over SEQ ID NO: 84 [0345] The phosphatase of SEQ ID NO: 84 was selected as the parent enzy me after screening variants as described in Examples 20 and 21 above. Libraries of engineered genes were produced using well established techniques (e.g., saturation mutagenesis, recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were produced in HTP as described in Example 1 and the cell paste was generated as described in Example 2. Cell lysis was performed as described in Example 4. Alkaline phosphatase activity was evaluated in the oligonucleotides 3’- dephosphorylation assay as described in Example 5. The HTP screening conditions are listed in Table 16.1.

[0346] Activity relative to SEQ ID NO: 84 (FIOP) was calculated as the percent dephosphorylated product formed by the variant over the percent product of SEQ ID NO: 84 (see Table 16.2).

Example 23

Oligonucleotide Deblocking Activity Improvements Over SEQ ID NO: 214.

[0347] The phosphatase of SEQ ID NO: 214 was selected as the parent enzyme after screening variants as described in Examples 22 above. Libraries of engineered genes were produced using well established techniques (e.g., saturation mutagenesis, recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were produced in HTP as described in Example 1 and the cell paste was generated as described in Example 2. Cell lysis was performed as described in Example 4. Alkaline phosphatase activity was evaluated in the oligonucleotides 3 ’-dephosphorylation assay as described in Example 5. The HTP screening conditions are listed in Table 17.1.

[0348] Activity relative to SEQ ID NO: 214 (FIOP) was calculated as the percent dephosphorylated product formed by the variant over the percent product of SEQ ID NO: 214 (see Table 17.2).

Example 24

Oligonucleotide Deblocking Activity Improvements Over SEQ ID: 232 [0349] The phosphatase of SEQ ID NO: 232 was selected as the parent enzy me after screening variants as described in Examples 23 above. Libraries of engineered genes were produced using well established techniques (e.g., saturation mutagenesis, recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were produced in HTP as described in Example 1 and the cell paste was generated as described in Example 2. Cell lysis was performed as described in Example 4. Alkaline phosphatase activity was evaluated in the oligonucleotides 3 ’-dephosphorylation assay as described in Example 5. The HTP screening conditions are listed in Table 18.1.

[0350] Activity relative to SEQ ID NO: 232 (FIOP) was calculated as the percent dephosphorylated product formed by the variant over the percent product of SEQ ID NO: 232 (see Table 18.2).

Example 25

Oligonucleotide Deblocking Activity Improvements Over SEQ ID NO: 562

[0351] The phosphatase of SEQ ID NO: 562 was selected as the parent enzyme after screening variants as described in Examples 24 above. Libraries of engineered genes were produced using well established techniques (e.g., saturation mutagenesis, recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were produced in HTP as described in Example 1 and the cell paste was generated as described in Example 2. Cell lysis was performed as described in Example 4. Alkaline phosphatase activity was evaluated in the oligonucleotides 3 ’-dephosphorylation assay as described in Example 5. The HTP screening conditions are listed in Table 19.1.

[0352] Activity relative to SEQ ID NO: 562 (FIOP) was calculated as the percent dephosphorylated product formed by the variant over the percent product of SEQ ID NO: 562 (see Table 19.2).

Example 26

Oligonucleotide Deblocking Activity Improvements Over SEQ ID NO: 656.

[0353] The phosphatase of SEQ ID NO: 656 was selected as the parent enzyme after screening variants as described in Examples 25 above. Libraries of engineered genes were produced using well established techniques (e.g., saturation mutagenesis, recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were produced in HTP as described in Example 1 and the cell paste was generated as described in Example 2. Cell lysis was performed as described in Example 4. Alkaline phosphatase activity was evaluated in the oligonucleotides 3 ’-dephosphorylation assay as described in Example 5. The HTP screening conditions are listed in Table 20.1.

[0354] Activity relative to SEQ ID NO: 656 (FIOP) was calculated as the percent dephosphorylated product formed by the variant over the percent product of SEQ ID NO: 656 (see Table 20.2).

Example 27

Oligonucleotide Deblocking Activity Improvements Over SEQ ID NO: 774.

[0355] The phosphatase SEQ ID NO: 774 was selected as the parent enzyme after screening variants as described in Examples 26 above. Libraries of engineered genes were produced using well established techniques (e.g., saturation mutagenesis, recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were produced in HTP as described in Example 1 and the cell paste was generated as described in Example 2. Cell lysis was performed as described in Example 4. Alkaline phosphatase activity was evaluated in the oligonucleotides 3 ’-dephosphorylation assay as described in Example 5. The HTP screening conditions are listed in Table 21.1 and Table 21.2.

[0356] Activity relative to SEQ ID NO: 774 (FIOP) was calculated as the percent dephosphorylated product formed by the variant over the percent product of SEQ ID NO: 774 (see Table 21.3).

[0357] While the invention has been described with reference to the specific embodiments, various changes can be made and equivalents can be substituted to adapt to a particular situation, material, composition of matter, process, process step or steps, thereby achieving benefits of the invention without departing from the scope of what is claimed.

[0358] For all purposes, each and every publication and patent document cited in this disclosure is incorporated herein by reference as if each such publication or document was specifically and individually indicated to be incorporated herein by reference. Citation of publications and patent documents is not intended as an indication that any such document is pertinent prior art, nor does it constitute an admission as to its contents or date.

Claims

CLAIMS What is claimed is:

1. A recombinant phosphatase, or a functional fragment thereof, comprising an amino acid sequence having at least 70%, 75%. 80%. 81%, 82%, 83%, 84%. 85%, 86%, 87%, 88%. 89%, 90%, 91%, 92%, 93%. 94%. 95%, 96%, 97%, 98%. 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of an even -numbered SEQ ID NO. of SEQ ID NOs: 2-18 and 24-1052, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 2-18 and 24-1052.

2. The recombinant phosphatase of claim 1. comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%. 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of an even-numbered SEQ ID NO. of SEQ ID NOs: 2-14 or SEQ ID NO: 18. 84, 214, 232, 562. 656, or 774. or to a reference sequence corresponding to an even- numbered SEQ ID NO. of SEQ ID NOs: 2-14, or SEQ ID NO: 18, 84, 214, 232, 562, 656, or 774.

3. The recombinant phosphatase of claim 1 or 2. wherein the phosphatase comprises an alkaline phosphatase of Thermoflexibacter. Pyrococcus. Thermotoga, Pseudothermotoga, or Bacillus.

4. The recombinant phosphatase of any one of claims 1-3, wherein the alkaline phosphatase is of Thermoflexibacter ruber, Pyrococcus furiosus, Thermotoga maritima, Thermotoga sp. 50_64, Pseudothermotoga lettingae, Thermotoga neapolitana, or Bacillus licheniformis .

5. The recombinant phosphatase of any one of claims 1-4, further comprising an N-tenninal deletion of up to 10, 25, 30, 40, 45, or 50 amino acids of the naturally occurring sequence.

6. The recombinant phosphatase of claim 5, wherein the N-terminal deletion removes or inactivates signaling sequences at the N-terminal sequence of the phosphatase.

7. The recombinant phosphatase of claim 5 or 6, wherein the N-terminal deletion preserves the capability of the phosphatase to form active dimeric structure.

8. The recombinant phosphatase of claim 1, comprising an amino acid sequence having at least 70%, 75%. 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to tire carboxyl tenninus of an even-numbered SEQ ID NO. of SEQ ID NOs: 12 and 24- 1052. or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 12 and 24-1052, wherein the amino acid comprises one or more amino acid differences relative to the reference sequence corresponding to amino acid residues 12 to the carboxyl tenninus of an even- numbered SEQ ID NOs: 12 and 24-1052. or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 12 and 24-1052.

9. The recombinant phosphatase of claim 1 or 8, comprising an amino acid sequence having at least 70%. 75%, 80%, 81%, 82%. 83%, 84%, 85%, 86%. 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%. 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of SEQ ID NO: 12, 18, 84, 214, 232. 562, 656, or 774, or to the reference sequence corresponding to SEQ ID NO: 12, 18. 84, 214. 232, 562, 656, or 774, wherein the amino acid comprises one or more amino acid differences relative to the reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of SEQ ID NO: 12. 18, 84, 214. 232, 562, 656, or 774, or to the reference sequence corresponding to SEQ ID NO: 12, 18, 84, 214, 232, 562, 656. or 774.

10. The recombinant phosphatase of claim 1 or 8, comprising an amino acid sequence having at least 70%. 75%, 80%, 81%, 82%. 83%, 84%, 85%, 86%. 87%, 88%, 89%, 90%. 91%, 92%, 93%, 94%, 95%, 96%, 97%. 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of an even-numbered SEQ ID NO. of SEQ ID NOs: 24-80, or to the reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 24-80, wherein the amino acid sequence comprises one or more amino acid differences relative to the reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of SEQ ID NO: 18, or to the reference sequence corresponding to SEQ ID NO: 18.

11. The recombinant phosphatase of claim 1 or 8, comprising an ammo acid sequence having at least 70%. 75%, 80%, 81%, 82%. 83%, 84%, 85%, 86%. 87%, 88%, 89%, 90%. 91%, 92%, 93%, 94%, 95%, 96%, 97%. 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of SEQ ID NO: 18, or to the reference sequence corresponding to SEQ ID NO: 18, wherein the amino acid sequence comprises one or more amino acid differences relative to the reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of SEQ ID NO: 18. or to the reference sequence corresponding to SEQ ID NO: 18.

12. The recombinant phosphatase of claim 10 or 11, wherein die amino acid sequence of the recombinant phosphatase comprises at least an amino acid difference at amino acid position 23, 25, 50, 56, 63, 99, 102, 136, 193, 195, 198, 224, 234. 235, 267, 306. or 345, or combinations thereof, wherein the amino acid difference is relative to the reference sequence corresponding to residues 12 to the carboxyl terminus of SEQ ID NO: 18. or to the reference sequence corresponding to SEQ ID NO: 18.

13. The recombinant phosphatase of any one of claims 10-12, wherein the amino acid sequence of the recombinant phosphatase comprises at least an amino acid difference 23Q/R, 25L/R, 50H, 56A, 63T, 99N, 102P, 1361, 193 A/G, 195D/E/S/T, 198R, 224E, 234E, 235D, 267Q, 306E, or 345R, or combinations thereof, wherein the amino acid difference is relative to the reference sequence corresponding to residues 12 to the carboxyl tenninus of SEQ ID NO: 18, or to the reference sequence corresponding to SEQ ID NO: 18.

14. The recombinant phosphatase of any one of claims 10-12, wherein the amino acid sequence of the recombinant phosphatase comprises at least an amino acid difference K23Q/R, K25L/R, K50H, C56A. K63T, K99N, K102P, K136I, K193A/G/D/E/S/T, K198R, K224E, K234E, K235D, K267Q, K306E, or K345R, or combinations thereof, wherein the amino acid difference is relative to the reference sequence corresponding to residues 12 to the carboxyl terminus of SEQ ID NO: 18, or to the reference sequence corresponding to SEQ ID NO: 18.

15. The recombinant phosphatase of claim 1, 8 or 9, comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%. 83%, 84%, 85%, 86%. 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of SEQ ID NO: 12, 84, 214, 232, 562, 656. or 774, or to a reference sequence corresponding to SEQ ID NO: 12. 84, 214. 232, 562, 656, or 774, wherein the amino acid sequence comprises one or more amino acid differences relative to the reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of SEQ ID NO: 12. 84, 214. 232, 562, 656, or 774, or to the reference sequence corresponding to SEQ ID NO: 12, 84. 214, 232, 562. 656, or 774.

16. The recombinant phosphatase of claim 1, 8 or 9. comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%. 83%, 84%, 85%, 86%. 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of an even-numbered SEQ ID NO. of SEQ ID NOs: 24-1052, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 24-1052, wherein the amino acid sequence comprises one or more amino acid differences relative to the reference sequence corresponding to amino acid residues 12 to the carboxyl tenninus of SEQ ID NO: 12, or to the reference sequence corresponding to SEQ ID NO: 12.

17. The recombinant phosphatase of claim 1, 8 or 9, comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%. 83%, 84%, 85%, 86%. 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of SEQ ID NO: 84, 214, 232, 562. 656, or 774, or to the reference sequence corresponding to SEQ ID NO: 84, 214, 232, 562, 656, or 774, wherein the amino acid sequence comprises one or more amino acid differences relative to the reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of SEQ ID NO: 12, or to the reference sequence corresponding to SEQ ID NO: 12.

18. The recombinant phosphatase of claim 1, 8 or 9, comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%. 87%, 88%, 89%, 90%, 91%, 92%,

93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of SEQ ID NO: 12, or to the reference sequence corresponding to SEQ ID NO: 12, wherein the amino acid sequence comprises one or more amino acid differences relative to the reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of SEQ ID NO: 12, or to the reference sequence corresponding to SEQ ID NO: 12.

19. The recombinant phosphatase of any one of claims 8-9 and 15-18, wherein the amino acid sequence of the recombinant phosphatase comprises at least an amino acid difference at amino acid position 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 , 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 56, 57, 60, 61, 63, 81, 82, 83, 89, 93, 95, 96, 98, 100, 101, 105, 108, 128, 138, 139, 141, 148, 150, 158, 169, 174, 175, 181, 202, 205, 206, 207, 211, 214, 215, 223, 224, 225, 226, 227, 228. 230, 231, 234, 235, 240, 241, 244, 247, 260, 261, 263. 266, 271, 291, 299, 301, 302, 306, 307, 336, 340. 341, 343, 345, 346, 347, 353, 357, 363. 367, 368, 371. 389, 390, 391, 392, or 393, or combinations thereof, wherein the amino acid difference is relative to the reference sequence corresponding to residues 12 to the carboxyl terminus of SEQ ID NO: 12. or to the reference sequence corresponding to SEQ ID NO: 12.

20. The recombinant phosphatase of any one of claims 15-18, wherein the amino acid sequence of the recombinant phosphatase comprises at least an amino acid difference or amino acid residue 11 A/D, 12-/D/E/G/Q/R/V/W, 13-/A/G/P/R/V/Y, 14-/E/F/I. 15-/E/Q/Y, 16-/D/L/P/V, 17- /A/D/S/T/V, 18-/D/G/I/L/P/Q/S/T, 19-/D/E/S/T/Y, 20-/A/G/N/Q, 21-/E/K/M/V, 22-/A/E/G/I/M/R, 23- /C/S/V, 24-/K/V, 25-/E/S/V, 26-. 27-/I, 28-/S, 29-/G/M, 30-/P/R/S, 31-/G/Q/R/T/V. 32-/K/T/V, 33-, 34-, 35-/F/P/R, 36-/C/G, 37-/D, 38-/D/R/S, 39-, 40-. 41-, 42-/C, 43-/G, 44-/K/V, 45-/V, 46-, 47-/E/K/P/T, 48- /N, 49-/H, 50-/A/N/P/R/T/V, 51R, 52S, 56P, 57F/R, 60A/G, 61C/S/V, 63V, 81S, 82G, 83L/W, 89H, 93D, 95A/F/L, 96P, 98V, 100F, 101V, 105S, 108S, 128L, 138N. 139A/E/I/K/V, 141P/Q/R, 148A/W, 150K/S, 158V, 169 A, 174V. 175P. 181G, 202Y, 205G, 206P, 207V. 21 IS. 214S/W, 215L, 2231, 2241. 225E, 226S, 227W, 228R. 230L. 231C/R, 234E/T, 235N, 240A, 241L. 244A/E/W, 247M. 260V, 261 A, 2631. 2661, 271A/V/W, 291A/L. 299S. 301M, 302L, 306H, 307A, 3361, 340D/H. 341L. 343G/K/M/Q/R. 345L, 3461. 347A, 353A/Q/T, 357A. 363H. 367A, 368G, 371L, 389L. 390D/H/M/R/W/Y, 391W. or 392G/L/N/R. or combinations thereof, wherein the amino acid difference is relative to the reference sequence corresponding to residues 12 to the carboxyl terminus of SEQ ID NO: 12, or to the reference sequence corresponding to SEQ ID NO: 12.

21. The recombinant phosphatase of any one of claims 15-18, wherein the amino acid sequence of the recombinant phosphatase comprises at least an amino acid difference or amino acid residue Gil A/D, C12-/D/E/G/Q/R/V/W, S13-/A/G/P/R/V/Y, P14-/E/F/I. T15-/E/Q/Y, E16-/D/L/P/V, K17-/A/D/S/T/V, K18-/D/G/I/L/P/Q/S/T. A19-/D/E/S/T/Y, K20-/A/G/N/Q, S21-/E/K/M/V. L22- /A/E/G/l/M/R, P23-/C/S/V, N24-/K/V. T25-/E/S/V. P26-. F27-/1, T28-/S, P29-/G/M, T30-/P/R/S, L31- /G/Q/R/T/V, A32-/K/T/V. E33-. Q34-. N35-/F/P/R. P36-/C/G. L37-/D. K38-/D/R/S, R39-, Q40-, W41-. A42-/C, E43-/G, P44-/K/V, I45-/V, E46-, S47-/E/K/P/T, Y48-/N, Q49-/H, I50-/A/N/P/R/T/V, K51R, N52S. A56P, K57F/R, N60A/G, G61C/S/V. P63V, A81S, A82G. M83L/W. K89H, E93D. C95A/F/L, S96P. I98V. L100F, I101V, A105S. A108S. N128L. K138N. S139A/E/I/K/V. K141P/Q/R, E148A/W, N150K/S, A158V, C169A, Q174V, K175P, E181G, F202Y. R205G. S206P, A207V, N211S, P214S/W, E215L. V223I, L224I. D225E. N226S, M227W, N228R, I230L, A231C/R. K234E/T. Q235N. G240A, F241L, Q244A/E/W, N247M, Q260V, S261A, K263I, L266I, Q271A/V/W, D291A/L, Q299S, T301M, I302L, K306H, V307A, L336I, N340D/H, I341L. E343G/K/M/Q/R, K345L, V346I, E347A, G353A/Q/T, S357A, Y363H, C367A, Q368G, K371L, F389L, G390D/H/M/R/W/Y, F391W, T392G/L/N/R, or S393A/E/G/I/K/L/M/Q/R, or combinations thereof, wherein the amino acid difference is relative to the reference sequence corresponding to residues 12 to the carboxyl terminus of SEQ ID NO: 12, or to the reference sequence corresponding to SEQ ID NO: 12.

22. The recombinant phosphatase of any one of claims 15-18, wherein the amino acid sequence of the recombinant phosphatase comprises at least an amino acid difference at amino acid position 19. 22, 50, 138, 139, 141, 158. 169, 244, 260, 306, 307, 340, 357, 390. 392, or 393. or combinations thereof, wherein the amino acid difference is relative to the reference sequence corresponding to residues 12 to the carboxyl terminus of SEQ ID NO: 12, or to the reference sequence corresponding to SEQ ID NO: 12.

23. The recombinant phosphatase of any one of claims 15-18 and 22. wherein the amino acid sequence of the recombinant phosphatase comprises at least an amino acid difference or amino acid residue 19-/D/E/S/T/Y, 22-/A/E/G/I/M/R, 50H/I/R, 138N, 139A/E/I/K/V, 141K/P/Q/R, 158V. 169A, 244A/E/W, 260Q/V, 306E/H, 307 A, 340D/H, 357A, 390D/H/M/R/W/Y. 392G/L/N/R, or 393A/E/G/I/K/L/M/Q/R, or combinations thereof, wherein the amino acid difference is relative to the reference sequence corresponding to residues 12 to the carboxyl terminus of SEQ ID NO: 12, or to the reference sequence corresponding to SEQ ID NO: 12.

24. The recombinant phosphatase of any one of claims 15-18 and 22, wherein the amino acid sequence of the recombinant phosphatase comprises at least an amino acid difference or amino acid residue A19D/E, L22G, I50R, K138N, S139A, K141P, A158V, C169A, Q244W, Q260V, K306H. V307A, N340D, S357A, G390H, T392L. or S393G, or combinations thereof, wherein the ammo acid difference is relative to the reference sequence corresponding to residues 12 to the carboxyl terminus of SEQ ID NO: 12, or to the reference sequence corresponding to SEQ ID NO: 12.

25. The recombinant phosphatase of any one of claims 15-18, wherein the amino acid sequence of the recombinant phosphatase comprises at least an amino acid difference at amino acid position 11, 13, 14, 16, 17, 18, 19, 20, 21, 28, 29, 31, 35, 42, 44, or 61, or combinations thereof, wherein the amino acid difference is relative to the reference sequence corresponding to residues 12 to the carboxyl terminus of SEQ ID NO: 12, or to the reference sequence corresponding to SEQ ID NO: 12.

26. The recombinant phosphatase of any one of claims 15-18, wherein the amino acid sequence of the recombinant phosphatase comprises at least an amino acid difference or amino acid residue 11D, 13G, 141, 16D, 17A/D/S/T/V, 18D/G/L/P/Q/S/T, 19D/S/T, 20A/G/N/Q, 21E, 28S, 29M, 31T, 35P, 42C, 44V, or 61S, or combinations thereof, wherein the amino acid difference is relative to the reference sequence corresponding to residues 12 to the carboxyl terminus of SEQ ID NO: 12, or to the reference sequence corresponding to SEQ ID NO: 12.

27. The recombinant phosphatase of any one of claims 1, 8, 9, and 15-19. comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to tire carboxyl terminus of an even-numbered SEQ ID NO. of SEQ ID NOs: 24-1052, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 24-1052, wherein the amino acid sequence comprises one or more amino acid differences relative to the reference sequence corresponding to amino acid residues 12 to the carboxy l terminus of SEQ ID NO: 84. 214, 232, 562, 656, or 774, or to the reference sequence corresponding to SEQ ID NO: 84, 214, 232. 562, 656, or 774.

28. The recombinant phosphatase of any one of claims 1, 8, 9, and 15-19. comprising an amino acid sequence haying at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to the carboxy l terminus of SEQ ID NO: 84, 214, 232, 562, 656, or 774, or to a reference sequence corresponding to SEQ ID NO: 84, 214, 232, 562, 656. or 774, wherein the amino acid sequence comprises one or more amino acid difference relative to the reference sequence corresponding to amino acid residues 12 to the carboxyl tenninus of SEQ ID NO: 84, 214, 232, 562, 656, or 774, or to the reference sequence corresponding to SEQ ID NO: 84, 214, 232. 562, 656, or 774.

29. The recombinant phosphatase of claim 27 or 28, wherein tire amino acid sequence of the recombinant phosphatase comprises at least an amino acid difference at amino acid position 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22. 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50. 51, 52, 56, 57, 60, 61, 63, 81, 82. 83, 89, 93, 95, 96, 98, 100. 101, 105, 108, 128, 138, 139, 141, 148, 150, 158. 169, 174, 175, 181, 202, 205, 206, 207, 211, 214, 215. 223, 224, 225, 226, 227, 228, 230, 231, 234, 235. 240, 241, 244, 247, 260, 261, 263, 266, 271, 291, 299. 301, 302, 306, 307, 336, 340, 341. 343, 345, 346. 347, 353, 357, 363, 367, 368, 371, 389. 390, 391, 392. or 393, or combinations thereof, wherein the amino acid difference is relative to the reference sequence corresponding to residues 12 to the carboxyl terminus of SEQ ID NO: 84, 214, 232. 562, 656, or 774, or to the reference sequence corresponding to SEQ ID NO: 84. 214, 232, 562. 656, or 774.

30. The recombinant phosphatase of claim 27 or 28, wherein tire amino acid sequence of the recombinant phosphatase comprises at least an amino acid difference or amino acid residue 11 A/D, 12- /D/E/G/Q/R/V/W. 13-/A/G/P/R/V/Y. 14-/E/F/I, 15-ZE/Q/Y, 16-/D/L/P/V, 17-/A/D/S/T/V, 18- /D/G/I/L/P/Q/S/T. 19-/D/E/S/T/Y, 20-/A/G/N/Q, 21-/E/K/M/V, 22-/A/E/G/I/M/R, 23-/C/S/V, 24-/K/V, 25-/E/S/V, 26-, 27-/I, 28-/S, 29-/G/M, 30-/P/R/S, 31-/G/Q/R/T/V, 32-/K/T/V, 33-, 34-, 35-/F/P/R, 36- /C/G. 37-/D, 38-/D/R/S, 39-. 40-, 41-, 42-/C, 43-/G. 44-/K/V, 45-/V, 46-. 47-/E/K/P/T, 48-/N, 49-/H, 50- /A/I/N/P/R/T/V, 51R, 52S, 56P, 57F/R, 60A/G, 61C/S/V, 63V, 81S, 82G, 83L/W, 89H, 93D, 95A/F/L, 96P, 98V, 100F, 101V, 105S, 108S, 128L, 138N, 139A/E/I/K/V, 141K/P/Q/R, 148A/W, 150K/S, 158V, 169A, 174V, 175P, 181G, 202Y. 205G, 206P, 207V, 211 S, 214S/W. 215L. 2231, 2241, 225E, 226S, 227W, 228R, 230L, 231C/R, 234E/T, 235N, 240 A, 241L, 244A/E/W, 247M. 260Q/V, 261A, 2631, 2661, 271A/V/W. 291A/L, 299S, 301M, 302L, 306H. 307A, 3361. 340D/H, 341L, 343G/K/M/Q/R, 345L, 3461, 347A, 353A/Q/T. 357A. 363H, 367A, 368G, 371L. 389L. 390D/G/H/M/R/W/Y, 391W. 392G/L/N/R/T, or 393A/E/G/I/K/L/M/Q/R, or combinations thereof, wherein the amino acid difference is relative to the reference sequence corresponding to residues 12 to the carboxyl terminus of SEQ ID NO: 84, 214, 232, 562. 656, or 774, or to the reference sequence corresponding to SEQ ID NO: 84, 214, 232, 562, 656, or 774.

31. The recombinant phosphatase of claim 15 or 17. wherein die amino acid sequence of the recombinant phosphatase comprises at least an amino acid difference at amino acid position 19, 22, 50, 138, 139, 141, 158, 169. 244, 260, 306. 307, 340, 357, 390, 392. or 393, or combinations thereof, wherein the amino acid difference is relative to the reference sequence corresponding to residues 12 to the carboxyl terminus of SEQ ID NO: 84, 214, 232. 562, 656, or 774, or to the reference sequence corresponding to SEQ ID NO: 84, 214. 232, 562, 656. or 774.

32. The recombinant phosphatase of any one of claims 27-31, wherein the amino acid sequence of the recombinant phosphatase comprises at least an amino acid difference or amino acid residue 19-/D/E/S/T/Y, 22-/A/E/G/I/M/R, 50H/I/R, 138N. 139A/E/I/K/V, 141K/P/Q/R, 158V. 169A, 244A/E/W, 260Q/V, 306E/H, 307A, 340D/H, 357A, 390D/G/H/M/R/W/Y. 392G/L/N/R/T, 393A/E/G/I/K/L/M/Q/R, or combinations thereof, wherein the amino acid difference is relative to the reference sequence corresponding to residues 12 to the carboxyl terminus of SEQ ID NO: 84, 214. 232, 562, 656. or 774, or to the reference sequence corresponding to SEQ ID NO: 84, 214. 232, 562, 656. or 774.

33. The recombinant phosphatase of claim 27 or 28, comprising an amino acid sequence having at least 70%. 75%, 80%, 81%, 82%. 83%, 84%, 85%, 86%. 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of an even-numbered SEQ ID NO. of SEQ ID NOs: 144-224, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 144-224, wherein the amino acid sequence comprises one or more amino acid differences relative to the reference sequence corresponding to amino acid residues 12 to the carboxyl tenninus of SEQ ID NO: 84, or to the reference sequence corresponding to SEQ ID NO: 84.

34. The recombinant phosphatase of claim 27 or 28, comprising an amino acid sequence having at least 70%. 75%, 80%, 81%, 82%. 83%, 84%, 85%, 86%. 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of SEQ ID NO: 84, or to the reference sequence corresponding to SEQ ID NO: 84, wherein the amino acid sequence comprises one or more amino acid differences relative to the reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of SEQ ID NO: 84, or to the reference sequence corresponding to SEQ ID NO: 84.

35. The recombinant phosphatase of claim 33 or 34, wherein tire amino acid sequence of the recombinant phosphatase comprises at least an amino acid difference at amino acid position 11, 1 , 16, 18, 19, 22, 27, 38, 44, 48, 50, 81. 82, 83, 95, 96, 100, 101, 150, 158, 174, 175, 202, 226, 230, 235, 241, 247, 261, 266, 299, 301, 302, 307, 336. 341, 346, 357, 368, or 389, or combinations thereof, wherein the amino acid difference is relative to the reference sequence corresponding to residues 12 to the carboxyl terminus of SEQ ID NO: 84. or to the reference sequence corresponding to SEQ ID NO: 84.

36. The recombinant phosphatase of claim 33 or 34, wherein tire amino acid sequence of the recombinant phosphatase comprises at least an amino acid difference or amino acid residue 11A, 15E, 16P, 181, 19E, 221, 271, 38D, 44K, 48N, 50A, 81S, 82G, 83L, 95F/L, 96P, 100F, 101V, 150K, 158V, 174V, 175P, 202Y, 226S, 230L, 235N, 241L, 247M, 261A, 2661, 299S, 301M, 302L, 307A, 3361, 341L, 3461, 357A, 368G, or 389L, or combinations thereof, wherein the amino acid difference is relative to the reference sequence corresponding to residues 12 to the carboxyl terminus of SEQ ID NO: 84, or to the reference sequence corresponding to SEQ ID NO: 84.

37. The recombinant phosphatase of claim 27 or 28, comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%. 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of an even-numbered SEQ ID NO. of SEQ ID NOs: 226-348, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 226-348, wherein the amino acid sequence comprises one or more amino acid differences relative to the reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of SEQ ID NO: 214, or to the reference sequence corresponding to SEQ ID NO: 214.

38. The recombinant phosphatase of claim 27 or 28, comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%. 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to amino acid residues 12 to the carboxy l terminus of SEQ ID NO: 214, or to the reference sequence corresponding to SEQ ID NO: 214, wherein the amino acid sequence comprises one or more amino acid differences relative to the reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of SEQ ID NO: 214, or to the reference sequence corresponding to SEQ ID NO: 214.

39. The recombinant phosphatase of claim 37 or 38, wherein the amino acid sequence of the recombinant phosphatase comprises at least an amino acid difference at amino acid position(s) 15, 15/50/150/174/175, 15/50/158/174/226/247/346, 15/50/158/226/247/357. 15/50/158/247/357, 15/50/226. 15/96, 15/96/175/247, 15/96/175/341, 15/96/226/357, 15/96/357, 15/158/174/175/357, 15/158/174/247, 15/158/174/301/346, 15/158/175/247, 15/158/175/247/307/341/346, 15/174, 15/174/175, 15/174/175/247, 15/174/175/247/346, 15/174/226/301, 15/174/341, 15/175/247, 15/175/247/301, 15/175/247/341/346, 15/175/247/346/357, 15/175/357, 15/247, 15/346, 50/174/175/247/357, 96/158/175/301, 96/174/175, 96/174/175/226, 96/174/175/301/341/346, 96/174/247/341, 96/174/247/346, 96/175. 158, 158/174/226/247, 158/174/247/307/357. 158/175, 158/175/226/247/301/341/346/363, 158/175/247/346. 158/175/247/346/357, 158/175/301, 158/247, 158/307/346, 174. 174/175/247, 174/175/357, 174/247, 174/301/341, 174/346, 174/357, 175, 226. 226/247, 226/247/346, 247/301/346. 247/341/346, 307/357. or 357, wherein the amino acid difference is relative to the reference sequence corresponding to residues 12 to the carboxyl terminus of SEQ ID NO: 214, or to the reference sequence corresponding to SEQ ID NO: 214.

40. The recombinant phosphatase of claim 37 or 38. wherein die amino acid sequence of the recombinant phosphatase comprises at least an amino acid difference(s) or amino acid residue(s) 15E, 15E/50A/150S/174V/175P, 15E/50A/158V/174V/226S/247M/346I, 15E/50A/158V/226S/247M/357A, 15E/50A/158V/247M/357A. 15E/50A/226S, 15E/96P, 15E/96P/175P/247M. 15E/96P/175P/341L, 15E/96P/226S/357A, 15E/96P/357A, 15E/158V/174V/175P/357A, 15E/158V/174V/247M. 15E/158V/174V/301M/346I, 15E/158V/175P/247M, 15E/158V/175P/247M/307A/341L/346I, 15E/174V, 15E/174V/175P. 15E/174V/175P/247M, 15E/174V/175P/247M/346I. 15E/174V/226S/301M, 15E/174V/341L. 15E/175P/247M, 15E/175P/247M/301M. 15E/175P/247M/341L/346I, 15E/175P/247M/346I/357A, 15E/175P/357A. 15E/247M, 15E/346I, 50A/174V/175P/247M/357A. 96P/158V/175P/301M. 96P/174V/175P, 96P/174V/175P/226S, 96P/174V/175P/301M/341L/346I, 96P/174V/247M/341L, 96P/174V/247M/346I, 96P/175P, 158V, 158V/174V/226S/247M, 158V/174V/247M/307A/357A. 158V/175P. 158V/175P/226S/247M/301M/341L/346I/363H, 158V/175P/247M/346I, 158V/175P/247M/346I/357A, 158V/175P/301M, 158V/247M, 158V/307A/346I, 174V, 174V/175P/247M. 174V/175P/357A, 174V/247M, 174V/301M/341L, 174V/346I, 174V/357A, 175P, 226S, 226S/247M. 226S/247M/346I, 247M/30 IM/3461, 247M/341L/346I, 307 A/357 A, or 357A, wherein the amino acid difference is relative to the reference sequence corresponding to residues 12 to the carboxy l terminus of SEQ ID NO: 214, or to the reference sequence corresponding to SEQ ID NO: 214.

41. The recombinant phosphatase of claim 27 or 28, comprising an amino acid sequence having at least 70%, 75%, 80%, 81%. 82%, 83%, 84%, 85%. 86%, 87%, 88%, 89%. 90%, 91%, 92%, 93%, 94%, 95%, 96%. 97%, 98%, 99%, or more sequence identity’ to a reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of an even-numbered SEQ ID NO. of SEQ ID NOs: 350-650, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 350-650, wherein the amino acid sequence comprises one or more amino acid differences relative to the reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of SEQ ID NO: 232, or to the reference sequence corresponding to SEQ ID NO: 232.

42. The recombinant phosphatase of claim 27 or 28. comprising an amino acid sequence having at least 70%. 75%, 80%, 81%, 82%. 83%, 84%, 85%, 86%. 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of SEQ ID NO: 232, or to the reference sequence corresponding to SEQ ID NO: 232, wherein the amino acid sequence comprises one or more amino acid differences relative to the reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of SEQ ID NO: 232. or to the reference sequence corresponding to SEQ ID NO: 232.

43. The recombinant phosphatase of claim 41 or 42, wherein tire amino acid sequence of the recombinant phosphatase comprises at least an amino acid difference at amino acid position(s) 12, 14, 15, 16, 17, 18, 19, 21. 22, 37, 43, 50. 57, 61, 89/95/141/169/234/306, 89/95/234/306/367, 89/96/367/371, 89/138/141/234/306/367/371, 89/138/234, 89/138/367, 89/141/169/234/306/371, 89/141/169/234/371. 89/141/169/306/367/371, 89/141/234/306/367/371, 89/141/306/367/371. 89/141/367, 89/141/371, 89/169. 89/169/234. 89/169/234/306/367/371, 89/169/234/367, 89/169/234/371, 89/169/306/367, 89/169/367, 89/169/371. 89/234. 89/234/306/371. 89/234/371. 89/306. 89/367/371. 89/371, 95/169/234/306/371, 96/141/234/306/371, 96/169/306, 138. 138/141, 138/141/169/234/367. 138/141/169/306. 138/141/234/306, 138/141/234/367, 138/141/234/371. 138/141/306/367, 138/141/306/371, 138/169/367/371, 138/234/367, 138/234/367/371, 138/306. 138/371, 139, 141, 141/169, 141/234, 141/234/306, 141/234/306/371. 141/234/371, 141/306/367. 141/306/371, 141/367. 169. 169/306, 205. 214, 224, 234. 234/306, 234/306/367. 234/306/367/371, 234/371, 244, 260. 271, 306, 306/367, 306/367/371. 306/371, 340, 341. 343, 367, 390, 391, 392. or 393, or combinations thereof, wherein the amino acid difference is relative to the reference sequence corresponding to residues 12 to the carboxyl terminus of SEQ ID NO: 232, or to the reference sequence corresponding to SEQ ID NO: 232.

44. The recombinant phosphatase of claim 41 or 42. wherein die amino acid sequence of the recombinant phosphatase comprises at least an amino acid difference(s) or amino acid residue(s) 12D/E/G/Q/R/V/W, 14E/F. 15E/Q/Y. 16L/V, 17S. 18G/L, 19Y, 21K/M/V, 22A/E/G/M/R. 37D, 43G. 50N/P/R/T/V, 57F/R, 61C/V. 89H/95A/141P/169A/234T/306H, 89H/95A/234T/306H/367A, 89H/96P/367A/371L, 89H/138N/141P/234T/306H/367A/371L, 89H/138N/234E, 89H/138N/367A, 89H/141P/169A/234E/371L, 89H/141P/169A/234T/306H/371L, 89H/141P/169A/306H/367A/371L. 89H/141P/234E/306H/367A/371L, 89H/141P/306H/367A/371L. 89H/141P/367A, 89H/141P/371L, 89H/169A, 89H/169A/234E, 89H/169A/234E/306H/367A/371L. 89H/169A/234E/367A, 89H/169A/234T, 89H/169A/234T/367A, 89H/169A/234T/371L. 89H/169A/306H/367A, 89H/169A/367A, 89H/169A/371L, 89H/234E/306H/371L, 89H/234T, 89H/234T/306H/371L, 89H/234T/371L, 89H/306H, 89H/367A/371L, 89H/371L, 95A/169A/234T/306H/371L, 96P/141P/234T/306H/371L, 96P/169A/306H, 138N, 138N/141P, 138N/141P/169A/234T/367A, 138N/141P/169A/306H, 138N/141P/234E/306H, 138N/141P/234E/367A, 138N/141P/234E/371L, 138N/141P/306H/367A, 138N/141P/306H/371L, 138N/169A/367A/371L, 138N/234T/367A, 138N/234T/367A/371L, 138N/306H, 138N/371L, 139A, 139E, 1391, 139K, 139V, 141P/169A, 141P/234E/306H. 141P/234E/306H/371L, 141P/234E/371L, 141P/234T, 141P/234T/371L, 141P/306H/367A. 141P/306H/371L, 141P/367A, 141Q/R, 169A, 169A/306H, 205G, 214W, 2241. 234E, 234E/306H/367A, 234T, 234T/306H, 234T/306H/367A/371L. 234T/371L. 244A/E/W, 260V. 271A/V/W. 306H, 306H/367A, 306H/367A/371L, 306H/371L, 340D/H. 341L. 343G/K/M/Q/R. 367A, 390D/H/M/R/W/Y, 391W, 392G/L/N/R, or 393A/E/G/1/K/L/M/Q/R. wherein the amino acid difference is relative to the reference sequence corresponding to residues 12 to the carboxyl terminus of SEQ ID NO: 232, or to the reference sequence corresponding to SEQ ID NO: 232.

45. The recombinant phosphatase of claim 27 or 28. comprising an amino acid sequence having at least 70%. 75%, 80%, 81%, 82%. 83%, 84%, 85%, 86%. 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of an even-numbered SEQ ID NO. of SEQ ID NOs: 652-740, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 652-740, wherein the amino acid sequence comprises one or more amino acid differences relative to the reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of SEQ ID NO: 562, or to the reference sequence corresponding to SEQ ID NO: 562.

46. The recombinant phosphatase of claim 27 or 28. comprising an amino acid sequence having at least 70%. 75%, 80%, 81%, 82%. 83%, 84%, 85%, 86%. 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of SEQ ID NO: 562, or to the reference sequence corresponding to SEQ ID NO: 562, wherein the amino acid sequence comprises one or more amino acid differences relative to the reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of SEQ ID NO: 562. or to the reference sequence corresponding to SEQ ID NO: 562.

47. The recombinant phosphatase of claim 45 or 46, wherein the amino acid sequence of the recombinant phosphatase comprises at least an amino acid difference(s) at amino acid position 12, 12/37/50/139/244/260/393, 12/37/57/139/244/260, 12/37/139/244/390/392/393, 12/37/139/393, 12/37/244/260/393, 12/37/244/392, 12/50/57/244/390/393, 12/50/139/244/260/392/393, 12/57/139/244/392, 12/139/244/260, 12/139/244/260/390/393. 12/139/244/260/392/393, 12/139/244/260/393, 12/139/244/390/392/393, 12/139/260/393, 12/260, 12/260/392, 12/390/392/393, 12/392. 37/50/57/139/244/260/392, 37/50/139/244/390/393. 37/57/139/244/390/393, 37/57/139/244/392/393. 37/57/139/390/392/393, 37/57/244/392. 37/139/223/244/393, 37/139/244/260/393, 37/139/244/390/392, 37/139/260/393, 50/139/244/260/390/392/393. 50/139/244/390/393, 50/244, 50/260. 57/244. 139/244/390/392/393. 139/244/392/393, 139/244/393. 139/260/393, 139/392, 244, 244/392, 260/390, or 392/393, wherein the amino acid difference is relative to the reference sequence corresponding to residues 12 to the carboxyl terminus of SEQ ID NO: 562. or to the reference sequence corresponding to SEQ ID NO: 562.

48. The recombinant phosphatase of claim 45 or 46. wherein the amino acid sequence of the recombinant phosphatase comprises at least an amino acid difference(s) or amino acid residue(s) 12D/37D/139V/393G. 12D/37D/244W/392L. 12D/50R/57R/244W/390H/393L, 12D/57R/139V/244W/392L. 12D/139A/244W/260V/390H/393L, 12D/139A/260V/393L. 12D/139V/244W/260V/393L, 12D/390H/392L/393G. 12D/392L, 12E. 12E/37D/50R/139V/244W/260V/393G, 12E/37D/57R/139A/244W/260V, 12E/37D/139V/244W/390H/392L/393G. 12E/37D/244W/260V/393L. 12E/50R/139V/244W/260V/392L/393G, 12E/139A/244W/390H/392L/393G, 12E/139V/244W/260V. 12E/139V/244W/260V/392L/393G. 12E/139V/260V/393L, 12E/260V. 12E/260V/392L. 37D/50R/57R/139A/244W/260V/392L. 37D/50R/139V/244W/390H/393G. 37D/57R/139A/244W/392L/393G, 37D/57R/139A/390H/392L/393G, 37D/57R/139V/244W/390H/393L, 37D/57R/244W/392L, 37D/139A/244W/260V/393L, 37D/139A/260V/393G, 37D/139V/223I/244W/393G, 37D/139V/244W/390H/392L, 50R/139A/244W/260V/390H/392L/393G, 50R/139V/244W/390H/393L, 50R/244W, 50R/260V, 57R/244W, 139A/260V/393L, 139V/244W/390H/392L/393G, 139V/244W/392L/393G, 139V/244W/393L, 139V/392L, 244W, 244W/392L, 260V/390H, or 392L/393G, wherein the ammo acid difference is relative to the reference sequence corresponding to residues 12 to the carboxyl terminus of SEQ ID NO: 562, or to the reference sequence corresponding to SEQ ID NO: 562.

49. The recombinant phosphatase of claim 27 or 28, comprising an amino acid sequence having at least 70%, 75%, 80%, 81%. 82%, 83%, 84%, 85%. 86%, 87%, 88%, 89%. 90%, 91%, 92%, 93%, 94%, 95%, 96%. 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of an even-numbered SEQ ID NO. of SEQ ID NOs: 742-894, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 742-894, wherein the amino acid sequence comprises one or more amino acid differences relative to the reference sequence corresponding to amino acid residue 12 to the carboxyl terminus of SEQ ID NO: 656, or to the reference sequence corresponding to SEQ ID NO: 656.

50. The recombinant phosphatase of claim 27 or 28, comprising an amino acid sequence having at least 70%, 75%, 80%, 81%. 82%, 83%, 84%, 85%. 86%, 87%, 88%, 89%. 90%, 91%, 92%, 93%, 94%, 95%. 96%. 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of SEQ ID NO: 656, or to the reference sequence corresponding to SEQ ID NO: 656, wherein the amino acid sequence comprises one or more amino acid differences relative to the reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of SEQ ID NO: 656, or to the reference sequence corresponding to SEQ ID NO: 656.

51. The recombinant phosphatase of claim 49 or 50, wherein the amino acid sequence of the recombinant phosphatase comprises at least an amino acid difference at amino acid position(s) 13, 22/141, 22/141/158, 22/158/340, 22/158/340/390/392. 23, 24, 25. 29. 30, 31, 32, 35. 36, 38, 44/50. 45/50. 47/50, 50, 50/51, 50/52, 50/56. 60, 63, 83, 98, 105, 128, 141/158/340, 141/340, 148, 150, 158. 158/340/371, 158/340/371/390/392, 181, 206, 207, 211. 215, 225, 227. 228, 231, 260, 291, 345, 347, or 353 wherein the amino acid difference is relative to the reference sequence corresponding to residues 12 to the carboxyl terminus of SEQ ID NO: 656. or to the reference sequence corresponding to SEQ ID NO: 656.

52. The recombinant phosphatase of claim 49 or 50, wherein the amino acid sequence of the recombinant phosphatase comprises at least an amino acid difference(s) or amino acid residue(s) 13A/P/R/V/Y, 22G/141K, 22G/141K/158V, 22G/158V/340D, 22G/158V/340D/390G/392T, 23C/S/V, 24K/V, 25E/S/V, 29G, 30P/R/S, 31G/Q/R/V, 32K/T/V, 35F/R, 36C/G. 38R/S, 44V/50I, 45V/50I, 47E/50I, 47K/50I. 47P/50I, 47T/50I, 501. 50I/51R, 50I/52S, 50I/56P, 60A/G, 63V, 83W, 98V. 105S. 128L, 141K/158V/340D, 141K/340D, 148A/W, 150S, 158V, 158V/340D/371L, 158V/340D/371L/390W/392N, 181G, 206P, 207V, 21 IS, 215L. 225E. 227W, 228R, 231C/R. 260Q, 291 A/L, 345L, 347A, or 353A/Q/T, wherein the amino acid difference is relative to the reference sequence corresponding to residues 12 to the carboxyl terminus of SEQ ID NO: 656, or to the reference sequence corresponding to SEQ ID NO: 656.

53. The recombinant phosphatase of claim 27 or 28, comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%. 83%, 84%, 85%, 86%. 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of an even-numbered SEQ ID NO. of SEQ ID NOs: 896-1052, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 896-1052, wherein the amino acid sequence comprises one or more amino acid differences relative to the reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of SEQ ID NO: 774, or to the reference sequence corresponding to SEQ ID NO: 774.

54. The recombinant phosphatase of claim 27 or 28, comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%. 83%, 84%, 85%, 86%. 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of SEQ ID NO: 774, or to the reference sequence corresponding to SEQ ID NO: 774, wherein the amino acid sequence comprises one or more amino acid differences relative to the reference sequence corresponding to amino acid residues 12 to the carboxyl terminus of SEQ ID NO: 774. or to the reference sequence corresponding to SEQ ID NO: 774.

55. The recombinant phosphatase of claim 53 or 54, wherein the amino acid sequence of the recombinant phosphatase comprises at least an amino acid difference(s) at amino acid position(s) 13, 13/25/31/105, 13/25/47, 13/25/47/56/105/260, 13/25/47/207/260/353, 13/25/47/207/353, 13/25/56/207/260/353, 13/25/260/353, 13/25/353, 13/32, 13/32/47/56/353, 13/32/93/105/260/353, 13/47, 13/47/56/105/207/353, 13/47/56/353, 13/47/93/207/353, 13/47/105/207/240/260/353, 13/47/207/260, 13/56/263/353, 13/56/353. 13/105, 13/207, 13/207/260/353, 13/207/353, 13/353, 25/31/32, 25/31/32/56/105/260/353, 25/32/207/353, 25/47, 25/47/105, 25/47/105/207, 25/47/353, 25/56. 25/56/105/260/353, 25/93/207/260/353, 25/105/207/260, 25/105/207/260/353, 25/207, 25/353, 31/47/49/105/207/260/353, 32/47/105/353, 32/56/105, 47. 47/56/105/139/260, 47/105, 47/105/207/353, 47/105/260/353, 47/105/353, 47/108/207/260/353, 47/139, 47/207/260/353, 47/207/353, 47/260, 47/260/353, 47/353. 56, 56/93/105/139/207, 56/105/207/260, 56/105/207/260/353, 56/105/207/353, 56/207. 56/207/260. 56/207/353. 56/214/353. 105, 105/139/207/353. 105/139/260/353, 105/207/353, 105/260/353, 105/353, 207, 207/260/353. 207/353, or 353, wherein the amino acid difference is relative to the reference sequence corresponding to residues 12 to the carboxyl terminus of SEQ ID NO: 774, or to the reference sequence corresponding to SEQ ID NO: 774.

56. The recombinant phosphatase of claim 53 or 54. wherein die amino acid sequence of the recombinant phosphatase comprises at least an amino acid difference(s) or amino acid residue(s) 13P, 13P/25E/31R/105S, 13P/25E/47P/56P/105S/260Q, 13P/25E/47P/207V/260Q/353Q, 13P/25E/47P/207V/353Q. 13P/25E/47T, 13P/25E/56P/207V/260Q/353Q, 13P/25E/260Q/353Q. 13P/25E/353Q, 13P/32T, 13P/32T/47T/56P/353Q, 13P/32T/93D/105S/260Q/353Q, 13P/47P, 13P/47P/56P/105S/207V/353Q, 13P/47P/93D/207V/353Q, 13P/47P/207V/260Q. 13P/47T, 13P/47T/56P/353Q. 13P/47T/105S/207V/240A/260Q/353Q, 13P/56P/263I/353Q, 13P/56P/353Q. 13P/105S. 13P/207V, 13P/207V/260Q/353Q, 13P/207V/353Q, 13P/353Q, 25E/31R/32T, 25E/31R/32T/56P/105S/260Q/353Q, 25E/32T/207V/353Q, 25E/47P/105S/207V, 25E/47P/353Q, 25E/47T, 25E/47T/105S. 25E/47T/353Q, 25E/56P, 25E/56P/105S/260Q/353Q, 25E/93D/207V/260Q/353Q, 25E/105S/207V/260Q, 25E/105S/207V/260Q/353Q. 25E/207V, 25E/353Q, 31R/47T/49H/105S/207V/260Q/353Q, 32T/47P/105S/353Q. 32T/56P/105S, 47P/56P/105S/139V/260Q, 47P/105S/207V/353Q. 47P/105S/353Q, 47P/108S/207V/260Q/353Q, 47P/207V/260Q/353Q, 47P/207V/353Q, 47P/260Q, 47P/260Q/353Q. 47P/353Q, 47T, 47T/105S, 47T/105S/207V/353Q, 47T/105S/260Q/353Q, 47T/139V, 47T/207V/260Q/353Q, 47T/353Q, 56P, 56P/93D/105S/139V/207V, 56P/105S/207V/260Q, 56P/105S/207V/260Q/353Q, 56P/105S/207V/353Q, 56P/207V, 56P/207V/260Q. 56P/207V/353Q, 56P/214S/353Q, 105S, 105S/139V/207V/353Q, 105S/139V/260Q/353Q, 105S/207V/353Q, 105S/260Q/353Q, 105S/353Q, 207V, 207V/260Q/353Q, 207V/353Q, or 353Q, wherein the amino acid difference is relative to the reference sequence corresponding to residues 12 to the carboxy 1 terminus of SEQ ID NO: 774, or to the reference sequence corresponding to SEQ ID NO: 774.

57. The recombinant phosphatase of claim 1, wherein the recombinant phosphatase comprises an amino acid sequence comprising residues 12 to the carboxyl terminus of an even-numbered SEQ ID NO. of SEQ ID NOs: 16-18 and 24-1052, or an amino acid sequence comprising an even- numbered SEQ ID NO. of SEQ ID NOs: 16-18 and 24-1052.

58. The recombinant phosphatase of any one of claims 1-57. wherein the recombinant phosphatase exhibits one or more improved properties as compared to a reference phosphatase having the amino acid sequence corresponding to SEQ ID NO: 12, 18, 84, 214, 232. 562, 656, or 774.

59. The recombinant phosphatase of claim 58, wherein the improved property is selected from i) increased phosphatase activity on an oligonucleotide having a 3 ’-phosphate group, ii) increased phosphatase activity on an 3’-phosphate-NTP, iii) increased solubility, iv) increased expression or protein yield, v) increased thermal stability , vi) increased resistance to proteolysis, and vii) increased resistance to inhibition by immobilization on epoxide activated poly aciy late resin, or any combination thereof, as compared to the reference phosphatase having the amino acid sequence corresponding to SEQ ID NO:

12, 18, 84, 214, 232, 562, 656, or 774.

60. The recombinant phosphatase of claim 58 or 59, wherein the reference phosphatase has the amino acid sequence

61 . The recombinant phosphatase of any one of claims 1 -60, wherein the recombinant phosphatase is purified.

62. A recombinant polynucleotide comprising a polynucleotide sequence encoding a recombinant phosphatase of any one of claims 1-57.

63. The recombinant polynucleotide of claim 62, wherein the polynucleotide sequence has at least 70%, 75%. 80%, 81%, 82%, 83%, 84%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference polynucleotide sequence corresponding to nucleotide residues 34 to the 3‘-terminal nucleotide of an odd numbered SEQ ID NO. of SEQ ID NOs: 1-13, or to a reference polynucleotide sequence corresponding to an odd numbered SEQ ID NO. of SEQ ID NOs: 1-

13. wherein the polynucleotide sequence encodes a phosphatase.

64. The recombinant polynucleotide of claim 62, wherein the recombinant poly nucleotide sequence has at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference polynucleotide sequence corresponding to nucleotide residues 34 to the 3 ’-terminal nucleotide of an even-numbered SEQ ID NO. of SEQ ID NOs: 15-17 and 23-1051, or to a reference polynucleotide sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 15-17 and 23-1051, wherein the polynucleotide sequence encodes a phosphatase.

65. The recombinant polynucleotide of claim 62, wherein the recombinant poly nucleotide sequence has at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference polynucleotide sequence corresponding to nucleotide residues 34 to the 3’-terminal nucleotide of SEQ ID NO: 11, 83, 213, 231, 561, 655, or 773, or to a reference polynucleotide sequence corresponding to SEQ ID NO: 11, 83, 213, 231. 561, 655, or 773, wherein the polynucleotide sequence encodes a phosphatase.

66. The recombinant polynucleotide of claim 62-65, wherein the recombinant polynucleotide sequence is codon-optimized for expression of the encoded recombinant phosphatase.

67. An expression vector comprising a recombinant polynucleotide of any one of claims 62- 66.

68. The expression vector of claim 67, wherein the polynucleotide is operably linked to a control sequence.

69. The expression vector of claim 68, wherein the control sequence comprises at least a promoter.

70. A host cell comprising an expression vector of any one of claims 67-69.

71. The host cell of claim 70, comprising a prokaryotic cell or eukaryotic cell.

72. The host cell of claim 71, comprising a bacterial cell, fungal cell, insect cell, or mammalian cell.

73. A method of producing an recombinant phosphatase in a host cell comprising culturing a host cell of any one of claims 70-72, under suitable culture conditions such that the recombinant phosphatase is produced.

74. The method of claim 73, further comprising recovering the recombinant phosphatase from the culture and/or host cells.

75. The method of claim 73 or 74, further comprising purifying the recombinant phosphatase.

76. A composition comprising a recombinant phosphatase of any one of claims 1-61.

77. The composition of claim 76, further comprising one or more of phosphatase substrates with a phosphate monoester or analog thereof.

78. The composition of claim 77. wherein the substrate phosphate monoester or analog thereof comprises NTP, NDP, NMP, 3‘-P-NTP, 3’-P-NDP, 3’-P-NMP, Np, NTP-a-S, NDP-a-S, NMP-a- S, 3’-P-NTP-a-S, 3’-P-NDP-a-S, 3’-P-NMP-a-S, NpS, or any combination thereof, wherein N represents a nucleoside.

79. The composition of claim 77. wherein the substrate phosphate monoester or analog thereof comprises a polynucleotide or oligonucleotide with a 5’-P, 3’-P, 5’-P(S), 3’-P(S), or any suitable combination thereof.

80. The composition of claim 79, wherein the polynucleotide or oligonucleotide is single stranded or double stranded.

81. The composition of claim 79 or 80, wherein the polynucleotide or oligonucleotide comprises at least a modified nucleoside or internucleoside linkage.

82. The composition of any one of claims 76-81, further comprising a buffer.

83. A method of cleaving a phosphate monoester or analog thereof, comprising contacting a substrate with a phosphate monoester or analog thereof with a recombinant phosphatase of any one of claims 1-61 under suitable conditions for cleaving of the phosphate monocstcr or analog thereof.

84. The method of claim 83, wherein contacting the phosphatase substrate is done in vitro.

85. The method of claim 83 or 84, wherein the substrate is NTP. NDP, NMP. 3’-P-NTP, 3’- P-NDP, 3’-P-NMP, Np. NTP-oc-S. NDP-oc-S. NMP-a-S, 3’-P-NTP-a-S, 3’-P-NDP-a-S, 3’-P-NMP-a-S. NpS, or any combination thereof.

86. The method of claim 83 or 84, wherein the substrate phosphate monoester or analog thereof comprises a polynucleotide or oligonucleotide with a 5’-P, 3’-P, 5’-P(S), 3’-P(S), or any suitable combination thereof.

87. The method of claim 86, wherein the polynucleotide or oligonucleotide is single stranded or double stranded polynucleotide or oligonucleotide.

88. The method of claim 86 or 87, wherein the polynucleotide or oligonucleotide comprises at least a modified nucleoside and/or modified intemucleoside linkage.

89. The method of any one of claims 83-88, wherein the suitable conditions comprise a temperature of 4-80 °C.

90. The method of any one of claims 83-89, wherein the suitable conditions comprise a pH between 7-10.