WO1998046770A2 - Stable biocatalysts for ester hydrolysis - Google Patents

Abstract

The instant invention encompasses isolated stable esterase enzymes characterized by the ability to remain stable at certain temperatures, substrate specificities, and activity profile; the expression vectors which can express, nucleic acids which encode for, and corresponding protein amino acid sequence of such proteins.

Description

Stable Biocatalysts for Ester Hydrolysis

This apphcauon claims pπoπty to U S. Application for Patent Seπal No 08/827.810. filed Apπl 11, 1997, which is hereby incorporated by relerence its entirety

ST_ATEMENT OF GOVERNMENT RIGHTS

The work disclosed in this apphcauon was supported in pan by Grant Number: NCI ₁-R43-CA63876-01 from the NIH-SBIR to ThermoGen Inc., therefore, the U.S. Government may have some πghts in the present invention

FIELD OF THE INVENTION

The instant disclosure is directed to the field of isolated stable biocatalysts that are suitable for enzymauc apphcauon in commercial pharmaceuucal and chemical synthesis, DNA vec_tors for the producuon of recombinant ester hydrolyzing proteins, host cells transformed by such vectors, and recomDinant ester hydrolyzing proteins produced oy such vectors and transformed cells.

B_ACKGROUND OF THE INVENTION

Esterases and Lipases. Esterases and lipases catalyze the hydiolysis of ester bonds to produce alcohols and carboxylic acids as shown below

Es_terases and lipases can be characteπzed bv different substrate specificiues. R gioup or chain length preference, and unique inhibitors ( 1. 2) The many esterases and hoases range from hydrolases such as the broad carboxyl esterases which preterenually hydrolvze esters with long carbon chain R groups, to choline esterases, and to acetyl esterases whⁱch ac_t on very specific substrates. In many cases, these hydrolases are also known to show stereo- and regio-selective preferences resulting from the chiral nature inherent in pro^teⁱn active si_tes. This preferential hydrolyuc activity make them useful for reacuons requⁱπng different regioselectivity and stereoselecuvity or for kinetic resoluuon methods on racemⁱc m_ixtures. For enzymes that demonstrate stereoselecuvity, if R* is a racemic mix^ture^, the product of enzyme catalyzed hydrolysis, R l , would be the most rapidly hydrolvzed stereo_isomer while _the remaining ester designated R*' would be the enπched antipode mⁱxed w_ith any remaining R l . The products can then be separated by chromatograph^y to provⁱde pure R l . The availability of a large pool oi esterases and lipases with varying specificities would be useful for screening the enzymes tor specific reactions, and developing opumal protocols for specific chemical synthesis The expedience of this process would facilitate ^the producuon scale-up of many useful pharmaceutical products In aqueous solvent systems, esterases and lipases carry out their natural reactions: the hydrolysis of ester bonds. In vitro, these enzymes can be used to cany out reactions on a wide vanety of substrates, including esters containing cyclic and acyclic alcohols, mono- and di-esters, and lactams (3). By carrying out the reactions in organic solvents (4, 5) where water is excluded, the reactions of esterases and lipases can be reversed. These enzymes can catalyze esteπfication or acylation reacuons to form ester bonds (3, 6, 7). This process can also be used in die transesteπfication of esters and in πng closure or opening reactions.

Optically pure chiral pharmaceuticals. Currently, the majoπty of synthetic chiral pharmaceuticals are sold as racemic mixtures However, due to advances in the synthesis of opucally pure (single isomer) chiral compounds, this situation is changing (7) Racemic drugs often contain one isomer which is therapeutically active and the other enantiomer which is at best inacuve and at worst a major cause of potenually harmful side effects. The non-useful isomer in a racemic drug is increasingly being viewed as a contaminant. Indeed, the FDA's Policy Statement for the Development of New Drugs recommends "that the pharmacokinetic profile of each isomer should be characteπzed in animals and later compared to the clinical pharmacokinetic profile obtained in Phase I" drug testing (8). Thus, pharmaceutical companies will need to develop a synthesis or separauon route to produce each pure isomer of each new synthetic drug.

Enzymatic synthesis of optically pure pharmaceuticals and intermediates. Since it is often very difficult to generate optically puie solutions of certain chiral molecules by classical chemical synthesis, new enzymatic biocatalysts will play a major role in this endeavor. In some cases, enzymes may be able to replace hazardous chemical syndiesis procedures with more environmentally-fπendly biological synthesis processes. It can also be much more cost effective to produce a pharmaceutical intermediate enzymatically if an enzyme can eliminate several chemical protection and deprotection steps at once (7). All six major classes of enzymes (oxidoreductases, transferases, hydrolases, lyases, isomerases, and hgases) have been useful in the synthesis of optically pure compounds as described in several detailed reviews (3, 7). The hydrolases have proven to be the most useful group of enzymes, due to the abundance of hydrolases, the information about them, their independence from cofactors, and the wide variety of substrates they can accept.

A survey of the literature shows many examples of mesophihc hydrolases particularly esterases and lipases used in chemical synthesis or chiral resolution These include esterases from pig (9, 10) and hoise (3) livers and a wide vanety of lipases Irom Aspergdlus sp, (11) Candida sp. ( 12- 16), Pseudomonas sp., (17-19), Rhizopus sp. (20) and others. Several lipases have been used in the synthesis of propranolol (7), a beta-adrenergic blocking agent used in the treatment ol angina and hypertension. Ibuprofen, a nonstearoidal antnnflammatory agent has been synthesized via stereo selective hydrolysis of its methyl ester using carboxyesteiase (7) While these enzymes have begun to demonstrate the utility of biocatalysts in chemical synthesis, there is still a profound need for a wider varie_ty of esterases and lipases which have varying substrate specificities, regioselectivities, and steroselectivities. In addition, since these enzymes need to be employed in a large-scale industnal setung, there is a need for them to have increased stability, higher thermotoleiance and a longer "shelf life"

Thermostable enzymes. Thermophihc organisms have already provided a rich souice of useful proteins that catalyze reacuons at higher temperatures and are stable for much longei penods of time (21, 22). One example is die DNA Polymerase I from Thermus aquanciis and its use in polymerase chain reaction (PCR) (23, 24) Thermophihc enzymes have become the most commercially successful enzymes in industry because ot their long-term stability and ease of use. The most successful enzyme to date, alpha-amylase, is used in corn processing and comes from the moderate thermophile B stearothermophilus (25) Another commercially successful industnal enzyme is subtilisin, a serine protease also found in vanous strains of Bacillus, has been widely used in laundry detergents and other cleaning soluuons.

The commercial success of these enzymes can be attπbuted to their ease of use In addition to funcuoning at high temperatures, thermostable enzymes generally posses an increased shelf life which markedly improves handling conditions, especially by those not u-ained in biochemistry to work with the specific range of conditions used for mesophihc enzymes If enzymes are to play a significant role in large scale processing of chemicals, they must be able to endure the harsh conditions associated with these processes Thermostable enzymes are easier to handle, last longer, and given the proper immobilization support should be reusable for muluple applications

Finally, the hydrophobic and electrostauc forces that allow these enzymes to survive high temperatures also allow them to generally function better in organic solvents (26- 1) While most enzymes lose a significant portion of their activity in organic solvents, thermostable enzymes may prove more tolerant to the denatuπng condiuons of many organic solvents. Highly thermostable esterases and lipases are necessary to expand the application of these biocatalysts in large scale industnal reactions.

Thermostable esterases and lipases To date, only one esterase and a lew lipases have been reported with moderately thermostable chaiacteπstics Tulin et al (32) reported a Bacillus stearothermophilus esterase cloned into Bacillus brevis which was stable up to 10 minutes at 70°C Sugihara et al.(33, 34) have isolated novel thermostable lipases irom two microorganisms, A Bacillus soil isolate and a Pseudomonas cepacia soil isolate The tormer lipase is stable up to 30 minutes at 65°C but rapidly inacuvated above this temperature The lipase from Pseudomonas cepacia was stable when heated for 30 minutes at 75°C and pH 6 5 but had only 10% ot its activity when assayed at this temperature. A thermoalcalophi c lipase (35) was identified from a Bacillus species MC7 isolated by continuous culture and had a half-life of 3 hours at 70^°C. Finally, Sigurgisladottir et al. (6) have reported the isolauon of one Thermus and two Bacillus strains which posses lipases active on olive oil up to 80°C, although there was no report on enzyme stability in this study.

These enzymes offer only limited vanations in substrate specificities and only moderate thermostability profiles. They do not address the need for different substrate specificities, the need to produce large scale quantities which can be economically commercialized, and many of them have only limited overall stability. In this patent application we have identified a series of esterases and lipases which offer a range of substrate specificities (including regioselectivity, stereoselecuvity), enhanced enzyme stability, and can be produced in large quantities for commercial use.

SUMMARY OF THE INVENTION The instant invenuon provides for the isolauon and characteπzation of commercial grade enzyme preparauons charactenzed by esterase activity, and corresponding to the data as disclosed in Table 1. In a preferred embodiment, die instant invention provides tor the isolation, and charactenzation of specifically punfied esterase which is characterized by esterase actiyity, and corresponding to the data as disclosed in Table 1. In a most preferred embodiment, the instant invenuon provides for proteins generated by recombinant DNA technology which have esterase activity. The instant invention encompasses lambda phage expression vectors which contain an insert that can be used for the production of recombinant ester hydrolyzing proteins of the instant invenuon, from a transformed cell host. The insert contained on die lambda phage expression vector may be used in, for example, a phage-plasmid hybnd expression vector or other suitable expression vector such as, but not limited to, plasmids, YACs, cos ids, phagemids, etc. In a preferred embodiment, a lambda expression vector is one of the vectors named in Table 7, or one which contains an insert which encodes for a substantially similar recombinant protein. The instant disclosure also provides for vectors which are capable of transforming a host cell, and which encode for recombinant ester hydrolyzing protems, the transformed host cells, and the recombinant ester hydrolyzing protein. Appropπate host cells include but are not limited to: E. coli, Bacilli, Thermus sp., etc. The recombinant ester hydrolyzing protein encoded by the vector is capable of hydrolyzing 5-bromo-4-chloro-3-indolyl-acetate (X-acetate). The recombinant ester hydrolyzing protein produced by the vector can be further charactenzed by a half-life stability comparable to that of a corresponding protein purified from the isolates. The recombinant ester hydrolyzing protein is also characterized by the ability to remain stable at temperatures comparable to, or better than that of the conesponding protein from the onginal isolates. Recombinant ester hydrolyzing protein encoded for by the vector can also be characterized by certain substrate specificities as discussed below, which are comparable to those of the conesponding punfied protein from the isolates In a preferred embodimen_t the vector is a vector named in Table 7 or 8, or one which contains an insert which encodes tor a substantially similar recombinant protein In a preferred embodiment of the instant invention, a vector which encodes specific recombinant ester hydrolyzing protein is one of the vectois named and listed in Table 8.

The instant invention is directed to the novel nucleic acids, and the proteins encoded for dierein, isolated from the expression vectors of the present invention. In particular, the present invenuon is directed towards die nucleic acid sequence for DNA insert of said vectors, and the die protein ammo acid sequence(s) expressible therefrom.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Enzyme Characteristics. Figure 4 depicts a sample activity profile which characteπzes and enzyme of the instant disclosure Graph 1 depicts the Temperature Profile ot the enzyme plotting relative esterase activity versus temperature Graph 2 depicts the Residual Esterase Activity of the listed enzyme plotting relative remaining activity versus ume in hours, at 25°C, 40°C, and 65°C Graph 3 depicts the pH profile for die listed enzyme plotung Relauve Esterase Ac vity versus pH. Data for enzymes are summaπzed in Tables 1, 2 and 10.

Figure 2. Kinetic analysis of ElOO. The enzyme displays normal Michaehs kinetics yielding linear data with both a) Lineweaver-Burke and b) Eadie-Hofstee analysis to give a Km = 7.2X 10^"5M and Vmax= 1.8xlO^~5Mmιιr 1 using p-NP as the substrate Figure 3. Temperature and pH profiles of ElOO. a) Temperature profile of ElOO Plot ot ElOO catalyzed hydrolysis ot p-nitrophenyl propπonate as a function of temperature Enzyme acuvity was determined upon exposure to different temperatures. Initial rates of nitrophenylpropπonate hydrolysis were determined in 50 mM borate Buffer pH 8 5 equilibrated to die desired temperature to which 0.25 mM substrate dissolved in CH3CN was added followed by enzyme. Rates were determined by momtoπng die change in absoibance at 405 nm and conected for the spontaneous hydrolysis of substrate substituting bovine serum albumin for enzyme, b) pH profile ot ElOO The effect of pH on the hydrolysis ot p- nitrophenyl propπonate catalyzed by ElOO The pH profile of the enzyme was determined by prepaπng different buffers appropπate for the desired pH's at 10 mM concentration Reacuons were performed by addition of the substrate (0.25 mM) dissolved in CH3CN to the buffer solution followed by the enzyme. Reactions were incubated for 5 minutes after which the reaction was terminated by addiuon of 0 1 mM PMSF dissolved in CH3CN The pH of the mixture is adjusted to 8.5 by addition of 0 1 M Tns-HCl. Absorbances are recorded at 405 nm and concentrations calculated based on the ε=17mM^~l ^cm~l for the product nitrophenol Formation of products is corrected for the spontaneous hydrolysis of the substrate. Figure 4. The tolerance of ElOO to the presence of organic cosolvents on the hydrolysis of p-nitrophenyl proprionate as determined by relative rates. Residual acuvity of the enzyme is determined in the presence of organic solvent by measuring the initial rate of enzyme catalyzed hydrolysis of pNP in the presence of various concentrations of CH3CN. Reactions are run in 50 mM Tris-HCl pH 8.5 at 37°C as described in determination of activity. Changes in absorbance are corrected for spontaneous hydrolysis of the substrate and the changes in extinction coefficient of the product in the presence of organic cosolvent.

Figure 5. Substrates used to screen stereo- and regioselectivity. Esterases are versatile biocatalysts in the sense that stereo- and regio-selectivity can be mediated by substrate structure which fall into four types. The compounds listed represent a range of different structural features encountered in common substrates with potential importance for the chemical intermediate industry. Several of the substrates are commercially available in entantio- or diastereomerically pure form and can be used in qualitadve screening procedures described in the text. Four classes of substrates most commonly associated with hydrolytic biocatalysts for chiral centers resolution are considered. A) Type I substrates position the desired product on the carboxylic acid side of the product, while Type II compounds the alcohol contains the requisite functionality. B) Type III and Type IV substrates can be considered subsets of Types I and II, but their unique properties dictate diat they be classified separately. Type III molecules require that the enzyme differentiates a prochiral substrate while Type IV compounds are meso structures. These last two substrate types demonstrate the synthetic importance of biocatalyst based resolution methods as these types of compounds are very difficult to selectively operate upon by other chemical means.

Figure 6. Nucleic acid sequence and translated protein amino acid sequence. The isolation and cloning of the genes encoding for the enzymes of the instant invention will result in DNA segments in which an open reading frame (ORF) may be found which corresponds to translated protein amino acid sequence. Alternative start codons are recognized in the an, however the encoded protein will comprise at minimum a core protein ORF. Figure 6A is an isolated nucleic acid sequence, and translated amino acid sequence which coπespond to E001 enzyme ORF, alternative start codons are underlined. Figure 6B is an isolated nucleic acid sequence, and translated amino acid sequence which correspond to E009 enzyme ORF, alternative start codons are underlined. Figure 6C is an isolated nucleic acid sequence, and translated amino acid sequence which correspond to E01 1 enzyme ORF, alternative start codons are underlined. Figure 6D is an isolated nucleic acid sequence, and translated amino acid sequence which correspond to E101 enzyme ORF, alternative start codons are underlined. Figure 6E is an isolated nucleic acid sequence, and translated amino acid sequence which corresponds to E019 enzyme ORF, alternative start codons are underlined. Figure 6F is an isolated nucleic acid sequence, and translated amino acid sequence which corresponds to E005 enzyme ORF, alternative start codons are underlined. Figure 6G is the cloned isolated nucleic acid sequence which contams the E004 ORF, alternative start codons are underlined. Figure 6H is the cloned isolated nucleic acid sequence which contams the E006 ORF, alternative start codons are underlined Figure 61 is the cloned isolated nucleic acid sequence which contains the E008 ORF, alternative start codons are underlined. Figure 6J is the cloned isolated nucleic acid sequence which contams the E010 ORF, alternative start codons are underlined Figure 6K is the cloned isolated nucleic acid sequence which contams the E013 ORF, alternative start codons are underlined. Figure 6L is the cloned isolated nucleic acid sequence which contains the E015 ORF, alternative start codons are underlined Figure 6M is the cloned isolated nucleic acid sequence which contams the E016 ORF, alternative start codons are underlined Figure 6N is the cloned isolated nucleic acid sequence which contams the E017 ORF, alternative start codons are underlined Figure 60 is the cloned isolated nucleic acid sequence which contams the E020 ORF, alternative start codons are underlined Figure 6P is the cloned isolated nucleic acid sequence which contams the E027 ORF, alternative start codons are underlined Figure 6Q, 6R, 6S, 6T and 6U are partial sequences

Figure 7.'Substrate Chain Length Specificity Figure 7A is a graph of data from a colorometnc esterase assay performed on the substrate bis-p-mtrophenyl-Carbonate Figure 7B is data from a colorometnc esterase assay performed on the substrate: p- nitrophenyl-Acetate Figure 7C the substrate bis-p-mtrophenyl-Propionate Figure 7D the substrate: bis-p-mtrophenyl-Butyrate Figure 7E the substrate: bis-p-mtrophenyl- Caproate. Figure the substrate: bis-p-nitrophenyl-Caprylate. Figure 7G the substrate bis-p-mtrophenyl-Laurate Note that E009 is an 80x dilution compared to the other enzymes in b, c, d, and f

Figure 8. Entanriomer Substrate Specificity Figure 8A summanzes the results of colorometnc esterase acUvity assays for entantiomer specificity Figure 8B-D reports quantitative colorometnc assay data m terms of minutes required for detectable color change.

Figure 9. Enzyme Activity against para-nitroanilide compounds Table lists the results of enzyme activity assay against vanous substrates Data is reported as normalized OD readings DETAILED DESCRIPTION OF THE INVENTION

The instant invention provides for isolated commercially useful protein preparations from themostable bactena which are selected for enzymatic activity, and charactenzed by apparent molecular weight, pH, and temperature stability. The isolated protein of the instant disclosure can be used as molecular weight markers for finding similar enzymes, as well as functionally as enzymes for carrying out biocatalysis. Commercial chemical synthesis of specific racemic products often require the use of such isolated enzyme preparations. The results of charactenzation assays demonstrate that the esterase enzymes descnbed have a range of optimal parameters. For instance, ElOO and E101 have optimal operating temperatures above 70°C as would be consistent with enzymes isolated from an extreme thermophile, and E001 -E021 have optimal commercial temperatures in the range of 40-50°C as would be consistent with enzymes isolated from the more moderate thermophihc organisms. Both groups, however, provide added stability and functionality as compared to other known esterases from thermophihc bactena. E001-E021 provide an optimal temperature environment for chemists who wish to work in less extreme temperature ranges, and also function well at room temperature The results also demonstrate that the enzymes descnbed posses a vanety of pH optima including some with no apparent preference under the conditions of the expenment, however the trend for most of the proteins is to have pH optima near or slightly below neutral.

The following examples are meant by way of illustration, and not limitation, as to the specific embodiments of the instant invention. One of ordinary skill in the art would understand that many equivalents to the instant inventions can be made with no more than routine expenmentation.

Example 1. Isolation and propagation of thermophihc organisms

Strains - Thermus sp. T351 (ATCC 31674) is available from the Amencan Type Culture Collection (ATCC). All isolated strains and cultures are grown on TT medium This medium consists of (per liter): BBL Polypeptone (8 gm), Difco Yeast Extract (4 gm), and NaCl (2 gm). Small scale cultures for screening are grown at 65°C at 250-300 rpm with 1 liter of medium in a 2 liter flask. Larger scale production of cells for enzyme punfication are grown in 17 liter fermentors (LH Fermentation. Model 2000 senes 1 ). The fermentors have a working volume of 15 liters and cultures were grown m TT broth, 250 rpm, 0 3 to 0.5 vvm (volumes air/volume media per minute) at 65°C Temperature is maintained by circulating 65°C water from a 28 liter 65°C water reservoir through hollow baffles with the stirred jars. E. coli strains are grown as descnbed in (37).

Enrichment Procedures for Newly Isolated Thermophiles Multiple stream sediments, composting organic materials, and soil samples are used to isolate new strains These samples are collected from numerous geographic sites ranging from the Midwes_{t t}o the Southeast. Samples (- 1 gm) are resuspended in 2 ml of TT broth and 50-100 μl of these samples were plated onto TT agar plates containing twice the usual amount of agar (3%)_. Agar is usually added to a final concentration of 1.5% for solid media This prevents highly motile microorganisms from overcrowding the plate at the expense of other microbes. Plates are incubated at 55°C or 65°C for one to two days and isolates then purified by numerous restreaks onto fresh plates for single colony isolation. The initial basis for differentiation is color, colony morphology, microscopic examination, temperature of growth, and lipase and esterase activities. Several hundred strains were initially isolated. 65 different microorganisms were chosen for further study.

Example 2. Methods for esterase identification and assay

Esterase Plate assay - Organisms are grown in liquid cultures on TT media at either 55°C or 65°C. Cells are pelleted by centrifugation (3,000 RPM for 20 minutes) and die supernatants saved to be tested. Pellets are washed with 2 volumes of 10 mM Tris HCl pH 8.0 three times after which the cell pellets are resuspended in fresh Tris buffer and disrupted by sonication. Cell debris is removed by centrifugation and the crude extracts were tested for esterase acuvity on an esterase screening plate. Briefly, fifty microliters of cell extract is transferred to a well on a microtiter plate consisting of 0.1 mg/ml of either 5-bromo-4-chloro-3-indolyl acetate or butyrate (for esterase acύviύes) suspended in 0.7% agarose and 0. IM Tris-HCl pH 8.0. Control wells consist of addition of either buffer, 20 U of Pig Liver Esterase (PLE), or 20 U of Porcine Pancreatic Lipase (PPL). Plates are incubated for sufficient time to allow full color development in control wells, usually about twenty minutes at 37°C. Dark wells represent posidve activity. Both cell extracts and culture supernatants are tested for esterase acuvity by this method.

Only cell extracts showed significant esterase acuvity.

Esterase Liquid assay and determination of specific activity - Protein concentrations are determined by the Pierce BCA assay using defined concentrations of bovine serum albumin as the standard. Protein concentrations are obtained from die calibrated absorbance of the sample soluύons at 562 nm and are expressed as milligrams of protein. Esterase activities are routinely measured by determining the rate of hydrolysis of p-nitrophenylproprionate (0.5 mM from a 10 mM stock dissolved in CH3CN) in 50 mM sodium phosphate buffer pH 7.0 equilibrated at 40°C and monitored at 346 nm (isosbestic point for the acid/carboxylate couple ε=4800). The specific acuvity is defined as the amount of p-nitrophenol produced in micromoles per minute per milligram of total protein.

Identification of extremely stable esterases. - Native (non denaturing) 10% polyacrylamide gels are run on crude extracts. After electrophoresis, the gels are equilibrated in pH 7.6 Trizma buffer and then stained for activity in either 0.15% X-acetate. The gels are then incubated at 55°C for up to 30 minutes. These gels can then be stained with an esterase activity stain containing either 5-bromo-4-chloro-3-indolyl acetate (X-acetate), 5-bromo-4- chloro-3-indolyl butyrate (X-butyrate) or 5-bromo-4-chloro-3-indoIyl caprylate (X-caprylate) and produced indigo precipitates. Two major bands were apparent in the lanes with Thermus crude extracts. A single small band of activity is seen in the E. coli control lanes. Esterases can be identified from Thermus sp. T351 and from several of the new isolates. Table 1 summarizes the activities which are found from these organisms.

TABLE 1. Summary of New Esterases and Strains Identified

Isolate-' Esterase Source Growth Temp (°C) Isolation w Specific

37 55 65 Temp (°C) (kD)² Activity-

SI E001 soil nd nd + 65 22 0.011

54 E002 compost - + + 65 28 0.87

50 E003 compost - + + 65 28 2.2

GP1 E004 soil nd nd + 65 36 0.3

C-l E005 compost nd nd + 65 28 2.3

55 E006 compost - + + 65 36 2.1

46 E007 compost - + + 65 28 0.3

30 E008 soil - + + 55 28 2.1

28 EO09 soil - + + 55 36 2.0

29 E010 soil - + - 55 46.5 2.3

31 E011 soil - + - 55 36 3.6

26b E012 soil - + - 55 28 5.2

27 E013 soil - + + 55 36 2.1

34 E014 soil - + +/- 55 36 0.8

62 E015 compost - + + 55 36 3.4

47 E016 compost - + + 65 28 0.8

49 E017 soil - + + 65 36 0.03

C-3 E018 compost nd nd + 65 36 0.077

4 E019 compost - + + 55 30 0 4

7 E020 compost - + + 55 28 1.6

32 E021/17b⁴ sou . + +/- 55 36 0.3

Thermus sp. T351 ElOO ATCC# 31674 nd + + 65 45 0.0032

Thermus sp. T351 E101 ATCC# 31674 nd + + 65 135 0.032

¹ Isolates GP1, 27,28,29,30,31.32,34,62 appear to be thermophihc Actinomyces.

2 Approximate molecular weight as determined by chromatography for E001-E021 or SDS-PAGE for ElOO and

E101. ^Specific activity is the amount of p-nitrophenol produced in micromoles per minute per milligram of total protein at 40°C after purificauon to homogeneity (tor ElOO and E101) or se i-punficauon (for E001 -

E021) as described in the Examples. ⁴E021 is also referred to as E017b.

Example 3. Procedure for purification of esterase activity to homogeneity.

Protein Isolation - A large batch cell culture is grown according to the methods described in Example 1 and the cell paste is collected by centrifugation and stored at -80°C. lOOg of cell paste is thawed in 200 ml of a stirred solution composed of 50 M phosphate buffer at pH 7.5 containing 200 mM KC1 and 0.1 M EDTA. Once dissolved, the suspension is allowed to warm to room temperature and then treated with lysozyme (0.1 mg/ml) for 2 hours. The solution is then sonicated to completely disrupt the cells. Settings used on a 375 watt Sonics & Matenals Vibra Cell sonicator with a standard 1/4" horn were 5 minutes of power setung 8 disrupuon with a 50% pulse rate. Alternative methods for cell disruption can include processing the cells through a device such as a french press, Gaullen homogemzer, microfluidizer or other homogemzer. Cell debns is removed by centnfugauon and proteins can be precipitated by NH₄SO₄ fractionation to 60% saturation. Precipitated protein is centrifuged and resuspended in minimal volume of 50 mM phosphate pH 6.5 containing 1 mM β-mercaptoethanol (BME). DEAE Purification - The protein solution is dialyzed against the resuspension buffer 3 umes using 10 Kd pore size dialysis tubing. The resulting protein solution is diluted two fold in the buffer and applied to a 100 ml bed volume DEAE column equilibrated in the same buffer. The column is washed with 200 ml equilibrauon buffer and men eluted with a linear gradient from 0 to 0.5 M NaCl. Q Resin purification - Active tractions isolated from DEAE punficauon are pooled and dialyzed against three changes of equilibration buffer and dialysate was applied to a 50 ml bed volume of sepharose Q resm equilibrated widi the buffer above. The column is washed with 100 ml of 50 mM phosphate pH 6.5 containing 0.1M KC1 and 1 mM BME and then eluted with 150 ml of a KC1 gradient from 0.1 M to 0.6M added to the above buffer. Ultrafiltration Concentration - Active fractions are pooled and concentrated using an Amicon Ultrafiltration system fitted with a 30 Kd cut off membrane.

Preparative SDS PAGE - Concentrated protein soluuons are loaded to a preparative 10% SDS-PAGE gel using the standard SDS loading buffer without boiling the sample. After development, die gel is treated with 0.7% agarose containing 0.1M phosphate pH 7.5 and 0.1 mg/ml 5-bromo-4-chloro-ιndoylacetate. The resulung blue band was excised from the gel, placed in dialysis tubing and die protein is recovered by electroeluuon in 0.05M Tπs buffer pH 8.5 for 1 hour. At this stage the protein is purified to homogeneity as observed by botii native- and SDS-PAGE stained with either coomassie or silver stain. Protein can be stored at 4°C for future use. Gel filtration - A gel filtrauon column can also be used as a further or substituted purification step.

Example 4. Method for commercial grade preparation of isolated Esterase.

For many industrial applications, a completely purified preparation of enzyme is neither required nor desired due to production cost considerations. A rapid, inexpensive protocol to produce a protein of interest in a form which is isolated to contain protein with significant esterase activity is desired. One such semi-puπfication procedure is described here. 50 g of cell paste is thawed in 100 ml of 50 mM Tris HCl buffer at pH 7.5 containing 0.1M NaCl and 0.01 mM EDTA. Cells are disrupted by sonication and the cell debns is removed by centnfugauon. The crude cell lysate is diluted by three fold with 50 mM Tris-HCl pH 7.5 and the mateπal is loaded to a DEAE cellulose column (bed volume 60 ml) equilibrated with the dilution buffer. The column is washed with three column volumes of dilution buffer followed by a salt gradient of 0-0.5M NaCl over 4 column volumes. Active fractions eluted from the ion exchange resin in the salt gradient window ot 0.25-0.35 M Fractions were assayed for activity as descnbed under determination of specific activity and those showing the highest activity were pooled and concentrated by ultrafiltration with 10 Kd moleculai weight cut off membrane. Concentrated enzyme samples are stored at 4°C for further use. In some instances, more than one ester hydrolysis activity may still be detected under long term exposure to substrate agarose overlays of proteins separated on native PAGE, indicating very small quantities of a second esterase activity which should not interfere with most industnal applications A further purification (such as an Ammonium sulfate salt precipitation, gel filtration, or other methods as descnbed in Example 3) can be applied it necessary The process can be scaled up or down as desired

Example 5. Method for determination of temperature profile.

Opumal temperature profiles for an esterase protein is performed by measunng the activity of the esterase diluted into 0. IM sodium phosphate buffer pH 7 0 equilibrated at 30°C, 35°C, 45°C, 55°C and 65°C respectively for five minutes. The temperature profile is then determined by measunng the rate of hydrolysis of p-mtrophenylpropnonate added to the equilibrated solution under reaction conditions described tor determination ot specific activity in Example 2 (modified by the vanous temperatures used in this experiment) Control reactions that substitute bovine serum albumin for esterase enzymes aie used to allow correction for temperature dependent autohydrolysis of the substrate The data is then plotted as relative acuvity versus the temperature of the reacuon

Example 6. Method for determination of enzyme stability.

The long term catalytic stability the esterase enzyme is evaluated by tesung the activity remaining after exposure to vanous temperatures. The enzyme stock solution is diluted into 0.1 M sodium phosphate buffer pH 7.0 and placed in a temperature bath equilibrated to 25°C, 40°C or 60°C respectively under sealed conditions to avoid concentration effects due to evaporation. Residual activity is then determined by removing aliquots at regular intervals and measunng the rate of hydrolysis of p-nitrophenyl-propnonate as described above Results are plotted as relauve acuvity vs. time. The results indicate that all enzymes tested retain most of the initial activity for at least 48 hours when exposed to temperatures up to and including 40°C. Activity does decrease at 60°C particularly foi enzymes isolated trom organisms with optimal growth temperatuies near 55°C. Figure 4 is an example ot the typical data obtained. Data for enzymes are summarized in tables 1, 2 and 10 Example 7. Method for determination of pH profile.

The pH profile of an esterase is determined as follows The rate of p-mtrophenylpiopπonate hydrolysis is determined under reaction conditions similar to those described for determinauon of specific acuvity in Example 2 with buffers of wide useful pH windows that overlap with at least one data point. For the purposes of these expeπments two buffers were selected that met the above cnteπa, Mes (useful range of 6-6.5) and Bis-tπs propane (useful buffer range 6.5-9) All pH tests were corrected for spontaneous autohydrolysis by subtraction of experimental runs from controls substituting bovine serum albumen for esterase. This control data treatment becomes especially important for pH's greater than 7 5

Example 8. Solvent effects on esterase activity

Industnal apphcauons for biocatalysts often require that enzymes funcuon under non- nauve and harsh condiuons. Exposure to elevated temperatures and pH fluctuations are possible challenges to enzyme activity, however the lack aqueous solubility of many compounds that may serve as substrate targets for biocatalysts is a significant challenge to the industrial organic chemist. Organic cosolvents are commonly used in reactions and isolated enzymes must be able to survive under condiuons of relatively high concentrations of cosolvent. Expenments are run in the presence of vanous organic solvents such as ethanol, acetonitπle, dimethylformamide, dioxane, toluene, hexane and detergents like SDS, tnton XlOO and Tween 20. Additional expeπments are also performed to test the activity of isolated catalysts in nearly anhydrous solvent conditions in which the enzymes will be lyophihzed from buffers and pH's of optimal activity

Example 9. Method for Protein Characterization by migration on Native PAGE The number of esterase enzymes in each semi-pure sample is determined from native gel PAGE using 4-15% acrylamide gradient (precast gels purchased from Bio-Rad laboratoπes) separaung proteins based on their charge to size rauo. The gel shows trace contamination with other enzymes capable of lndoylacetate hydrolysis that could not be detected easily with the HPLC because of column diluuon effects. What is clear from the gel expeπments is that most of the samples have a single major acuvity band or zone mat have similar migration characteπstics .

Example 10. Determination of Relative Molecular Weight by Chromatography.

The estimated nauve molecular weights tor the protein of interest is determined by separauon on a Pharmacia Superdex S200 FPLC column fitted to a Hitachi HPLC 6200 system. Proteins were separated by isocratic elution in 0.05 M sodium phosphate buffer at pH 7.0 containing 0.1 M NaCl. The solvent flow rate was maintained at 0.5 ml/min and protein was detected by UV at 280 nm Esterase active fracuons were detected initially by 5- bromo-3-chloro-3-ιndolyl-acetate plate assay with follow-up assay of most active fractions by p-mtrophenyl-propπonate hydrolysis (both methods are descnbed in Example 2) Molecular weights are estimated by companson to standard elution profiles (plotted as the log of molecular weight vs. lime in minutes) generated by use of the following proteins: β- amylase 200 Kd, alcohol dehydrogenase 150 Kd, bovine serum albumin 66 Kd, carbonic anhydrase 29 Kd, cytochrome c 12.3 Kd.

Example 11. Characterization of substrate specificities.

Substrate preference of esterases for hydrolytic activity on vanous esters can be determined as follows A gnd of molecules is prepared on microtiter plates by dissolving each substrate (0.1 M final concentration) in CH3CN and mixing with 0.1M phosphate buffer pH 7.5. Partially punfied enzymes is dien added to the wells and the reaction mixture is incubated for 30 minutes. Crude lysates can also be tested this way. Plates are checked after 10, 20 and 30 minutes to determine relauve acuviues. For expeπments with noncolored substrates, reactions are run in test tubes under the same conditions as descnbed for the colored substrates except that the reacuons are extracted tiiree umes with dichloromethane The organic layers are combined, dned with MgSO4 and concentrated to 0.1 ml 111 a nitrogen stream. The concentrates are then spotted to silica gel TLC plates and developed in a solvent mixture of 80.20.0.01 hexane:ethyl etheπaceuc acid. TLC plates are visualized with UV and

I2

Example 12. Rapid Screen Assay for quick substrate specificity characterization.

A new metiiod was developed to rapidly screen for esterase activity based on the mechanism of the enzyme catalyzed hydrolysis reaction wherein the pH of the system is reduced by the release of protons upon ester hydrolysis The proton flux in the reaction can be monitored by use of indicator dyes tiiat have pH-dependent color transiuons in the desired pH range of enzyme acuvity. The best indicators tested are phenol red for enzymes that function optimally at slightly elevated pHs (starting point pH 8.5) or bromothymol blue (starung point pH 7.2) for enzymes that operate well at more neutral conditions

The indicator reacuons are monitored by one of two methods. Spectroscopic studies are performed by measunng the UV Vis maxuna of a 0.001% soluuon of either phenol red or bromothymol blue dissolved in different pH buffers at 5 mM concentration Hydrolytic reacuons are then performed by adding the substrate (0 1 mM final concentration) to a 5 mM buffer soluuon (sodium phosphate pH 7.2 for bromotiiymol blue indicator and sodium borate pH 8.5 for phenol red indicator) and equilibrating the temperature at 25°C for five minutes followed by initiation of the reaction by addition ot 0 1U target enzyme

An alternative method for monitonng the hydrolytic reactions is useful for broad screening applications. 5 mM buffer containing 0.001 % indicator dye and substrates dissolved in CH3CN, DMF or DMSO to an organic solvent composition of no more than 10% is added to a stirred 24 well microtiter tray The temperature is allowed to equilibrate for five minutes at 25°C after which the reaction is initiated by addition of 0.1 U of the esterase. Reaction progress is monitored by solution color changes upon which, aliquots of

NaOH are added to return the reaction color to the starting point. Reactions are determined to be complete when no further color change is detected after prolonged incubation. Product formation is verified by TLC analysis of reactions acidified with 0.1 M HCl, extracted with ethyl acetate, dried with Na2SO4 and concentrated under a stream of N2. For testing substrates in which enzyme-based chiral resolution is being screened, products are separated and isolated by chiral phase HPLC and enantiomeric purity is determined by integration of peak areas for each isomer. Rapid assay of a variety of hydrolytic activities, in this cases esterases. is determined in a microtiter plate experiment using several different enzymes and substrates. Accurate comparison of commercially available enzymes can be insured by using the same specific activity for each enzyme determined from the total protein and the initial rate of hydrolysis of the common substrate p-nitrophenyiproprionate. The data are recorded as the time required to visualize a pH dependent color change for the given indicator dye. Control experiments using BSA as the protein source cause no change in indicator color and establish that pH changes in solution are the result of an enzyme catalyzed hydrolysis. Control tests of reaction solutions containing enzymes and indicators without substrates established that color changes in the solutions are not the result of buffer salts or the enzymes alone.

Studies performed to determine whether the microtiter plate format was amenable to small scale preparative chemistry are performed as follows. Using racemic phenethylacetate and pig liver esterase. reactions are run and titrated with aliquots of 0.1N NaOH to maintain original solution color until no further color changes occuπed at which point the reactions are stopped. Products are isolated and tested by TLC and compared to total amount of base added to verify the extent of the reaction. Phenethyl alcohol is separated from starting acetyl ester by flash column chromatography followed by analysis by chiral phase HPLC. The enantiomeric excess of the hydrolysis products is determined from the peak integration and compared to an identical reaction run in the absence of indicator dye. The results from these experiments suggest that inclusion of indicator dye has no effect on the stereoselectivity of esterase catalyzed resolution of phenethylacetate.

In order to test the assay for usefulness in a broad-based enzyme screening method, seven organisms isolated from various sources in the environment were tested for their ability to produce enzymes that would catalyze the hydrolysis of a group of structurally diverse compounds. Table 2 shows the results of these studies. TABLE 2. Substrate Specificity.

Results- are reported as the amount of time required to change indicator color. The data is indicative of variable substrate specificity between different environmental isolates. Of particular note is die suggestion of stereoselectivity as determined from the relative rates of hydrolysis for substrate enantiomers. Control reacuons are similar to those described above in die substrate specificity studies with commercially available enzymes.

Example 13. Further characterization of substrate specificities.

Depicted in Figure 10 are examples of the substrates that can be tested with each enzyme activity. These molecules have been chosen specifically because of their importance as intermediates in the synthetic literature with die potential for industrial application. Experiments can be performed with crude lysates or proteins isolated from media broth in cases where the activities are known to rapidly assess the likely reaction chemistry including substrate preference and stereochemistry. All structure activity tests are compared to standard mesophile biocatalysts such as pig liver esterase. The reactions are monitored by TLC analysis to compare the products to standards purchased from commercial sources or prepared by chemical means (for example, base-catalyzed hydrolysis of esters).. Investigations of stereochemical preference by each esterase can be evaluated by one of two methods. In the first method, standard single stereoisomers of commercially available entantiomerically pure substrate esters are hydrolyzed by each enzyme and the relative rates of hydrolysis for each antipode are used as diagnostic qualitative determinants of potential chiral selectivity. In the second method, those molecules not commercially available as single stereoisomers are hydrolyzed as racemates using kinetic resolution methods (running the reaction generally less than 50% completion). The products of the reaction are isolated and analyzed for their enantiomeric excess (ee) by chiral phase HPLC (Diacel Chiraicel OD or OB) or ^lH NMR of the corresponding diasteriomers prepared by derivatizing products to

Mosher derivatives (alcohol products) or menthyl derivatives (carboxylate products).

Diastereomeric ratios determined from the NMR spectra are based on corresponding peak integrations and compared to either literature values or standards obtained from commercial sources using of chiral shift reagents when necessary. Optical rotations and absolute configurations of die products are then determined by polarimetric analysis and compared to values found in the literature or determined from standards obtained from commercial suppliers.

Example 14. Characterization of Proteins E001 - E021/17b

Strains from the identified sources as listed in Table 1 were isolated by growth in TT media at 65°C as described in Example 1 (ie. SI from soil, etc.). Specific esterase hydrolytic activity was identified by the methods described in Example 2 and the isolated esterase protein assigned the identifier as listed in Table 1 (ie. E001 etc.) To prepare enzyme, a 15 liter culture of isolate is grown and the cells are spun down and collected as described in Example 1. The cells are lysed and a isolated preparation of was purified according to the procedures outlined in Example 4. The protein was characterized using the methods described in Example 5 to determine the temperature profile. Example 6 to determine protein stability, and Example 7 to determine the pH profile, and the results are shown in Figure 4. The protein was characterized by migration on Native gradient PAGE as described in Example 9 and the data is shown in Figure 2. The specific activity was determined as described in Example 2 and the molecular weight was determined by chromatography as described in Example 10 and are presented in Table 1. Substrate specificity for several proteins has been demonstrated and are shown in Table 2. Thus the identified and characterized esterases have been demonstrated to be useful, and to posesses unique activity at commercially useful purity. Certain results are summarized in Table 10.

Example 15. Characterization of ElOO

Purification of ElOO - ElOO is purified from Thermus sp. T351 over 300 fold by a series of four steps described in Example 3: DEAE purification, Q Resin purification, Ultrafiltration concentration, and preparative SDS PAGE. The specific activity could not be measured in the crude lysate since there was a secondary esterase activity present (E101). The secondary activity could be completely removed from the target esterase during the first chromatographic step in which the secondary esterase passed through the DEAE column unbound. For purification of various technical grades of ElOO, DEAE purification alone is sufficient to yield ElOO enzyme substantially purified away from any other contaminating activity. Q Resin purification and ultrafiltration allow for higher purity product to be produced as required by specific applications. A final SDS PAGE purification step allows the protein to be purified to homogeneity for determination of molecular characteristics.

Protein Characterization - The active band is collected by electroelution on a preparative

SDS-PAGE gel and rerun on 10% SDS-PAGE under denaturing conditions. This shows a single band with a relative molecular mass of about -45 Kd. Unboiled samples run on the same SDS-PAGE gels show multiple bands in approximate increments of the proposed monomeric molecular mass. Additionally, the nonboiled sample can be stained for activity, however only bands corresponding to multimeric forms of the enzyme are found to retain activity beginning with dimeric species. The specific activity of the purified protein is approximately S^xlO^Mmin-'mg-' using 4-methyl-umbelliferyl-butyra:e (MUB) as the substrate. Measurement of EI00 Enzyme Activity - Esterase activity is measured by monitoring the hydrolysis of p-nitrophenylproprionate (pNP), or in some cases MUB. Each substrate is dissolved in acetonitrile and added to the reaction mixture (100 μM final concentration) which contain 50 mM Tris HCl pH 8.5 adjusted for temperature dependent pH variation. Reactions are thermally equilibrated at 37°C for 5 minutes prior to initiation of the reaction by addition of 10 μL of enzyme sample, while control reactions substituted equivalent amounts of BSA. The reaction is monitored spectrophotometrically at 405 nm ε=17mM^~1cnr ¹ for pNP and 360 nm ε=7.9 mM-'cπr¹ for MUB.

The rates of enzyme catalyzed hydrolysis are corrected for the spontaneous hydrolysis of the substrate. Protein concentrations are determined by either the absorbance at 280 nm or by Lowery assay. Crude activity is determined by a colorimetric assay based on the hydrolysis of 5-bromo-4-chloro-3-indoyl esters suspended in a 0.7% agar matrix on microtiter plates. A 0.1 mg/ml solution of the indolyl derivative is dissolved in a minimal volume of acetonitrile and added to a warm solution of 0.7% agar containing 0.1M phosphate buffer pH 7.5. 10 μL of this solution is distributed to microtiter plates which, when cooled, could be used with as much as 100 μL of enzyme sample and incubated at temperatures from ambient to >65°C.

ElOO was effectively inhibited when exposed to tosyl fluoride but was unaffected by the presence of either metal ions, chelating agents or reducing molecules Table 3. TABLE 3. Inhibition by reaction components on the hydrolysis of p- nitrophenylprorionate by ElOO.

Additive (concentration) Relative Rate^{3 (%)}

None 100

PMSF (O. lmM) 0

BME (lO mM) 99

DTT(lmM) 101

CaCl₂(10 mM) 108

MgCl₂(10 mM) 95

ZnCl₂(10 mM) 90

EDTA(lmM) 96

Reaction conditions are those described in the general expeπmental above except for the addition of specified components. Relative rates are corrected for the spontaneous rate of hydrolysis of the uncatalyzed reaction.

Substrate specificity of ElOO - The substrate specificity was tested as outlined as according to Example 11, and die results from the structure activity experiments for ElOO are shown in summary Table 4. ElOO displays a broad substrate specificity catalyzing the hydrolysis of a number of nitrophenyl, coumaryl and alkyl esters. The enzyme displays hydrolytic activity towards both straight chain and aromatic moieties on the carboxylate side of substrates however, carboxylate R groups of long alkyl chains >C8 or tiiose containing naphthyl leaving groups are not substrates. The enzyme displays no significant activity towards either casein or milk as assayed by clearing zones on agar plates.

TABLE 4. Substrate Activity of ElOO

Substrate ElOO Control

I-acetate^a ++

I-butyrate^a ++

I-caprylate^a +

N-acetate^a -

U-acetate^a ++ +/-

U-stearate^a — pN-acetate^a ++ pN-proprionate^a ++ oN-proprionate^a ++ oN-caprylate^a + oN-palmitate^a +- oN-stearate^a -

Me-PA^b +

Et-PA^b + isoProp-PA^b +

Structure activity assay of partially purified esterase ElOO from Thermus species. (++) highest activity as determined by (a) color formation in less then 10 min DΓ significant product formation on (b)TLC. The remaining activity measurements follow the order: + > +/- > - > - -. Structure abbreviations are as follows: I, chloro-bromo-indoyl, N, a-napthyl, U, methylumbelliferyl, pN, p-nitrophenyl, oN, o-nitrophenyl, PA, phenylacetate. Determination of Kinetic Characteristics - Kinetic characteristics are determined by measunng the concentration dependent initial rates of enzyme catalyzed hydrolysis ot nitrophenyl propπonate. Reactions are run at pH 8.5 in 50 mM Tns-HCl buffer equilibrated to 37°C and initiated by addition of enzyme. Rates are determined from the absorbance changes due to formation of product nitrophenol at 405 nm Rates are corrected for the spontaneous hydrolysis of substrate dunng die course of the reacuon. Concentration vs. rate data are analyzed by both double reciprocal plots and by HanesWolff plots to determine Km, Vmax and Vmax/Km The kineuc characteπstics of ElOO determined from plots of the initial rates of hydrolytic reacuons are shown in Figure 6.

Determination of Temperature Profile and Optimal p for ElOO - The temperature profile of the enzyme is determined as shown in Figure 7a. Enzyme activity is observed to steadily increase to the limit of the assay, over 70°C, (where the background signal from autohydrolysis of the substrate became too high and is no longer correctable) as the temperature of me reacuon is elevated and suggests tiiat the low end for opumal acuvity for ElOO is greater than 70°C. ElOO displays a basic pH profile witii a low end opumal acuvity observed to be approximately 9.0, the limit of substrate stability at 37°C (Figure 7b). Determination of Enzyme Stability in the Presence of Organic Solvents - ElOO is tested for tolerance to organic solvent composition using the polar aprotic cosolvent acetomtπle as a preliminary system, the enzyme retained 50% of its activity in a solvent mixture of 20 vol % organic cosolvent (Figure 8).

N-Termιnal Sequencing of ElOO - Punfied proteins are run on 10% SDS-PAGE gels and then transferred to PVDF membranes by electroblotung Membranes are washed witii seveial changes of doubly disulled water to remove any remaining SDS or other contaminants and tiien stained with coomassie blue. Membranes were then destained with several changes of 50:40:10 MeOH:H 0:AcOH followed by one wash of 10% MeOH. Membranes are then air dried and then submitted for sequencing. The N-terminal sequence of ElOO was determined at the University of Illinois Urbana Champaign genetic engineenng facility.

The N-terminus of ElOO was determined by automated sequencing of the polypeptide punfied by 10% SDS-PAGE and transferred to a PVDF support. The sequence obtained was. MKLLEWLK7EV, where the letters refer to the standard amino acid single letter code and me "?" refers to an indeterminate amino acid. Thus, ElOO has been demonstrated to be a useful esterase with unique activity at commercially useful punty.

Example 16. Characterization of E101

E101 is one of two esterase activities that are isolated from Thermus sp T351. E101 can be punfied away from a second esterase, ElOO, in an early punfication step. Purification ofElOl - A Thermus sp. T351 supernatant prepared as descnbed in Examples 1 and 2 is fractionated with NH₄SO₄ and the piecipnated proteins are collected between 20- 60% saturation. Pellets are redissolved in 30 ml of buffer (50 mM Tns-HCl pH 8.0. 1 mM BME) and dialyzed against the same buffer using 30 Kd cutoff dialysis tubing. Dialysate is loaded to 100 ml bed volume of DEAE res equilibrated with the buffer above and the column was washed with 150 ml of the equilibration buffer. Active protein is observed in the load and wash fractions, pooled, and concentrated with the use of an Amicon concentratoi fitted with a YM30 membrane. Concentrated proteins are then loaded directly to a 25 ml bed volume of sepharose SP resin equilibrated with the above buffer Active fractions appear in the load and wash fractions which are pooled and concentrated as above. Concentrate is then loaded to a Sephracryl HR200 gel filtration column ( 1x40 cm) and 0.5 ml fractions aie collected at a flow rate of 2 ml/hr Active fractions are collected and analyzed by SDS- PAGE. In order to perform N-terminal sequencing, fractions considered to be homogeneous are concentrated and submitted to a protein sequencing service center The enzyme is stored at 4°C for future use.

E101 can be purified over 35 fold by these methods and possesses characteristics dramaucally different from ElOO, the other esterase which is isolated from this suain Attempts to use ion exchange chromatography result in subtractive punfication since in no instance was the protein retained Resins investigated include DEAE, Q sepharose, CM cellulose, SP sepharose and hydroxyappatite under conditions that vaπed from pH 6 0 to 9.0, and buffers from phosphate to borate including Tns and Hepes After two ion exchange steps the protein is punfied to homogeneity by gel filtration chromatography however, the protein appears to have an interaction with the column as retention is considerably longer than the molecular weight would suggest The molecular weight of the protein appears to be approximately 135 Kd with a monomer mass of -35 Kd as determined from native and denatunng SDS-PAGE respectively

El 01 Characteristics - The specific activity of the enzyme is ten fold greater than observed for ElOO with 4-methyl-umbelhferyl butyiate (MUB) as the substrate. E101 is inhibited by PMSF but is insensitive to metal ions or metal ion chelators. The specific activity ot the punfied protein was found to be 3.2x10 ^s mol min 'mg^-1 and was determined from initial rates of hydrolysis using methyl umbelliferyl butyrate as a substrate Table 5 outlines the inhibitory effect of vanous substances on E101 activity TABLE 5. The inhibitory effect of reaction components on the hydrolysis of p-nitrophenylprorionate by ElOl.

Additive (concentration) Relative Rate³

None 100 %

PMSF (O.lmM) 0

BME (lO mM) 96

DTT(lmM) 98

CaCl₂(10 mM) 102

MgCl₂(10 mM) 97

ZnCl₂(10 mM) 100

EDTA(lmM) 93

Reaction conditions are those descnbed in the general expenmental above except for the addition of specified components. Relative rates are conected for the spontaneous rate of hydrolysis of the uncatalyzed reaction.

Substrate specificity of El 01 - The substrate specificity of E 101 was determined as descnbed in Example 11. The results from the structure activity expenments for ElOl are shown in Table 6. The hydrolytic activity of the enzyme is similar to that observed for ElOO and has no observable protease activity toward milk or casein.

TABLE 6. Substrate Activity of ElOl

Substrate ElOl Control

I-acetate^a ++

I-butyrate^a ++

I-caprylate^a +

N-acetate^a -

U-acetate^a ++ +/-

U-stearate^a +/- pN-acetate^a + pN-propnonate^a + oN-proprionate^a ++ oN-caprylate^a +/- oN-palmitate^a +/- oN-stearate^a -

Me-PAb ++

Et-PA^b +-. isoProp-PA^b +

St cture activity assay of partially punfied esterase ElOl from Thermus species (++) highest activity as determined by (a) color formation in less then 10 min or significant product formation on (b)TLC. The remaining activity measurements follow the order: + > +/- > - > - -. Structure abbreviations are as follows: I, chloro-bromo-indoyl, N, a-napthyl, U, methylunmbelliferyl, pN, p-nitrophenyl, oN, o-nitrophenyl, PA, phenylacetate.

Thus, ElOl has been demonstrated to be a useful esterase with unique activity at commercially useful purity. Example 17. Cloning of Esterase.

General Cloning Strategy - The λ ZAP cloning system from Stratagene™ can be used for the library constructions and detection of esterase activity. Other cloning systems can also be used to yield similar results. The usual efficiency of cloning in λ vectors vary from 10⁵ to 10⁷ hybrid clones per mg of cloned DNA and is sufficient to produce a representative gene library from a convenient amount of size-selected chromosomal DNA fragments. We have found that detection of esterase activity in phage plaques, as opposed to bacterial colonies, is more efficient due to the easier access of substrate to the enzyme. Phages are generally less sensitive to the toxic action of cloned proteins and are also able to survive at the temperatures up to 70°C. The ability of the cloning system to tolerate elevated temperatures and potential toxicity of me cloned proteins is necessary for the detection of the activity of thermophilic proteins, such as the esterases described here.

Isolation of DNA for Construction of gene banks - Genomic DNA is prepared from a culture of the appropriate strain containing the esterase of interest as described in Example 1. Cells of different strains are grown to late log phase in 100 ml TT broth (8 g Polypeptone (BBL 11910), 4 g yeast extract, 2 g NaCl, per liter) at 55°C or 65°C overnight shaking at 250 RPM. Cells are recovered by centrifugation and the pellet is resuspended in 5 ml of lysis buffer (10 mM Tris-HCL, pH 7.0, 1 mM EDTA, and 10 mM NaCl). Lysozyme is added to a final concentration of 2 mg/ml. Cells are incubated at 37°C for 15 minutes followed by the addition of SDS to 1 %. The lysate is gently extracted three times with phenoL/chloroforrn/iso-amyl alcohol (25/24/1) and the DNA spooled from a 95% ethanol overlay of the aqueous phase.

One of ordinary skill would find other methods for preparation of DNA which are well known in the art (37). For example, fresh colonies of a strain containing the esterase of interest are inoculated in 50 ml of TT media in 250 ml Erlenmeyer flask and incubated at 55°C for 24 hours at 200 rpm in a New Brunswick Environmental Shaker. The cells are harvested by centrifugation at 3000 g for 15 min., resuspended in 5 ml of GTE buffer (50 mM Glucose, 25 mM Tris-HCl pH 8, 10 mM EDTA) and treated with 2 mg/ml of lysozyme at 37°C for 10 min. Lysozyme- generated spheroplasts are lysed by the addition of 1% SDS and partially deproteinased by addition of 100 μg ml of proteinase K at 24°C for 10 min. Chromosomal DNA is further purified by three phenol/chloroform extractions, precipitated witii 2.5 volumes of ethanol and resuspended in 1 ml of TE (10 mM Tris pH 8.0; 1 mM EDTA), after washing in 20 ml of 75% ethanol. The extracted fraction consists of DNA fragments larger than 50 kb, with a concentration of about 0.5 ng μl, as detected by gel electrophoresis using a 0.7% agarose gel run at 10 V/cm for 4 hours.

Construction of Gene Libraries - Genomic DNA is partially digested with the restriction enzyme Sau3A and then ligated to predigested Lambda ZAP Express (Stratagene Cloning Systems) Products of ligauon reactions are packed in vitro using λ packaging extracts which are purchased from Promega. This vector accommodates DNA up to 12 kb in length and allows identification of clones botii by expression off the T3 and T7 promoters and by probe hybndization to plaques. The library is retained and screened for esterase activity Othei methods for generating genomic DNA hbranes are also well known in me art.

Five samples of 10 μg of chromosomal DNA of each ot the strains prepared as descnbed above, are treated witii different concentrations of Sau3A restπcuon endonuclease (New England BioLabs) according to the manufacturer's instructions for 30 mm at 37°C in a volume of 50 μl each. The concentration of Sau3A is vaned from 0 1 u to 0.002 u/ μg of the digested DNA in separate tubes. The reactions are stopped by heat inactivauon of the endonuclease at 70°C for 10 minutes and analyzed by gel electrophoresis on a 0.7% agarose gel run at 10 V/cm for 4 hours (a typical digestion pattern is obtained, data not shown) Fractions with an average fragment size of 5 kb are chosen for cloning. For native strains containing E001, E002, E003, E006. EO07, E008, E009, E010, E012, E016, E020 these are the second of the five samples of digested chromosomal DNA with the concentration of Sau3A of about 0.02 u/μg of the DNA. For the rest of the strains, the proper degree of partial digestion is achieved in e first test tube with 0.1 u of Sau3 A /μg of the DNA Fifty ng of chromosomal DNA fragments are hgated with equimolar amounts of dephosphonlatyed BamHI-arms of the lambda ZAP phage vector (Stratagene) in 5 μl with 1 unit of ligase (New England Biolabs). Ligauon reactions are performed at 18°C for 8 hours and stopped by heat inactivauon at 70°C for 10 mm. One μl of the ligauon reaction, containing approximately 10 ng of DNA insert, is used for in vitro packaging with 10 μl of lambda proheads (produced by Promega Corp) The packaging reaction is performed at 28°C for 90 min, combined with 100 μl of an overnight culture of E. coli XL1 Blue and plated using 2 ml of 0.7% top agar (0 8%NaCl, 10 mM MgSO4) per plate onto five 90-mm Petn plates containing LB media. Seπal dilutions of die packaging mixture are produced in order to determine the cloning efficiency which is generally about 1.0 x 10⁷ hybrid phages/μg of cloned DNA. Cloning efficiencies for each individual strain vaned, the size of the library generated fell within a range of 0.5 to 2.5 x 10^ from which two to twelve positive clones were analyzed (data not shown) Hybπd phages from one plate are harvested to collect the amplified library, which is stored in 3 ml of LB media with 25% glycerol The four other pπmary plates are treated with indicator agar containing 5-bromo-4-chloro-3- indolyl-acetate (X- Acetate) as descnbed below, to find hybnd plaques carrying esteiase genes Screening of gene banks for esterase activity - The products of the above packaging reacuons are infected into E coli XL1 blue MRF' (Stratagene) Pnmary plaques of an unamplified gene library are screened for enzyme activity by overlaying the plates with top agar containing X-Acetate for 30 minutes at 65°C The concentration of substrate in the indicator overlay is diluted from a 4% stock in ethanol or N,N-dιmethyl formamide to a concentrauon generally between 0.1 and 1% (usually about 0.4% is used) in ihe final solution. O_ther suitable substrates may be substituted in this procedure including, but not limited to, 5- bromo-4-chloro-3-indolyl-butyrate (X-butyrate), 5-bromo-4-chloro-3-indolyl-proprionate

(X-proprionate), 5-bromo-4-chloro-3-indolyl-stearate (X-stearate), 4-methylumbelliferyl- acetate (MUA), 4-methylumbelliferyl-butyrate (MUB), 4-methylumbelliferyl-proprionate

(MUP), or other 5-bromo-4-chloro-3-indolyl- or 4-methylumbelliferyl- esters which may be either synthesized or purchased from a commercial vendor such as Sigma Chemical. In order to inactivate background endogenous esterase activity from E. coli, the plates are preheated at 65^°C for 20 minutes. Hybrid phages surviving mis procedure are picked and re-screened three times. The extracts are then analyzed for the presence of a protein band with the same mobility as the native protein as described below. The lambda ZAP cloning system permits an excision of smaller plasmid vector to simplify the insert characterization. While other methods may be employed for screening gene banks for esterase activity, i.e. isolation, purification, and N-terminal sequencing of protein; creation of degenerate nucleotide probes from N-terminal sequence; screening of gene bank with degenerate probes, the instant method is efficient and uniquely suited for the purpose of isolation of promising clones.

In particular, the four primary plates with phage colonies generated during the cloning described above, are incubated at 65°C for 30 min. in order to inactivate some of the potential E. coli esterase activities. Approximately two ml of 0.7% top agar (0.8% NaCl, 10 mM MgSO₄) containing about 1 mg/ml of the colorimetric esterase substrate X-Acetate or other substrate (including but not limited to X-butyrate, X-proprionate, X-stearate, and 4- methyl-umbelliferyl based substrates) is overlaid onto each plate. Expression of cloned esterases can be detected by blue halos around phage colonies (or fluorescent halos in the case of die 4-methylumbelliferyl substates). A typical result for this process can yield a ratio of 1: 3000 positive colonies to hybrid phages.

Between two and twelve primary positive phage plaques are generally picked up from each set of plates, resuspended in 50 μl of LB medium, and s eaked onto a lawn of E. coli XL1 Blue using sterile paper strips. These purified phage plaques are then overlaid by indicator agar containing X-Acetate as before, and positive plaques were selected as in primary screening experiment. Three rounds of such purification by restreaking are generally sufficient to produce a pure hybrid phage clone expressing esterase activity. All these clone candidates demonstrate significant esterase activity in the X-Acetate plate assay. Several clone candidates from each strain are chosen for further analysis, each representing the progeny of single primary phage plaque. Testing Protein Profiles Produced by Phage Clones - Production and analysis of protein from the phage clones is performed as follows, but alternative methods are possible: A single plaque from each clone is resuspended in 20 μl of an overnight culture of E. coli XL1 Blue (grown in LB medium with the presence of 10 mM of MgSO₄), incubated for 20 min at 24°C in one well of a 96-well microtiter plate to allow adsorption, transferred into 15-ml test tube containing 2 ml of LB, and grown overnight at 37°C in a New Brunswick Environmental Shaking incubator set at approximately 300 rpm. Cell debris can be removed by centrifugation at 12,000 g for 10 min. Phage lysates from the clones are then subjected to 4- 15% gradient Native polyacrylamide gel electrophoresis (PAGE) for comparison to the native proteins purified from the original organisms. Precast gradient gels are purchased from BioRad Laboratories (catalog number 161-0902) and used according to the manufacturer's instructions for native gels . An esterase preparation from the original strain, purified by HPLC to a single protein band is used as a control on the same gel. Alternatively, a native protein preparation which has not been purified to homogeneity but is purified to a single esterase activity can be used as a control. Protein bands possessing an esterase activity can be detected by applying an X-Acetate overlay and incubating at room temperature for 5-20 min. The relative mobility of the clone candidates can be compared to the native esterase protein.

The data generated for 107 hybrid phage clone candidates from 20 strains are summarized in Table 7, which shows the results of the typical comparison of the esterase activities detected in lambda clones compared to the host strain. For each gene library screened, there is at least one clone candidate expressing an esterase protein with the mobility of the protein purified from the original strain. Several of the λ clone candidates express esterase activities which have mobilities that are different from die major component of the esterase specimens purified from the original strains. Similar sized bands possessing esterase activity are observed in die native organism as minor components (data not shown). These cloned ester hydrolyzing activities are given names depicted in Table 7. Excision of the Plasmid Vector from the Phage - The lambda ZAP vector allows the phage clone to be conveniently converted into a plasmid vector to allow better physical characterization of the DNA insert and regulated expression of cloned genes. Induction of M13-specific replication by co-infection with the heiper phage results in excision of a multicopy plasmid carrying the cloned insert. 10 μl phage stocks of the lambda hybrids (with about 10⁷ Colony Forming Units (CFU)) and 1 μl of Exassist M13 helper phage (about 10¹⁰ CFU) are used to infect 20 μl of an overnight culture of die E. coli XL1 Blue grown in LB. After 20 min at 24°C, the cell suspension is transfeπed from one of the wells of a 96-well microtiter plate into a 15-ml culture tube, diluted with 2 ml of LB, grown overnight at 37°C and 300 rpm, heated at 65°C for 10 min, and cleared by centrifugation at 3000 g for 20 min. Excised plasmids packed in M13 particles are transduced into a lambda resistant strain, XLOLR, that does not permit the development of the Ml 3 helper phage. Ten μl of excised phage lysate are mixed with 30 μl of the overnight culture of the E. coli XLOLR strain in one well of 96-well microtiter plate, incubated for 20 min at 37°C to allow adsorption, diluted with 100 μl of LB, and incubated at 37°C for 40 min to express the kanamycin (Km) resistance marker (neo) of the plasmid. Cells are plated onto two LB plates supplemented with 40 mg/ml Km. One of the plates also contains 50μl of a 4% X-Acetate stock solution. Preliminary experiments are performed by growing plates at 37°C to demonstrate that a significant phenotypic segregation occurs with the transductant E. coli colonies expressing cloned thermophihc esterases. In an extreme case of the CΕ020 strain, very few colonies not expressing any esterase activity could be re-streaked from primary transductant colonies, which actively expressed esterase activity. Because of this segregation and apparent instability of plasmids containing die active clones, protocols for manipulation of most of the esterase clones needed to be modified as compared with the standard protocol of plasmid excision recommended by Stratagene. It was possible that the instability was due to the function of the cloned protein expressed in the cell, thus it was hypothesized that lowering the growth temperature might overcome the segregation problem, since the esterases were from tiiermophilic organisms and may not be as active at the lower temperatures.

Therefore, to overcome the problem of instability due to the activity of the esterase containing plasmids, cultivation of E. coli cells harboring thermophilic esterases is performed at 28°C and 30°C, with the result tiiat the effective phenotypic segregation is reduced. Thus, in the event that a cloned thermophilic esterase activity is lethal or partially lethal to the host cell, the growth temperature of the strain should be lowered to 30°C or even room temperature. The recombinant strains harboring plasmids with active esterase proteins often exhibited a phenotypic segregation of the esterase activity on X-acetate plates. This segregation could be due to plasmid or insert loss if the esterase activity had toxic properties to the cell. To overcome this cells could be grown at lower temperatures (presumably reducing the activity of the cloned thermophilic esterases). Thus strains can be plated with X-Acetate at 28°C and 37°C. Yellow colonies of faster growing segregants are visible at both temperatures, but contra-selection at 37°C is much stronger. After determining that temperature makes a large difference in stability of the clone phenotype, further experiments are carried out by plating all plasmid based clones at 26°C, generally for 48 hours. E. coli cells are plated in a medium containing X-Acetate to detect expression of cloned esterase by the plasmid, and a degree of segregation in or between primary colonies. Thus, growth of the transformed cells at a temperature which reduces the activity of the cloned esterase is important to the effective isolation of productive plasmids. In the specific case, eight bacterial colonies derived from each of the phage clones are picked from the plates without X-Acetate, transfeπed into 100 ml of LB supplemented with 40 mg/ml Km in a 96-well plate and grown overnight. Progeny of these colonies are analyzed by a spot-test using X-Acetate containing agar. Several plasmid clones derived from each phage are chosen for further study by picking ones producing brightest blue halos and least amount of the esterase" segregants.

Selection for the Stable Plasmid Variants - Since it is determined that the plasmid-based vectors carrying esterase genes are often unstable, stable variants of the plasmids are isolated. One method for such isolation is as follows. E. coli cells carrying excised plasmids are purified using LB plates supplemented with Km and a limited amount of X-Acetate to reduce any potential negative growth impacts from production ot the somewhat lethal indole product ot the colonmetπc reaction. Colonies aie selected by their phenotype (in general giving a modest growth rate and intensive blue color) and grown in 2 ml of LB with Km in 15 ml test tube tor 48 hours to reach ODgoo of about 1.0 and harvested by centnfugauon at 12,000 g tor 1 mm. Cell pellets are resuspended in 500 ml of 0.1 M Phosphate buffer pH 7.0 and sonicated using a Somes & Mateπals Vibra Cell 375 Watt sonicator at 4°C. Somcation is performed using a microtip, 40% max capacity, 50% time pulse for 45 sec. Lysates are centπfuged at 12,000 g for 5 min and tested for its relative esterase activity Variants with the highest activity are selected for the next round of growth and analysis. Three rounds ot plating followed by growth in liquid medium and acuvity assays are performed to verify the stability of the clones.

Deviauons in specific esterase activity among vanants trom the same plasmid lineage can be reduced to a factor of three from over a factor of 100 by this procedure Stabilization of the activity generally occurs at the level corresponding to the highest activity values detected in the first round ot stabilization This could mdicate that E. coli host mutations are being selected which allow higher tolerance of the cloned protein, rathei than simply suppressed activity of cloned toxic gene.

Physical Characterization of Plasmid Clones - Plasmid DNA is extracted from E. coli cells using a standard alkali lysis procedure, or other procedures known in the art (37). The DNA is digested with a seπes of restnction endonucleases such as ΕcoRI. BamHI, Hindlll. PstI, ΕcoRV, and Xbal to establish digestion pattern of the clone and to determine a size of the cloned DNA fragment The physical map patterns for the pioduction clones weie determined The insert sizes tor each clone are calculated from this data and is summarized in Table 8.

Table 7. Cloned Esterase Candidates and Analysis

Recomb. Specific

Acuvity Esterases Denvauve Acuve Acuvity in

Nauve in phage Idenufied Primary Clone Plasmid Plasmid Stabilized clone

# Strain lysate? in Phage Name Name Derivative U/mg Lysate

1 SI + E001 lambdaTGE 1.1 pTGEl. l + 1536

2 SI + E001, E022 1ambdaTGE 1.2 pTGEl.2 +

3 SI + E001, E022 1ambdaTGE 1.3 pTGEl.3 +

4 S I + E001 lambdaTGE 1.4 pTGE1.4 +

5 S I + E001 lambdaTGE 1.5 pTGE1.5 + 1489

6 SI nt nt lambdaTGE 1.6 pTGEl.6 +

7 S I nt nt lambdaTGE 1.7 pTGE1.7 +

8 S I + E022 lambdaTGE 1.8 pTGEl.8 -

9 54 + E002 lambdaTGE 2.1 pTGE2.1 + 8300

10 54 + E023 lambdaTGE 2.2 pTGE2.2 nt 550

11 54 + E023 lambdaTGE 2.3 pTGE2.3 +

12 54 + E002 lambdaTGE 2.4 pTGE2.4 + 2530

13 54 + E002 lambdaTGE 2.8 pTGE2.8 -

14 50 E003 lambdaTGE 3.1 pTGE3.1 -

15 50 + EO03 ambdaTGE 3.2 pTGE3.2 + 2610

16 50 + E003 ambdaTGE 3.3 pTGE3.3 +

17 50 + E003 lambdaTGE 3.4 DTGE3.4 +

18 GP1 + E004 ambdaTGE 4.1 DTGE4.1 -

19 GP1 + E024 ambdaTGE 4.2 DTGE4.2 +

20 GP1 + E004 lambdaTGE 4.3 3TGE4.3 + 320

21 GP1 + E004 lambdaTGE 4.4 3TGE4.4 -

22 GP1 + E004 ambdaTGE 4.5 3TGE4.5 nt

23 GP1 + E004 ambdaTGE 4.6 DTGE4.6 + 490

24 C- l + E005 ambdaTGE 5.1 DTGE5.1 -

25 C-l + E025 [ambdaTGE 5.2 DTGE5.2 +

26 C-l + E005 ambdaTGE 5.3 3TGE5.3 + 984

27 C-l - ambdaTGE 5.4 DTGE5.4 nt

28 C-l + E0O5 ambdaTGE 5.5 DTGE5.5 nt

29 55 + E006 ambdaTGE 6.1 pTGEό. l -

30 55 +/- E026 lambdaTGE 6.2 pTGE6.2 -

31 55 + E006 lambdaTGE 6.3 pTGE6.3 + 230

32 55 + E006 lambdaTGE 6.4 pTGE6.4 -

33 55 + E006 lambdaTGE 6.5 pTGE6.5 -

34 55 + E006 ■ambdaTGE 6.6 pTGE6.6 -

35 46 +- . ** lambdaTGE 7.1 pTGE7.1 + 210

36 46 +- *** lambdaTGE 7.2 pTGE7.2 +

37 30 + E008 lambdaTGE 8.1 pTGEδ. l -

38 30 + E008 lambdaTGE 8.2 pTGE8.2 -

39 30 + E008 lambdaTGE 8.3 pTGE8.3 +

40 30 + E008 lambdaTGE 8 4 pTGE8.4 +

41 30 + E008 lambdaTGE 8.5 pTGE8.5 + 330 Table 7. Continued

Recombinant Speαfic

Acuvity Esterases Deπvauve Acuve Acuvity in

Nauve in phage Identified in Primary Clone Plasmid Plasmid Stabilized clone

# Strain lvsate⁹ Phaee Lvsate Name Name Derivative U/me

42 28 . lambdaTGE 9.1 pTGE9.1 +

43 28 - lambdaTGE 9.2 pTGE9.2 -

44 28 + E009 lambdaTGE 9.3 pTGE9.3 + 512

45 28 + E009 lambdaTGE 9.4 pTGE9.4 + >270

46 28 + E009 lambdaTGE 9.5 pTGE9.5 -

47 28 + E009 lambdaTGE 9.6 pTGE9.6 +

48 28 + E009 lambdaTGE 9.7 pTGE9.7 +

49 29 - lambdaTGE 10.1 pTGElO. l -

50 29 - lambdaTGE 10.2 pTGE10.2 -

51 29 + E010 lambdaTGE 10.3 pTGE10.3 + 546

52 29 - lambdaTGE 10.4 pTGE10.4 + >600

53 29 + E010 lambdaTGE 10.5 pTGE10.5 +

54 29 + E010 lambdaTGE 10.6 pTGE10.6 -

55 29 - lambdaTGE 10.7 pTGElO.7 -

56 29 + E010 lambdaTGE 10.8 pTGE10.8 +

57 31 - lambdaTGE 11.1 pTGEl l. l +

58 31 - lambdaTGE 11.2 pTGEll.2 -

59 31 + E011 lambdaTGE 11.4 pTGE11.4 +

60 31 + E011 lambdaTGE 11.9 pTGE11.9 +

61 31 + E011 lambdaTGE 11.10 pTGEl l.10 + 1052

62 31 - lambdaTGE 11.7 pTGEl l.7 +

63 26b + lambdaTGE 12.1 pTGE12.1 +

64 26b + lambdaTGE 12.2 pTGE12.2 + >600

65 26b + lambdaTGE 12.3 pTGE12.3 +

66 26b + lambdaTGE 12.4 pTGE12.4 +

67 26b + E029 lambdaTGE 12.5 pTGE12.5 -

68 26b + E029 lambdaTGE 12.6 pTGE12.6 -

69 27 + E013 lambdaTGE 13.1 pTGE13.1 +

70 27 + E013 lambdaTGE 13.2 pTGE13.2 + 428

71 27 + E013 lambdaTGE 13.3 pTGE13.3 + 33

72 27 + E013 lambdaTGE 13.4 pTGE13.4 +

73 34 - lambdaTGE 14.2 pTGE14.2 -

74 34 + E014 lambdaTGE 14.3 pTGE14.3 + 460

75 34 - lambdaTGE 14.4 pTGE14.4 -

76 34 + E014 lambdaTGE 14.5 pTGE14.5 + >1200

77 34 + E027 lambdaTGE 14.6 pTGE14.6 + >900

78 34 - lambdaTGE 14.7 pTGE14.7 +

79 34 + E014 lambdaTGE 14.8 pTGE14.8 -

80 34 + E014 lambdaTGE 14.9 pTGE14.9 +

81 62 + E015 lambdaTGE 15.1 pTGE15.1 +

82 62 + E015 lambdaTGE 15.2 pTGE15.2 +

83 62 + E015 lambdaTGE 15.3 pTGE15.3 +

84 62 + E015 lambdaTGE 15.4 pTGE15.4 +

85 62 + E015 lambdaTGE 15 5 pTGE15.5 +

86 62 + E015 lambdaTGE 15.6 pTGE15.6 + Table 7. Continued

Recombinant Specific

Acuvity Esterases Deπvative Acuve Acuvity in

Nauve in phage Idenufied in Primary Clone Plasmid Plasmid Stabilized clone

# Strain lvsate⁹ Phase Lvsate Name Name Deπvauve U/mε

87 62 + E015 lambdaTGE 15.7 pTGE15.7 +

89 62 + E015 lambdaTGE 15.9 pTGE15.9 + 4700

90 47 + E016 lambdaTGE 16.1 pTGElό. l + 600

91 47 + lambdaTGE 16.2 pTGE16.2 +

92 47 + E016 lambdaTGE 16.3 pTGE16.3 + >1200

93 47 + lambdaTGE 16.4 pTGE16.4 +

94 47 + E016 lambdaTGE 16.5 pTGE16.5 +

95 47 + lambdaTGE 16.6 pTGE16.6 +

96 47 + lambdaTGE 16.7 pTGE16.7 +

97 C-3 + lambdaTGE 18.1 pTGE18.1 + nt

98 C-3 + lambdaTGE 18.2 pTGE18.2 -

99 4 + E019 lambdaTGE 19.1 pTGE19.1 + >120

100 4 + E019 lambdaTGE 19.2 pTGE19.2 +

101 4 + E019 lambdaTGE 19.3 pTGE19.3 +

102 4 + E019 lambdaTGE 19.4 pTGE19.4 + 1960

103 4 + E019 lambdaTGE 19.5 pTGE19.5 -

104 4 + E019 lambdaTGE 19.6 pTGE19.6 +

105 7 - lambdaTGE 20.1 pTGE20.1 +

105 7 - lambdaTGE 20.2 pTGE20.2 +

106 7 + E020 lambdaTGE 20.3 pTGE20.3 + 2470

107 7 + E028 lambdaTGE 20.4 pTGE20.4 +

108 7 - lambdaTGE 20.5 pTGE20.5 +

109 7 + E020 lambdaTGE 20.6 pTGE20.6 +

1 10- 32 - lambdaTGE 21.1 - pTGE21.1- +

104 21.5 21.5

105 32 + E017b lambdaTGE 21.6 pTGE21.6 +

106 32 + E017b lambdaTGE 21.8 pTGE21.8 + 930

107 32 + E017b lambdaTGE 21.9 pTGE21.9 +

No protein detected by activity s cttaniinn.

Table 8. Production Clone Data

Producuon Selected Recombinant Approx. Specific Acuvity in Enzyme Production plasmid Strain Name DNA Insert Typical

Size ¹ Recombinant (kb) Crude Extract*- (U/mg) recEOOl pTGEl. l CE001 3.5 1,536 recE001.5 pTGE1.5 CE001.5 nt nt recE002 _PTGE2.1 CE002 2.5 8,300 recE003 pTGE3.2 CE003 4.1 2,610 recE004 pTGE4.6 CE0O4 3.4 490 recE005 pTGE5.3 CE005 1 9 984 recEOOό pTGE6.3 CE006 6 230 recE007 pTGE7.1 CE007 3.7 210 recE008 pTGE8.5 CE008 3.2 330 recE009 pTGE9.4 CE009 4.5 270 recEOlO pTGE10.3 CE010 2.5 546 recEOl l pTGEl l.10 CE011 2 4 1,052 recE029 pTGE12.2 CE029 4.2 600 recE013 pTGE13.2 CEO 13 2.2 428 recE014 pTGE 14 3 CE014 2 5 460 recE015 pTGE15.9 CEO 15 3 5 4,700 recEOlό pTGElό.l CEO 16 2 600 recE016.3 pTGE16.3 CE016.3 1.8 1,200 recE017b pTGE21.8 CE017b 3.8 930 rccE019 pTGE19.4 CE019 3.7 1,960 recE020 pTGE20.3 CE020 2.7 2,470 recE022 pTGEl .8 CE022 nt nt recE023 pTGE 2.2 CE023 3.7 550 recE024 pTGE4.2 CE024 ni nt recE025 pTGE5.2 CE025 nt nt recE027 pTGE14.6 CE027 2.6 900 recE028 DTGE204 CE028 2 5 nt

¹ Insert sizes are esumated from the agarose gel The estimated insert size is based on a vector size of 4.5 kb and the accuracy which could be achieved anaiyzing each of the six digesuon patterns.

²Specific acuvity is calculated as the amount of p-nitrophenol produced in micromoles per minute per milligram of total protein as descnbed in Example 2. The numbers reported here are trom a typical producuon batch and may vary Generation of the tag sequences for PCR identification of esterase containing inserts

The DNA sequences of die ends of the insert fragment carrying esterase genes can be determined by sequencing the ends of the inserts using standard T7 and S6 pπmers to produce unique tags of the cloned DNA. Sequence analysis can be earned out to design PCR pπmers which can uniquely amplify the DNA inserts from both the clones and the host organisms. These tags can be potentially used to generate this DNA fragment from the chromosome of the studied organisms using PCR technique.

Screening of the Cosmid library with an oligonucleotide probe - For cloning of enzymes which cannot be cloned by activity, other methods are used. A degenerative probe is prepared to the N-terminal sequence of the protein and hybridized to plaques from the recombinant phage bank. Alternatively, degenerate PCR amplification probes can be made using the N-terminal sequence or sequences obtained from the n-termini of internal protein fragments which have been obtained after proteolytic digestion of the enzyme. Using these sequences, a probe can be made from an amplified region between the N-terminus and an internal fragment or between two internal fragment sequences to identify a clone carrying the DNA encoding for die enzyme of interest.

Example 18. OVERPRODUCTION AND OVEREXPRESSION OF ESTERASES

Production of recombinant esterase - The production strains used are listed in Table 8. Cloned enzymes are produced from E. coli. strain XLOLR. Alternatively, any suitable E. coli host may be used, including but not limited to HB101, C600, TGI and XL 1 -Blue.

Several media can be used to produce cloned esterases. LB (10 gm/1 tryptone. 5gm/l yeast extract and lOgm/l NaCl) and Ternfic Broth ( 12gm/l tryptone. 24gm/l yeast extract and

4mL/l glycerol supplemented with 100 ml of a sterile solution of 0.17 M KH2PO4. 0.72 M K2HPO4 after autoclaving) have been tested and the results from optimal growth conditions for die production strains listed in Table 9 below. Each media is supplemented with 10-50 μg ml kanamycin.

Optimal production media depends on a number of factors, including media cost and specific activity of the produced proteins. TB media is a richer media and therefore mote expensive. For instance, in die case of CE009, while more total units are produced in a single fermentation run, not enough is produced to justify the higher cost of the media. In addition, the specific activity is higher for the LB media preparation.

Fermentation production is run in 17 L Fermentors (15 L working volume/LH

Fermentation) at 30°C, 600 RPM, and 0.5 vvm air flow. The seed train is established as follows. A loopful of a frozen production culture is used to inoculate 50 ml of production media in a 250 ml Erlenmeyer flask. The flask is incubated at 30°C for two days (250RPM) and then used to inoculate a 1 liter flask with 250 ml of production media. This flask is incubated 1 day at 30°C and 250 RPM. The 1 liter flask is used to inoculate the termentoi Production of substantially purified preparations from a cell paste of strains producing the recombinant enzymes are earned out similar to the methods descnbed in Example 4 and the specific protocols descnbed in Examples 14-34 for the native proteins.

Table 9. Preferred media for Strains CE001-CE010.

LB TB

Specific Total Specific Total Current

Acuvity Cell mass Total Acuvity Cell mass Total Growth media

Strain (U/mg) (g) Units ttJ/mg) (g) Units of choice*

CE001 213 0.41 4500 138 0.84 6725 TB

CE002 98 0.52 1625 101 0.93 4575 TB

CE0O3 272 0.42 4200 22 0.87 1025 LB

CE004 208 0.47 3650 28 0.90 1350 LB

CE00S 123 0.40 3675 125 1.00 7600 TB

CE0O6 85 0.42 2125 71 0.62 2175 LB

CE007 9 0.39 225 19 0.75 500 TB

CE008 71 0.51 2775 45 0.80 2350 LB

CE009 109 0.42 2650 74 0.81 3050 LB

CE010 418 0.42 2200 225 0.95 8375 TB

^♦Given current media costs

Optimization of esterase production - Further optimization of esterase production is performed by media studies in shake flasks followed by further optimization at the 1 liter to 20 liter scale. Depending on the enzyme, final fermentauon conditions can involve either a fed-batch or continuous fermentation process. Since the esterase activity being analyzed is intracellular, the use of a clear or defined media such as TT media is necessary. Organisms of interest are grown and cell pellets are collected by centrifugation. Pellets are disrupted by sonication and enzymes can be purified using the standard techniques of ion exchange and gel permeation chromatography described in Examples 3 and 4. Growth conditions including media composiuon, pH, and temperature are opύmized at the small scale (ie. shake flasks, and 1 liter fermentors) to give the highest cell density while retaining the highest amount of enzyme.

Isolation of High-production mutants - Several simple mutagenesis schemes are used to try and isolate high-producing mutants of the different activities of interest. These include mutagenesis with uv-light or chemical mutagens such as ethylmethane sulfanoate (EMS) ot N-methyl-N-mtro-ZV-nitrosoguanidine (MNNG) The cells are treated with varying concentrations of the mutagen (or varying exposure times with uv light) according to methods descnbed in Miller (38) Optimal concentrations of the different mutagens with different organisms vary. In general, killing concentrations allowing 80% survival for EMS, approximately 50% survival for MNNG, or 10-50% survival tor uv light are desired

Mutagenized cultures are then grown up, allowing the mutagen to wash out and plated onto solid media.

Mutants are identified by applying an esterase plate screen to the cells. For example witii an esterase screen, an agar overlay containing a coloπmetπc or fluorogenic substrate such as 5-bromo-4-chloro-3-ιndolyl-acetate or 4-methyulumbellιferyl acetate will be applied

Colonies which show a significant increase in activity by hydrolysis of the substrate will be identified.

Candidate mutants are then analyzed by native polyacrylamide gel electrophoresis and compared to the parental strain. Standard assay methods descnbed in Example 2 or the rapid esterase/lipase screen descnbed in Example 12 can then be applied to identify any differences in amounts of enzyme activity If a production level increase is large an increased band on either a Native or SDS polyacrylamide gel after coomassie staining may be seen Strains with multiple activities can also be differentiated in this way, veπfying that the increase is in d e enzyme of interest It is then confirmed that the mutants have unalteied kinetic and substrate properties as the parental enzyme. The majoπty of mutauons identified by dns approach are expression mutauons which can be isolated in eidier a promotei region, repressor molecule, or other controlling element. Most mutations m the enzyme structural genes will likely inacuvate the enzyme, however, an enhanced activity may also be isolated If it is apparent that the mutauon increases the acuvity of the desired protein band but not the intensity of the band on a coomassie stained gel, the mutant is lecharacteπzed to determine if it is a more efficient biocatalyst

Example 19. Esterase Screening Kit A large subset of enzymes can be packaged into an easy to use screening kit to rapidly analyze a large number of enzymes at once. The kits are formulated to eliminate as many potential errors as possible and each enzyme is provided in a lyophihzed form if possible near its optimal buffer and reaction conditions.

Many different formats for the kit aie possible, from a senes of glass vials, to varying size microtiter plates constructed of different plastic materials The microtiter plate is favored because of its ease of handling and manipulating. Most mictotiter plates are made ot polystyrene however, which will not stand up to most organic solvents. For expenments which utdize aqueous solvent, the polystyrene is not a problem Other more tolerant plastics such as polypropylene are available and are ideal for the kit. Large size 24-well microtiter plates which allow 3 ml of sample to be assayed (allowing enough sample for multiple TLC or HPLC analysis) have been developed Other formats may also be useful for different applications.

Each kit is prepared by addition ot a stu bai, buffer (0 IM Na phosphate pH 7 0) and 1U of each enzyme to each well of a 24 well polypropylene tray (Tomtec). Enzymes aie ahquotted into each well or vial in set amounts so that it can be assured that an equal amoun_t of activity is provided for companson The entire kit is then lyophihzed, sealed with heat seal foil (3M) and labeled. Separate expenments found that there was no significant loss in enzyme activity when proteins were lyophihzed in the kit trays as suggested by earlier experiments compaπng glass to plastic. In addition to enzymes, each kit contains toui control wells that are composed of buffers at pH's trom 6-9 since it was found that some ot the substrates tested tend to be unstable in buffered solutions which can confuse positive results with autohydrolysis. The rest of the kit is composed of an instruction manual, a data sheet, a sample preparation vial a glass eye dropper and a plastic eye dropper The kit is formulated in such a way that only solvent and substrate need be added to each well. The rapid-screen indicator dye mediod descnbed in Example 12 can also be included in each well or vial. This makes a preliminary qualitative determination of enzyme effectiveness simple and fast.

Example 20. Cloning and Characterization of Recombinant Proteins

The cloning and characteπzauon of recombinant proteins from strain isolates which produced the native isolated protein (as listed in Table 1) was earned out as descnbed in Example 37 Lambda expression vectors were isolated as described above (specific named isolates are shown in Table 7) E. coli clones harboπng the excised hybnd phage-plasmids were deπved as summanzed in Table 7, and were finally selected for esterase activity by subsequent screening, which after 3 rounds of stabilizing procedure was calculated to approximate units of activity per mg of total cell protein obtained. Esterase activity stain gel used to screen positive phage library candidates for the recombinant proteins allowed the identification of alternauve recombinant proteins as well. Production of the recombinant protein is earned out as descnbed above, using TB for the media and puπfying the enzyme as descnbed for d e native (nonrecombinant) protein in Example 4

Example 21. Sequencing of Recombinant Proteins

The isolauon and cloning of the genes encoding for the enzymes ot the instant invenuon results in DNA segments in which an open reading frame (ORF) may be found which corresponds to translated protein amino acid sequence Sequencing of the DNA inserts which contain the corresponding nucleic acid sequence which encode for the protein enzymes can be conducted by the usual methods, either manually or using automated apparatus. Once obtained, analysis of the nucleic acid sequence can reveal the presence ot alternative start codons, a phenomenon lecogmzed in the art, however the encoded protein enzyme will compπse at minimum a coie protein ORF. Figure 6A is an isolated nucleic acid sequence, and translated amino acid sequence which correspond to E001 enzyme ORF, alternative start codons are underlined. Figure 6B is an isolated nucleic acid sequence, and translated amino acid sequence which correspond to E009 enzyme ORF, alternative start codons are underlined. Figure 6C is the cloned isolated nucleic acid sequence which contams the E011 ORF, alternative start codons are underlined. Figure 6D is the cloned isolated nucleic acid sequence which contains the ElOl ORF, alternative start codons are underlined. Figure 6E is the cloned isolated nucleic acid sequence which contains the E019 ORF, alternative start codons are underlined. Figure 6F is the cloned isolated nucleic acid sequence which contams the E005 ORF, alternative start codons are underlined Figure 6G is the cloned isolated nucleic acid sequence which contains the E004 ORF, alternative start codons are underlined Figure 6H is the cloned isolated nucleic acid sequence which contams the E006 ORF, alternative start codons are underlined Figure 61 is the cloned isolated nucleic acid sequence which contains the E008 ORF, alternative start codons are underlined Figure 6J is the cloned isolated nucleic acid sequence which contains the E010 ORF, alternative start codons are underlined. Figure 6K is the cloned isolated nucleic acid sequence which contains the E013 ORF, alternative start codons are underlined. Figure 6L is the cloned isolated nucleic acid sequence which contains the E015 ORF, alternative start codons are underlined. Figure 6M is the cloned isolated nucleic acid sequence which contains the E016 ORF, alternative start codons are underlined. Figure 6N is the cloned isolated nucleic acid sequence which contams the E017 ORF, alternative start codons are underlined Figure 60 is the cloned isolated nucleic acid sequence which contains die E020 ORF, alternative start codons are underlined. Figure 6P is the cloned isolated nucleic acid sequence which contains the E027 ORF, alternative start codons are underlined. Figure 6Q contams the nucleic acid sequence of the 5' end, and Figure 6R contains the 3' end of the insert which contams the E003 Figure 6S contains the nucleic acid sequence of the 5' end, and Figure 6T contains me 3' end of the insert which contains the E004 ORF Figure 6U contams the nucleic acid sequence of the 3' end of the insert which contains the E014 ORF. These nucleic acid sequences allow one of ordinary skill in the art, practicing routine methods to complete charactenzation of the full length nucleic acid sequence of the insert, the detection of clones via hybndization, and the creation of amplification pnmers for detecting, amplifying and generating full length homologous genes Table 10. ThermoCat™ E001-E020 Spec comparison

Specific Tempβralurβ PH | Hall ϋfe (hours)

BiocaiaJyst Activity MW Opt. lUsβfui Range Opt. | 50% Range 40^βC 60°C

E001 0.5 u/mg 22 ItDai 45'C RT-55'C 7 5 broad . ++ 34

E002 1.0 u/mg 28 kOai 45*C RT-60*C 7 0 broad ♦ ♦ . 30

E003 0.5 u mg 28 kOai 45'C HT-60'C 7 0 broad ♦ + ♦ 60

E004 0.6 u/mg 3β Oai 45*C RT-60 C 6.5 <6.0-8.0 ♦ ++ 1 0

E005 6.7 u/mα 28 kOai 45*C RT-60^»C 7 0 broad ♦ + + 1 5

E006 3.6 u mg 36 kOa_ 45^ΦC RT-60^»C 6.5- 7.0 broad ♦ +♦ 30

E007 2.7 u/mg 28 kOai 35*C RT-60^#C 7 0 <6.0-8.0 >480 90

E008 1.5 u mg 28 tcDat 40*C RT-55*C 6.5- 7.0 <6.0-8.0 5 0 < 1

E009 1.3 u mg 36 kOai 45»C RT-50'C 6.5- 7.0 <6.0-8.0 ♦ ♦ + < 1

E010 4 9 u mg 46 kOai 45*C RT-55'C 6 5 <6.0-8.0 ♦ ♦ . < 1

E01 1 6.2 u/mg 36 kOai 45*C RT-60'C 6.5 - 7.0 <β.0-8.0 . . ♦ 4

E012 10.7 u/mg 28 kOat 45"C RT-60*C <-6.0 <6.0- 7.5 . + + 240

E0t 3 5.3 u/mg 36 kOai 45'C RT-60*C 7 0 <6.0-8.0 >480 6

E014 0.9 u/mg 36 kOat 45'C RT-50*C 7 0 <6.0-8.0 . + ♦ < 1

E015 3.0 u/mα 36 kOai 45*C RT-60'C >9.0 7 5 >9.0 ♦ ♦ + 6

EOlβ 1.2 u mg 28 kOai 45*C RT-60 C nd nd ♦ + ♦ 240

E017b 0.4 u/mg 36 kDal 40*C RT-50*C >9.0 7.5->9.0 . ♦♦ 4

EOlβ 0.2 u mg nd nd nd nd nd 120 30

E019 0.9 u mg 30 kOai 45'C RT-βO'C >9.0 broad nd 25

E020 1 3.9 u/mα 28 kD4 45*C RT-60'C I broad broad | ♦♦ + 1 2

'broad pH range refers to > 50% activity through all pH tested (6.0-8.5)

Example 22. Ester Chain Length Specificity Characterization

The enzymes ot the instant invention can be further charactenzed by testing tot enzymatic specificty for substrate esters of different chain length. Such assays can be conducted using die methods descnbed above, selecung die appropnate substrates. Figure 7 depicts die result of colormetric esterase acuvity assays of the vanous enzymes. The graphed data was obtained where the reacuon condiuons were estimated to be approximately 0.1 U/l ml reacuon, wim 500 ug/ml substrate, where 1 Unit (U) is calculated for each enzyme stock preparaύon in relauon to esterase activity where 1 Unit is the amount of enzyme needed to hydrohze approximately 1 umol of p-nitrophenyl propπonate per minute. The data is reported as approximate maximum OD4i()_nm dunng incubation.

Figure 7A graphs data using the substrate bis-p-nitrophenyl-carbonate. The highest activity was found with enzyme E019, which showed an OD4 iθnm of 0.30 atter 4 houts 5 incubauon. Figure 7B graphs data using the substrate p-nitrophenyl-acetate. The highest activity was found with enzyme E020, which showed an OD4iθn of 3 571 atter 400 seconds incubation. Figure 7C graphs data using the substrate bis-p-nitrophenyl-propionate The highest activity was found with enzyme E003, which showed an OD4iϋnm of 1 4 ^{Jt lt;ι} 600 seconds incubauon. Figure 7D graphs data using the substrate bis-p-nitrophenvl butyrate. The highest activity was found with enzyme E020, which showed an OD4iQ_nm of

1.19 after 160 seconds incubation. Figure 7E graphs data using the substrate bis-p- mtrophenyl-caproate. The highest acuvity was found with enzyme E009, which showed an OD4iθnm of 0-37 after 560 seconds incubation. Figure 7F graphs data using the substrate bis-p-nitrophenyl-caprylate. The highest activity was found with enzyme E003, which showed an OD4iθnm of 0.07 after 360 seconds incubation. Figure 7G graphs data using the substrate bis-p-nitrophenyl-laurate. The highest activity was found with enzyme E016, which showed an OD4iθnm of 0.1 1 after 480 seconds incubauon.

Example 23. pH Dependent Assay for Entantiomer Esterase Specificity

The enzymes of the invention can be further characterized by testing for enzymatic specificity for specific entanuomer substrate esters of different chiral structure. Such assays can be performed using the methods descnbed above, selecting the appropnate substrate. The results of screening are depicted in Figure 8. Figure 8A summanzes the results of colorometnc esterase activity assays for entamomer specificity. Figure 8B depicts quantita ve colorometnc assay data results in terms of minutes required for detectable color change, indicating pH change. The numbers report time in minutes following addition of enzyme. NH indicates no hydrolysis was detected after 3 days, and o/n indicates no hydrolysis after overnight incubauon (approximately 6-15 hours). Substrates 1, 2, 4, 6, 8, and 9 were dissolved to a concentrauon of 40 mM in a 25 mM KPi buffer, pH=7 4, containing -0.005% of bromothymol blue. Substrates 3, 5 and 7 were dissolved to a concentration of 10 mM in a 5 mM KPi buffer, pH=7.4, containing -0.005% of bromothymol blue and up to 10% MeCN as cosolvent. The esterases tested were added in the amount of 1 U per well, as determined by hydrolysis of PNP-propionate. The control reaction was the substrate solution, widi no added enzyme.

Example 24. Characterization of Enzyme Activity against para-nitroanilide compounds

The enzymes of the invention can be further characterized by testing for enzymatic specificity for alternative substrates which are similar to esters. Such assays can be performed using the methods described above, selecting the appropπate substrates. The enzymes of the invention were charactenzed against the ani des and esters listed below and the results depicted in Figure 9. The assays were performed according to the general formula:

Test reactions were run in microtiter plates with each reaction in a total volume oϊ about 100 ul. Each reaction consisted of about 75 ul of pH7.0 phosphate buffer, 5 ul of 5mM substrate, and 20 ul of enzyme adjusted to 50 U/ml (where 1 U is approximatly the amount needed to hydrolize 1 uM of p-nitrophenyl-propionate in 1 minute). The final reaction mixture contained about 1U enzyme and 0.25 mM substrate in each well. The reactions were incubated for about 2.5 hours at 37C. Control reactions, lacking enzyme, were run in adjacent wells. A control containing no substrate was also run on each plate. Following incubation, the plates were read at 405 nm in a BIORAD Model 3550 microplate reader. Values of the controls were subtracted from the experimental well values to determine net acuvity.

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Claims

We Claim:

1. An isolated nucleic acid having the nucleic acid sequence of figure 6E (E019).

2. A nucleic acid segment having the nucleic acid sequence of an open reading frame encoded for by the nucleic acid of claim 1.

3. An expression vector nucleic acid construct having an operably linked expressible nucleic acid insert which is a nucleic acid of claim 2.

4. A host cell transformed witii the expression vector construct of claim 3.

5. A protein expressed by an expression vector construct of claim 3.

6. An isolated nucleic acid having the nucleic acid sequence of figure 6F (E005).

7. A nucleic acid segment having the nucleic acid sequence of an open reading frame encoded for by the nucleic acid of claim 6.

8. An expression vector nucleic acid construct having an operably linked expressible nucleic acid insert which is a nucleic acid of claim 7.

9. A host cell transformed with die expression vector construct of claim 8.

10. A protein expressed by an expression vector construct of claim 8.

11. An isolated nucleic acid having the nucleic acid sequence of figure 6G (E004).

12. A nucleic acid segment having the nucleic acid sequence of an open reading frame encoded for by the nucleic acid of claim 11.

13. An expression vector nucleic acid construct having an operably linked expressible nucleic acid insert which is a nucleic acid of claim 12.

14. A host cell transformed with the expression vector construct of claim 13.

15. A protein expressed by an expression vector construct of claim 13.

16. An isolated nucleic acid having the nucleic acid sequence of figure 6H (E006).

17. A nucleic acid segment having the nucleic acid sequence of an open reading frame encoded for by the nucleic acid of claim 16.

18. An expression vector nucleic acid construct having an operably linked expressible nucleic acid insert which is a nucleic acid of claim 17.

19. A host cell transformed with the expression vector construct of claim 18.

20. A protein expressed by an expression vector construct of claim 18.

21. An isolated nucleic acid having the nucleic acid sequence of figure 61 (E008).

22. A nucleic acid segment having the nucleic acid sequence of an open reading frame encoded for by the nucleic acid of claim 21.

23. An expression vector nucleic acid construct having an operably linked expressible nucleic acid insert which is a nucleic acid of claim 22.

24. A host cell transformed with the expression vector construct of claim 23.

25. A protein expressed by an expression vector construct of claim 23.

26. An isolated nucleic acid having the nucleic acid sequence of figure 6J (E010).

27. A nucleic acid segment having the nucleic acid sequence of an open reading frame encoded for by the nucleic acid of claim 26.

28. An expression vector nucleic acid construct having an operably linked expressible nucleic acid insert which is a nucleic acid of claim 27.

29. A host cell transformed with the expression vector construct of claim 28.

30. A protein expressed by an expression vector construct of claim 28.

31. An isolated nucleic acid having the nucleic acid sequence of figure 6K (E013).

32. A nucleic acid segment having the nucleic acid sequence of an open reading frame encoded for by the nucleic acid of claim 31.

33. An expression vector nucleic acid construct having an operably linked expressible nucleic acid insert which is a nucleic acid of claim 32.

34. A host cell transformed with die expression vector construct of claim 33.

35. A protein expressed by an expression vector construct of claim 33.

36. An isolated nucleic acid having the nucleic acid sequence of figure 6L (E015).

37. A nucleic acid segment having the nucleic acid sequence of an open reading frame encoded for by the nucleic acid of claim 36.

38. An expression vector nucleic acid construct having an operably linked expressible nucleic acid insert which is a nucleic acid of claim 37.

39. A host cell transformed with the expression vector construct of claim 38.

40. A protein expressed by an expression vector construct of claim 38.

41. An isolated nucleic acid having the nucleic acid sequence of figure 6M (E016).

42. A nucleic acid segment having the nucleic acid sequence of an open reading frame encoded for by the nucleic acid of claim 41.

43. An expression vector nucleic acid construct having an operably linked expressible nucleic acid insert which is a nucleic acid of claim 42.

44. A host cell transformed with the expression vector construct of claim 43.

45. A protein expressed by an expression vector construct of claim 43.

46. An isolated nucleic acid having the nucleic acid sequence of figure 6N (E017).

47. A nucleic acid segment having the nucleic acid sequence of an open reading frame encoded for by the nucleic acid of claim 46.

48. An expression vector nucleic acid construct having an operably linked expressible nucleic acid insert which is a nucleic acid of claim 47.

49. A host cell transformed with the expression vector construct of claim 48.

50. A protein expressed by an expression vector construct of claim 48.

51. An isolated nucleic acid having die nucleic acid sequence of figure 60 (E020).

52. A nucleic acid segment having die nucleic acid sequence of an open reading frame encoded for by the nucleic acid of claim 51.

53. An expression vector nucleic acid construct having an operably linked expressible nucleic acid insert which is a nucleic acid of claim 52.

54. A host cell transformed with the expression vector construct of claim 53.

55. A protein expressed by an expression vector construct o f claim 53.

56. An isolated nucleic acid having the nucleic acid sequence of figure 6P (E027).

57. A nucleic acid segment having the nucleic acid sequence of an open reading frame encoded for by the nucleic acid of claim 56.

58. An expression vector nucleic acid construct having an operably linked expressible nucleic acid insert which is a nucleic acid of claim 57.

59. A host cell transformed with the expression vector construct of claim 58.

60. A protein expressed by an expression vector construct of claim 58.

61. An isolated nucleic acid having the nucleic acid sequence of figure 6Q (E003- 5') at the 5' end and the nucleic acid sequence of figure 6R (E003-3') at the 3' end of a contiguous nucleic acid molecule, separated by intervening nucleic acids.

62. A nucleic acid segment having the nucleic acid sequence of an open reading frame encoded for by the nucleic acid of claim 61.

63. An expression vector nucleic acid construct having an operably linked expressible nucleic acid insert which is a nucleic acid of claim 62.

64. A host cell transformed with the expression vector construct of claim 63.

65. A protein expressed by an expression vector construct of claim 63.

66. An isolated nucleic acid having the nucleic acid sequence of figure 6S (E004- 5') at the 5' end and the nucleic acid sequence of figure 6T (E004-3') at the 3' end of a contiguous nucleic acid molecule, separated by intervening nucleic acids.

67. A nucleic acid segment having the nucleic acid sequence of an open reading frame encoded for by the nucleic acid of claim 66.

68. An expression vector nucleic acid construct having an operably linked expressible nucleic acid insert which is a nucleic acid of claim 67.

69. A host cell transformed with die expression vector construct of claim 68.

70. A protein expressed by an expression vector construct of claim 68.

71. An isolated nucleic acid having the nucleic acid sequence of figure 6U (BOMS') at the 3' end of the molecule.

72. A nucleic acid segment having the nucleic acid sequence of an open reading frame encoded for by the nucleic acid of claim 71.

73. An expression vector nucleic acid construct having an operably linked expressible nucleic acid insert which is a nucleic acid of claim 72.

74. A host cell transformed with the expression vector construct of claim 73.

75. A protein expressed by an expression vector construct of claim 73.