EP0915987A2

EP0915987A2 - POLY-($g(a)-L-ASPARTIC ACID), POLY-($g(a)-L-GLUTAMIC ACID) AND COPOLYMERS OF L-ASP AND L-GLU, METHOD FOR THEIR PRODUCTION AND THEIR USE

Info

Publication number: EP0915987A2
Application number: EP98922651A
Authority: EP
Inventors: David A. Tirrell; Michael Del Grosso; Rainer Siegmeier; Andreas Bommarius
Original assignee: Donlar Corp; University of Massachusetts Amherst
Current assignee: Donlar Corp; University of Massachusetts Amherst
Priority date: 1997-04-21
Filing date: 1998-04-06
Publication date: 1999-05-19
Also published as: WO1998048032A3; WO1998048032A2

Abstract

The invention provides Poly-(α-L-aspartic acid) (= poly-L-α-Asp), Poly-(α-L-glutamic acid) and new copolymers of L-Asp and L-Glu with essentially α-linkages as well as salts of said polymers and copolymers. A method for producing said polyamino acids comprises employing an oligonucleotide construct encoding the sequence of the desired polymer, cloning the oligonucleotide into a plasmid, transformation of host cells with the plasmid and expression of the genetic information. Because of their good biological degradability the new poly-L-α-amino acids can be used as co-builders, sequestering agents and corrosion inhibitors.

Description

Poly- (α-L-aspartic acid) , Poly- (α-L-glutamic acid) and copolymers of L-Asp and L-Glu, method for their production and their use

Introduction and background

The invention refers to Poly- (α-L-aspartic acid) (= poly- α-Asp) , Poly- (α-L-glutamic acid) and new copolymers of L-Asp and L-Glu with essentially α-linkages as well as salts of said polymers and copolymers. The terms polymer and copolymer comprise 5 to approx 200 amino acid units. The invention also refers to a method for producing said polymers and copolymers by means of a microbiological process. A further object of the invention is directed to the use of said polymers and copolymers as sequestering and dispersing agent.

Polyaspartic acids (poly-asp) produced chemically from L- or DL-aspartic acid or suitable precursors, their salts as well as copolymers with other copolymerizable compounds are used as water-soluble sequestering agent and dispersing agents .

Disadvantages of the poly-aspartic acids and salts as well as relevant copolymers currently used are their incomplete biodegradability as well as their discoloration to a dark product stemming from the harsh conditions of their manufacture. For use in the laundry detergent industry the dark discoloration is a major disadvantage. The task addressed by this application is to provide new polymers of amino acids which feature improved biodegradability together with improved or at least not worsened abilities for complexation of divalent metal ions such as calcium or magnesium. Furthermore, there is demand for polymers which are colorless or nearly colorless in solid condition or in aqueous solution.

Polyamino acids and their salts are accessible in various methods of chemical production. Different methods of production and a few properties of polyamino acids are described, e.g., in Nachr. Chem. Tech. Lab., 1996, 44, 1167 - 1170 as well as in Hydrophylic Polymers, Performance with Environmental Acceptance, editor: J. Edward Glass, ACE, Washington, 1996, Commercial Poly (Aspartic Acid) and It's Uses, K.C. Low, A. P. Wheeler, L.P. Koskan, 99-111.

According to the above, sodium salts of polyaspartic acids are obtained via the thermal polymerization of aspartic acid: To this end maleic acid anhydride is converted via maleic acid into fumaric acid, which is aminated in a following chemical or enzymatic method step to the ammonium salt of aspartic acid. The free aspartic acid isolated therefrom is subjected to a solid-phase polymerization to the primary polymerization product. In a modified method the course of the reaction as well as the product properties can be influenced by the addition of suitable catalysts such as phosphoric acid. Alternatively, maleic acid anhydride or maleic acid anhydride derivatives such as maleic acid ammonium salts, maleic amide acid, maleic amide acid ammonium salts can be thermally polymerized to a primary polymerization product in the presence of nitrogen- containing compounds such as ammonia but also of ammonium salts such as ammonium carbonate. The primarily formed polymerization products are distinguished by polysuccinimide structural elements which result in subsequent hydrolysis in polyaspartic acids with α- and β- linked aspartic acid units. The α/β ratio can be determined via NMR spectroscopic methods. An α/β ratio of approximately 30:70, which can be influenced only slightly, results for all polyaspartic acids obtained according to the thermal polymerization methods and alkaline hydrolysis described here.

The main production methods described up to the present determine the structural parameters such as, e.g., the molecular weight, the linearity and the properties correlating therewith as well as the action in various applications and the biological degradability.

Besides the a. m. chemical processes there exist only a few biological routes to amino acid polymers:

The EP-patent application 256 423 of Chisso discloses a process for the manufacture of ε-poly-L-lysine, derived from Streptomyces albulus subspecies lysinopolymerus No. 346-D. The process for the manufacture of of ε-poly-L- lysine is characterized by the addition of sugar to the culturing medium. The EP-A 0 557 954 refers to a process for the manufacture of ε-poly-L-lysine with immobilized bacterial cells under aerobic conditions.

An isolated γ-polyglutamate hydrolase is known from the EP- A 559 175 and US 5,356,805. Takeda Chemical discloses in the EP-A 410 638 a process to the manufacture of polyglutamic acid. The microorganisms used for the polymerisation of L-glutamic acid were bacteria such as Bacillus subtilis or Bacillus licheniformis . The product contains poly-α-L-glutamic acid with a wide spectrum of Glu-units in the polymer.

The State of the Art concerning biological routes to other poly-amino acids is very limited: Tirrell et al . have done some work - see „Biomolecular Materials", Special Report, Chemical & Engineering News, Dec. 19, 1994, p. 40-51 and „Synthesis of biopolymers : proteins, polyesters, polysaccharides and polynucleotides", Current Opinions in Solid State & Materials Science 1996, Vol. 1, p. 407-411.

Poly-L-Asp of the desired length and purity has not been synthesized before through biological methods. The closest example in the literature (G. Zhang, M.J. Fournier, T.L.

Mason and D.A. Tirrell, Macromolecules 1992, 25, 3601-3603) is a 76-mer with the structure Asp-Glu- (Glu₁₇-Asp) ₄-OH.

Poly-α-L-Glu has not been synthesized either directly through microbiological methods. For chemically generated poly-Glu the same observations and arguments hold as for chemcally generated poly-Asp.

Summary of the invention

We have found Poly- (α-L-aspartic acid) (= Poly (α-L-Asp) _m) and Poly- (α-L-glutamic acid) (= Poly (α-L-Glu) _m) with a polymerisation degree m selected within 5 to 200. The polymerisation degree is well defined and only dependent on the length of the polynucleotide which is used in the microbiological manufacturing process . We further have found copolymers on the basis of L-Asp and L-Glu with essentially only α-linkages and well defined position of the L-amino acids over the length of the copolymer, the polymerisation degree (= number of amino acid units) of which is selected of 5 to 200.

The task to find the a. m. polymers consisting of poly-α-L- Asp, poly-α-L-glu or copolymers of both amino acids that feature improved properties with respect to biological degradability and discoloration is solved by employing a oligonucleotide construct that encodes the desired sequence of Asp polymers (or Asp/Glu polymers) . The amino acid Glu is encoded by two codons , GAA and GAG. In certain organisms such as E. coli , Glu is preferably encoded by GAA. Asp is encoded by two codons as well, GAC and GAT. The latter is preferred in many organisms such as E. coli . The oligonucleotide construct encoding for the desired Asp length (or Asp/Glu length) is cloned into a plasmid. Cells of the desired host cell line, such as Escherichia coli , Bacillus subtilis ox Corynebacterium glutamicum, are transformed with the altered plasmids, and the genetic information expressed. Detailed description of the invention

The procedure to obtain the poly-amino acids follows that described in the^' publication of G. Zhang, M.J. Fournier, T.L. Mason and D.A. Tirrell, Macromolecules 1992, 25, 3601- 3603, and is described below.

Fermentations in complex media, DNA treatments and transformations are conducted as described in Sambrook, Frisch and Maniatis (J. Sambrook et al . , Molecular Cloning. A Laboratory Manual, Cold Spring Harbor Press, 1989) .

The oligonucleotide duplex which encodes the desired Glu or Asp or Glu/Asp amino acid sequence of the predetermined number of amino acid units of each polymer chain is synthesized on a DNA synthesizer. The oligonucleotides are purified by electrophoresis on a polyacrylamide denaturing gel, annealed, enzymatically phosphorylated at the 5' termini, and ligated with BamUI -digested, phosphatase- treated plasmid pUC18. The sequence of the insert is verified by a technique such as the Sanger dideoxy sequencing strategy. E. coli strains are transformed with the recombinant plasmid and marked by an appropriate technique such as insertional inactivation of a gene or resistance against antibiotics.

Should a Glu/Asp sequence be desired which by itself is a multimer of a simpler Glu/Asp sequence the recombinant plasmid is digested and affords a DNA fragment encoding the desired poly-Glu/Asp sequence. This DNA „monomer" is purified on a polyacrylamide gel and self-ligated in head- to-tail fashion with T4 DNA ligase to yield a population of multimers. A portion of the ligation mixture is analyzed on a 1.5% agarose gel. The ligation mixture is cloned into a plasmid such as pUC803 or pUC18. E. coli cells are then transformed with the recombinant plasmids . A plasmid containing the repeat units of the DNA monomer is isolated, and the nucleotide sequence confirmed by sequencing of the double-stranded DNA. The BamRI segment is recovered, purified and inserted into a suitable expression vector. The ligation mix is used to transform E. coli cells, and the presence and orientation of the insert are checked by digestion with restriction enzymes BamHI and Aval , respectively.

Individual colonies of the expression host, which contained recombinant plasmid of the correct sequence and orientation, either as monomer or in repeating units, were used to inoculate a preculture of LB medium with an antibiotic. After growth the culture was used for inoculation of the main culture (also containing antibiotics) in fermentation media such as, e.g., LB or 2xYT. Protein expression was achieved by the addition of an inductor (IPTG = isopropylthiogalactopyranoside) toward the end of the logarithmic growth phase. The rate of cell growth is normal prior to induction but declines as protein product appears in whole cell lysates analyzed on a 12% SDS-polyacrylamide gel. As a control, no such product was found in crude lysates of cells lacking the artificial coding sequence. The cells were harvested two hours later by centrifugation.

After cell lysis, e.g. in a homogenizer, the fusion protein is purified by standard techniques such as affinity chromatography, precipitation in organic solvent, and the like. Electrophoretic purification on a non-denaturing polyacrylamide gel affords a product which migrates as a single band at the expected molecular weight and which yields amino acid analyses consistent with the desired sequence .

The new aminoacid polymers and copolymers have many fold usefulness in any kind of water treatment and relevant processes :

• Use as co-builder in detergents:

In the formulation of modern universal detergents co- builders are considerably significant for supporting the actual builders [skeletal substances] . Their task is to eliminate the calcium- and magnesium ions stemming in part from the contamination and in part from the water and to force the surfactant action therewith.

Polyaspartic acid Na-salts have a dispersing action which reinforces the primary washing power. Polyaspartic acids and their salts are used in washing [detergent] - and cleaning agents. The detergents can be powdery or also be present in liquid form. The composition of the washing- and cleaning agent formulations can be very different. Washing- and cleaning agent formulations customarily contain 2 to 50 % by weight surfactants and optional builders. This data applies both to liquid and to powdery detergents. Washing- and cleaning agent formulations customary in Europe, the USA and Japan are to be found, e.g., in Chemical and Engn. News, vol. 67, 35 (1989) in table form. Further data about the composition of washing- and cleaning agents can be gathered from Ullmanns Encyklopadie der technischen Chemie, Verlag Chemie, Weinheim, 1983, 4 edition, pp. 63 - 160. The use of polyaspartic acid in washing- and cleaning agents is described, among other places, in WO 95/16020, WO 95/16726, DE 44 30 520, DE 44 28 638, DE 44 28 597.

• Use in water treatment :

Inhibitors are used in cooling-water circuits, in the treating of boiler and feed water and in the desalination of see water for avoiding and eliminating precipates and coatings . Polyaspartic acids prevent and/or delay the crystallization of alkaline-earth salts such as calcium carbonate, calcium sulfate, etc. The action thereby is far below the concentrations necessary for complex formation (threshold effect) .

• Use as corrosion inhibitor:

Polyaspartic acid, synthezised by thermal condensation of L-aspartic acid, is described as an inhibitor of corrosion in Little et al . „Corrosion Inhibi tion by thermal Polyaspartate", surface reactive peptides and polymers, pp. 263-279, ACS Symposium Series 444 (1990) . Thermal polyaspartate binds to surfaces of mild steel and moderately supresses both anodic and cathodic corrosion reactions .

• Use in petroleum production [from wells]

In the production of petroleum, especially in the North See, the oil is won with the aid of see water. The see water contains sulfate. The petroleum is accompanied by formation water containing barium ions and strontium ions. If the formation water and the see water mix, poorly soluble Ba sulfates and Sr sulfates form which may possibly clog boreholes and pipelines. Polyaspartic acids prevent and/or delay the crystallization of the precipitates so that the undesired coating formations do not occur.

• Use for desalination in natural gas production:

In the winning of natural gas relatively dry natural gas flows through saturated salt brines and absorbs moisture. This produces a concentration and possibly a supersaturation of the salt brine, which crystallizes salts out, especially NaCl and KC1. The crystallizates clog the porous sandstone, which lets the natural gas through only very poorly or not at all any more.

Natural gas standing under pressure has a residual moisture content in the winning process. Salts are dissolved in this residual moisture. During the winning [extraction] process, especially during the expansion of the gas, a critical pressure is dropped below which results in a crystallizing out of the salts and a clogging of the porous sandstone .

Polyaspartic acids prevent and/or delay the crystallization of the salts in both instances on account of their dispersing properties.

Examples and reference examples

Reference example 1 : Polyaspartate starting with L- aspartic acid

133 g L-aspartic acid were placed in a 1 1 round-bottomed flask and heated in a rotary evaporator at a pressure of 20-100 mbar to 230-250 °C for 4 h. The powdery polysuccinimide produced was dissolved in sodium hydroxide solution (50 % by weight) under agitation at 30-50 °C and the solution adjusted to pH 9-11. After the water was distilled of in the rotary evaporator under reduced pressure polyaspartic acid Na salt was obtained as solid.

Reference example 2 : Polyasparate [sic - aspartate? Here and below.] starting from rac-aspartic acid

Production analogous to the method starting from L-aspartic acid (see section 4.1)

Reference example 3 : Polyasparate starting from maleic acid anhydride and ammonia solution

Maleic acid anhydride was added to a solution of concentrated NH₃ solution in a round-bottomed flask and the reaction mixture concentrated by evaporation in a rotary evaporator to dryness and the residue heated at 230-250 °C in the rotary evaporator for 4 h. The powdery polysuccinimide produced was dissolved in sodium hydroxide solution (50 % by weight) under agitation at 30-50 °C and the solution adjusted to pH 9-11. After the water was distilled of in the rotary evaporator under reduced pressure polyaspartic acid Na salt was obtained as solid.

Reference example 4 : Polyasparate starting from monoammonium maleate

Maleic acid anhydride was charged into a cone, ammonia solution in a round-bottomed flask. A monoammonium maleate slurry was obtained thereby. The precipitate was filtered off and pre-dried. The solid monoammonium maleate was now polymerized analogously to section 4.1.

Example 1 Poly- (α-L-asp) ₄₈

Concerning the principles of the production of poly-α-L amino acids the following literature is referred to: Zhang et al . , Macromolecules 1992, 25, pp. 3601-3603 and Panitch et al . , Macromolecules 1997, pp. 42-49.

General remarks :

Fermentation in full medium, DNA treatments and transformations were carried out as described in Sambrook,

Frisch and Maniatis (J. Sambrook et al . , Molecular Cloning.

A Laboratory Manual, Cold Spring Harbor Press, 1989) .

Amino acid analyses were carried out on an apparatus like, for example, the Applied Biosystems 420/130A derivatizer/analyzer device.

Preparation of the synthetic DNA

Oligonucleotides were prepared by means of β- cyanoethylphosphorus amidite chemistry on a synthesizer like Biosearch Model 8700 and purified by means of 10 % denaturing polyacrylamid gel electrophoresis . The purified oligonucleotides were annealed at 80 °C and allowed to cool off for several hours until room temperature. The double strand was phosphorylated by T4-polynucleotide kinase, precipitated in ethanol and dried in a vacuum.

Cloning and amplification:

The double strand was ligated into a plasmid like pUC18 which had been cleaved by restriction endonucleases like, e.g., Eco RT or Bam HI and transformed in cells like, e.g., E. coli DH5αF' . The cells were cultivated at 37 °C on a medium like 2xYT under the addition of ampicillin (about 0.2 mg/ml) , isopropyl-β-D-thiogalactopyranoside (IPTG) (0.025 mg/ml) and a chromogenic substrate like 5-bromo-4- chloro-3-indolyl-β-D-galactopyranoside (called X-Gal here) which turned blue in the case of cells with active β- galactosidase gene but remained white in the case of cells which had lost the corresponding activity by inserting inactivation of the β-galactosidase gene.

Plasmid DNA from the white transformants is sequenced in order to verify the identity of the inserted DNA, e.g. by sequenase 2.0 of Amersham Life Sciences. After isolation of the recombinant plasmid from the 2xYT culture medium the DNA was digested by the restriction endonuclease BanI and the fragments separated by non-denaturing PAGE and the interesting DNA monomer washed down.

Polymerization of the DNA monomer and cloning of the multimer

In the case of Asp/Glu sequences representing multimers of a shorter Asp/Glu monomer the purified DNA monomer is self- ligated with T4 DNA ligase in order to produce a distribution of multimers. The multimers are separated by electrophoresis and ligated into a dephosphorylated, high copy number cloning vector digested with BanI. The recombinant plasmid was transformed into a strain like E. coli. Transformants are analyzed by analysis of the restriction enzyme digestion pattern and the desired DNA chain length (with the desired number of repeating units) selected. Construction of the bacterial expression vector

The recombinant plasmids from the transformants were digested with restriction endonucleases like BamHI , the multimer fragments separated on 1 % agarose gel electrophoresis and recovered by extraction e.g. in phenol, phenol/chloroform or ethanol. Transformants were checked by digestion with nucleases like Aval for the presence and orientation of multimers. Transformants with correct sequence were used for the transformation of the expression host.

Fermentation and protein expression

In both the cases of monomeric or multimeric Asp/Glu sequence, fermentation is conducted by vigorous aeration in a YT medium to an optical density of OD₆₀₀ = 1.0, synthesis of the fusion protein is induced by the addition of isopropyl-β-D-thiogalactopyranoside (IPTG) at 0.4 mM. Cells are harvested 2 h after induction, e.g. by centrifugation at 4000 g for 20 min at 4°C. The pellet from the centrifugation is re-suspended in water and stored at -20 °C.

Protein purification

The fusion protein was obtained by centrifugation after the thawed [defrosted] cells had been treated successively twice with EDTA/surfactant , then with lipase, finally with organic solvents (chloroform/methanol) and were finally washed with water. An affinity chromatography then selectively follows, e.g., via glutathione-linked sepharose . Cleaving of the fusion protein

The fusion protein is preferably cleaved by the bromocyanogen method (cf . B.J. Smith, Methods in Biology, New Protein Techniques, Humana, Clifton, NH, 1988) . After the cleaving the solvent is drawn off by vacuum evaporation and the insoluble portion recovered by centrifugation and dried by lyophilization.

Example 2

Biological degradability of synthetically and fermentatively produced polyaspartates

In the determination of the aerobic biological degradability of organic substances in aqueous medium according to DIN EN 29888 (Zahn-Wellens method) a defined substance concentration with microorganisms (activated sludge) from a municipal sewage treatment plant is inoculated and subsequently aerated. The incubation takes place at room temperature in the dark. During the test time the decrease of substance concentration is followed via summation parameters like COD (chemical oxygen demand) and/or TOC (total organic carbon) . The degradability is defined as the decrease in COD or in TOC (expressed in per cent) during the test period, corrected by the particular blank value and relative to the initial concentration used.

Biological degradability according to Zahn-Wellens determined as CSB elimination after 28 days. Clarification sludge from the Hanau sewage treatment plant . Starting from L-aspartic acid from reference example 1 85

Starting from rac-aspartic acid from reference example 2 40

Starting from maleic acid anhydride/NH₃ from reference example 3 30

Starting from monoammonium maleate from reference example 4 45

Starting from fermentative poly- (α-L-Asp) ₄₈ from example 1 95 - 100 %

Claims

1. Poly- (╬▒-L-amino acids) selected from the Group Poly(╬▒- L-aspartic acid) _m , Poly (╬▒-L-glutamic acid)_n and

Poly (╬▒-L-Aspartic/glutamic acid)_m, wherein m is a whole number selected from 5 to 200.

2. Process for the manufacture of Poly-╬▒-L-amino acids selected from the Group Poly (╬▒-L-aspartic acid)_m , Pol (╬▒-L-glutamic acid)_n and Poly (╬▒-L-Aspartic/glutamic acid)_m, wherein m is a whole number selected from 5 to 200 comprising the steps a) the synthesis an oligonucleotide of lenght 3 x m, wherein m is the number of Asp or Glu or the sum of Asp and Glu-moieties coding for Asp and/or Glu b) the incorporation of the oligonucleotide into a plasmid together with a promoter c) the expression of the gene in a microorganism selected from the Group of E. coli, Corynebacterium glutamicum, Aspergillus niger, Bacillus subtilis d) the fermentation of the respective organism e) the isolation of the oligo-L-Asp (or oligo- (L- Asp) _m- (L-Gllu) _n) product .

3. Process as in claim 2, wherein the polymerisation degree m is precisely controlled by the length (1) of the oligonucleotide (m = 3 1) .

4. Process as in claim 2 or 3 , wherein m is selected from whole numbers between 10 and 150.

5. Use of Poly- (╬▒-L-amino acid) according to claim 1 as Co-builder in detergent-bleaching and cleaning- compositions .

6. Use of Poly- (╬▒-L-amino acids) according to claim 1 as sequestering agent in the water treatment .

7. Use of Poly- (╬▒-L-amino acids) according to claim 1 as corrosion inhibitor to suppress anodic and cathodic corrosion reactions.