Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P21/00—Preparation of peptides or proteins
  • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof

Definitions

• 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 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 .
• 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.
• sodium salts of polyaspartic acids are obtained via the thermal polymerization of aspartic acid:
• 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.
• suitable catalysts such as phosphoric acid.
• 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 ⁇ - and ⁇ - linked aspartic acid units.
• the ⁇ / ⁇ ratio can be determined via NMR spectroscopic methods.
• An ⁇ / ⁇ 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.
• the EP-patent application 256 423 of Chisso discloses a process for the manufacture of ⁇ -poly-L-lysine, derived from Streptomyces albulus subspecies lysinopolymerus No. 346-D.
• the process for the manufacture of of ⁇ -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 ⁇ -poly-L-lysine with immobilized bacterial cells under aerobic conditions.
• An isolated ⁇ -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- ⁇ -L-glutamic acid with a wide spectrum of Glu-units in the polymer.
• Poly- ⁇ -L-Glu has not been synthesized either directly through microbiological methods.
• chemically generated poly-Glu the same observations and arguments hold as for chemcally generated poly-Asp.
• the polymerisation degree is well defined and only dependent on the length of the polynucleotide which is used in the microbiological manufacturing process .
• the task to find the a. m. polymers consisting of poly- ⁇ -L- Asp, poly- ⁇ -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.
• 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.
• a Glu/Asp sequence 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 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.
• 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 :
• 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.
• washing- and cleaning agents can be gathered from Ullmanns Encyklopadie der ischen 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.
• 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) .
• Polyaspartic acid synthezised by thermal condensation of L-aspartic acid, is described as an inhibitor of corrosion in Little et al .
• 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 .
• 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.
• 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.
• Amino acid analyses were carried out on an apparatus like, for example, the Applied Biosystems 420/130A derivatizer/analyzer device.
• Oligonucleotides were prepared by means of ⁇ - 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.
• 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 DH5 ⁇ F' .
• restriction endonucleases like, e.g., Eco RT or Bam HI
• cells like, e.g., E. coli DH5 ⁇ F' .
• the cells were cultivated at 37 °C on a medium like 2xYT under the addition of ampicillin (about 0.2 mg/ml) , isopropyl- ⁇ -D-thiogalactopyranoside (IPTG) (0.025 mg/ml) and a chromogenic substrate like 5-bromo-4- chloro-3-indolyl- ⁇ -D-galactopyranoside (called X-Gal here) which turned blue in the case of cells with active ⁇ - galactosidase gene but remained white in the case of cells which had lost the corresponding activity by inserting inactivation of the ⁇ -galactosidase gene.
• ampicillin about 0.2 mg/ml
• IPTG isopropyl- ⁇ -D-thiogalactopyranoside
• X-Gal 5-bromo-4- chloro-3-indolyl- ⁇ -D-galactopyranoside
• 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.
• 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.
• 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.

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