Polyasparaginic acid homopolymers an copolymers, biotechnical production and use thereof

The invention relates to the production of polyaspartic acid homo- and copolymers by biotechnological processes and to the use of the resulting products (for influencing the crystallization or agglomeration behavior of sparingly soluble salts or solids in aqueous systems).

BACKGROUND OF THE INVENTION
 1. Field of the Invention
 The invention relates to the production of polyaspartic acid homo- and
 copolymers by biotechnological processes and to the use of the resulting
 products (for influencing the crystallization or agglomeration behavior of
 sparingly soluble salts or solids in aqueous systems).
 2. Description of the Related Art
 Crystallization and agglomeration processes are, as biological
 mineralization, among the fundamental processes of animate nature. Thus,
 they are involved, for example, in the structure of skeletons or shells in
 living organisms. In nature, these mineralization processes are controlled
 by naturally occurring proteins and polysaccharides. (S. Weiner, Biochem.
 22, (1983), 4139-45; C. S. Sikes, A. P. Wheeler, in Chemical Aspects of
 Regulation of Mineralisation., Eds. C. S. Sikes , A. P. Wheeler University
 of South Alabama Publ. Services (1988), 15-20).
 Unfortunately, both in nature and in the industrial sector, unwanted
 mineralization processes also occur and result in tenacious, troublesome
 deposits and encrustations such as, for example, dental plaque, organ
 concretions or, in the industrial sector, encrustations on heat exchanger
 surfaces or cooling towers particle agglomerations in pigment dispersions,
 encrustations on hard (for example glass metal) and soft (textile)
 surfaces. In the past, various proposals have been made for exploiting
 this natural action principle for industrial problems. Thus, the U.S. Pat.
 Nos. 4,534,881, 4,585,560, 4,587,021 describe the inhibition of calcium
 carbonate deposits by protein fractions, polysaccharide fractions or
 polyamino acid fractions from calcium carbonate-forming organisms such as
 crustaceans etc.
 In addition, the inhibition of mineral deposits by polyanionic hydrophobic
 polypeptides with a block copolymer structure and related phosphorylated
 polypeptides is claimed in the literature (U.S. Pat. No. 4,868,287). The
 polypeptides used are prepared by methods of peptide chemistry. WO
 92/17194 states that an improved synthesis of these polypeptides is
 provided.
 Since the proteins described above acquire their polyanionic
 characteristics through a high aspartic acid content, aspartic acid homo-
 and copolymers are also claimed for this purpose. These polyaspartic acids
 are, however, all obtained by chemical synthesis. Thus, for example, a
 polyaspartic acid sodium salt can be prepared by thermal polycondensation
 of aspartic acid to polysuccinimide and subsequent basic hydrolysis.
 (Kovacs et al. J. Org. Chem, 26 (1961)1084-1091). Further applications
 claim the preparation and use of polyaspartic acids by thermal
 polycondensation of aspartic acid in the presence of acidic catalysts such
 as phosphoric acid. In addition, polyaspartic acids are also prepared by
 thermal polymerization starting from aspartic acid precursors such as
 maleic acid ammonium salt (EP 0 256 366), maleic amide (EP 0604 813) and
 maleic anhydride, and ammonia-releasing compounds.
 BRIEF SUMMARY OF THE INVENTION
 The present invention now describes biological methods for producing
 aspartic acid homo- and copolymers and the use of the resulting polymers
 for inhibiting mineral deposits and dispersing solid particles.
 To date, three different polyamino acids have been found in nature,
 poly-.gamma.-glutamate, poly-.SIGMA.-lysine and
 poly-.alpha.-arginylaspartate (cyanophycin).
 Poly-.gamma.-glutamate is produced by various Gram-positive bacteria such
 as, for example, Bacillus licheniformis, Bacillus subtilis natto or
 Bacillus anthracis. poly-.SIGMA.-Lysine is produced by Streptomyces
 albulus.
 Poly-.alpha.-arginylaspartate is produced by many cyanobacteria such as,
 for example, Spirulina platensis, Aphanocapsa PCC 6308 or Anabena
 cylindrica. Synthesis takes place by the non-ribosomal pathway, resulting
 in a polypeptide which has a polydisperse molecular weight distribution
 and is stored in the form of cyanophycin granules inside cells.
 To date, only the biotechnological production of poly-.gamma.-glutamate
 using Bacillus subtilis or Bacillus licheniformis is disclosed in the
 patent literature. (JP 1-174397 (1989), JP 43-24472 (1969) and U.S. Pat.
 No. 2,895,882).
 DETAILED DESCRIPTION OF THE INVENTION
 We have now found that aspartic acid homo- and copolymers can be produced
 using various cyanobacteria via the intermediate cyanophycin. The
 resulting polymers have the following structures.
 ##STR1##
 R.sub.1 : .tbd.OH or arginyl
 n: 5-400
 If the total of all the R.sub.1 radicals corresponds to 100%, then the
 proportion of R.sub.1.dbd.OH is between 5% and 100%, preferably 30%-100%
 and particularly preferably 70% to 100%. The molecular weight of the
 polymers is generally between 1000 and 100,000, preferably between 2000
 and 50,000, particularly preferably between 2000 and 30,000.
 The total n of all repeating units depends on the cleavage conditions to
 which the intermediate cyanophycin is subjected. Arginine elimination can
 take place both with acid and with base. If an acidic hydrolysis is
 carried out, stoichiometric amounts of acid in relation to the
 incorporated arginine are necessary because the acid is trapped as
 arginine salt. It is possible to employ as acid all mineral acids such as,
 for example, hydrochloric acid, sulfuric acids, phosphoric acids and lower
 fatty acids of C.sub.1 -C.sub.5. The hydrolytic cleavage can moreover take
 place under pressure using carbonic acid or CO.sub.2. Depending on the
 concentration of the acid employed and on the reaction conditions,
 depolymerization by hydrolytic cleavage of the polyaspartate chain may
 also take place, in addition to the arginine elimination. However, the
 unwanted depolymerization can be minimized by suitable choice of the
 reaction conditions, such as dilute acid, moderate reaction times,
 temperatures not exceeding 100.degree. C.
 However, the hydrolysis can also advantageously be carried out under basic
 conditions, because the polyaspartate chain is more stable under these
 conditions. The reaction is carried out at a pH.gtoreq.8.5, preferably
 9-12, and at temperatures between 20.degree. C. and 150.degree. C.,
 preferably 50.degree. C.-120.degree. C. After the hydrolysis, the reaction
 product is removed by filtration from the unreacted cyanophycin and the
 alkali-insoluble arginine. Suitable as base for the alkaline hydrolysis
 are all metal hydroxides or carbonates which make pH values&gt;8.5 possible
 in aqueous medium. Alkali metal and alkaline earth metal hydroxides are
 preferred.
 The cyanophycin employed for the hydrolytic formation of the aspartic acid
 homo- and copolymers is obtained by fermentation of cyanobacteria such as,
 for example, Aphanocapsa PCC 6308, Anabena cylindrica or Spirulina
 platensis. A possible biosynthetic pathway is described in the
 experimental part.
 The aspartic acid homo- and copolymers obtained as products were
 characterized by elemental analysis, amino acid analysis and NMR
 spectroscopy. The molecular weight was determined with the aid of aqueous
 GPC. In addition, for industrial applications, the products were tested
 for their ability to inhibit mineral deposits such as calcium carbonate,
 calcium sulfate, calcium phosphate, calcium oxalate and barium sulfate,
 and for their dispersing capacity for solid particles. The calcium
 carbonate inhibiting capacity was carried out inter alia by a method of C.
 S. Sikes, A. P. Wheeler in Chemical Aspects of Regulation of
 Mineralisation, pp. 53-57, University of South Alabama Publication Series
 (1988). The products are completely biodegradable owing to their natural
 polypeptide structure based on .alpha.-linked L-aspartic acid.
 They can be employed, for example, as cobuilders in detergents and
 cleaners, for inhibiting and dispersing deposits in cooling and heating
 circulations for diminishing and dispersing deposits, and for reducing
 corrosion and inhibiting gas hydrates in petroleum and natural gas
 production.

EXAMPLE 1
 Culture conditions, extraction and purification of cyanophycin:
 The cyanobacterium Aphanocapsa PCC6308 is incubated in a 10 l fermenter
 (batch culture) with 9 l of BGII medium under phototrophic conditions
 (6000 lux, l/d cycle 12/12) at 30.degree. C. and supplied with air (200
 ml/min). Before the cells reach the stationary phase (after 14 days with
 an optical density OD.sub.665 of about 1.6), 10 mg/l L-arginine and/or 200
 mg/l NaNO.sub.3 and 5 mg/ml chloramphenicol are added to the medium, and
 then the cell suspension is incubated for a further 48 h with reduced
 light (600 lux) and at lower temperature (20.degree. C.). The cells are
 harvested by centrifugation at 10 000 xg and washed twice in distilled
 water. The cell pellet (about 25 g wet weight, about 3 g dry matter) is
 taken up in 100 ml of an aqueous buffer solution (pH 7.2). The cells are
 disrupted by ultrasound treatment and then stirred at 4.degree. C. for 15
 h. The crude cyanophycin is pelleted by centrifugation at 30 000 xg. The
 crude cyanophycin is resuspended in 60 ml of H.sub.2 O. The supernatant (S
 1000) obtained by three fractional centrifugations at 1 000 xg is
 subjected to a centrifugation at 30 000 xg, and the pellet obtained in
 this way is dissolved in 0.1N HCl (yield: about 1000 mg of native
 cyanophycin). The native cyanophycin dissolved in 0.1N HCl is finally
 purified by retritration three times in 1N NaOH (yield: about 150 mg of
 cyanophycin).
 The strain Aphanocapsa (=Synechocystis) PCC6308 was originally isolated in
 1949 and G. C. Gerloff from a lake in Wisconsin (USA) and was described
 for the first time by Gerloff et al. in 1950 (Gerloff, G. C., Fitzgerald,
 G. P. & Skoog, F. 1950. The isolation, purification and nutrient solution
 requirements of blue-green algae. In Proceedings of the Symposium on the
 Culturing of Algae, pp. 27-44. Dayton, Ohio, U.S.A.: Charles F. Kettering
 Foundation).
 The strain Synechocystis PCC 6308 used in the present application was
 deposited in the name of Bayer AG, 51368 Leverkusen, on Feb. 19, 1998 at
 the DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH,
 Mascheroder Weg 16, D-38124 Brunswick with the number DSM 12037.
 EXAMPLE 2
 Basic hydrolysis of cyanophycin
 500 mg of cyanophycin from Example 1 are suspended [lacuna] 5 ml of water.
 75 mg of NaOH (100%) are added, and the mixture is heated at 90.degree. C.
 with stirring for 12 h. The mixture is then cooled to room temperature and
 filtered. The residue remaining is a mixture of arginine and unreacted
 cyanophycin. The filtrate contains the sodium salts of the aspartic acid
 homo- and copolymers.
 Determination of the calcium carbonate inhibiting capacity by modification
 of the NACE.sup.1) method: TM 0374-90
 .sup.1) NACE: National Association of Corrosion engeneers
 Starting materials:
 Solution 1:
 12.15 g of calcium chloride dihydrate, analytical grade
 3.68 g of magnesium chloride hexahydrate, analytical grade made up to 1000
 ml of solution with distilled water.
 Solution 2:
 7.36 g of sodium bicarbonate, analytical grade made up to 1000 ml of
 solution with distilled water.
 Solutions 1. and 2. must each be made up freshly and saturated with
 CO.sub.2 before use thereof.
 100 ml of solution 1. are mixed with 1,2,3,5,10 ppm inhibitor (active
 substance) based on the complete test mixture. Then 100 ml of solution 2.
 are added.
 The test mixture is then mixed by shaking in a closed vessel and stored in
 a waterbath preheated to 70.degree. C. for 16 h. (For comparison, a sample
 without added inhibitor is included in the test series.) After this time,
 all the samples are removed simultaneously from the waterbath and cooled
 to 30.degree. C. 5 ml portions are taken from all the samples, filtered
 through a 0.45 .mu.m filter and acidified with hydrochloric acid for
 stabilization.
 The calcium content in the filtrate is determined by titration with an
 indicator or by atomic absorption spectroscopy.
 The inhibiting capacity is calculated as follows:
 ##EQU1##
 a: amount of calcium in the sample
 b: amount of calcium in the blank after heat treatment
 c: amount of calcium in the blank before heat treatment