WO2002012508A2 - Method for improved production of cyanophycin and the secondary products thereof - Google Patents

Abstract

The invention relates to thermostable cyanophycin synthetases and a method for the improved production of cyanophycin and/or the secondary products thereof.

Description

Process for the improved production of cyanophycin and its derivatives

The present invention relates to thermostable cyanophycin synthetases, transformed organisms containing such an enzyme and a process for the improved production of cyanophycin and / or its secondary products, for example polyaspartic acid or arginine.

Multi-L-Arginyl-Poly-L-aspartate (cyanophycin) is a branched polypeptide that contains aspartic acid and arginine in an approximately equimolar ratio. The chemical structure corresponds to a poly-α-aspartate backbone with arginine side residues, which are linked to almost all β-carboxyl groups of the backbone via peptide bonds.

DE-A 198 25 509 describes the identification, cloning and heterologous expression of the gene for the cyanophycin synthetase from Synechocystis sp. PCC 6803. The enzyme activity is determined here by means of a radioactive test in which L-fU- ¹ C] -arginine is incorporated into cyanophycin from Synechocystis sp. PCC 6308 as a “primer” (precursor). The temperature optimum of the enzyme is here at 28 ° C. ,

DE-A 197 09 024 describes the extraction and purification of cyanophycin from Synechocystis sp. PCC 6308 known, the synthesis is carried out at 20 ° C.

DE-A 198 13 692 only discloses the isolation of the cyanophycin synthase gene from Synechocystis sp. PCC 6803 or Anabaena variahilis sp. ATCC 29413. Process engineering features for the production of cyanophycin are not described here. FEMS Microbiology Letters 181 (1999) 229-236 describes the production of cyanophycin with Synechococcus sp. MA 19 known. However, the synthetic enzyme is not described for this.

A disadvantage of the production of cyanophycin according to the processes known hitherto is that a relatively narrow temperature range, generally below 35 ° C., must not be exceeded for optimum product yield. This represents a considerable restriction of the degrees of freedom within the process control for the production of cyanophycin and / or its secondary products.

It is therefore desirable to produce cyanophycin and / or its secondary products at substantially higher temperatures than previously described, combined with greater flexibility in process control and significantly improved product yields.

This object is achieved by the present invention.

The present invention relates to cyanophycin synthetases with increased stability and activity in the temperature range of> 35 ° C., characterized in that they

a) an amino acid sequence according to SEQUENCE PROTOCOL 1 encoded by an isolated nucleotide sequence according to SEQUENCE PROTOCOL 2, an allele, homologue or derivative of this nucleotide sequence or a nucleotide sequence hybridizing therewith and from Synechocystis sp. PCC

6308, or

b) that it codes an amino acid sequence in accordance with SEQUENCE PROTOCOL 3 by an isolated nucleotide sequence in accordance with SEQUENCE PROTOCOL 4 an allele, homologue or derivative of this nucleotide sequence or one with this have hybridizing nucleotide sequence and from Synechococcus sp. MA 19 originate.

In a preferred variant of the present invention, the cyanophycin synthetases according to the invention have an optimum temperature in the range from 35 ° C. to 55 ° C., preferably in the range from 35 ° C. to 50 ° C.

The cyanophycin synthetases according to the invention are thermostable enzymes.

The present invention also relates to isoenzymes of the invention

Cyanophycin synthetases. This includes enzymes with the same or comparable substrate and activity specificity, but which have a different primary structure. In addition, the present invention also includes modified forms of the cyanophycin synthetases. According to the invention, this includes enzymes in which there are changes in the sequence, for example at the N- and / or C-terminus of the polypeptide or in the region of conserved amino acids, but without impairing the function of the enzymes. These changes can be made by exchanging one or more amino acids according to known methods.

A special embodiment variant of the present invention comprises variants of the cyanophycin synthetases according to the invention, the substrate specificity of which is weakened or enhanced, for example by amino acid exchange, compared to the respective starting protein. The same applies to the stability of the enzymes according to the invention in the cells, which are, for example, more or less susceptible to degradation by proteases.

The present invention furthermore relates to polypeptides with the function of a cyanopyhcin synthetase, the amino acid sequence of which has been modified in such a way that they are resistant to compounds having a regulatory action, for example those which they contain their activity-regulating metabolic end products are desensitive (feedback-desensitive).

According to the invention, an isolated nucleotide sequence or an isolated nucleic acid fragment is to be understood as a polymer from RNA or DNA which can be single or double-stranded and optionally can contain natural, chemically synthesized, modified or artificial nucleotides. The term DNA polymer also includes genomic DNA, cDNA or mixtures thereof.

According to the invention, alleles are functionally equivalent, i.e. H. to understand essentially equivalent nucleotide sequences. Function-equivalent sequences are those sequences which, despite a different nucleotide sequence, for example due to the degeneracy of the genetic code, still have the desired functions. Functional equivalents thus include naturally occurring variants of the sequences described here, as well as artificial, e.g. B. obtained by chemical synthesis and possibly adapted to the codon use of the host organism nucleotide sequences. In addition, functionally equivalent sequences include those which have a modified nucleotide sequence which, for example, imparts a desensitivity or resistance to inhibitors to the enzyme.

A functional equivalent is also understood to mean, in particular, natural or artificial mutations in an originally isolated sequence which continue to show the desired function. Mutations include substitutions, additions, deletions, exchanges or insertions of one or more nucleotide residues.

Also included here are so-called sense mutations, which can lead to the exchange of conserved amino acids at the protein level, but which do not lead to a fundamental change in the activity of the protein and are therefore function-neutral. This also includes changes in the nucleotide sequence that are at the protein level the N- or C-terminus of a protein affect, but without significantly affecting the function of the protein. These changes can even have a stabilizing influence on the protein structure.

Furthermore, such nucleotide sequences are also, for example, by the present

Includes invention, which is obtained by modification of the nucleotide sequence, resulting in corresponding derivatives. The aim of such a modification can e.g. B. further narrowing down the coding sequence contained therein or e.g. also be the insertion of further restriction enzyme recognition sites. Functional equivalents are also those variants whose function is weakened or enhanced compared to the original gene or gene fragment.

In addition, artificial DNA sequences are the subject of the present invention, as long as they impart the desired properties as described above and can be incorporated or appended into the gene of the cyanophycin synthetase according to the invention. Such artificial DNA sequences can be determined, for example, by back-translating proteins created using computer-aided programs (molecular modeling) or by in-vitro selection. Coding DNA sequences are particularly suitable which are obtained by back-translating a polypeptide sequence in accordance with the codon sequence specific for the host organism.

Usage. The specific codon usage can easily be determined by a person familiar with molecular genetic methods by computer evaluations of other, already known genes of the organism to be transformed.

According to the invention, homologous sequences are to be understood as those which are complementary to and / or hybridize with the nucleotide sequences according to the invention. According to the invention, the term hybridizing sequences includes substantially similar nucleotide sequences from the group of DNA or RNA which, under known stringent conditions, enter into a specific interaction (binding) with the aforementioned nucleotide sequences. This also includes short nucleotide sequences with a length of, for example, 10 to 30, before moves 12 to 15 nucleotides. According to the invention, this also includes so-called nucleotide primers or probes.

According to the invention, the preceding (5'- or upstream) and / or subsequent (3'- or downstream) coding regions (structural genes) are also

Sequence areas included. In particular, this includes sequence areas with a regulatory function. You can influence the transcription, the RNA stability or the RNA processing as well as the translation. Examples of regulatory sequences include a. Promoters, enhancers, operators, terminators or translation enhancers.

The present invention furthermore relates to a gene structure comprising at least one of the previously described nucleotide sequences coding for a cyanophycin synthetase and to regulatory sequences which are operatively linked thereto and which control the expression of the coding sequences in the host cell.

An operational link is understood to mean the sequential arrangement of, for example, the promoter, coding sequence, terminator and, if appropriate, further regulatory elements in such a way that each of the regulatory elements is its own

Can perform function in the expression of the coding sequence as intended. These regulatory nucleotide sequences can be of natural origin or can be obtained by chemical synthesis. In principle, any promoter which can control gene expression in the corresponding host organism is suitable as the promoter. According to the invention, this can also be a chemically inducible promoter by means of which the expression of the genes underlying it in the host cell can be controlled at a specific point in time. A promoter that can be induced by IPTG (isopropyl-β-thiogalactopyranoside) is mentioned here as an example. A gene structure is produced by fusing a suitable promoter with the nucleotide sequence according to the invention using common recombination and cloning techniques as are known in the literature. To connect the DNA fragments to one another, adapters or linkers can be attached to the fragments.

In addition, the present invention relates to a vector containing at least one nucleotide sequence of the type described above coding for a cyanophycin synthetase specific for the production of cyanophycin, regulative nucleotide sequences operatively linked thereto and additional nucleotide sequences for selecting transformed host cells, for replication within the host cell or for Integration into the corresponding host cell genome. Furthermore, the vector according to the invention can contain a gene structure of the aforementioned type.

As vectors are those that are in microorganisms, such as. B. bacteria,

Mushrooms and / or plants are replicated. Known vectors are, for example, pBluescript (Stratagene, 11099 North Torney Pines Red., La Jolla, CA 92 037, USA) or pEKO (Eikmanns, BJ. Et al., Gene, 1991, 102: 93-98). However, this list is not limiting for the present invention.

Using the nucleic acid sequences according to the invention, appropriate probes or nucleotide primers can be synthesized and used to amplify and isolate, for example, genes from other single or multicellular organisms, preferably bacteria, fungi, algae or plants, using the PCR technique.

The present invention thus also relates to a probe for identifying and / or isolating genes coding for proteins involved in the biosynthesis of cyanophycin, preferably further thermostable cyanophycin synthetases, this probe being produced on the basis of the nucleic acid sequences of the type described above and one for detection suitable Contains marker. The probe can be a partial section of the sequence according to the invention, for example from a conserved area which, for. B. has a length of 10 to 30 or preferably 12 to 15 nucleotides and can hybridize specifically with homologous nucleotide sequences under stringent conditions. Suitable markings are well known from the literature.

The present invention further relates to the transfer of the nucleic acid sequence according to the invention or a part thereof coding for a cyanophycin synthetase, an allele, homolog or derivative thereof or a nucleotide sequence hybridizing with these sequences into a heterologous host system. This also includes the transfer of a gene construct or vector according to the invention into a heterologous host system.

According to the invention, a heterologous host system is a single-cell or multi-cell system

To understand organism. Examples of these are microorganisms, fungi, lower or higher plants, tissues or cells thereof. According to the invention, bacteria are preferred, particularly preferred the enterobacterium genus and in particular the Escherichia coli species or coryneform bacteria, in particular the genus Corynebacterium or Brevibacterium, especially Corynebacterium glutamicum.

The nucleotide sequence coding for a thermostable cyanophycin synthetase according to the invention is transferred into one of the host systems mentioned above by known methods. Examples of methods for DNA transfer into suitable host systems are transformation, electroporation, conjugation or agrobacterial-mediated DNA transfer or “particle bombardment”. These lists are only used to explain the present invention and are not limiting.

A transformed single or multicellular organism resulting from a successfully carried out nucleic acid transfer thus differs from that corresponding non-transformed organism in that it contains additional nucleic acids of the type according to the invention and can express them accordingly.

The present invention thus also relates to a transformed single or multicellular organism containing a cyanophycin synthase according to the invention and / or a vector containing a cyanophycin synthetase of the type described above.

The present invention further relates to a method for providing a cyanophycin synthetase according to the invention of the type described above, the nucleotide sequence coding for the enzyme being isolated from a single- or multicellular organism, optionally operatively linked to regulatory structures and or in one for heterologous expression suitable vector is cloned, possibly transferred to a heterologous host system, expressed there and then isolated from this host system and, if necessary, purified and / or enriched.

It is also conceivable to directly isolate an amount of cyanophycin synthetases of the type described above sufficient for the synthesis of cyanophycin from an organism, without prior enrichment in a heterologous one

System.

Furthermore, the enzymes of the cyanophycin synthetases of the type described above can then be used, for example, in an in vitro system for the synthesis of cyanophycin and / or its secondary products.

In particular, polyaspartic acids can be modified with the aid of a cyanophycin synthetase of the type described above in such a way that graft copolymers are formed when the polyaspartic acids are used as primers in the enzymatic polymerization reaction (Table 1, Table 2). In Vitro Synthesis of graft copolymers of cyanophycin

Table 1: Influence of polymers containing aspartic acid on the incorporation of arginine by the cyanophycin synthetase from Synerhocystis sp. PCC 6308

Compound used as primer Incorporation of arginine [% of maximum]

Cyanophycin 100

Without primer 0.2

Poly-α, β-D, L-aspartic acid 97

Mw: -3500

Poly-α, β-D, L-aspartic acid 49

Mw: -7500

Poly-α, β-D, L-aspartic acid 25

Mw: -19000

The amount of arginine incorporated is determined after overnight incubation using the method described below (cyanophycin synthetase enzyme test, see below). The polymeric primers are added to the reaction mixture at a final concentration of 0.87 mg / ml.

The polyaspartic acids are produced by chemical synthesis from maleic acid and ammonia or from aspartic acid. Table 2: Influence of aspartic acid-containing polymers and N-acetylglucosamine on the incorporation of arginine by the purified cyanophycin synthetase from Synechococcus sp. MA 19.

c. The amount of arginine incorporated is determined after overnight incubation using the method described below (cyanophycin synthetase enzyme test). The substances used as primers are used in a final concentration of 1.0 mg / ml.

The present invention also relates to a process for the preparation of cyanophycin and / or its secondary products, a cyanophycin synthetase and / or a vector and / or a transformed single or multicellular organism of the type described above being used. However, the present invention includes not only the production of cyanophycin and / or its secondary products in a living host system, but also the in vitro synthesis of cyanophycin using an isolated cyanophycin synthetase of the type described above.

The process according to the invention for the production of cyanophycin is characterized in that the enzyme-catalyzed synthesis takes place in a temperature range from 20 ° C. to 60 ° C., preferably in a range from 35 ° C. to 55 ° C. The influence of temperature on the activity and stability of the enzymes Synechocystis sp. PCC 6308 and from Synechococcus sp. MA 19 is shown in FIGS. 2 to 5. As can be seen in FIG. 5, the stability of the enzyme from Synechococcus sp. MA 19 can also be increased. Umg ^"1 means the unit of specific activity in units per mg protein; in the case of cyanophycin synthetase, 1 U = 1 nmol arginine is incorporated in cyanophycin pro

Minute ie Umg ^"1 = nmol ^• min ^{" 1 •} mg ^"1 .

The process according to the invention is advantageously characterized in that the process is less susceptible to faults due to the wide temperature range, in particular above 28 ° C., allows greater variability in the process control and thus delivers an improved product yield. The production of cyanophycin and / or its secondary products according to the invention is thus substantially more reproducible and more economical than the previously known processes.

At the molecular level, the cyanophycin synthetase according to the invention catalyzes an ATP-dependent chain extension reaction (elongation). This requires both monomers, ATP, Mg ²⁺ , K ⁺ , a sulfhydryl reagent and small amounts of cyanophycin as a primer. The dependence of the cyanophycin synthetases according to the invention on their substrates, cosubstrates, arginine-analogous compounds and other substances of the different enzyme preparations are shown in Table 3, Table 4 and Table 5.

FIG. 7 shows in particular the influence of arginine and aspartic acid-like compounds on the by the purified cyanophycin synthetase from Synechocystis sp. PCC 6308 catalyzed incorporation of arginine and aspartic acid into

Cyanophycin shown. This clearly shows that on the one hand canavanine and lysine instead of arginine and on the other hand aspartic acid-β-methyl ester and asparagine instead of aspartic acid can be incorporated into the polymer. Table 3: Dependence of the cyanophycin synthetase activity in the soluble cell fraction on substrates, cosubstrates and the arginine analogue canavanine ^d .

Enzyme activity of Synechocystis sp. PCC6308 recombinant E. coli [% of maximum]

Complete 00 100 Without enzyme <0.2 <0.2

Heat-inactivated extract ³ <0.2 <0.2 Without ATP <0.2 <0.2 Without cyanophycin 2.5 0.9 Without L-aspartic acid 26.0 15.5 Without L-arginine ^f 77.0 55.2 L-glutamic acid instead of L-arginine S 60.0 <0.2 L-glutamic acid instead of L-aspartic acid ^h 36.0 1.6 L-glutamic acid as the only amino acid substrate 27.0 0.9

Plus 1 μM canavanine n.b. 40.0 Plus 10 μM canavanine n.a. 28.4 Plus 100 μM canavanine n.a. 12.1

The activities were determined as the rate of incorporation of the amino acid monomers in cyanophycin within 15 min after the start of the reaction in the soluble cell fraction of Synechocystis PCC6308 and of IPTG-induced E. coli TOP 10 {pSK :: cphA _co ). The activities, which corresponded to 100% in each case, were 9.1 nmol arginine min ^"1 (g protein) ^_1 for Synechocystis PCC6308 and 26.6 nmol arginine min ^{" 1} (g protein) ^_1 for E. coli.

The soluble cell fraction was incubated at 95 ° C for 2 min. f. The activity was determined using 0.1 mM L [U- ¹ C] aspartic acid as substrate.

G. The activity was determined using 0.01 mM L [U- ¹ C] glutamic acid as substrate.

H. The activity was determined with 0.1 mM L [U- ¹⁴ C] glutamic acid as substrate.

n. b., not determined.

Table 4: Incorporation of different radiolabeled amino acids in cyanophycin by purified cyanophycin synthetase from Synechocystis sp. PCC 6308.

i. The complete reaction mixture is described below

Cyanophycin synthetase enzyme test listed. k. The final concentrations of the radiolabeled compounds are as follows: L- [U- ¹⁴ C] arginine, 0.5 mM; L- [U- ¹⁴ C] aspartic acid; 5 mM; L- [U- ³ H] - canavanine, 1 mM; L- [4,5- ³ H] lysine, IMM; L- [U- ¹⁴ C] -glutamic acid, 0.5 mM (if used instead of L-arginine), 5 mM (used instead of L-aspartic acid).

Table 5: Incorporation of different radiolabeled amino acids in cyanophycin by purified cyanophycin synthetase from Synechococcus sp. MA 19.

1. The complete reaction mixture is described below

Cyanophycin synthetase enzyme test listed. m. The final concentrations of the radiolabeled compounds were as follows: L- [U- ¹⁴ C] aspartic acid, 10 µM; all other 5 μM.

n. n. d., undetectable.

Without the addition of L-aspartate to the reaction mixture, the initial rate of cyanophycin synthesis in extracts from Synechocystis sp. PCC6308 drastically. The absence of L-arginine only leads to a moderate decrease in cyanophycin synthesis. The time course of the incorporation of these amino acid monomers into the cyanophycin is shown in FIG. 1.

This means that L-aspartate is only added to the polymer end. The presence of both monomers is required for further elongation.

The present invention further relates to an improved cyanophycin synthetase enzyme test combined with an improved separation of the enzymatically formed cyanophycin from the reaction mixture. The cyanophycin formed is first washed after its precipitation from the reaction mixture and the precipitate (pellet) is then resuspended in an acidic aqueous medium. The cyanophycin goes into solution and can be removed from the supernatant and used directly for determining the concentration in a scintillation counter. This procedure is distinguished from the known methods in that a large number of washing steps are dispensed with, no transfer of insoluble cyanophycin is required, the amount of radioactive waste is drastically reduced, and the cyanophycin by solubilization under acidic conditions of others Impurities in the precipitate are cleaned and a homogeneous cyanophycin solution is available, from which a much more precise and reproducible determination can be made by means of scintillation counting than from a dispersion, as was previously customary. A Comparative comparison of the isolation of cyanophycin from the reaction mixture is shown in flow diagrams in FIG. 6.

The present invention further relates to the use of a vector containing a cyanophycin synthetase according to the invention of the aforementioned type for producing a transformed single or multicellular organism as described above. The use of such a transformed single- or multi-cell organism for the production of a cyanophycin synthetase according to the invention and / or for the production of cyanophycin and / or its secondary products is also included in the present invention. In addition, a cyanophycin synthetase isolated according to the invention can also be used for the in vitro production of cyanophycin and / or its secondary products. In addition, the use of cyanophycin for the production of food supplements and / or agents in the field of agriculture and / or crop protection is covered in the present invention. Further areas of application of cyanophycin and / or its secondary products can be seen in the paper, textile, pigment, lacquer, ceramic, building material or detergent industry and in the areas of water and wastewater treatment.

Areas of application of the secondary products of cyanophycin are agents in the field of agriculture and / or crop protection, in the paper, textile, pigment, varnish, ceramic, building material or detergent industry as well as in the areas of water and waste water treatment.

The present invention is characterized in more detail by the following examples, which, however, are not limiting for the invention: General methods:

The cultivation of microorganisms such as Escherichia coli or Corynebacterium glutamicum and the production of cell extracts was carried out according to standard methods described in Sambrook, J. et al. (1998, Molecular Cloning: A Laboratory Manual, 2 ^nd Edition, Cold Spring Harbor Laboratory Press, NY.) Or according to the manufacturer's instructions. E. coli TOP 10 (Invitrogen, San Diege, USA) and C. glutamicum wild type DSM 20 300 are used as bacterial strains. The cultivation of blue-green algae, such as. B. Synechocystis PCC 6308 was carried out as described in Rippka R. et al. (J. Gen. Microbiol, 1979, 111: 1-61). For the isolation, manipulation and transfer of DNA, the polymerase chain reaction

(PCR) and the procedures for analyzing proteins by means of SDS polyacrylamide gel electrophoresis (SDS-PAGE) are also based on Sambrook, J. et al. (1998, Molecular Cloning: A laboratory Manual, 2 ^nd Edition, Cold Spring Harbor Laboratory Press, NY.) Referenced.

Cloning of the genes cph A and cph B from Synechocystis PCC 6308

The genes cphA coding for the cyanophycin synthetase and cphB coding for the cyanophycinase were gradually extracted from the total DNA of Synechocystis PCC6308 by three polymerase chain reactions (PCR) using the following

Oligonucleotides (primer) amplified: primer Pl [5'-

ATGGG (AGCT) CA (CT) AT (ACT) GT (ACT) GA (AG) CA (CT) GT-3 '] and primer P2 [5'-A (AG) (AGCT) GC (AG) TT (AGCT ) GC (AGT) ATCAT (AG) AA-3], both of which had been derived from the conserved amino acid sequence MGHTVΕHV and PFMIANA of the cyanophycin synthetases from Anabaena variabilis (Ziegler et al.

1998, Eur. J. Biochem. 254, 154, 1998) and Synechocystis PCC6803 (translation product of the open reading frame slr 2002 from the gene bank CyanoBase, Kaneko et al. 1996, DNA Res. 3.109; www.kazusa.or.jp/cyano); Primer P3 [5'-ATGGG (AGCT) CA (CT) CA (CT) GATAT (CT) GC (AGCT) GG-3 '], which had been derived from the conserved amino acid sequence MGHHMIAG

A. variabilis cyanophycinases (Ziegler et al. 1998, Eur. J. Biochem. 254,154, 1998) and Synechocystis PCC6803 (translation product of the open reading frame slr 2001, CyanoBase); Primer P4 (5'-TTGGCGATCATAAAAGGCGC-3 '), the sequence of which is complementary to the central region of Synechocystis sp. PCC6308 cphA gene; Primer P5 (5'-GCGCAAGTATCTTCATCGATACCAA-3 '), the sequence of which is complementary to the central region of Synechocystis sp. PCC6308 cphB gene and primer P6 (5-TGGCGGCGGTGTATGAAAAC-3 '), the sequence of which part of the Synechocystis sp. PCC6308 represents cphA gene. For the heterologous expression of cphA in E. coli, a genomic region comprising the 3 'end of cphB, the intergenic region between cphB and cphA and the complete cphA gene was created using primer P3 (see above) and primer P7 (5'-

TTCAGAATTCACTACTCACTATTC-3 '), the sequence of which is complementary to the 3' end of cphA, amplified. The primers were obtained from MWG-Biotech AG (Ebersberg). Vent DNA polymerase (New England Biolabs, Schwalbach, Taunus) was used for the PCR according to the information in the PCR application manual (Biochemica 1995, Boehringer Mannheim). After electrophoretic separation, the PCR products were isolated from agarose gels, ligated with EcoRV-linearized vector pBluescript SK- (Stratagene Cloning Systems, San Diego, Calif, USA) and according to E. coli TOP 10 (Invitrogen, San Diego, Calif, USA) transferred.

Cyanophycin synthetase enzyme test

The method is a modification of the method described by Ziegler et al. in Eur. J. Biochem. 254, 154 (1998). The reaction mixture contained in a total volume of 0.125 ml 50 mM Tris, 20 mM MgCl ₂ , 20 mM KC1, 4 mM ATP,

10 mM 2-mercaptoethanol, 0.1 mM L-aspartic acid, 0.034 mM chloramphemcol, 0.01 mM L- [U-14C] -arginine (Amersham Pharmacia Biotech, Freiburg; specific radioactivity: 10.3 Gbq / mmole), 0.87 mg cyanophycin per ml (isolated from Synechocystis sp.PCC6308) and 0.2 to 0.5 mg protein from the soluble cell fraction. After incubation for usually 15 minutes at a temperature of usually 28 ° C, 1 ml of ice-cold water was added to the reaction mixture to stop the reaction. The insoluble cyanophycin under these conditions was separated by centrifugation (15 min, 14000 x g) and washed twice with 1 ml each of 50 mM Tris-HCl, 2 mM EDTA (pH 8.2). The pellet was resuspended in 1.0 ml of 1.5 M HC1. The suspension was centrifuged again.

0.5 ml of the supernatant, which contained the cyanophycin soluble under these conditions, was removed and mixed with 5 ml of LSC scintillation cocktail HYDROLUMA (J. T. Baker, Deventer, the Netherlands). The radioactivity was measured with a scintillation measuring device model LS 6500 (Beckman Instruments GmbH, Munich).

Purification and determination of cyanophycin

The isolation of cyanophycin from living cells of Synechocystis PCC 6308 or heterologous systems is carried out according to the method of Simon, R.D. (Biochim

Biophys Acta, 1976, 422: 407-418). A colorimetric quantification of the cyanophycin is carried out using Sakagushi reagent according to a method described by Allen, MM (Methods Enzymol., 1988, 167: 207-213). The amino acid composition of cyanophycin is determined by means of high pressure liquid chromatography (HPLC). Legend for the figures and tables:

1: SEQUENCE LIST 2 of the nucleotide sequence of the cyanophycin synthetase gene according to the invention coding for the cyanophycin synthetase according to the invention from Synechocystis PCC 6308.

2: SEQUENCE PROTOCOL 1 of the amino acid sequence of the thermostable cyanophycin synthetase according to the invention derived from the corresponding cyanophycin synthetase gene of the Synechocystis PCC 6308.

Fig. 1: Time course of the incorporation of the amino acid monomers in cyanophycin. The built-in content of L-arginine was determined at certain times (min) in a reaction mixture totaling 125 μl, which contains the following components: 1.25 nmol radioactive L [U- ¹⁴ C] arginine, 12.5 nmol unlabeled L-aspartate, 500 nmol ATP, 0.38 mg soluble cell protein of the in

Presence of IPTG cultured, transformed E. coli strain TOP 10 and 0.11 mg cyanophycin as a primer (Δ). The content of L [U- ¹⁴ C] asparagine, which is incorporated in the absence of L-arginine, was measured in the presence of 0.11 mg (D) or 0.05 mg ( ^• ) cyanophycin.

2 and 3: Influence of temperature on the by the purified cyanophycin synthetase from Synechocystis sp. PCC 6308 catalyzed incorporation of arginine in cyanophycin. Fig. 3 a) cyanophycin synthetase activity as a function of the incubation temperature. The enzyme activity was determined at a time interval of 4 min after the start of the reaction at the indicated

Temperature determined. Fig. 3b) Time course of the incorporation of arginine in cyanophycin at 28 ° C (-D-) and at 50 ° C (- • -). The amount of arginine incorporated was determined at the times indicated in 125 μl reaction mixture. Fig. 4: Temperature profile (- • -) and heat stability (- ♦ -) of the purified cyanophycin synthetase from Synechococcus sp. MA 19. For the temperature profile, the enzyme activities were determined at a time interval of 15 min after the start of the reaction at the specified temperature. The heat stability of the enzyme was determined as the activity (measured at 28 ° C.) after every 30 min incubation of the enzyme solution at the temperatures indicated.

5: Stability of the purified cyanophycin synthetase from Synechococcus sp. MA 19 at 50 ° C as a function of the incubation time in absence (- ♦ -) and

Present (- ■ -) of 1 M ectoin. The enzyme preparation was incubated at 50 ° C and the activity was determined at the indicated times (at 28 ° C).

Fig. 6: Flow diagrams of the isolation of cyanophycin from the reaction mixture in comparison (A) according to Simon, R.D. (Biochim Biophys Acta, 1976, 422: 407-418), (B) according to Ziegler, K. et al. (Eur. J. Biochem., 1998, 254: 154-159) and (C) by the method according to the invention. The insoluble cyanophycing granules are shown as spheres; Transfer steps are shown by curved arrows.

SEQUENCE LIST 4 of the nucleotide sequence of the cyanophycin synthetase gene according to the invention coding for the cyanophycin synthetase from Synechococcus sp MA 19 according to the invention.

SEQUENCE LIST 3 of the nucleotide sequence of the thermostable cyanophycin synthetase according to the invention derived from the corresponding cyanophycin synthetase gene of Synechococcus sp MA 19.

7a and 7b: Influence of connections that have structural similarity

Have aspartic acid and arginine. Fig. 7a) incorporation of aspartic acid (Hatched columns) and arginine (shaded columns) after adding 5 mM of the specified compound to the otherwise complete reaction mixture. Fig. 7b) Influence of the specified compounds when these in a concentration of 5 mM instead of aspartic acid (hatched columns) or in a concentration of 0.5 mM instead of arginine in the

Reaction mixture were given.

The abbreviations of the amino acids are determined according to the standard one-letter method.

Claims

claims: 1. Cyanophycin synthetases with an activity within a temperature range of 35 ° C to 55 ° C, characterized in that they a) an amino acid sequence according to SEQUENCE PROTOCOL 1 encoded by an isolated nucleotide sequence according to SEQUENCE PROTOCOL 2 has an allele, homologue or derivative of this nucleotide sequence or a nucleotide sequence hybridizing with it and originating from Synechocystis PCC 6308 or b) that they have an amino acid sequence according to SEQUENCE PROTOCOL 3 encoded by an isolated nucleotide sequence according to SEQUENCE PROTOCOL 4, an allele, homolog or derivative of this nucleotide sequence or a nucleotide sequence hybridizing with this and from Synechococcus sp. MA 19 originate. 2. Vector containing at least one nucleotide sequence coding for a cyanophycin synthetase specific for the production of cyanophycin according to claim 1. 3. Transformed single or multicellular organism containing a cyanophycin synthetase according to claim 1 and / or a vector according to claim 2. Transformed single or multicellular organism according to claim 3, characterized in that it is a microorganism, a fungus, a lower or higher plant, tissue or a cell thereof. 5. Transformed organism according to claim 4, characterized in that it is a bacterium of the genus of enterobacteria or coryneform bacteria. 6. The method for providing a cyanophycin synthetase according to claim 1, characterized in that the nucleotide sequence coding for the enzyme is operatively linked to regulatory structures and / or cloned into a vector suitable for heterologous expression, transferred into a heterologous host system, expressed and out this host system is isolated and purified and / or enriched. 7. A process for the preparation of cyanophycin and the derived products to be produced therefrom, characterized in that a cyanophycin synthetase according to claim 1 and / or a vector according to claim 2 and / or a transformed single or multicellular organism according to one of claims 3 to 5 is used becomes. 8. Use of a vector according to claim 2 for the production of a transformed single or multicellular organism according to one of claims 3 to 5. 9. Use of a transformed single or multicellular organism according to one of claims 3 to 5 for the production of a cyanophycin synthetase according to claim 1 and / or for the production of cyanophycin. 10. Use of a cyanophycin synthetase according to claim 1 for the production of cyanophycin. 11. Isoenzymes and modified forms of cyanophycin synthetase, characterized in that they are obtained by modification of the cyanophycin synthetase from Synechocystis PCC 6308. 12. Isoenzymes and modified forms of the cyanophycin synthetase according to claim 11, characterized in that they are obtained by amino acid exchange. 13. Isoenzymes and modified forms of the cyanophycin synthetase according to claim 12, characterized in that the amino acid exchange takes place by modification of the nucleotide sequence of the underlying gene. 14. Artificial DNA sequences, characterized in that these are incorporated or appended to the cyanophycin synthetase gene according to claim 1. 15. Heterologous host systems containing a nucleic acid sequence or a part thereof coding for a cyanophycin synthetase, an isoenzyme or a modified form thereof according to claims 1 and 11. 16. Use of cyanophycin prepared according to claim 10 for the synthesis of polyaspartic acid or arginine. 17. Use of the cyanophycin synthetase in vitro for the production of polyaspartic acid-cyanophycin graft polymers. 18. Use of cyanophycin for the production of nutritional supplements and / or agents in the field of agriculture and / or Plant protection and / or in the paper, textile, pigment, paint, ceramic, building material or detergent industry as well as in the areas of water and wastewater treatment. 19. Use of secondary products of cyanophycin for the production of Funds in the field of agriculture and / or crop protection and / or in the paper, textile, pigment, lacquer, ceramic, building material or detergent industry as well as in the areas of water and waste water treatment.