Method for obtaining monatin

FIELD: biotechnologies.

SUBSTANCE: invention represents the method for obtaining 2S,4R-monatin or its salt providing the contact of 4R-IHOG with aminotransferase of L-amino acid in presence of L-amino acid and method for 2S,4R-monatin or its salt obtaining with provision of 2S,4R-monatin isomerisation. The present invention also reveals aminotransferases of L-amino acid for performing the methods of monatin obtaining and method for obtaining such L-aminotransferases providing the cultivation of bacterial cell, into which there input is an expression vector containing polynucleotide that codes the presented aminotransferase.

EFFECT: invention allows obtaining 2S,4R-monatin and 2R,4R-monatin with improved output due to the use of revealed aminotransferases of L-amino acid.

27 cl, 6 dwg, 23 tbl, 48 ex

 

The text descriptions are given in facsimile form.

1. The method of obtaining the 2S,4R-Sonatina or its salts, providing contacts 4R-IHOG with normal L-amino acids in the presence of L-amino acids, with the formation of 2S,4R-Sonatina, where in which aminotransferase L-amino acid consists of an amino acid sequence that is 90% or more identical to the amino acid sequence represented by the sequence SEQ ID NO:2, SEQ ID NO:48, SEQ ID NO:53, SEQ ID NO:61, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:95, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:101, SEQ ID NO:103, SEQ ID NO:105, SEQ ID NO:107, SEQ ID NO:109 or SEQ ID NO:111.

2. The method of receiving according to claim 1, additionally providing for the engagement of the keto-acid decarboxylase degradation keto-acid, while keto-acid is formed from L-amino acids under the action of aminotransferases L-amino acids.

3. The method of receiving according to claim 1, in which L-amino acid is L-aspartate.

4. The method of receiving according to claim 3, additionally providing for the contacting of oxaloacetate with oxaloacetate for the irreversible formation of pyruvate, with oxalate the t is formed from L-aspartate under the action of aminotransferases L-amino acids.

5. The method of receiving according to claim 1, in which aminotransferase L-amino acids derived from a microorganism belonging to the genus Arthrobacter, the genus Bacillus, the genus Candida, the genus Corynebacterium, the genus Lodderomyces, the genus Micrococcus, the genus Microbacterium, genus Nocardia, genus Pseudomonas, genus Rhizobium, genus Stenotrophomonas, the genus Dietzia, the genus Ochrobactrum, the genus Brevundimonas, the genus Burkholderia, a genus Carnimonas, the genus Yarrowia, the genus Clostridium, the genus Deinococcus, the genus Eubacterium, genus Lactobacillus, genus Methanothermobacter, the genus Phormidium, the genus Pyrococcus, the genus Rhodococcus, the genus Saccharomyces, the genus of Saccharophagus, the genus Sinorhizobium, the genus Thermoanaerobacter, the genus Thermotoga, or the genus Thermus.

6. The method of receiving according to claim 5, in which aminotransferase L-amino acids derived from a microorganism belonging to the species Arthrobacter sp., Bacillus altitudinis, Bacillus cellulosilyticus, Bacillus pumilus, Bacillus sp., Candida norvegensis, Candida inconspicua, Corynebacterium ammoniagenes, Corynebacterium glutamicum, Lodderomyces elongisporus, Micrococcus luteus, Microbacterium sp., Nocardia globerula, Pseudomonas chlororaphis, Pseudomonas citronocllolis, Pseudomonas fragi, Pseudomonas putida, Pseudomonas synxantha, Pseudomonas taetrolens, Pseudomonas sp., Rhizobium radiobacter, Rhizobium sp., Stenotrophomonas sp., Dietzia marts, Ochrobactrum pseudogrignonense, Brevundimonas diminuta, Burkholderia sp., Carnimonas sp., Yarrowia lypolytica, Clostridium cellulolyticum, Deinococcus geothermalis, Eubacterium rectale, Lactobacillus acidophilus, Methanothermobacter thermautotrophicus, Phormidium lapideum, Pyrococcus horikoshii, Rhodococcus erythropolis, Saccharomyces cerevisiae, Saccharophagus degradans, Sinorhizobium meliloti, Thermoanaerobacter tengcongensis, Thermotoga maritima, or Thermus thermophilus.

7. The method of receiving according to claim 1, in which aminotransferase L-amino acids contains one or more mutations in amino acid residues selected and the group, consisting of amino acid residues in position 39, 109, 128, 150, 258, 287, 288, 289, 303, 358 and in position 431 in the amino acid sequence represented by the sequence SEQ ID NO:2.

8. The method of receiving according to claim 7, in which one or more mutations in amino acid residues selected from the group consisting of
i) replacement of lysine at position 39 arginine;
ii) replacement of the serine at position 258 glycine;
iii) replacement of the glutamine at position 287 glutamic acid;
iv) replacement of threonine at position 288 with glycine;
v) replacement of isoleucine at position 289 alanine;
vi) replacement of the aspartic acid at position 109 glycine;
vii) replacement of histidine at position 150 tyrosine;
viii) replacement of phenylalanine at position 303 with leucine;
ix) replacement of the aspartic acid at position 358 tyrosine;
x) substitution of serine at position 431 threonine; and
xi) replacement of the glutamic acid at position 128 glycine.

9. The method of receiving according to claim 1, in which the 4R-IHOG is brought into contact with the normal L-amino acids using the transformant that expresses the specified aminotransferase L-amino acids.

10. The method of receiving according to claim 1, further providing for condensation of indole-3-pyruvate and pyruvate, with the formation of 4R-IHOG.

11. The method of receiving according to claim 1, in which indole-3-pyruvate and pyruvate condense by contacting indole-3-pyruvate and pyruvate with aldolase.

12. The method of obtaining of claim 10, in which at least part of the pyruvate used in the formation of 4R-IHOG, derived from pyruvate formed from oxaloacetate under oxaloacetate.

13. The method of obtaining of claim 10, additionally providing for the deamination of tryptophan to form indole-3-pyruvate.

14. The way of getting item 13, in which the tryptophan will dataminimum by contacting tryptophan enzyme involved deamination.

15. The method of obtaining of claim 10 or 13, in which the receipt of 2S,4R-Sonatina or its salt is carried out in a single reactor.

16. The method of obtaining 2R,4R-Sonatina or its salts, including the following (I) and (II):
(I) implementation of the method according to claim 1 with the formation of 2S,4R-Sonatina; and
(II) isomerization of 2S,4R-Sonatina with the formation of 2R,4R-Sonatina.

17. The way of getting item 16, in which the isomerization 2S,4R-Sonatina carried out in the presence of an aromatic aldehyde.

18. The way of getting item 16, in which the salt is a sodium salt or potassium salt.

19. Aminotransferase L-amino acids, which is a protein selected from the group consisting of the following (a) and (b):
(A) a protein consisting of an amino acid sequence that is 90% of the sludge is more identical to the amino acid sequence, represented by the sequence SEQ ID NO:2, and possessing an activity of aminotransferases L-amino acids;
where the protein of (A) contains as the main mutations of one or more mutations in amino acid residues selected from the group consisting of:
i) replacement of lysine at position 39 arginine;
ii) replacement of the serine at position 258 glycine;
iii) replacement of the glutamine at position 287 glutamic acid;
iv) replacement of threonine at position 288 with glycine;
v) replacement of isoleucine at position 289 alanine;
(B) a protein consisting of amino acid sequences containing a mutation from one to thirty amino acid residues selected from the group consisting of deletions, substitutions, additions and / or insertions of amino acid residues in the amino acid sequence represented by the sequence SEQ ID NO:2, and possessing an activity of aminotransferases L-amino acids
where the protein of (b) contains as the main mutations of one or more mutations in amino acid residues selected from the group consisting of:
i) replacement of lysine at position 39 arginine;
ii) replacement of the serine at position 258 glycine;
iii) replacement of the glutamine at position 287 glutamic acid;
iv) replacement of threonine at position 288 with glycine;
v) replacement of isoleucine at position 289 alanine.

20. Polynucleotide, which encodes an aminotransferase L-amino acids is about p.19.

21. The expression vector containing polynucleotide according to claim 20.

22. The bacterial cell, which introduced the expression vector according to item 21.

23. The method of obtaining L-aminotransferase, providing for the cultivation of bacterial cells by article 22 of the environment, obtaining aminotransferase L-amino acids.

24. The method of obtaining the 2S,4R-Sonatina or its salts, providing contacts 4R-IHOG with normal L-amino acid according to claim 19 in the presence of L-amino acids, with the formation of 2S,4R-Sonatina.

25. The method of obtaining 2R,4R-Sonatina or its salts, including the following (I) and (II'):
(I) implementation of the method according to paragraph 24 with the formation of 2S,4R-Sonatina; and
(II') isomerization 2S,4R-Sonatina, with the formation of 2R,4R-Sonatina.

26. The way of getting A.25, in which isomerization 2S,4R-Sonatina occurs in the presence of an aromatic aldehyde.

27. The way of getting A.25, in which the salt is a sodium salt or potassium salt.



 

Same patents:

FIELD: chemistry.

SUBSTANCE: neutral carbon source used is glucose, which is converted to aniline under the action of Escherichia coli or Streptomyces griseus bacteria. The glucose is obtained from plants. The stabiliser, vulcanisation accelerator or modified natural rubber is prepared from aniline obtained as described above.

EFFECT: invention improves environmental friendliness of methods of preparing a stabiliser, vulcanisation accelerator and modified natural rubber, which saves oil resources.

6 cl, 3 dwg, 5 ex

FIELD: medicine.

SUBSTANCE: method for producing 4'-hydroxy-L-isoleucine or 4,4'-dihydroxy-L-isoleucine or their salts by: enzymatic conversion in the presence of dioxygenase recovered from the bacterium belonging to a group including Pantoea and Pseudomonas geni, or by cultivation of a first producer bacterium possessing activity of dioxygenase recovered from the second bacterium belonging to a group including Pantoea and Paseudomonas geni, in a nutrient medium containing L-isoleicine and/or 4-hydroxy-L-isoleucine or thier salts and recovery of the end product from the culture fluid.

EFFECT: invention enables high-effective production of said compounds and their salts.

13 cl, 24 dwg, 5 tbl, 11 ex

FIELD: chemistry.

SUBSTANCE: disclosed is an enzymatic method of producing electroconductive polymers, specifically polyaniline, polypyrrole, polythiophene and substituted derivatives thereof. The method involves use of a salt of a transition metal complex as an enzymatic reaction accelerator, having redox potential in the range of 0.35-0.95 V. The salt is selected from a group comprising cyanide complexes of molybdenum, osmium, ruthenium, tungsten and iron. The process is carried out at pH 2.5-5.5 and temperature 0-30C and the oxidant used is molecular oxygen.

EFFECT: method enables to carry out high-speed oxidative polymerisation using low concentration of enzymatic reaction accelerator and an acid dopant.

3 dwg, 12 ex

FIELD: medicine.

SUBSTANCE: microorganisms were obtained by destruction of SpeE gene, and, at least, one gene, taken from SpeG, ArgI or PuuP in microorganism, which has metabolic way of putrescine synthesis. In addition, claimed is method of said microorganisms obtaining and method of putrescine production by their culturing. Claimed mutant microorganisms can be applied for large-scale production of putrescine for industrial application.

EFFECT: possibility of microorganisms to increased putrescine production.

30 cl, 5 dwg, 2 tbl, 8 ex

FIELD: medicine.

SUBSTANCE: method of preparing a composition containing (meth)acrylamide polymer provides polymerisation of (meth)acrylamide and mixed monomer containing (meth)acrylamide. (Meth)acrylamide is produced by a biocatalytic reaction or an enzymatic process, and the biocatalyst contains the nitrile hydrase enzyme. An aqueous solution of (meth)acrylamide contains a cell material and/or ingredients of an enzymatic broth. The cell material and/or the ingredients of the enzymatic broth are not removed from the aqueous solution of (meth)acrylamide. Said method is used to prepare the composition applied as flocculants.

EFFECT: invention allows producing high-molecular water-soluble or swelling polymers.

13 cl, 4 tbl, 3 ex

FIELD: chemistry.

SUBSTANCE: method of producing cyclopropyl-condensed inhibitors of dipeptidyl peptidase IV involves using BOC-protected amine with structural formula (3) , obtained through reductive amination of acid with formula (1) by treating the said acid with ammonium formate, nicotinamide adenine dinucleotide, dithiothreitol and partially purified concentrate of phenyl alanine dehydrogenase and formate dehydrogenase (PDH/FDH) enzymes and without separation - by treating the obtained amine of formula (2) with ditertbutyl dicarbonate, obtaining BOC-protected amine.

EFFECT: cutting on costs.

13 cl, 7 ex

FIELD: technological processes.

SUBSTANCE: method stipulates for cultivation of root hairs B.vulgaris with application of previously sterilised nutrient medium. Then inoculation of root hairs is performed in the range of 0.1-0.5 g of raw weight /40 ml of cultural medium, and growth continues for 2-24 days. Then exponential phase of root hairs culture growth is selected, and dioxyphenylalanine and dopamine is extracted from root hairs with dissolver.

EFFECT: method allows preparing dioxyphenylalanine and dopamine with high output.

6 cl, 2 tbl, 3 ex

FIELD: biotechnology, microbiology.

SUBSTANCE: method involves culturing halophilic microorganisms in the presence of encapsulated adsorbent and/or antioxidant up to the stationary growth phase in preparing the seeding material. Then in submerged culturing fresh medium is added by portions in the amount 1/3 of the final volume of cultural fluid followed by contacting cultural fluid with an encapsulated adsorbent and/or antioxidant, and additional feeding with dry or concentrated medium is carried out. The amount of dissolved oxygen is maintained at the level 5-10% of the equilibrium level. Invention provides increasing the level of accumulation of biomass, to enhance the specific content of bacteriorhodopsin in halophilic microorganisms biomass and the total yield of bacteriorhodopsin at the minimal content of carotinoids in biomass, and to reduce consumption of nutrient medium also.

EFFECT: improved preparing method of biomass.

1 tbl, 10 ex

FIELD: biotechnology, microbiology.

SUBSTANCE: method involves using an encapsulated adsorbent and/or antioxidant in preparing seeding material and submerged culturing. In preparing seeding material the culturing process is carried out in freshly prepared nutrient medium up to the stationary growth phase. Submerged culturing is carried out by simultaneous inoculation of bioreactor with seeding material and contacting the content of bioreactor with encapsulated adsorbent and/or antioxidant. The oxygen content in bioreactor is maintained at the level 5-10% of the equilibrium level. Invention provides increasing yield of halophilic microorganisms biomass up to 42 g/l and with the content of bacteriorhodopsin up to 1.6 g/l and in practically absent of carotinoids in biomass. Invention provides reducing time for preparing the unit mass of the end product and to decrease consumptions for production of bacteriorhodopsin.

EFFECT: improved preparing method of biomass.

1 tbl, 10 ex

FIELD: biotechnology, cytokines, pharmacy.

SUBSTANCE: invention relates to a biotechnological method for preparing medicinal agents. Proposed method involves destruction of cells of yeast producer of interleukin-2 (IL-2) wherein synthesis of required protein (IL-2) is carried out, but this producer doesn't secrete this protein, and the following separation of water-insoluble fraction. In the process for preparing water-insoluble components of disturbed cells are retained. The interleukin-2 preparation obtained by this method shows less cost, it is active in oral administration and shows higher activity in some cases as compared with IL-2 preparations purified from components of yeast cells fraction.

EFFECT: improved preparing method, improved and valuable properties of interleukin-2.

13 cl, 2 tbl, 10 ex

FIELD: biotechnologies.

SUBSTANCE: invention pertains to the versions of antibodies against IL-6 receptor, variable regions of light and heavy chains of which are modified by introduction of amino-acid replacement. There revealed is a pharmaceutical composition for treating the diseases associated with IL-6 and containing the said version of antibody.

EFFECT: invention allows efficient treatment of the diseases associated with IL-6.

8 cl, 48 dwg, 16 tbl, 20 ex

FIELD: biotechnologies.

SUBSTANCE: method involves cultivation in the appropriate conditions of yeast Saccharomyces cerevisiae and release of target protein; besides, release is directed with leader polypeptide, which has amino acid sequence SEQ ID NO1 and representing a variant of a pro-area of leader polypeptide of protein PpPIRl Pichia pastoris.

EFFECT: invention enlarges the range of methods for obtaining target protein in yeast Saccharomyces cerevisiae and increases possibilities for effective synthesis of such proteins.

2 dwg, 4 ex

FIELD: biotechnologies.

SUBSTANCE: invention describes two-stranded RNA modified with lipids and including antisense thread, the nucleotide sequence of which is complementary to target sequence in the target gene, and a sense thread, the nucleotide sequence of which is complementary to antisense thread. A two-stranded lipid is connected to the first nucleotide on the 5'-end of sense thread, either directly or through a linker. RNA has high stability to action of nuclease and is effectively absorbed with a cell, as well as generates excellent effect of RNA interference.

EFFECT: improvement of efficiency.

15 cl, 14 dwg, 3 ex

FIELD: biotechnologies.

SUBSTANCE: invention also refers to an expression vector and a transformant containing polynucleotide, as well as a production method of lipid composition or composition based on fat acids using the transformant.

EFFECT: invention allows producing a new ferment with improved properties, which has glycerol-3-phosphatocyltransferase activity.

11 cl, 4 dwg, 14 tbl, 4 ex

FIELD: biotechnologies.

SUBSTANCE: recombinant hybrid inhibitor of angiogenesis represents a protein shown in dwg. 1. This protein includes amino acid sequence of plasminogen of a human being from amino acid 82 to 341 and sequence Cys-Asp-Cys-Arg-Gly-Asp-Cys-Phe-Cys, which are covalently connected to each other. An inhibitor production method involves expression of its gene in cells of E. coli producer strain, which are transfected with recombinant plasmid DNA pBSRK13 with a physical map presented in dwg. 2, which has the size of 4155 pairs of bases. This plasmid includes a gene coding the recombinant hybrid inhibitor of angiogenesis, as well as a gene of signal peptide OmpA, lac-operator, a gene of stability to kanamycin, replicative origin pUC ori and a gene coding the lac-operator under control of promoter T7. A target protein is extracted from periplasmatic area of bacterial cells by affine and gel-filtration chromatography. The invention can also be used in medicine for creation of new medicinal agents with antiangiogenic therapeutical effect.

EFFECT: invention allows producing a new protein having antiangiogenic activity and increased selectivity of action in relation of tumoral endothelium.

2 cl, 3 dwg, 1 tbl, 5 ex

FIELD: biotechnologies.

SUBSTANCE: genetic structure pAd-SM is produced, being built by homological recombination of the vector pAdEasy-1, containing the main part of genome of adenovirus and plasmid pAdTrack-CMV, where cDNA fragments of human genes SOX2 and C-MYC are placed, being connected with a nucleotide sequence that codes P2A-peptide. The plasmid pAd-SM produced as a result of homological recombination contains a fragment SOX2-P2A-C-MYC under control of a constitutive promotor CMV.

EFFECT: possibility to produce adenovirus particles for simultaneois delivery of genes and expression of proteins SOX2 and C-MYC in human cells, pAd-SM contains a gene that codes a fluorescent protein EGFP, under control of a CMV promotor, which makes it possible to track transduction of cells and elimination of virus DNA from a cell.

3 cl, 1 dwg

FIELD: biotechnologies.

SUBSTANCE: method is proposed to produce a polypeptide, including cell cultivation, which intensely expresses a bicarbonate carrier and has a transferred DNA, which codes the desired polypeptide.

EFFECT: invention makes it possible for the cell to produce the specified polypeptide and the appropriate cell.

12 cl, 15 dwg, 6 ex

FIELD: chemistry.

SUBSTANCE: invention relates to compounds of general formula III and their pharmaceutically acceptable salts, A represents (C1-C6)alkyl-O-, phenyl-(C1-C6)alkyl-O-; aryl, selected from phenyl, naphthyl, and which is possibly substituted by 1-3 substituents, given in the invention formula; or heteroaryl, which has four or five carbon atoms and one heteroatom, selected from oxygen, nitrogen and sulphur, which is possibly substituted by 1-3 substituents, given in the invention formula; B represents phenyl, possibly substituted by 1-3 substituents, where substituents are selected from (C1-C6)alkyl, (C3-C7)cycloalkyl, (C1-C6)alkyl-O-, hydroxy, amino and halogeno; and R1 and R2 independently represent (C1-C6)alkyl, phenyl-(C1-C6)alkyl-, hydroxy-(C1-C6)alkyl, (C3-C7)cycloalkyl, (C2-C6)alkenyl or (C2-C6)alkynyl; on condition that R1 is different from R2; where absolute configuration of asymmetric R1 and R2 -carrying carbon atom is mainly R-configuration. Invention also relates to pharmaceutical composition, possessing ability to modulate gene expression, methods of modulation of gene expression in host cell, method of regulating expression of endogenous or heterologous gene in transgenic subject, method of regulating transgenic expression in transgenic subject, method of polypeptide production and to method of obtaining formula IV compound. Method includes stages: a) interaction of formula V compound with formula IV compound with obtaining formula VII compound; b) reduction of formula VII compound with obtaining formula VIII compound, b) interaction of formula VIII compound with formula B-CO-LG compound, where B has values, given above, and LG is leaving group representing -F, -Cl or -Br, with formation of formula IX compound, d) removal of group R7CO2- from formula IX compound with obtaining formula X compound, e) interaction of formula X compound with formula A-CO-LG compound, where A has values, given above, and LG is leaving group, representing -F, -Cl or -Br, with obtaining formula IV compound ( compounds of formulas V, VI, VII, VIII, IX, X are given in the invention formula).

EFFECT: obtaining formula III compounds, possessing ability to modulate gene expression.

19 cl, 4 ex, 2 tbl, 78 ex

FIELD: biotechnology.

SUBSTANCE: antibodies are used as pharmaceutical means for treatment or prevention of inflammatory diseases.

EFFECT: invention enables to obtain antibodies with effective neutralising activity against NR10.

7 cl, 33 dwg, 19 tbl, 10 ex

FIELD: biotechnology.

SUBSTANCE: isolated recombinant adenosine deaminase is described, which comprises polypeptide SEQ ID NO: 1 or a version polypeptide SEQ ID NO: 1 of isolated recombinant adenosine deaminase, where the version polypeptide SEQ ID NO: 1 comprises one or more amino acid substitutions selected from the group consisting of: Gin instead Lysl98; Ala instead Thr245; and Arg instead of Gly351, and DNA encoding it. The conjugate of polyalkylene oxide with the said adenosine deaminase for treatment of adenosine deaminase-mediated diseases is presented where adenosine deaminase comprises from 11 to 17 chains of polyalkylene oxide with a molecular weight of 5 kDa for ADA protein. The methods of purification of the recombinant adenosine deaminase are proposed, including protein purification using ion exchange chromatography, or protein purification using hydrophobic interaction chromatography. Also the preparations of recombinant adenosine deaminase produced by these methods are provided.

EFFECT: invention enables to obtain recombinant adenosine deaminase, having increased stability.

14 cl, 1 tbl, 10 ex

FIELD: medicine, genetics, biochemistry.

SUBSTANCE: invention relates to new NOS-variants or mutants that comprise structural modifications in site Akt-dependent phosphorylation. Modified NOS-proteins or peptides, in particular, human proteins or eNOS-peptides having change of amino acid residue corresponding to S/T in motif of the consensus-sequence RXRXXS/T of NOS-polypeptide of wild type and nucleic acid molecules encoding thereof can be used in genetic therapy and proteins and NOS-peptides can be used in screening methods of agents modulating activity of NOS. The advantage of invention involves the creature of new NOS-variants or mutants that can be used in genetic therapy.

EFFECT: valuable medicinal properties of mutants.

25 cl, 1 tbl, 9 dwg, 3 ex

Up!