Recombinant strain escherichia coli zv1 as carrier of burkholderia pseudomallei chromosomal dna cloned sequence determining synthesis of protein 32 kda and resistance to pefloxacin and streptomycin
FIELD: microbiology, molecular biology, genetic engineering.
SUBSTANCE: invention relates to designing recombinant strains of E. coli carrying the cloned sequences of genome of meliodosis pathogen, B. pseudomallei, and determining different spectra of the medicinal resistance. Method involves transformation of E. coli JM107 competent cells with Kpn I-fragments of chromosomal DNA of the strain B. pseudomallei 56770 SMR2 ligated with Kpn I-restricts of vector pUC19 followed by selection of clones showing the combined resistance to antibacterial preparations of different classes. Then method involves carrying out the plasmid screening of the recombinant clones and hybridization analysis for detection of chromosomal DNA fragment by using B. pseudomallei chromosome sequences as a probe. Then method involves assay of the presence of the expression product of chromosomal DNA cloned sequence by immunoblotting method with immunoglobulins of specific meliodosis antsera. The prepared recombinant strain ZV1 is deposited in the State Collection of pathogenic microorganisms "Mikrob" at number KM167 and it shows resistance to pefloxacin and streptomycin. Use of the invention provides carrying out investigations of molecular-genetic bases of the multiple medicinal resistance of B. pseudomallei and to study the functional role of separate proteins in formation of the polyresistance in the meliodosis pathogen.
EFFECT: valuable properties of strain.
1 dwg, 1 tbl, 2 ex
The invention relates to Microbiology and molecular biology and concerns the construction of recombinant strains of E. coli carrying the cloned sequence of the genome of the pathogen melioidosis Century pseudomallei determining different spectra of drug resistance. The resulting strain E. coli ZV1 is a producer of recombinant plasmid DNA pJla1 with a cloned fragment of chromosomal DNA .pseudomallei determining the synthesis of the protein of 32 kDa and resistance to pefloxacin (PfR) and streptomycin (SmR). The strain is designed to investigate the molecular genetic basis of multidrug resistance Century. pseudomallei. The strain deposited in the Public Collections of Pathogenic Bacteria "Microbe" (GKPB "M") under number KM167.
Characteristic and important biological property of the pathogen melioidosis close to him and microorganisms is a high natural resistance to the effects of broad-spectrum antimicrobial compounds and protective factors of the immune system of the host [Vorachit M, Chongtrakool P., Arkomsean S., Boonsong S. Antimicrobial resistance in Burkholderia pseudomallei // Acta Trop. - 2000. - Vol.74. - P.139-144; Brett P., Woods, D. Pathogenesis of and immunity to melioidosis // Acta Trop. - 2000. - Vol.74. - P.201-210]. The presence of these properties determines the ability .pseudomallei to long and often asymptomatic, persistence in the infected host and creates significant is radnoti for the successful treatment of disease.
The study of the molecular basis for the implementation of various types of drug resistance .pseudomallei started relatively recently, however, and based on the obtained data it can be argued that the causative agent of melioidosis has an extremely vast genetic potential for the formation of either spectrum resistance. So, identified the chromosomal operon amrAB-oprA related to the multiple determinants of resistance of the transport type and defines high level of stability Century pseudomallei to aminoglycosides and macrolides [Moore, A., DeShazer D., Reckseidler, S., Weissman, A., Woods, D. E. Effluxediated aminoglycoside and macrolide resistance in Burkholderia pseudomallei. // Antimicrob. Agents Chemother. - 1999. - Vol.43. - P.465-470]. Identified and characterized chromosomal genes waaF, waaE, udg, gmhD, lytB, homologous components of the systems of LPS biosynthesis and exopolysaccharides from different gram-negative bacteria and are responsible for resistance Century. pseudomallei to the effects of cationic peptides and polymyxins [Burtnick .N., Woods, D.E. Isolation of polymyxin B-susceptible mutants of Burkholderia pseudomallei and molecular characterization of genetic loci involved in polymyxin resistance. // Antimicrob. Agents Chemoter. - 1999. - Vol.43. - P.2648-2656]. The presence in the genome of C. pseudomallei R-determinants of different classes of evidence and published the preliminary results of the project to sequence the genome of C. pseudomallei [Holden M, Titball R., Peacock, S., et. al. Genomic plasticity of the causative agent of melioidosis, Burkholderia pseudomallei // Proc. Nal. Acad. Sci. USA. - 2004. - Vol.101. P.14240-14245].
It should be noted that research aimed at deciphering the mechanisms of formation resistance .pseudomallei, can serve as a basis for understanding basic biochemical characteristics of an organism, determining its increased metabolic plasticity, adaptability to the effects of specific and nonspecific inhibitors and protective host factors, as well as to provide the necessary material to develop new approaches to the use of antibiotics in the treatment of melioidosis infection. The possibility of studying the mechanisms of drug resistance are determined by the presence of molecular-biological methods and model systems to identify and analyze sequences of the genome, determining the resistance. Necessary elements here are special strains carrying genetic determinants of altered sensitivity, methods of molecular analysis R-determinants of various types, as well as the cloning of the sequence determinants of resistance and analysis of their expression in heterologous systems.
Today there are a number of reports on the cloning and analysis of gene expression .pseudomallei determining resistance to antimicrobial compounds of different classes. Thus, in E. coli klonirovannyi blaA gene BPS.pseudomallei encoding β-lactamase, is able to hydrolyze penicillins and individual cephalosporins [Cheung So, But R., Woo P., et al. Cloning and expression of class A β-lactamase gene blaABPSin Burkholderia pseudomallei // Antimicrob. Agents Chemother. - 2002. - Vol.46. - P.1132-1135]. Based on the analysis of published sequences of the genome of the pathogen melioidosis (www.sanger.ac.uk/Projects/B_pseudomallei) the authors have designed specific primers containing sequences of the restriction sites EcoRI and XbaI, amplified chromosomal sequence blaABPSCentury pseudomallei and cloned in E. coli in the vector pBK-CMV. Recombinant plasmids pBKCMV01 and pBKCMV07 stably replicated in strains of Escherichia coli. Amino acid sequence of recombinant β-lactamase with an isoelectric point pI of 7.7 and a molecular weight 29.1 kDa had a high degree of homology with β-lactamases of class a, such as PenA Usersa, BlaI Yersinia enterocolitica, CTX-M-18 Klebsiella pneumoniae, SFO-1 Entero-bacter cloacae, FONTA-4 Serratia fonticola and OXY Klebsiella oxytoca.
In many respects, a similar approach was used for cloning and analysis of gene β-lactamase D-class .pseudomallei [Niumsup P., Wuthiekanun V. Cloning of the class D β-lactamase gene from Burkholderia pseudomallei and studies on its expression in ceftazidime-susceptible and - resistant strains // J. Antimicrob. Chemother. - 2002. - Vol.50. - P.445-455]. Strains of E. coli carrying the recombinant plasmid with the cloned PCR fragments of the genome Century pseudomallei, homologous g is us β -lactamases of class D, had a high level of resistance to ceftazidime and oxacillin. Analysis of cloned sequences allowed us to identify a gene, designated Okha-42 and encoding β-lactamase extended-spectrum homologous some oxacillin-gidrolizuut enzymes, such as OXY-22 Ralstonia pickettii, AsbB, AmpS and AmpH different species of Aeromonas, OXA-18 Pseudomonas aeruginosa and OKHA-9 Klebsiella pneumoniae.
Work on cloning sequences of the genome .pseudomallei determining multiple resistance to antimicrobial compounds of various classes, much less reflected in modern scientific periodicals. We should mention the study of structural and functional organization of plasmids .pseudomallei, which was carried out cloning and analysis of expression in E. coli fragments 12.95 TPN plasmids RRM [Zakharov IS, Viktorov D.V., Zamaraev VS Cloning and expression 7.55 TPN fragment of plasmid RRM Burkholderia pseudomallei in Escherichia coli. // Mol. Genet. microbiol. virusol. - 2002. No. 2. - P.19-22]. It is established that the recombinant clones carrying 7.55 TPN Cloned fragment RRM, vysokofrekvencny to individual aminoglycosides (streptomycin, kanamycin, gentamicin and fluoroquinolones (pefloxacin, ofloxacin). Two proteins of the outer membrane 23 and 27 kDa, missing the original strain of E. coli and capable of forming a multimeric complex 120 kDa,were detected by Western-blotting in the strain with the recombinant plasmid pSI 9, vector part of which, however, has undergone significant recombination of perestroika. According to the dot - and southern-hybridization analysis, was observed integration of the cloned sequence in the chromosome in E. coli clones carrying recombinant plasmids pSI 2 pSI and 4.
The aim of the invention is the construction of E. coli strain carrying a stably can replicate the recombinant plasmid with the cloned sequence of the chromosomal DNA Century pseudomallei determining resistance to antimicrobial compounds of different classes.
As the source of chromosomal DNA was used strain .pseudomallei 56770 SMR2 with a high level of resistance to cephalosporins, fluoroquinolones, aminoglycosides, β-lactams and different stable preservation of the phenotype of resistance with long-term cultivation in non-selective conditions. Chromosomal DNA was isolated by the method of neutral lysis [Maniatis T., Fritsch E., Sambrook D. Molecular cloning. - Lane. from English., M.: Mir, 1984. - 392 C.] of the 24-hour culture of the pathogen melioidosis grown on Nutrient-arape (Difco) with pefloxacin (60 μg/ml) at 37°C.
Chromosomal DNA Century pseudomallei 56770 SMR2 and the vector pUC19 was subjected to complete hydrolysis by endonuclease kpni restriction sites and after the resultant deposition rates ligated at a ratio of 25:1. In several series of experiments transformed ligase mix comp is competent cells E. coli JM107 received standard calcium method [Maniatis T., Fritsch E., Sambrook D. Molecular cloning. - Lane. from English., M.: Mir, 1984. - 392 S.], with subsequent plating on a dense nutrient medium Antibiotic medium 2 (Difco)containing Pfx 0.4 mg/ml
Transformants resistant to pefloxacin additionally investigated for resistance to aminoglycosides (kanamycin, streptomycin, gentamicin), β-lactams (ampicillin) and carbapenems (imipenem).
For plasmid screening used alkaline method for isolating plasmid DNA [Birnboim, H., Doly J. A rapid alkaline extraction method for screening recombinant plasmids //Nucleic Acids Res. -1979. - Vol.7. - P.1513] with subsequent detection of plasmid replicons by electrophoresis in 0.8% agarose gel.
For detection of the cloned fragment of chromosomal DNA .pseudomallei was used for hybridization analysis. Probe (sequence chromosome .pseudomallei 56770 SMR2) were labeled with bio-11-dUTP in PCR using forward and reverse primers pUC/M13 with the following parameters of reactions: denaturation 95°C - 30 s, annealing at 50°S - 40 s, elongation 72°C - 50 C. To visualize the results of hybridization used a set of "non-radioactive DNA identification" (NGO "Silex", Russia).
For detection of the products of expression of the cloned fragment of chromosomal DNA Century pseudomallei used the method of Western blotting [Towbin H., Staehelin T. Electrophoretic transfer of proteins from polyacrylamide gels to nirocellulose sheets: procedure and some applications // Proc. Natl. Acad. Sci. USA. - 1979. - N 76. - P.4350-4354] using antibodies polyclonal goat serum to cells .pseudomallei labeled with alkaline phosphatase.
The recombinant strain E. coli ZV1 (pJla1)obtained by transformation of the cells of E. coli JM107 Cloned fragments of chromosomal DNA Century pseudomallei 56770 SMR2, legirovannye with the vector pUC19, has resistance to pefloxacin (PfR) and streptomycin (SmR), loses a token resistance vector (ArSretains its basic cultural-morphological and biochemical properties of E. coli JM107. The strain E. coli ZV1 (pJla1) is the carrier vysokonapornogo plasmid replicon pJlal size 6.1 TPN output Level (synthesis) of plasmid DNA pJlal in strain is 0.25 mg/l in culture density OD/600=0.5. According to the restriction and hybridization analysis, plasmid pJlal shall be composed of ori replication of the vector pUC19, and the fragment cloned chromosomal DNA .pseudomallei approximately 5.5 TPN Product of expression of the cloned fragment is a protein with a molecular mass of 32 kDa interacting specifically to immunoglobulins melioidosis antisera in Western-blotting.
Example 1. The test recombinant strain E. coli ZV1 (pJla1) no expression of antibiotic resistance compared to the recipient strain E. coli JM107.
The resistant strains to antibiotics is determined by the serial dilution method on nutrient dense environments assessing the IPC product for 5×104M.K. Bacterial suspension prepared from 24 h agar cultures grown at 37°C. the results of the comparative analysis of the levels of resistance of the recombinant strain E. coli ZV1 (pJla1) and the source of the E. coli strain JM107 to antibiotics of different classes given in the table.
As follows from the obtained data, the recombinant strain E. coli ZV1 (pJla1) has resistance to individual antibiotics aminoglycoside (streptomycin) and fluoroquinolone (pefloxacin) rows.
Example 2. Analysis of the products of expression of the cloned sequences of chromosomal DNA .pseudomallei.
Bacterial cells grown for 24-48 h at 37°on nutrient agar, resuspended in lyse buffer (0.0625 M Tris-HCl pH 6.8, 2% SDS, 10% glycerol, 5% β-mercaptoethanol), heated 5 min at 100°and remove nelsonbay material by centrifugation at 5000 g. 10-15 µl of the supernatant analyzed by electrophoresis in polyacrylamide gel. For electrophoresis using gels (11% polyacrylamide, 0.1% SDS)prepared according to the method of Laemmli [Laemmli U. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. - Nature. - 1970. - V.227. - P.680-685]. Electrophoresis is carried out at stabilizirovannom a current of 5 mA for 18 h in a vertical machine SE400 (HSI). To control the speed of fractionation in the way the s make 0.004% bromophenol blue. For staining gels use Kumasi G-250 (Serva).
Electrophoretic transfer of proteins to nitrocellulose membrane VA (LKB) with a pore size of 0.22 μm is conducted according to Towbin [Towbin H., Staehelin T. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications // Proc. Natl. Acad. Sci. USA. - 1979. - N 76. - P.4350-4354] in the vertical cell THE (HSI) for 18 h at stable voltage of 45 V and the temperature control with a 10°C. After electroblotting membrane was washed in TBST buffer (10 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.05% tween-20) three times for 2 min, blocked in 2% BSA in TBST for 15 h at 4°repeat the washing step and incubated for 1.5 h at room temperature with specific IgG labeled with alkaline phosphatase, breeding 1:128 in TBST with 0.5% BSA. Then the membrane was washed five times for 2 min in TBST, activate the enzyme, incubare membrane 2 min in AR buffer (100 mM Tris-HCl pH 9.5,100 mM NaCl, 5 mM MgCl2) and treated with substrate solution (0.02% bromo-chloro-indolyl-phosphate, 0.02% nicrosini tetrazole buffer AR).
In Western blot turns (drawing) revealed a specific interaction melioidosis immunoglobulins with drug abdelmassih proteins E. coli E. coli ZV1 (protein a 32 kDa) and drugs used as positive controls (obselete proteins .pseudomallei 56770, B. thailandensis E264). The drug abdelmassih proteins of E. coli strain JM107 not contain components, specifically in aimogasta with immunoglobulins melioidosis antisera.
The strain Escherichia coli ZV1 KM167 (national collection of pathogenic bacteria "Microbe") is a carrier of recombinant plasmid DNA J1 1 with a cloned fragment of the chromosomal DNA of Burkholderia pseudomallei determining the synthesis of the protein of 32 kDa and resistance to pefloxacin (RfR) and streptomycin (SmR).
FIELD: biotechnology, molecular biology, microbiology, genetic engineering.
SUBSTANCE: invention relates to a method for preparing an immunogenic polypeptide inducing immune response that represents the protective response against infection with Bacillus anthracis. Proposed immunogenic polypeptide comprises from one to three domains of the full-scale Protective Antigen (PA) from B. anthracis or their variants and at least one of indicated domains represents domain 1 or domain 4 from PA or its variant. These variants of immunogenic polypeptide and full-scale PA are produced as result of expression in E. coli. Also, invention proposes a vector for expression in bacterial cells that comprises nucleic acid encoding abovementioned immunogenic polypeptide. Also, invention the developed method for prophylaxis of infection caused by B. anthracis based on administration of sufficient amount of immunogenic polypeptide. Also, invention proposes a vaccine for prophylaxis of infection caused by B. anthracis that comprises the effective amount of immunogenic polypeptide and a suitable carrier. Invention provides preparing the effective agent used for prophylaxis of infection caused by B. anthracis.
EFFECT: improved preparing method and valuable properties of polypeptide and vaccine.
22 cl, 5 dwg, 3 tbl, 6 ex
FIELD: biotechnology, in particular gene engineering, pharmaceutical and food processing industry.
SUBSTANCE: DNA sequence (1341 n.p.) encoding fatty acid -desaturase (447 amino acid residue, 57 kD) of nematode Caenorhabditis elegants is isolated and characterized. Obtained DNA-sequence is expressed in bacterium and yeast cells to produce Biologically active enzyme recombinant form. Said recombinant form is capable to catalyze conversion of dihomo-γ-linolenic acid to arachidonic acid and eicosatetraenoate to eicosapentaenoate.
EFFECT: method for large-scale production of polyunsaturated fatty acid.
15 cl, 4 dwg, 1 ex
FIELD: biotechnology, microbiology, genetic engineering.
SUBSTANCE: invention proposes a new recombinant plasmid pR752 (5269 pair bases) comprising genetic construction under control of bacteriophage T5 promoter and encoding a module polypeptide consisting of 6 histidine residues, hemoglobin-like protein of E. coli, modified fragment of large T-antigen SV-40, translocation domain of diphtheria toxin, spacer sequence (Gly-Ser)5 and human epidermal growth factor (6 His-HMP-NLS-Dtox-(Gly-Ser)5-EGF) and designated for the directed transfer of photosensitizers into target-cell nuclei. By transformation of the strain E. coli M15 (rep 4) with plasmid pR752 the recombinant strain E. coli VKPM B-8356 as a producer of new polypeptide vector is prepared. The usage of this new strain is able to enhance the effectiveness of effect of photosensitizers by some orders. Invention can be used in medicinal-biological industry in preparing agents providing the directed transport of photosensitizing agents into tumor cell nuclei.
EFFECT: valuable biological and medicinal properties of polypeptide.
3 dwg, 4 ex
FIELD: biotechnology, genetic and protein engineering.
SUBSTANCE: invention reports construction of plasmid DNA pES6-1 based on plasmid pET22b(+) and DNA fragment comprising a sequence of artificial gene encoding human interferon β-1b providing expression of human recombinant interferon β-1b. Also, the strain Escherichia coli BDEES6 (BL21(DE3)/pES6-1) as producer of human recombinant interferonβ-1b is prepared. Invention provides enhancing yield of human recombinant interferon β-1b. Invention can be used in medicine and pharmaceutical industry.
EFFECT: valuable biological and medicinal properties of strain.
2 cl, 2 dwg, 2 ex
FIELD: genetic engineering, molecular biology, biochemistry.
SUBSTANCE: recombinant plasmid DNA pTES-His-OPH is constructed for expression of polypeptide eliciting properties of organophosphate hydrolase comprising Cla I/Hind III fragment of plasmid pTrcTEGF, fragment of plasmid pTES-OPH with nucleotide sequence that encodes amino acid sequence of the matured form of organophosphate hydrolase, and nucleotide sequence encoding 6 histidine residues that is located by 5'-end of nucleotide sequence encoding organophosphate hydrolase. Based on indicated plasmid the strain Escherichia coli TSKMIBKH-29 - a producer of polypeptide eliciting properties of organophosphate hydrolase is obtained. Applying the invention provides preparing polypeptide with properties of organophosphate hydrolase by simplified technology and this polypeptide elicits the improved catalytic effectiveness of action with respect to thio-containing phosphoric acid triesters. Invention can be used for carrying out hydrolysis of organophosphate compounds.
EFFECT: valuable biochemical properties of producer.
2 cl, 4 dwg, 2 tbl, 4 ex
FIELD: biotechnology, genetic engineering, pharmaceutical industry.
SUBSTANCE: plasmid DNA pET23-a(+)/PrxVIhumΔ178 with molecular weight of 19691.61 Da is constructed. DNA contains RNA-polymerase T7 promoter; replication initiation site; genetic marker which determinates resistance of cells transformed by said plasmid to ampicillin; and nucleotide sequence encoding N-terminal fragment of human peroxiredoxine VI containing 177 of amino acid residues. E.coli strain BL21/DE3/pET23-a(+)/PrxVIhumΔ178 being producer of N-terminal fragment of human peroxiredoxine VI is obtained by transformation of E.coli cells with plasmid DNA pET23-a(+)/PrxVIhumΔ178. Method of present invention makes it possible to obtain human peroxiredoxine VI fragment having reduced molecular weight, improved tissue permeability, and antioxidant activity of full-scale peroxiredoxine.
EFFECT: human peroxiredoxine VI fragment with improved tissue permeability.
2 cl, 3 dwg, 4 ex
FIELD: genetic and tissue engineering, biotechnology, medicine, agriculture.
SUBSTANCE: invention relates to the development of simple with constructive relation peptide vector (PGE-κ) consisting of polypeptide sequence of epidermal growth factor (EGF) and modified sequence of signal peptide T-antigen SV-40. New vector PGE-κ is able to provide the selective delivery of genetic material in target-cell cytoplasm carrying external receptors to EGF and the following its transport across nuclear membrane. Also, invention proposes a method for preparing peptide vector PGE-κ involving its expression as a fused protein "mutant thioredoxine-linker-vector" and cleavage of product expressed in E. coli in the linker region with specific protease. Invention provides preparing the recombinant strain E. coli B-8389 VKPM as a producer of the fused protein comprising PGE-κ. Proposed vector shows simple structure, absence of toxicity and immunogenicity and these properties provide its usefulness for the directed genetic modification of epithelial, embryonic and tumor cells in vivo.
EFFECT: improved preparing method, valuable medicinal properties of vector, improved genetic modification.
7 cl, 12 dwg, 4 tbl, 16 ex
FIELD: biotechnology, genetic engineering, amino acids.
SUBSTANCE: invention relates to a method for preparing L-methionine by culturing the microorganism strain transformed with plasmid comprising gene yjeH or gene with homology with its by above 30% and used as a producer of L-methionine. Prepared L-methionine is isolated from medium. The claimed invention provides preparing L-methionine with high degree of effectiveness.
EFFECT: improved preparing method.
11 cl, 1 tbl, 6 ex
SUBSTANCE: dipeptide is produced from L-amino acid ester and L-amino acid using protein with dipeptide production enzyme activity. Said protein is obtained by culturing of transformed bacterial cell.
EFFECT: simplified method for dipeptide production of decreased cost.
11 cl, 1 dwg, 4 tbl, 14 ex
FIELD: biotechnology, in particular antibacterial protein salivaricine B belonging to lantibiotics.
SUBSTANCE: antibacterial protein is expressed by strain Streptococcus salivarius K12 (DSM N 13084), has molecular weight of about 2733 Da, contains N-terminal amino acid sequence (AS) representing in SEQ ID NO:1, as defined in description, or AS representing in SEQ ID NO:3, and has bactericide activity against S. pyogenes. Both protein and microorganism producing thereof are useful as active ingredients in antibacterial and antibacterial therapeutic compositions, wherein the latter one may additionally contain salivaricine A2 (AS SEQ ID NO:5, as defined in description). Also invention relates to polynucleotide, encoding protein (salivarius B) optionally including nucleotide sequence (NS) (NS SEQ ID NO:2), as defined in description). Polynucleotide optionally includes DNA sequence encoding antibacterial protein, representing genome part of strain K12 (DSM 13084). Strain S. salivarius K12 (DSM N 13084) and S. salivarius K30 (DSM N 13085) being producers of protein salivaricine B also are described.
EFFECT: new protein having both antibacterial and bactericide activity.
29 cl, 3 dwg, 1 tbl
SUBSTANCE: l-cysteine is produced by cultivation of bacteria belonging to genus Escherichia transformed with DNA encoding mutant serine-acetyl transferase, wherein said serine-acetyl transferase has one or more mutations in natural sequence of serine-acetyl transferase from E.coli in sites from 89 to 96, and reduced feedback inhibition by L-cysteine.
EFFECT: high effective method for L-cysteine production.
6 cl, 5 dwg, 3 tbl, 4 ex
FIELD: genetic engineering, biotechnology.
SUBSTANCE: invention proposes a fused protein of beta-galactosidase and cellulose-binding domain of endoglucanase CelD from Anaerocellum thermophilium. The recombinant plasmid DNA pLACspCBD is constructed providing its expression in E. coli cells. The strain E. coli as a producer of indicated fused protein is obtained. A method for preparing this fused protein in immobilized form is developed that involves treatment of cellulose with hydrolyzate of the indicated strain of E. coli. Using the invention provides simplifying processing of dairy foodstuffs. Invention can be used in food processing industry.
EFFECT: valuable properties of strain and protein, improved preparing method.
5 cl, 1 dwg, 5 ex
FIELD: biotechnology, microbiology, amino acids.
SUBSTANCE: invention relates to a method for preparing L-histidine that is prepared by culturing microorganism of genus Escherichia with the enhanced activity of any enzyme taking part the conversion of 5'-phosphoribosyl-4-carboxamide-5-aminoimidazole to inosine-5'-monophosphate. Then the prepared L-histidine is isolated from the cultural fluid. The claimed invention provides preparing L-histidine with the high degree of effectiveness.
EFFECT: improved preparing method.
11 cl, 2 tbl, 2 ex
FIELD: biotechnology, microbiology, amino acids.
SUBSTANCE: method involves culturing bacterium of genus Escherichia modified by such manner that activity of enzyme transaldolase in cell of such bacterium is increased. Prepared and accumulated L-histidine is isolated from cultural fluid. The claimed invention provides preparing L-histidine with the enhanced degree of effectiveness.
EFFECT: enhanced yield of L-histidine.
8 cl, 1 tbl, 2 ex
FIELD: biotechnology, microbiology, amino acids.
SUBSTANCE: invention relates to a method for preparing L-amino acids, for example, L-arginine by culturing of microorganism as a producer of L-arginine belonging to genus Escherichia. Indicated microorganism is modified and it comprises inactivated operon nir. Then accumulated L-arginine is isolated from the cultural fluid. The claimed invention provides preparing L-arginine with the high degree of effectiveness.
EFFECT: improved preparing method.
4 cl, 1 tbl, 2 ex
FIELD: biotechnology, biochemistry, amino acids.
SUBSTANCE: L-glutamic acid is produced in culturing corynebacterium possessing ability to produce L-glutamic acid. This microorganism shows reduced ability for synthesis of trehalose or its absence as result of destruction of gene encoding trehalose-6-phosphate synthase, or gene encoding maltooligolysyltrehalose synthase, or by addition of mutations in these genes. Invention provides preparing L-glutamic acid with high degree of effectiveness.
EFFECT: improved method and enhanced effectiveness for preparing amino acid.
6 cl, 1 tbl, 3 ex
FIELD: biotechnology, microbiology, amino acids.
SUBSTANCE: invention represents a method for preparing L-cysteine by using bacterium belonging to genus Escherichia wherein production of L-amino acid by indicated bacterium is increased due to enhancing expression of genes of cluster cysPTWAM. Microorganisms are cultured in nutrient medium containing thiosulfate followed by isolation of prepared and accumulated L-cysteine from cultural medium. Invention provides preparing L-cysteine with high degree of effectiveness.
EFFECT: improved preparing method.
9 cl, 2 tbl, 5 ex
FIELD: biotechnology, microbiology.
SUBSTANCE: invention relates to a method for preparing inosine and 5'-inosinic acid that are prepared by using microorganism Escherichia coli. Production of inosine by indicated microorganisms is elevated due to the enhanced activity of protein encoded by gene yijE. Invention provides increasing yield of inosine and 5'-inosinic acid.
EFFECT: improved preparing method, valuable properties of strain.
8 cl, 3 dwg, 2 tbl, 3 ex