30 results about "L-Lactate dehydrogenase" patented technology

The invention relates to an unmarked gene knock-out method of pediococcus acidilactici DQ2 based on homologous recombination. The method comprises the following steps: temperature sensitive-type shuttle plasmid pSET4E and knock-out plasmid containing homologous fragments at upstream and downstream parts of target genes to be knocked out are constructed, the knock-out plasmid is subjected to electrotransformation into pediococcus acidilactici, and single commutators generating homologous recombination for the first time and double-exchange mutant strains generating homologous recombination for the second time are screened and identified. The method disclosed by the invention realizes the unmarked gene knock-out of pediococcus acidilactici for the first time, the obtained knock-out bacterial strain does not carry any resistant gene, can be taken as a original strain for subsequent and reconstruction, and also can be used for large-scale industrial production in a safe mode. The method is used for respective knock-out of L-lactate dehydrogenase gene and d-lactate dehydrogenase gene of the pediococcus acidilactici DQ2 (a preservation number is CGMCC NO.7471), the obtained knock-out bacterial strains are respectively named as pediococcus acidilactici ZP26 and TY112, the preservation numbers are CGMCC NO.8665 and CGMCC NO.8664 respectively, and optically pure D-lactic acid and L-lactic acid are respectively generated.

The invention relates to genetic engineering bacteria for producing D-lactic acid and a construction method and application thereof. The strain of C.glutamicum Res 167 delta ldh deleted in L-lactate dehydrogenase gene (ldh) is used as a starting strain to over express exogenous D-lactate dehydrogenase gene so as to obtain C.glutamicum Res 167 delta ldh / ldhA. The genetic engineering bacteria are preserved in China General Microbiological Culture Collection Center with the preserving registration number of CGMCC No.4041. By utilizing the genetic engineering means, the expression of the exogenous D-lactate dehydrogenase gene is realized in the corynebacterium glutamicum deleted in L-lactic acid metabolic pathway, and the genetic engineering bacteria producing high optical purity D-lactic acid are successfully constructed. When the engineering bacteria are used for producing lactic acid through fermentation, the yield of the D-lactic acid is over 40g / L, and the purity is over 99 percent. The invention has important significance for the industrial production of the D-lactic acid, and has wide application prospect.

A corynebacterium acetoacidophilum strain and a method for producing succinic acid therefrom belong to the technical field of bioengineering. The invention discloses a corynebacterium acetoacidophilum strain YF / delta ldh and a method for producing succinic acid therefrom. The corynebacterium acetoacidophilum strain is an ldh (L-lactate dehydrogenase) missing bacterium built by gene knockout technology, is preserved in china center for type culture collection on February 29th, 2012, and encoded as CCTCC NO.M2012041. The strain is high in acid yield, highly resistant to sodions and high in foodsafety, can accumulate succinic acid more than 95g / L by being culture for 48 hours in the anaerobic environment using glucose as substrate and sodium bicarbonate as carbon dioxide donor, and can accumulate succinic acid 136g / L by being cultured for 94 hours.

The invention relates to an isobutanol synthetic bacterium genome dimension metabolic network model and a molecular modification method. According to the method, the necessary path for the thallus growth and the isobutanol synthesis is calculated by a network module, the metabolic network model is subjected to the element mode analysis, the standard deviation coefficient of each gene is calculated, and the isobutanol biosynthesis yield of different genes is determined; the two key genes including an L-lactate dehydrogenase gene 1dh and a pyruvate dehydrogenase complex E2 subunit coding gene pdhC which are most important to the isobutanol biosynthesis are predicted according to a principle that the standard deviation coefficient is smaller than 0.35, the standard deviation coefficients of the two genes are respectively 2.5 to 3 and 1.5 to 2; and through the determination of the two genes, the isobutanol yield can be improved, and 0.5-0.6C-mol / C-mol glucose can be reached. The key genes which are most important to the isobutanol biosynthesis are obtained through utilizing the relative flux value and are used as modification target spots, the molecular modification of the isobutanol synthetic bacterium is guided, and the isobutanol yield is improved.

The invention discloses an engineering bacterium, and an application thereof in the production of caffeic acid, and belongs to the technical field of bioengineering. The engineering bacterium providedby the invention is a recombinant bacterium capable of producing caffeic acid at a low cost; the recombinant bacterium can simultaneously express four enzymes which are tyrosine phenol lyase, tyrosine ammonia lyase, L-lactate dehydrogenase and NADH oxidase respectively; and the recombinant bacterium knocks out a phenolic substance-decomposing gene, and can achieve enhanced expression of any one or more of a lactic acid transporter gene, a catechol transporter gene and a coenzyme synthesis-related gene. The engineering bacterium has the advantages of realization of the efficient production ofcaffeic acid, simple process, few impurities and great industrial application values.

The invention discloses an engineering bacterium, and an application thereof in the production of Danshensu by using a cheap substrate, and belongs to the technical field of bioengineering. The engineering bacterium provided by the invention is a recombinant bacterium capable of producing Danshensu at a low cost; the recombinant bacterium can simultaneously express four enzymes which are tyrosinephenol lyase, L-amino acid oxidase, L-lactate dehydrogenase and alpha-hydroxycarboxylic acid oxidase respectively; and the recombinant bacterium knocks out a phenolic substance-decomposing gene, and can achieve enhanced expression of any one or more of a lactic acid transporter gene, a catechol transporter gene and a coenzyme synthesis-related gene. The engineering bacterium has the advantages ofrealization of the efficient production of Danshensu, simple process, few impurities and great industrial application values.

A yeast having a human- or frog-origin gene encoding L-lactate dehydrogenase transferred thereinto. By producing lactic acid by using the above-described gene and a method of producing lactic acid with the use of the enzyme, it is possible to efficiently produce lactic acid which is widely usable. As a result, lactic acid can be provided at a lower cost.

The invention discloses engineering bacteria for expressing L-lactate dehydrogenase subjected to orthogenetic evolution, which is engineering escherichia coli capable of heterologously expressing NAD (Nicotinamide Adenine Dinucleotide) independent L-lactate dehydrogenase subjected to orthogenetic evolution. The engineering bacteria can highly express corresponding mutant protein on the basis of commercial escherichia coli C43 (DE3) and through transporting an expression vector pETDuet-1-mlldD containing the mutant NAD independent L-lactate dehydrogenase, and has the S-mandelic acid degrading activity. The invention also discloses an application of the engineering bacteria in the resolution of racemic mandelic acid. The racemic mandelic acid is conducted through taking intact cells of the constructed engineering bacteria as a catalyst, and meanwhile, homochiral R-mandelic acid and benzoyl formic acid are produced. The engineering bacteria has the advantages of high substrate utilization rate, high product concentration, high optical purity, simplicity and convenience in future extraction, and the like.

The invention relates to genetic engineering bacteria for producing D-lactic acid and a construction method and application thereof. The strain of C.glutamicum Res 167 delta ldh deleted in L-lactate dehydrogenase gene (ldh) is used as a starting strain to over express exogenous D-lactate dehydrogenase gene so as to obtain C.glutamicum Res 167 delta ldh / ldhA. The genetic engineering bacteria are preserved in China General Microbiological Culture Collection Center with the preserving registration number of CGMCC No.4041. By utilizing the genetic engineering means, the expression of the exogenous D-lactate dehydrogenase gene is realized in the corynebacterium glutamicum deleted in L-lactic acid metabolic pathway, and the genetic engineering bacteria producing high optical purity D-lactic acid are successfully constructed. When the engineering bacteria are used for producing lactic acid through fermentation, the yield of the D-lactic acid is over 40g / L, and the purity is over 99 percent. The invention has important significance for the industrial production of the D-lactic acid, and has wide application prospect.

The invention discloses a preparation method of bacillus subtilis engineering bacteria for high yielding of alanine, wherein the method comprises that alanine-synthesized branched metabolism genes L-lactate dehydrogenase gene ldh and acetate kinase gene ackA on a B.subtilis 168 chromosome are knocked out by using a homologous recombination method, and a mutant strain is obtained. The single-gene mutant strain has the preservation number of IBL23-ldh, the construction of the mutant strain is achieved by knocking out a part of the lactate dehydrogenase gene of bacillus subtilis; a double-gene mutant strain is obtained by again knocking out the acetate kinase gene based on the single-gene mutant strain and has the number for IBL23-ldh / ackA.

A corynebacterium acetoacidophilum strain and a method for producing succinic acid therefrom belong to the technical field of bioengineering. The invention discloses a corynebacterium acetoacidophilum strain YF / delta ldh and a method for producing succinic acid therefrom. The corynebacterium acetoacidophilum strain is an ldh (L-lactate dehydrogenase) missing bacterium built by gene knockout technology, is preserved in china center for type culture collection on February 29th, 2012, and encoded as CCTCC NO.M2012041. The strain is high in acid yield, highly resistant to sodions and high in foodsafety, can accumulate succinic acid more than 95g / L by being culture for 48 hours in the anaerobic environment using glucose as substrate and sodium bicarbonate as carbon dioxide donor, and can accumulate succinic acid 136g / L by being cultured for 94 hours.

The present invention relates to a method for preparing a D-lactic acid-producing strain modified to inhibit L-lactate dehydrogenase (L-LDH) activity and to introduce D-lactate dehydrogenase (D-LDH) activity in an L-lactic acid-producing strain, a mutated D-lactic acid-producing strain prepared by the above method, and a method for producing D-lactic acid including the steps of culturing the strain and recovering D-lactic acid from the culture media.

The invention relates to p-carboxyl propionamidophenyl silicon oxide, a co-immobilized L-lactic dehydrogenase compound and a preparation method thereof. The compound is formed by using p-carboxyl propionamidophenyl silicon oxide with regularly-arranged carboxyl groups as a skeleton, and grafting L-lactic dehydrogenase and cofactor NADH. The novel organic / inorganic composite two-dimension lamellar compound overcomes the disadvantages that conventional montmorillonite and hydrotalcite lamellar materials are difficult to control a layer space environment; an application scope of functional enzyme is greatly broadened by developing and synthesizing the organic / inorganic composite lamellar compound with the functional enzyme; and a series of novel biological functional composite materials with various performances are developed.

The invention provides a method for quickly distinguishing lactic acid bacterium strains. The method includes the steps that firstly, a culture solution of lactic acid bacterium strains to be detected is centrifuged, and supernate is taken and diluted by 20-25 folds; secondly, the supernate, water, a glycylglycine buffer solution, NAD+ and D-GPT are mixed for 2-4 min before D or L-lactic dehydrogenase is added according to the table 1, a light-absorbing value A1 is read, then D-or L-LDA is added for reacting for 4-6 min, and a light-absorbing value A2 is read; thirdly, a light-absorbing value change value (A2-A1) of samples is subtracted by a contrasted light-absorbing value change value (A2-A1) to obtain deltaAD-lactic acid or deltaAL-lactic acid, the deltaAD-lactic acid or deltaAL-lactic acid is substituted into the following formula to calculate the concentration of D-lactic acid or L-lactic acid of the lactic acid bacterium strains to be detected, and please see the formula in the description. The lactic acid bacterium strains to be detected are distinguished according to the concentration ratio of D-lactic acid and L-lactic acid of the lactic acid bacterium strains to be detected.

The present invention relates to a homocysteine ​​assay kit and its preparation method and detection method. The homocysteine ​​assay kit includes reagent R1 and reagent R2. Reagent R1 is prepared from the following components: Aqueous solution: phosphate buffer, 1.8~2.2mL / L Triton X‑100, 0.11~0.16g / L L‑serine, 805~815KU / L lactate dehydrogenase, 0.5~0.75g / L NADH and 0.8-1.2mL / L ProcLin300; reagent R2 is an aqueous solution prepared from the following components: phosphate buffer, 1.8-2.2mL / L Triton X-100, 0.05-0.10g / EDTA dihydrate disodium salt in L, ProcLin300 in 0.8-1.2mL / L, cystathionine-β-synthase in 20.0-22.0KU / L and cystathionine-β-synthase in 10.0-14.0KU / L ‑Decomposing enzymes. The homocysteine ​​determination kit forms a reagent R1 and a reagent R2 by screening each component containing a specific ratio, and has a low detection limit, high sensitivity, wide linear range as a whole, and good stability.