30 results about "L-Lactate dehydrogenase" patented technology

The invention relates to an L-lactate dehydrogenase gene-deleted engineering bacterium and a construction method and application thereof. Upstream and downstream sequences of ldh1 and ldh2 of L-lactate dehydrogenase (ldh) gene are subjected to polymerase chain reaction amplification, cloned segments are connected to a gene knock-out vector, the constructed knock-out vector is inoculated into corynebacterium glutamicum, and the gene engineering bacterium C.glutamicum Res167 delta ldh is obtained by silencing and screening L-lactate dehydrogenase (ldh) genes in the strain through a homologous recombination method. By the homologous recombination method, the L-lactate dehydrogenase genes in the corynebacterium glutamicum are silenced, so that the gene engineering bacterium for producing pureD-lactic acid is obtained. When the engineering bacterium is used for lactic acid fermentation production, the yield of the D-lactic acid is more than 20g/L, and the purity is over 99 percent. The invention has important significance for industrial production of the D-lactic acid and has wide application prospect.

The invention discloses escherichia coli with coexpression of L-lactate dehydrogenase and formate dehydrogenase. The escherichia coli is characterized in that L-lactate dehydrogenase genes ldhL from bacillus coagulans and formate dehydrogenase genes fdh from candida boidinii are introduced into the escherichia coli, wherein the register number of GenBank of nucleotide sequences of the L-lactate dehydrogenase genes ldhL is KF386111; the register number of GenBank of nucleotide sequences of the formate dehydrogenase genes fdh is AJ011046. The invention further discloses a construction method and an application of the reconstructed escherichia coli. The method has the characteristics of being simple to operate, low in cost, high in product synthesis efficiency and high in optical purity. Thus, an effective way for biosynthesis of L-phenyllactic acid is provided.

Yeast includes an introduced gene coding a Homo sapiens- or frog-derived L-lactate dehydrogenase.

It is possible to produce lactic acid, which has a variety of applications, efficiently and more cost-effectively by using the yeast and the method of producing lactic acid by using the yeast.

The invention discloses a vector for knocking out an L-lactic dehydrogenase 1 gene and a construction method of the vector. The construction method comprises the following steps: completely splicing upstream and downstream DNA fragments of a gene which does not contain an L-lactic dehydrogenase encoding gene by using an overlapping extension process, also cloning the spliced fragments to a sub-vector pMD18-T and transforming the spliced fragments to escherichia coli DH5alpha competent cells, screening constructed sub-cloning vectors by virtue of blue-white spots, performing double enzyme digestion to obtain a target strip, then connecting the target strip to a pG+host9 plasmid, and performing bacterial colony PCR and double enzyme digestion identification to successfully obtain a recombinant vector. According to the vector and the construction method thereof disclosed by the invention, a recombinant plasmid of which the L-lactic dehydrogenase encoding genes are knocked out is constructed by using an overlapping extension PCR process, so that excessive base sequences introduced by enzyme digestion and connection can be avoided; and the recombinant vector disclosed by the invention can be used for producing D-lactic acid with high optical purity.

An economical and expedient method is disclosed for the preparation of alpha-hydroxy-carboxylic acids or salts thereof in very high enantiomeric purity which comprises oxidizing a corresponding alpha-amino-carboxylic acid or salt thereof using an amino acid deaminase followed by reducing the corresponding alpha-keto-carboxylic acid or salt produced using a D- or L-lactate dehydrogenase in the combination with an electron donor and an enzyme/substrate system for recycling the electron donor. The resulting alpha-hydroxy-carboxylic acids, hydrates, and salts thereof are valuable components and intermediates in the preparation of chiral compounds, especially pharmaceuticals. This invention also relates to the use of alpha-amino-carboxylic acids, hydrates, and salts thereof and a coupled enzyme system in the production of alpha-hydroxy-carboxylic acids, hydrates, and salts thereof.

The invention provides a sialic acid assay kit, comprising a reagent R1 and a reagent R2, wherein the reagent R1 is prepared from the following components with corresponding concentrations: 8-20g/L tris(hydroxymethyl) aminomethane, 5-15ml/L HCL, 0.5-5KU/L neuraminidase, 0.5-5KU/L lactate dehydrogenase and 0.1-2mmol/L NADH; the reagent R2 is prepared from the following components with correspondingconcentrations: 8-20g/L tris(hydroxymethyl) aminomethane, 5-15ml/L HCL, 0.5-5KU/L N-acetylneuraminic acid aldolase, 25-100g/L sucrose and 0.6-1.2 ml/L triethanolamine. The invention belongs to the technical field of biological detection. The sialic acid assay kit provided by the invention significantly enhances the anti-interference ability while improving the stability, can significantly reducethe interference of bilirubin, hemoglobin, triglyceride and the like, and is good in accuracy when being used for the determination of sialic acid.

The invention discloses an engineering bacterium, and an application thereof in the production of levodopa, and belongs to the technical field of bioengineering. The engineering bacterium is a recombinant Escherichia coli capable of producing pure levodopa at low cost; the recombinant Escherichia coli simultaneously expresses exogenous L-lactate dehydrogenase, NADH oxidase and tyrosine phenol lyase, and is obtained by knocking out an aromatic compound-degrading gene from host Escherichia coli; and the recombinant Escherichia coli can achieve enhanced expression of any one or more of a lactic acid transporter gene, an ammonia ion transporter gene, a catechol transporter gene, an NAD synthesis gene and a pyridoxal phosphate synthesis gene. A bacterium can be applied to the production of levodopa, and a method for producing the levodopa has the advantages of simple production process, few impurities, easily available raw materials and good industrial application prospect.

The invention discloses an engineering bacterium, and an application thereof in the production of pyruvic acid, and belongs to the technical field of bioengineering. The engineering bacterium simultaneously expresses exogenous L-lactate dehydrogenase and NADH oxidase, is obtained by knocking out a pyruvic acid absorption gene from host Escherichia coli, and can achieve enhanced expression of a lactic acid transporter gene, a pyruvic acid transporter gene and an NAD synthesis gene. The double-enzyme co-expression engineering bacterium is constructed on the basis of reconstructing Escherichia coli transportation and a coenzyme synthesis system in order to realize efficient production of pyruvic acid and reduce the generation of impurities.

The invention discloses a method for preparing recombined bacillus alcalophilus. According to the method, bacillus alcalophilus for producing L-lactic acid is used as original strain, and by means of genetic engineering operation, the original strain is made not to express L-lactic dehydrogenase but to express D-lactic dehydrogenase. The invention further discloses recombined bacillus alcalophilus prepared with the method, application of the recombined bacillus alcalophilus, and a method for preparing D-lactic acid. When the recombined bacillus alcalophilus prepared with the method for preparing the recombined bacillus alcalophilus is applied to preparation of D-lactic acid, the optical purity of the obtained D-lactic acid is higher than 99.8%, the conversion rate is 94% or more, and the yield can reach 142 g/L.

The invention discloses genetic engineering compound bacteria and an application thereof in biosynthesis of PGA (phenylglyoxylic acid). The genetic engineering compound bacteria contain a novel L-lactate dehydrogenase gene LhLDH, an existing D-mandelate dehydrogenase gene and encoded genes thereof as well as a novel mandelate racemase gene HrMR, the genetic engineering compound bacteria are utilized for production and application of PGA synthesized through whole-cell catalysis of racemic mandelic acid, the production process is simple, the conditions are mild, the efficiency is high and the cost is low. The genetic engineering compound bacteria have the benefits as follows: the constructed genetic engineering compound bacteria can realize high yield of D-mandelate dehydrogenase, L-lactatedehydrogenase and mandelate racemase; on the basis of whole-cell catalysis, the genetic engineering compound bacteria can efficiently oxidize racemic mandelic acid for production of PGA without addition of coenzymes. Through the genetic engineering compound bacteria, the conversion rate of racemic mandelic acid can be 96% or above, the purity of PGA can be 99% or above, and the genetic engineeringcompound bacteria have good industrial prospects.

The invention discloses an L-lactate dehydrogenase mutant with the improved catalysis efficiency and a building method thereof, and belongs to the technical field of genetic engineering. On the basisof L-lactate dehydrogenase, the L-lactate dehydrogenase molecular structures are modified through a site-specific mutagenesis biological technology; one strain of L-lactate dehydrogenase escherichia coli engineering bacterium is obtained; the mutant enzyme specific enzyme activity is improved by 6.1 times; the catalytic activity is improved by 4.2 times. The limitation problem of low catalysis activity of the L-lactate dehydrogenase is solved; a novel idea is provided for the L-lactate dehydrogenase molecular modification.

The invention discloses an L-lactate dehydrogenase mutant with improved catalytic efficiency and application thereof, and belongs to the technical field of genetic engineering. On the basis of L-lactate dehydrogenase, the molecular structure of L-lactate dehydrogenase is transformed through a site-directed mutagenesis biotechnology to obtain engineered L-lactate dehydrogenase escherichia coli, thespecific activity of mutant enzymes is increased by 5.2 times, and the catalytic activity is increased by 3.8 times. The L-lactate dehydrogenase mutant solves the limitation problem of low catalyticactivity of L-lactate dehydrogenase and provides a new idea for the molecular transformation of L-lactate dehydrogenase.

The invention discloses an engineering bacterium, and an application thereof in the production of p-hydroxycinnamic acid, and belongs to the technical field of bioengineering. The engineering bacterium provided by the invention is a recombinant bacterium capable of producing p-hydroxycinnamic 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 phenol transporter gene and a coenzyme synthesis-related gene. The engineering bacterium can realize the efficient production of p-hydroxycinnamic acid, and a method for producing the p-hydroxycinnamic acid has the advantages of simple process, few impurities and great industrial application values.

The invention discloses an engineering bacterium, and an application thereof in the production of p-hydroxyphenyllactic acid by using a cheap substrate, and belongs to the technical field of bioengineering. The engineering bacterium constructed in the invention can simultaneously express four enzymes which are tyrosine phenol lyase, L-amino acid oxidase, alpha-hydroxycarboxylic acid oxidase and L-lactate dehydrogenase respectively. The knockout or enhanced expression of a related gene on the genome of the Escherichia coli can promote the transportation of the substrate and reduce the decomposition of the product. The method for producing the p-hydroxyphenyllactic acid has the advantages of simple process, few impurities and great industrial application values.

The invention discloses an engineering bacteria and an application in preparation of L-alanine, which belongs to the technical field of bioengineering. The recombinant Escherichia coli of the presentinvention simultaneously express exogenous L-lactate dehydrogenase and L-alanine dehydrogenase, and enhances the expression of a lactate transporter gene and a NAD synthase gene on the basis of the host Escherichia coli. The method constructs the double-enzyme co-expressed engineering bacteria on the basis of engineering Escherichia coli transport and coenzyme synthesis system, and realizes efficient preparation of L-alanine by using cheap raw materials.