40 results about "Formate dehydrogenase H" patented technology

The invention discloses an alcohol dehydrogenase mutant and application thereof to synthesis of diaryl chiral alcohol and belongs to the technical field of biological engineering. The alcohol dehydrogenase mutant disclosed by the invention has good catalytic activity and stereoselectivity, and a series of chiral diaryl alcohol with R- and S- configurations can be prepared through efficient catalysis. According to the alcohol dehydrogenase mutant, alcohol dehydrogenase is coupled with glucose dehydrogenase or formate dehydrogenase, and can be used for synthesizing various antihistamine drugs, i.e., a chiral diaryl alcohol intermediate. Compared with an existing report, a method for preparing the diaryl chiral alcohol through asymmetric catalysis of the alcohol dehydrogenase has the advantages of simplicity in operation, high substrate concentration, complete reaction and high product purity and has a very good industrial application prospect.

The invention discloses a phosphinothricin dehydrogenase mutant, genetic engineering bacteria and a one-pot multi-enzyme synchronous directed evolution method. The phosphinothricin dehydrogenase mutant is obtained by mutating the 164th amino acid from alanine to glycine, mutating the 205th arginine to lysine and mutating the 332nd threonine to alanine of phosphinothricin dehydrogenase derived fromPseudomonas fluorescens, and an amino acid sequence is as shown in SEQ ID No.1. The genetically engineered bacteria are obtained by introducing a gene of the phosphinothricin dehydrogenase mutant into a host cell. An encoding gene of glucose dehydrogenase or an encoding gene of formate dehydrogenase can also be introduced into the host cell to perform simultaneous directed evolution to overexpress the double genes. The one-pot multi-enzyme synchronous directed evolution method of the invention can screen out the genetically engineered bacteria with greatly improved activity. Compared with catalytic processes such as transaminase, the L-PPT preparation method of the invention has a relatively simple process, a high conversion rate of raw materials, a conversion rate of up to 100%, and highstereo-selectivity.

A thermophilic microorganism lacks lactate dehydrogenase activity and preferably contains an active pyruvate formate lyase pathway. The thermophilic microorganism contains a gene encoding an NAD-linked formate dehydrogenase. The gene encoding an NAD-linked formate dehydrogenase is preferably a codon optimised version of the gene encoding a thermostable NAD-linked formate dehydrogenase. DNA constructs allow stable expression of the gene encoding an NAD-linked formate dehydrogenase in the thermophilic microorganism. The DNA constructs are based upon use of an insertion sequence to achieve stable expression or recombination to insert the gene encoding an NAD-linked formate dehydrogenase into the lactate dehydrogenase gene, thus achieving gene knockout and new functionality in a single step. The microorganisms are useful in fermentation of sugars to produce ethanol.

An objective of the present invention is to provide polypeptides capable of retaining a strong enzyme activity of formate dehydrogenase in the presence of an organic solvent and to provide the uses thereof. Formate dehydrogenase mutant polypeptides, which are resistant to organic solvents, were constructed by substituting cysteines at position 146 and / or at position 256 in the amino acid sequence of Mycobacterium vaccae-derived formate dehydrogenase by site-directed mutagenesis. The polypeptides have strong activities of formate dehydrogenase in the presence of an organic solvent. The mutants are useful for the production of alcohols using ketones as raw material, etc. <IMAGE>

The invention provides a formate dehydrogenase having high specific activity, small Km values for formate and NAD, broad temperature and pH ranges of stability, broad temperature and pH ranges for action, and capable of regenerating a coenzyme with good efficiency in case of being present in an enzymatic reduction reaction system without inactivated.The invention provides the enzyme mentioned above which has characteristics suited for industrial application by screening for soil formate dehydrogenase-producing microorganisms. The invention further provides a DNA containing the gene coding for the enzyme of the invention, a recombinant DNA constructed using a vector, and a transformant obtained by using this plasmid. The invention further provides a process for producing the formate dehydrogenase of the invention by using any microorganism of the genus Thiobacillus or a transformant constructed by using a formate dehydrogenase gene derived from above strain of microorganism, and a process for regenerating a coenzyme by using said enzyme in an enzymatic reduction system.

The invention discloses an alcohol dehydrogenase mutant, and an application thereof in the synthesis of bisaryl chiral alcohols, and belongs to the technical field of bioengineering. The alcohol dehydrogenase mutant has excellent catalytic activity and stereoselectivity, and achieve efficiently catalyzed preparation of a series of chiral bisaryl alcohols with R- and S- configurations. Alcohol dehydrogenase is coupled with glucose dehydrogenase or formate dehydrogenase in order to synthesize multiple antihistamine chiral bisaryl alcohol intermediates. Compared with existing reports, the methodfor preparing the bisaryl chiral alcohols by asymmetric catalytic reduction of the alcohol dehydrogenase has the advantages of simplicity in operation, high substrate concentration, completeness in reaction, high product purity and great industrial application prospect.

The invention relates to an NADP+ dependent formate dehydrogenase mutant with significantly improved substrate affinity and coenzyme affinity and a coding nucleic acid of the mutant, and a recombinantexpression vector and a recombinant expression transformant containing a nucleic acid sequence. The formate dehydrogenase mutant is a derived protein constituted by a new amino acid sequence formed by substituting one or more amino acid residues of 30th arginine, 124th isoleucine, 146th glycine, 216th glycine, 261st proline, 262nd serine, 287th alanine, 361st arginine or 381st glutamine in an amino acid sequence shown in SEQ ID No.2 with other amino acid residues; the substrate affinity and the coenzyme affinity of the derived protein are higher than those of formate dehydrogenase constitutedby the amino acid sequence shown in SEQ ID No.2. Compared with the prior art, the mutant has the advantages of high substrate affinity and coenzyme affinity, and has a good application prospect.

The invention discloses a method for measuring the activity of formate dehydrogenase in plant leaves and evaluating the activity of formate dehydrogenase. The activity of formate dehydrogenase in the leaf extracting solution is eliminated in a high-temperature treatment manner, and according to the chemical property of formic acid, a potentiometric titration method is adopted to quantitatively evaluate the activity of formate dehydrogenase in plant leaves through measuring the reduction value of the formaldehyde content in the plant fresh extracting solution and the inactivated extracting solution and the acidity change difference in the plant fresh extracting solution and the inactivated extracting solution before and after the formaldehyde is added. Experimental results show that when the ratio of the plant mass to the extracting solution is 1: 4 and the formaldehyde concentration is 350 mg.L <-1>, the activity of the formate dehydrogenase is sequentially as follows: the activity of the scindapsus aureus is greater than that of peony chlorophytum comosum, and the activity of the peony chlorophytum comosum is greater than that of aloe; when the formaldehyde concentration is 500-750 mg.L <-1>, the activity of the formate dehydrogenase is as follows: the activity of the peony chlorophytum comosum is greater than that of the scindapsus aureus, and the activity of the formate dehydrogenase is not detected in the aloe leaf extracting solution.

The invention discloses a glufosinate-ammonium dehydrogenase mutant, an engineering bacterium, an immobilized cell and application. The mutant is obtained by carrying out single mutation or multiple mutations on the 145th site, the 384th site, the 348th site, the 292th site, the 202 site, the 70th site and the 78th site of a glufosinate-ammonium dehydrogenase amino acid sequence shown in SEQ ID No.2. According to the immobilized cell of the co-expression engineering bacterium of the glufosinate-ammonium dehydrogenase mutant and the formate dehydrogenase, the L-glufosinate-ammonium is obtained through catalytic reduction by using a 2-carbonyl-4-(hydroxymethylphosphonyl)-butyric acid substrate, the asymmetric synthesis of the L-glufosinate-ammonium is realized, an expensive chemical resolution reagent is not needed, the thermal stability and the operation stability of the L-glufosinate-ammonium are improved, and the repeated use is realized. The enzyme activity of the immobilized cells is 162.5 U / g, the enzyme activity recovery rate is 78.1%, and the immobilized cells can be recovered through filtration and can be repeatedly used for more than 20 batches to keep 100% conversion rate, so that the production cost is greatly reduced.

The invention provides a kind of alcohol dehydrogenase and its encoding gene and its catalytic synthesis ( R )-phenylethylene glycol, the amino acid sequence of the alcohol dehydrogenase is shown in sequence 1 in the sequence listing, and its gene sequence is shown in sequence 2 in the sequence listing. alcohol dehydrogenase gene mladh In catalytic synthesis ( R )‑Phenylglycol. Alcohol dehydrogenase MLADH and formate dehydrogenase FDH were used to construct a dual-enzyme coupled catalytic system. In the system, the enzyme activity ratio of alcohol dehydrogenase MLADH and formate dehydrogenase FDH was 1:5-10. The invention constructs a double-enzyme coupling system of alcohol dehydrogenase MLADH and formate dehydrogenase FDH, and realizes the in-situ regeneration of coenzyme NADH. In the present invention, soybean oil is introduced into the system using phosphate buffer salt as the water phase as the organic phase, which reduces the inhibition of the catalytic reaction by the substrate and the product, promotes the efficiency of the catalytic conversion, and the conversion rate reaches 99%. e.e. The value is above 99%.

The invention relates to a genetically engineered bacterium capable of efficiently producing 1,3-propanediol and a construction method and application thereof. The present invention strengthens the expression of 1,3-propanediol oxidoreductase in Klebsiella pneumoniae to enhance the enzymatic activity of 1,3-propanediol oxidoreductase, and simultaneously constructs a NADH regeneration pathway to reduce the accumulation of 3-hydroxypropionaldehyde, Enhances the conversion of 3-hydroxypropionaldehyde to 1,3-propanediol, thereby increasing the yield of 1,3-propanediol. The enzymatic activity of the 1,3-propanediol oxidoreductase of the genetically engineered bacteria constructed by the present invention is 3.25-3.5 times that of the starting strain; the enzymatic activity of the formate dehydrogenase is 0.414-0.45 U / mg. Using the genetically engineered bacteria constructed by the present invention to carry out fed-batch fermentation for 29 hours, the yield of 1,3-propanediol is increased by 43.89%-97.93% compared with the starting strain, and the synergistic effect of the double genes significantly increases the yield of 1,3-propanediol and yield.

The invention discloses a method for synthesizing L-2-aminobutyric acid by an enzymatic method. According to the method, 2-ketobutyric acid is catalyzed by the aid of alanine dehydrogenase and formate dehydrogenase to produce the L-2-aminobutyric acid. The method particularly includes the steps: uniformly mixing 0.5-1.5mol of 2-ketobutyric acid, 0.5-1.5mol of ammonium formate, o-0.5g / L of NAD (nicotinamide adenine dinucleotide) and 20g / L of formate dehydrogenase and alanine dehydrogenase co-expression wet cells (or 10-30g / L of formate dehydrogenase wet cells, 10-30g / L of alanine dehydrogenase wet cells) in a rector; adjusting a pH (potential of hydrogen) value to be 7.0-9.0; performing catalytic reaction for 16-20h at the temperature ranging from 30 DEG C to 37 DEG C to obtain the L-2-aminobutyric acid. The method is rapid in reaction, reverse oxidation deamination activity is low, and product loss is greatly reduced.

The invention relates to an NADP+ dependent formate dehydrogenase mutant with significantly improved substrate affinity and coenzyme affinity and a coding nucleic acid of the mutant, and a recombinantexpression vector and a recombinant expression transformant containing a nucleic acid sequence. The formate dehydrogenase mutant is a derived protein constituted by a new amino acid sequence formed by substituting one or more amino acid residues of 30th arginine, 124th isoleucine, 146th glycine, 216th glycine, 261st proline, 262nd serine, 287th alanine, 361st arginine or 381st glutamine in an amino acid sequence shown in SEQ ID No.2 with other amino acid residues; the substrate affinity and the coenzyme affinity of the derived protein are higher than those of formate dehydrogenase constitutedby the amino acid sequence shown in SEQ ID No.2. Compared with the prior art, the mutant has the advantages of high substrate affinity and coenzyme affinity, and has a good application prospect.

A process for preparing ethyl (3R, 5S)-dihydroxy-6-benzyloxy hexanoate via stereoselective microbial reduction is provided, which includes that ethyl 3,5-diketo-6-benzyloxy hexanoate is used as a substrate and a diketoreductase is used as biocatalyst. Specifically, the process includes these following steps: (1) the substrate, diketoreductase and cofactor regeneration system mediated by formate dehydrogenase are added to the reaction liquid and conducted to shake and react for at least one hour, and the reaction liquid is the mixture of organic solvent and buffer solution; (2) the product of step (1) is separated and purified to obtain the single enantiomer of (3R, 5S)-dihydroxy compound.

The present invention relates to a recombinant microorganism for producing formic acid, which has a formate dehydrogenase 1 alpha subunit (FDH1α)-encoding endogenous gene deleted therefrom and an FDH1-encoding exogenous gene introduced thereinto, and a method for production of formic acid by using the microorganism.

Improved yeast cell with increased succinic acid production based on yield and titer. Increased activity of one or more enzymes involved in the pentose phosphate pathway, reducing flux through phosphoglucose isomerase, increasing flux to cytoplasmic acetyl-CoA, installation of a malic enzyme, and / or installation of a formate dehydrogenase leads to increased production of succinic acid.

The invention discloses a more effective glycerol utilization method, namely: knocking out the gene encoding formate dehydrogenase and pyruvate dehydrogenase complex in Klebsiella pneumoniae (Klebsiella pneumoniae), and utilizing the obtained recombinant bacteria that simultaneously produce 1,3-propanediol and acetoin. Compared with the existing technology, when using the biotransformation glycerin of the present invention, not only can two high value-added products be produced simultaneously, but also the efficiency of product separation and purification is significantly improved.

The invention relates to a recombinant engineering bacterium and application thereof in efficient conversion of L-pantoic acid lactone, the recombinant engineering bacterium is induced to efficiently express L-pantoic acid lactone dehydrogenase, ketone pantoic acid lactone reductase and formate dehydrogenase, and the L-pantoic acid lactone is efficiently converted to generate DL-pantoic acid lactone. According to the recombinant engineering bacterium, the purity and quality of DL-pantoic acid lactone, the reaction efficiency and the resource utilization rate are remarkably improved, the production cost is reduced, and the recombinant engineering bacterium has the advantages of being easy and convenient to operate, environmentally friendly and the like.