135 results about "Formate dehydrogenase" patented technology

The invention discloses a formate dehydrogenase mutant with improved enzyme activity and stability as well as a construction method of the formate dehydrogenase mutant and belongs to the field of gene engineering technology. According to the mutant, on the basis of amino acid shown in SEQ ID No.2, alanine in the 10th site is mutated into cysteine. The enzyme ratio and the enzyme activity of the obtained mutant are increased by 1.3 times by comparison with those before mutation, the half-life period (t1/2) at the temperature of 60 DEG C is prolonged by 6.8 times by comparison with that before mutation, the tolerance of cupric ions is improved by 30 times than that before mutation, the stability under the condition that pH is equal to 4 is improved by 2.0 times, and the catalytic efficiency is improved by 1.4 times. The formate dehydrogenase mutant and the construction method indicate that an amino acid residue in the 10th site is mutated into cysteine, and then cysteine forms a correct disulfide bond with a 30th cysteine residue of natural formate dehydrogenase, so that the stability and the catalytic efficiency of the enzyme are improved, and the industrial application potential of the enzyme is improved.

The invention relates to the field of biotechnology in the pharmaceutical industry, and discloses the preparation of an immobilized whole-cell catalyst and its application in the synthesis of a Puley intermediate (R)-2-hydroxy-4-phenyl-butyric acid. The recombinant cells containing the D-type lactate dehydrogenase gene and the formate dehydrogenase gene are established and fixed to prepare the immobilized whole cell catalyst. Using this catalyst to synthesize the intermediate (R)-2-hydroxy-4-phenyl-butyric acid of lisinopril, the reaction conditions are mild, the action is specific, and there are no by-products. It is used in medicine and food industries. value.

Nucleotide sequences encoding formate dehydrogenases D & E and a method for preparing succinic acid using the same, more particularly, formate dehydrogenases D & E converting formate to carbon dioxide and hydrogen, fdhD and fdhE nucleotide sequences encoding the formate dehydrogenases D & E, recombinant vectors containing the nucleotide sequences, microorganisms transformed with the recombinant vectors, and a method for preparing succinic acid using the transformed microorganism.

It is intended to obtain anaerobic microbial cells in an amount sufficient for hydrogen generation reaction, impart a hydrogen generation function to an aerobic microorganism within a short time and provide an industrial advantageous method of producing hydrogen. The above object can be established by providing a highly efficient microbial hydrogen production method characterized by comprising culturing a microorganism having a formate dehydrogenase gene and a hydrogenase gene under aerobic conditions, culturing the resulting microbial cells under anaerobic conditions in a liquid culture medium containing a formic acid compound, and then using the thus obtained cells for hydrogen generation.

This invention relates to a test kit for diagnosing hepatic and biliary diseases. The test kit has such advantages as high stability, high accuracy and high sensitivity. The test kit comprises two reagents. Reagent 1 comprises oxidized thio coenzyme, buffer solution, preservative, and anti-interference agent. Reagent 2 comprises 3alpha-HSD, reduced coenzyme, buffer solution, preservative, complexing agent, polymer accelerator, stabilizer, and one of formate dehydrogenase-formic acid-NAD, glutamate dehydrogenase-glutamic acid-oxidized nicotinamide coenzyme, glucose-6-phosphate dehydrogenase-glucose-oxidized nicotinamide coenzyme, glucose dehydrogenase-xylose-oxidized nicotinamide coenzyme, lactate dehydrogenase-alpha-ketoglutarate-oxidized nicotinamide coenzyme, and glycerol dehydrogenase-ethylene glycol-oxidized nicotinamide coenzyme.

A process for production of cyclopropyl-fused pyrrolidine-based inhibitors of dipeptidyl peptidase IV is provided which employs a BOC-protected amine of the structure

prepared by subjecting an acid of the structure

to reduce amination by treating the acid with ammonium formate, nicotinamide adenine dinucleotide, dithiothreitol and partially purified phenylalanine dehydrogenase/formate dehydrogenase enzyme concentrate (PDH/FDH) and without isolating treating the resulting amine of the structure 2

with di-tert-butyl dicarbonate to form the BOC-protected amine.

The invention relates to a method for preparing alpha-aminobutyric acid by utilizing series connection amino acid dehydrogenase and codon optimization formate dehydrogenase recombinant escherichia coli. First, codon optimization is performed on a Candida boidinii formate dehydrogenase gene sequence according to the codon preference of the escherichia coli. After codon optimization on formate dehydrogenase, the expression quantity of formate dehydrogenase in the escherichia coli can be improved remarkably, the yield of alpha-aminobutyric acid is increased, and in the escherichia coli, co-expression formate dehydrogenase and L-amino acid dehydrogenase can promote circulation of cofactors in mycetome without needing the adding of any exogenous cofactors. In addition, the process of utilizing whole-cell transformation bulk chemical L-threonine to produce alpha-aminobutyric acid is simple and quick, and the cost is low. In a 5 L fermentation tank, the yield of alpha-aminobutyric acid obtained through the method can be 81.5 g/L, and an actually effective strategy is provided for industrial production of alpha-aminobutyric acid.

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.

The invention discloses a method for producing a phenyllactic acid through whole-cell transformation of phenylalanine, and belongs to the technical field of enzyme engineering. The method successfullyconstructs an L-amino acid deaminase, L-lactic dehydrogenase and formate dehydrogenase co-expressed strain. Phenyllactic acid of 30 g/L is produced through the whole-cell transformation of phenylalanine, and the transformation rate is 100%. The establishment of a whole-cell transformation system solves the problems of complicated steps, low yield and environment pollution in the chemical synthesis of the phenyllactic acid as well as the problem of low transformation rate in the production of the alpha-oxoglutarate through the enzymatic conversion, the phenyllactic acid is produced through a one-step method at high yield without pollution, and a certain theoretical foundation is laid for the subsequent industrial production.

The invention discloses a method of immobilizing enzyme by means of coordination of graphene oxide and metal ions and belongs to the technical field of enzyme immobilization. In the immobilization, transition-state metal ions are coordinated with a carboxyl group on the graphene oxide, and through the coordination, formate dehydrogenase is immobilized. The graphene oxide in a lamellar structure is an excellent immobilization carrier, of which the surface has functional groups, such as epoxy groups, carbonyl groups, carboxyl groups and the like, so that the carrier is large in support area. Through the coordination effect of the metal ions, the graphene oxide is uniformly dispersed and support load of the enzyme is increased. In addition, through a coordination interaction with a histidine label of the enzyme, the enzyme is immobilized on the graphene oxide. The method is simple in processes, has mild preparation conditions and high immobilization rate. The immobilized enzyme is significantly improved in temperature stability, pH stability and repeatedly usage stability, and is high in enzyme activity recovery rate.

The invention relates to a thermostability enhanced formate dehydrogenase mutant and a preparation method thereof. The mutant is derived from wild type formate dehydrogenase of Candida boidinii, can catalyze coenzyme NADH cyclic regeneration, shows higher thermostability compared with the wild type formate dehydrogenase, and has one or more mutation characteristics of I98V, V152I, A154D and D158R. The recombinant formate dehydrogenase mutant provided by the invention has better thermostability, and is suitable for cyclic regeneration of NADH under high temperature. Compared with the formate dehydrogenase discovered so far, the formate dehydrogenase mutant has the activity that is not significantly reduced in the range of 50DEG C-55DEG C, and has better thermostability, thus being more suitable for industrial application.

The invention provides a preparation method of (R)-2-(2,5-difluorophenyl) pyrrolidine or salt thereof. N-protected pyrrolidone and a 2,5-fluorophenyl magnesium chloride Grignard reagent are subjectedto a Grignard reaction, and a compound represented as the formula 3 is obtained; the compound represented as the formula 3 is subjected to deprotection and ring closure under the acidic condition, anda compound represented as the formula 4 or salt of the compound is obtained; under enzyme catalysis, formic acid or formate is utilized as a hydrogen donor, a cofactor is adopted, cofactor cycle is realized, and the compound represented as the formula 4 or the salt of the compound is selectively reduced to synthesize an optically pure compound, namely, (R)-2-(2,5-difluorophenyl) pyrrolidine or the salt thereof; enzyme is a combination of recombinant imine reductase and formate dehydrogenase, and the cofactor is oxidized/reduced type nicotinamide adenine dinucleotide phosphate or oxidized/reduced type nicotinamide adenine dinucleotide. The method is safe in process and simple to operate, and yield and optical purity of a product are both higher.

The invention discloses an enzyme combination for producing L-phosphinothricin. The enzyme combination comprises glutamate dehydrogenase and a coenzyme regenerating enzyme, wherein the coenzyme regenerating enzyme is alcohol dehydrogenase, formate dehydrogenase and phosphite dehydrogenase. The invention also discloses a production method of L-phosphinothricin, 4-(methylhydroxyphosphinyl)-2-oxobutyric acid is used as a raw material, NH<4><+>, a coenzyme NADP<+>/NADP, and a coenzyme regeneration substrate are added, and then the enzyme combination is used for catalysis, wherein the glutamate dehydrogenase is used to catalyze a reaction of 4-(methylhydroxyphosphinyl)-2-oxobutyric acid to obtain L-phosphinothricin, and the coenzyme regenerating enzyme is used to reduce NADP<+> to NADP. According to the enzyme combination and the production method of L-phosphinothricin provided by the invention, by-products produced are very easy to remove, a post-treatment process of the product is simplified, the total yield of the product is greater than 95%, and the production cost of L-phosphinothricin is reduced, so that the method is a green, environment-friendly, and low-carbon process route, and is suitable for large-scale industrial production applications.

The invention discloses a method for detecting TMAO (trimethylamine oxide) by an enzyme method and application thereof. The detection method utilizes a multi-enzyme combination system containing TMAOTDM (demethylase), FADH (formaldehyde dehydrogenase), FDH (formate dehydrogenase) and diaphorase, so as to perform fluorescence determining on the TMAO; the method can be used for developing a TMAO detection kit. The method for detecting the TMAO by the enzyme method has the advantages that the defect of failure to directly determine based on dehydrogenase and diaphorase coupling because of no participation of NAD dependence dehydrogenase in the metabolism path of the TMAO is overcome; by adopting the multi-enzyme coupling of TDM-FADH-FDH-diaphorase, the system for fluorescence determining ofthe TMAO is constructed, and one TMAO molecule is decomposed into two fluorescence signals; the sensitivity is high, the detection is rapid and stable, the repeatability is good, the operation is simple and convenient, and the cost is lower.

The invention discloses a biotransformation method for L-glufosinate. The biotransformation method comprises the following steps: composing a reaction system from a solvent and 2-carbonyl-4-(hydroxymethylphosphoryl)butyric acid used as a substrate, and then adding a biocatalyst, coenzyme, an additive and an ammonium salt into the reaction system for a biotransformation reaction so as to obtain a transformed solution containing L-glufosinate, wherein the biocatalyst is the whole cell of a genetically engineered bacterium co-expressed by glufosinate dehydrogenase derived from Saccharomyces cerevisia and formate dehydrogenase derived from Candida boidinii; the coenzyme is NADP+; and the ammonium salt is ammonium formate. The biotransformation method is simple in process flow, free of specialrequirements on equipment and suitable for industrial production. HPLC-MS and HPLC are employed for monitoring until the substrate is fully utilized.

The invention discloses a single cell factory capable of efficiently synthesizing L-phenylglycine as well as construction and application of the single cell factory and belongs to the technical fieldof microorganisms. Firstly, efficient expression of leucine dehydrogenase obtained from Bacillus cereus in escherichia coli is realized, and site-directed mutation is carried out to obtain a mutant N71S with a remarkably improved reduction property; a mutant enzyme and a formate dehydrogenase mutant are co-expressed in the escherichia coli to form an intracellular in-situ co-factor NADH (Nicotinamide Adenine Dinucleotide) circulating system; the expression amount of the formate dehydrogenase mutant is optimized and controlled through a promoter and an RBS (Ribosomal Binding Site) sequence to successfully construct a recombinant escherichia coli single cell factory; the single cell factory is subjected to whole-cell conversion to prepare the L-phenylglycine. The method disclosed by the invention has the advantages of simple and rapid conversion process, low cost, no byproduct and easiness for separation and purification; when conversion is carried out in a 5L fermentation tank for 4h, the yield of the L-phenylglycine can reach 105.7g/l, the conversion rate is 93.3 percent and the space-time yield of the L-phenylglycine is 26.3g/L; an actually practical and effective strategy is provided for industrial production of the L-phenylglycine.

The invention discloses a method for preparing chiral (S)-acetoin by virtue of whole-cell biological catalysis. The method comprises the following steps: firstly carrying out coexpression on butanedione reductase genes and formate dehydrogenase genes in escherichia coli, so as to obtain recombinant escherichia coli; and carrying out asymmetric reduction reaction in a water phase by taking a resting cell of the recombinant escherichia coli as a whole-cell biocatalyst, butanedione as a substrate and formic acid or formate as a coenzyme NADH regenerated hydrogen donor, so as to generate chiral (S)-acetoin. A whole coenzyme regeneration system of the cell is utilized, and only a proper amount of formic acid or formate needs to be added as the hydrogen donor, so that the transfer ability of a thallus is improved; expensive coenzyme NADH does not need to be added, and the yield of chiral (S)-acetoin can reach up to 46.1g/L; the process is simple and efficient, the production cost is low, and the problems of low efficiency and high cost of traditional conversion are solved; and the method has an extremely high economic value and is applicable to large-scale business application.

The invention belongs to the field of asymmetrically preparing chiral medicine intermediates in a biocatalysis method, and particularly relates to a method for preparing 3R, 5S-dyhydroxy-6-benzyloxy-ethyl hexanoate with single optical purity via reduction by taking 3, 5- dicarbonyl-6-benzyloxy-ethyl hexanoate as a substrate and taking double carbonyl reductase as a biocatalyst. The method comprises the following steps: (1) adding the substrate, the double carbonyl reductase and a coenzyme cyclic regeneration system mediated by formate dehydrogenase in a reaction liquid, and oscillating for reaction for at least 1 hour, wherein the reaction liquid is the mixed solution of an organic solvent and a buffer solution; and (2) separating and purifying the product obtained in step (1) to obtain a single optical isomer 3R, 5S- dihydroxyl compound. The invention adopts the double carbonyl reductase and is simultaneously combined with the nonaqueous phase solvent system and the coenzyme regeneration system to synthesize the 3R, 5S-dyhydroxy-6-benzyloxy-ethyl hexanoate via catalysis, the optical purity ee and de values are both larger than 99.5%, the concentration of the substrate reaches 150g/L, and the method has wide practical value.

The invention relates to promotion of synthesis of (R)-styrene glycol by coupling (R)-carbonyl reductase and formic dehydrogenase, which belongs to the technical field of asymmetrical transformation of biological catalysis. The invention provides a recombinant Escherichia coli, namely E.coli Rosetta/ pETDuet-rcr-fdh, with a preservation number of CCTCC NO: M208123, wherein the (R)-special carbonyl reductasei and the formic dehydrogenase are coupled by a co-expression mode; asymmetrical transformation reaction is catalyzed by utilizing a cultured recombinant strain and taking 2-hydroxyacetophenone as a substrate; and the optical purity of the (R)-styrene glycol reaches 100 percent e.e., and the yield of the (R)-styrene glycol reaches 85.9 percent by optimizing the biotransformation reaction conditions. The invention utilizes double-enzyme coupling technology to solve the problem of limitation of regeneration cycle of coenzyme under the condition of high-concentration substrate, provides an effective path for high-efficiency and low-cost preparation of the (R)-styrene glycol, and lays a foundation for industrial application of the biosynthesized (R)-styrene glycol.

The invention provides a coenzyme regeneration system which comprises formate dehydrogenase (FDH), leucine dehydrogenase (LDH) and ammonium formate. The invention further provides an establishment method of the coenzyme regeneration system. The establishment method mainly includes the steps of 1), heterogeneous expression of formate dhydrogenase and leucine dehydrogenase and 2), establishment of the coenzyme regeneration system. The coenzyme regeneration system is high in coenzyme regeneration efficiency and suitable for large-scale industrialized application, the establishment method is simple and convenient, and cost of coenzyme reaction is lowered greatly.

The invention relates to a hollow fibrous membrane reactor integrating a gas distribution function and an enzyme catalysis function and an application method of the reactor. A hollow fibrous membrane is used as a gas distributor, the hollow fibrous membrane for distributing the gas and a hollow fibrous membrane of an immobilized enzyme are assembled into a same component, the bottom end of the membrane component and the bottom ends of all fibrous membranes in the component are sealed, the top end of the membrane component and the top end of the hollow fibrous membrane of the immobilized enzyme are sealed, and the top end of the hollow fibrous membrane for distributing the gas is open and is used for introducing CO2 gas; the hollow fibrous membrane reactor is applied to a formate dehydrogenase (FDH) catalytic CO2 methanoic acid synthetic system, by controlling the upstream gas rate, the number of the hollow fibrous membrane for distributing the gas and the micro-structural parameters of the membrane, the quantity and the size of bubbles, the retention time and space distribution in a solution can be adjusted, so that the gas-liquid contact time and the gas-liquid contact area can be increased, the liquid phase can be effectively disturbed, the rapid and efficient dispersion and mixing between the gas phase and the liquid phase and inside the liquid phase can be realized, and the mass transfer effect can be improved.

The invention discloses a process method for preparing single optical pure L-homophenylalanine by using L-homophenylalanine dehydrogenase as a biological catalyst. The method particularly comprises the following steps of: adding a substrate, ammonium formate, the L-homophenylalanine dehydrogenase and a formate dehydrogenase mediated coenzyme recycling and regenerating system into reaction liquid; after reacting under an oscillating or stirring condition, filtering from the reaction system to directly obtain an L-homophenylalanine product; and circulating the filtrate for the next reaction, wherein the reaction temperature is between 15 and 40 DEG C. The L-homophenylalanine is prepared by coordinating the L-homophenylalanine dehydrogenase with the coenzyme recycling and regenerating system and by adopting a simple filtering circulation process, wherein the substrate concentration reaches 560 mM; the NAD+ concentration of the required coenzyme is low; the coenzyme can be recycled and reused for many times, so that the cost of the coenzyme can be ignored; the yield excesses 95 percent; and important application value is achieved.