39 results about "Formate dehydrogenase H" patented technology

The invention relates to a preparation method and application of a sludge petroleum degrading complex enzyme. The preparation method comprises the following steps: (1), performing cell disruption on acinetobacter calcoaceticus, centrifuging, and taking a supernatant to obtain a petroleum degrading enzyme solution 21#; (2), mixing the petroleum degrading enzyme solution 21# and formate dehydrogenase to obtain the sludge petroleum degrading complex enzyme. For the first time, after a petroleum-degrading enzyme system derived from acinetobacter calcoaceticus is mixed with the formate dehydrogenase in proportion, the sludge petroleum degrading complex enzyme can be applied to degradation and repair of petroleum in high-concentration petroleum-contaminated sludge, so that immobilization of adsorbents such as diatomite is not required, and the petroleum in the sludge can be reduced in a short time. The sludge petroleum degrading complex enzyme has high-efficiency petroleum contamination treatment capacity, has low production cost, and has a wide application prospect.

The invention relates to a construction method and application of a formate dehydrogenase engineering strain. The construction method comprises the following steps: (1) extracting candida boidinii genome DNA as a template; (2) with the genome DNA as the template, performing PCR amplification to obtain an fdh gene segment; (3) performing double digestion on the fdh gene segment and connecting to a plasmid pET28a(+) to obtain a plasmid pET28a(+)-fdh; (4) transforming the plasmid pET28a(+)-fdh into an escherichia coli BL21(DE3) competent cell to obtain a recombinant strain BL21(DE3)-fdh for expressing fdh. According to the construction method provided by the invention, a primer pair is degenerate primers designed according to the fdh sequence of multiple strains of candida boidinii registered in NCBI (National Center of Biotechnology Information); An fdh gene can be amplified by taking the genome DNA of most candida boidinii strains as the template while the influence of individual base difference in the gene sequence is avoided.

The invention relates to a genetically engineered bacterium for producing free fatty acid under the assistance of formate, the genetically engineered bacterium is recombinant saccharomyces cerevisiae containing a formate dehydrogenase gene FDH, a 1, 5-diphosphate carboxylase/oxygenase gene RuBisCO and a ribulose phosphate kinase gene PRK, and the genetically engineered bacterium is subjected to chassis microbial modification of knockout of related genes of an FFAs catabolism pathway. The genetically engineered bacterium is safe and non-toxic, can enable microorganisms to produce FFAs by utilizing carbon dioxide and a derivative formic acid thereof through fermentation, and is environment-friendly, relatively low in production cost and wide in application prospect.

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.

The invention relates to a genetic engineering strain for efficiently producing 1,3-propanediol, a construction method for the genetic engineering strain and application of the genetic engineering strain. According to the genetic engineering strain, the construction method therefor and the application of the genetic engineering strain, the enzyme activity of 1,3-propanediol oxidoreductase is improved through strengthening expression of the 1,3-propanediol oxidoreductase in Klebsiella pneumoniae, meanwhile, a NADH regeneration way is constructed, thus, the accumulation of 3-hydroxypropanal is lowered, the conversion of the 1,3-propanediol from the 3-hydroxypropanal is strengthened, and thus, the yield of the 1,3-propanediol is increased. The enzyme activity of the genetic engineering strain, i.e., the 1,3-propanediol oxidoreductase, constructed by the method is 3.25 to 3.5 times that of a starting strain; and the enzyme activity of formate dehydrogenase is 0.414U/mg to 0.45U/mg. Throughcarrying out fed-batch fermentation for 29 hours by employing the genetic engineering strain constructed by the method, the yield of the 1,3-propanediol is increased by 43.89%-97.93% compared with that of the starting strain; and due to a dual-gene synergistic effect, the output and yield of the 1,3-propanediol are increased remarkably.

The invention discloses a formate dehydrogenase mutant and application thereof. The formate dehydrogenase mutant comprises mutation of at least one amino acid in the 54th site, the 121st site and the 228th site of an amino acid sequence as shown in SEQ ID No.1. Compared with conventional wild type formate dehydrogenase, the formate dehydrogenase provided by the invention has higher reductase activity on NMN, the enzyme activity of the formate dehydrogenase reaches about 80-886 times that of the wild type, the formate dehydrogenase can efficiently reduce the NMN into NMNH, and the formate dehydrogenase can be used in the form of crude enzyme without purification or can be used only after partial purification due to the high catalytic activity. Therefore, the production cost can be greatly reduced, and the method is suitable for large-scale industrial production and has better market competitiveness.

Disclosed are a phosphinothricin dehydrogenase mutant, a recombinant bacterium and a one-pot multi-enzyme synchronous directed evolution method. The phosphinothricin dehydrogenase mutant, with an amino acid sequence as shown in SEQ ID No.1, is obtained by mutating alanine at position 164 to glycine, arginine at position 205 to lysine, and threonine at position 332 to alanine in a phosphinothricin dehydrogenase derived from Pseudomonas fluorescens. The recombinant bacterium is obtained by introducing a gene encoding the phosphinothricin dehydrogenase mutant into a host cell. The host cell can also incorporate a gene encoding a glucose dehydrogenase or a gene encoding a formate dehydrogenase to undergo synchronous directed evolution to achieve double gene overexpression. The one-pot multi-enzyme synchronous directed evolution method of the present invention can screen recombinant bacteria with greatly improved activity. Compared with other catalysis processes such as the transaminase method, the method for preparing L-PPT of the present invention features relatively simple process, high conversion of raw materials of up to 100%, and high stereo selectivity.

The invention relates to the field of biochemical engineering, and discloses a carbonyl reductase mutant as well as a construction method and application thereof.The 143rd leucine in an amino acid sequence shown in SEQ ID NO.2 is mutated into alanine, the 136th leucine is mutated into alanine, and 135th glycine is mutated into isoleucine, so that the enzyme activity of the carbonyl reductase mutant is improved by 8 times; and the e.e. Value is increased to 99%, coenzyme circulation is successfully realized by coupling with formate dehydrogenase, and finally, the conversion rate and the e.e. Value of a 450 g/L substrate within 12 hours can be over 99% when the final concentration of thalli is 20 g (dcw)/L, so that the problems of low substrate concentration, low conversion rate, low yield, low e.e. Value, relatively high production cost and the like are effectively solved.

The invention discloses a method for reducing an NAD analogue by using formic acid and application of the method. In the method, a reducing agent is a formic acid compound, a catalyst is formate dehydrogenase capable of utilizing the formic acid compound, and while the formate dehydrogenase oxidizes the formic acid compound, the NAD analogue is converted into a reduction state. The method can be used for producing a reduction-state NAD analogue or a deuterated reduction-state analogue. The reducing power can also be provided for the enzymatic reaction consuming the reduction-state NAD analogue; the reduction-state NAD analogue can be used as a coenzyme to be applied to enzyme-catalyzed reduction reactions of malic enzyme ME-L310R/Q401C, D-lactic dehydrogenase DLDH-V152R, saccharomyces cerevisiae alcohol dehydrogenase and the like, and wide application of the NAD analogue is facilitated. The reduction method of the NAD analogue can be used for regenerating the reduced NAD analogue for preparing malic acid or lactic acid under a mild condition; and can also be used as a redox force to regulate the metabolic intensity of malic acid or lactic acid in microorganisms.

The invention relates to the technical field of industrial catalysis, in particular to a preparation method of reduced nicotinamide adenine dinucleotide, which comprises the following steps: 1) screening the activity of formate dehydrogenase at high temperature; 2) screening the activity of formate dehydrogenase under an acidic condition; (3) according to the results of the step (1) and the step (2), formate dehydrogenase with relatively high activity under a high-temperature condition and an acidic condition is selected for later use; respectively weighing ammonium formate and NAD (Nicotinamide Adenine Dinucleotide) into a flask, adding pure water, stirring and dissolving in a water bath, and regulating the pH to be acidic by using sodium hydroxide; finally, formate dehydrogenase obtained through screening is added for reaction, sodium hydroxide is used for maintaining the pH value to be acidic, and the reduced nicotinamide adenine dinucleotide is prepared. According to the method, the reaction can be completed in a short time, so that hydrolysis of a product under an acidic condition is avoided, and carbonate generated under an alkaline condition is also avoided; after the reaction, no large amount of carbonate exists, and the purification cost is low.

The invention relates to an efficient preparation method of D-pantoic acid, which specifically comprises the following steps: (1) valine, methanol and compound enzyme are mixed with water, and the compound enzyme comprises L-amino acid deaminase, methanol dehydrogenase, aldolase, formate dehydrogenase and ketopantoic acid reductase; (2) ammonium formate is added, and D-pantoic acid is prepared through an enzyme catalytic reaction under the conditions that the temperature is 25-55 DEG C and the pH value is 4-7; wherein the molar ratio of the valine to the methanol is (0.9 to 1.1): (0.9 to 1.1). According to the method, valine and methanol are used as substrates for fermentation and conversion to generate D-pantoic acid, the raw materials are cheap and easy to obtain, and the reaction cost is low.