16 results about "Formic dehydrogenase" patented technology

The invention discloses an enzyme catalytic synthesis method of a luliconazole chiral intermediate, which comprises the following steps: adding a recombinant escherichia coli wet cell for expressing alcohol dehydrogenase, a recombinant escherichia coli wet cell for expressing formate dehydrogenase, a buffer solution, a substrate, a cosolvent, ammonium formate and a coenzyme into a reaction container, reacting at 25-35 DEG C for 2-24 hours, extracting, separating, and carrying out rotary evaporation, thereby obtaining the luliconazole chiral intermediate. The luliconazole chiral intermediate is obtained; the substrate is 2, 2 ', 4'-trichloroacetophenone; the alcohol dehydrogenase is a mutant of AKR3. The method is mild in reaction condition, environmentally friendly, high in regioselectivity and stereoselectivity, high in conversion rate, high in chiral purity, small in enzyme dosage, low in preparation cost and suitable for industrial production.

The application discloses a carbonyl reductase mutant, a combined enzyme and application of the combined enzyme in synthesis of S-configuration boselike, and belongs to the technical field of biological catalysis and pharmacy. The amino acid sequence of the wild-type carbonyl reductase mutant provided by the application is shown as SEQ ID NO:1, and the mutant is obtained by mutation of the wild-type carbonyl reductase, and the mutation mode is that F at the 147th position is mutated into L, V at the 163rd position is mutated into Y, L at the 176th position is mutated into E, and P at the 200th position is mutated into R. By using the mutant, a catalytic system is formed by combining the mutant with formic acid dehydrogenase (FDH), and optical pure S-configuration boselike can be efficiently and highly selectively prepared under high substrate concentration of 250-320 g / L, separation and purification problems caused by generation of a large amount of soluble by-products in catalysis of other combined enzymes are avoided, a post-treatment process is obviously simplified, and production cost and environmental burden are reduced.

The invention discloses an enzymatic synthesis method of a melogabalin intermediate and related alcohol dehydrogenase, racemic 3-ethyl bicyclo [3.2. 0] hept-3-ene-6-ketone is used as a substrate, a genetically engineered bacterium for producing alcohol dehydrogenase is used as an enzyme catalyst, a genetically engineered bacterium for producing formate dehydrogenase, ammonium formate and coenzyme are matched, an enzymatic reaction is carried out under a stirring condition, and the melogabalin intermediate is obtained. And carrying out purification and separation on a reaction product to obtain the melogabalin intermediate (1R, 5S)-3-ethyl bicyclo [3.2. 0] hept-3-ene-6-ketone. The method has the advantages of simple reaction, few steps, mild reaction, less pollution and the like, and has great industrial application potential and commercial value.

This invention relates to a formate dehydrogenase mutant and its applications, belonging to the field of enzyme engineering. The formate dehydrogenase mutant is derived by mutating one or more of the following positions in the amino acid sequence shown in SEQ ID NO.1: tryptophan at position 128 is mutated to lysine, alanine at position 225 is mutated to valine, tryptophan at position 595 is mutated to cysteine, asparagine at position 750 is mutated to cysteine, aspartic acid at position 759 is mutated to valine, and serine at position 888 is mutated to glutamic acid. The formate dehydrogenase mutant provided by this invention has significant advantages in substrate affinity, catalytic activity, and enzyme stability, thereby increasing its value in industrial applications.

The invention relates to a formate dehydrogenase mutant and application thereof, and belongs to the field of enzyme engineering and carbon dioxide reduction. The invention provides a formate dehydrogenase mutant. The formate dehydrogenase mutant comprises a formate dehydrogenase catalytic subunit FdhA, an electron transport subunit FdhB and a membrane anchoring protein subunit FdhC, compared with formate dehydrogenase, the formate dehydrogenase mutant has the advantages that a membrane anchor protein subunit FdhC is deleted, or the membrane anchor protein subunit FdhC and an electron transport subunit FdhB are deleted. Compared with the existing formate dehydrogenase, the formate dehydrogenase mutant provided by the invention has high activity and can tolerate oxygen at the same time. The formate dehydrogenase mutant provided by the invention shows huge potential value in the carbon dioxide immobilization industry.

The invention relates to a formate dehydrogenase mutant and application thereof, and belongs to the field of enzyme engineering. The formate dehydrogenase mutant is obtained by mutating one or more of the following positions of an amino acid sequence shown as SEQ ID NO.1: the 128 tryptophan is mutated into lysine, the 225 alanine is mutated into valine, the 595 tryptophan is mutated into cysteine, the 750 asparagine is mutated into cysteine, and the 759 asparagine is mutated into valine. Serine at the 888th site is mutated into glutamic acid. The formate dehydrogenase mutant provided by the invention has the advantage that the substrate affinity, the catalytic activity, the enzyme stability and the like are obviously improved, and the value of the formate dehydrogenase mutant in industrial application is improved.

The invention belongs to the technical field of bioengineering, and relates to a modified mannitol dehydrogenase and application thereof, the mannitol dehydrogenase AsMDH is subjected to amino acid mutation in an amino acid sequence shown as SEQ ID NO.1, and the amino acid mutation site is at least one of the 37th site, the 75th site, the 176th site, the 185th site, the 197th site, the 225th site, the 244th site and the 300th site. The obtained recombinant strain containing the mutant and a strain of formate dehydrogenase containing CbFDH plasmid are subjected to double-enzyme coupling, so that the efficiency of producing D-mannitol by taking D-fructose as a raw material is remarkably improved. After 150g / L of fructose is added for reaction for 8 hours, the conversion rate of a double-enzyme system of the recombinant strain containing the Y224F mutant reaches 90.12%, and the conversion rate of a double-enzyme system of the recombinant strain containing the Q300L mutant reaches 86.23%. The cost of the recombinant strain is reduced, the enzyme activity is improved, and the conversion efficiency of D-mannitol is improved.

The application discloses a mutant D-amino acid dehydrogenase and a method for preparing D-phenylalanine through a multi-enzyme cascade, wherein the mutant D-amino acid dehydrogenase is obtained by site-directed mutagenesis of wild-type amino acid dehydrogenase of symbiotic bacillus licheniformis; the multi-enzyme cascade preparation method is to use cheap L-phenylalanine as raw material, first obtain DL-phenylalanine through catalysis of alanine racemase, and then convert L-phenylalanine into D-phenylalanine through L-amino acid deaminase, mutant D-amino acid dehydrogenase and formic acid dehydrogenase. The concentration of D-phenylalanine product obtained by the method reaches 80-120 g / L, and the conversion rate can reach 95-98 %, so that the method not only shortens the production cycle and has high product concentration, but also is easy to industrialize.

The application belongs to the technical field of bioengineering, and relates to a formic acid dehydrogenase mutant and application thereof Cl The amino acid sequence of the FDH is shown in SEQ ID NO. 1, and the amino acid mutation is at least one of positions 52, 87, 111, 124, 177 and 243. As The double-enzyme coupling is performed on the strain containing the mannitol dehydrogenase of the MDH plasmid, and thus the efficiency of producing D-mannitol from D-fructose is significantly improved. The double-enzyme system of the recombinant strain containing the L124F mutant has a catalytic conversion rate of 92.3% within 10 hours, which is obviously better than the conversion rate before the improvement, and provides potential for the industrial development of mannitol.

The present application relates to the technical field of biology, and particularly relates to a complex enzyme, a co-immobilized enzyme and application thereof. The present application provides a complex enzyme, a co-immobilized enzyme and application thereof, and a preparation method of Bose factor, wherein the preparation method takes 1-C-(beta-D-pyranosyl) acetone as an initial substrate, selectively synthesizes high-purity S-Bose factor in one step under the action of a carbonyl reductase, introduces formic acid dehydrogenase to regenerate an NADH system, takes NAD and sodium formate as substrates, cyclically generates NADH and carbon dioxide, reduces the accumulation of intermediate products, and finally stably realizes the generation of 380 g / L products, the reaction conversion rate is greater than 99%, the ee value is 100%, the immobilized enzyme is used for 15 times, the enzyme activity retention rate is above 85%, and compared with the free enzyme, the cost is reduced by more than 5 times.

The invention discloses formate dehydrogenase as well as a preparation method and application thereof, the amino acid sequence of the formate dehydrogenase is as shown in SEQ ID NO: 1, or the formate dehydrogenase has at least 95% sequence identity with the amino acid sequence as shown in SEQ ID NO: 1, and compared with the amino acid sequence as shown in SEQ ID NO: 1, the formate dehydrogenase contains amino acid residue difference at one or more sites of I127, C150, A203 or T344. The formate dehydrogenase disclosed by the invention can be used as a coenzyme regeneration enzyme to participate in an oxidoreductase catalysis reaction depending on a reduced coenzyme NADH, and relatively high enzyme activity and good thermal stability are kept in the reaction process, so that the regeneration efficiency of the reduced coenzyme NADH is maintained, and the generation amount of a product obtained by oxidoreductase catalysis is increased.

The purpose of the present invention is to provide a means with which it is possible to electrochemically measure a coenzyme in real time. Provided is an enzyme electrode including an electrode base material and a formic acid dehydrogenase β subunit (FoDH1B) fixed to the electrode base material via at least one compound selected from the group consisting of (1) and (2): (1) aromatic compounds having an aromatic hydrocarbon ring skeleton or a nitrogen-containing heterocyclic skeleton; and (2) compounds represented by general formula (2a). Formula 2a: HS-(CH2)m-OH [where m is an integer of 1-10].

The invention discloses a construction method of formic acid nutritional escherichia coli, and belongs to the technical field of bioengineering. According to the invention, E.coli MG1655 is used as an original strain, and a formic acid nutritional type recombinant strain E.coli FAFdP is successfully constructed through systematic metabolic engineering transformation. Metabolic transformation of the strain mainly comprises three key modules: 1) a formic acid-tetrahydrofolic acid conversion module which is responsible for initial activation of formic acid; 2) a glycine splitting system module for realizing integration and metabolic flux regulation of a C1 unit; 3) a formate dehydrogenase energy supply module for providing necessary reducing power support for formic acid metabolism. OD600 of the constructed recombinant strain E.coliFGFdP reaches 0.91 after shake-flask culture, and the recombinant strain E.coliFGFdP can be used as an excellent strain of artificial formic acid nutritional escherichia coli and has a good application prospect in the fields of chemical engineering, degradable materials and food.

The present application belongs to the technical field of bioengineering, and relates to a reformed mannitol dehydrogenase and application thereof As The amino acid sequence of the MDH is shown in SEQ ID NO. 1, and the amino acid mutation is at least one of positions 37, 75, 176, 185, 197, 225, 244 and 300. Cb The strain containing the formic acid dehydrogenase of the FDH plasmid is coupled with the strain containing the mannitol dehydrogenase, and then the efficiency of producing D-mannitol from D-fructose is significantly improved. After adding 150 g / L of fructose for 8 h, the conversion rate of the double-enzyme system of the recombinant strain containing the Y224F mutant reaches 90.12%, and the conversion rate of the double-enzyme system of the recombinant strain containing the Q300L mutant reaches 86.23%. The present application reduces the cost of the recombinant strain, improves the enzyme activity, and improves the conversion efficiency of D-mannitol.

The invention belongs to the technical field of bioengineering, and relates to a formate dehydrogenase mutant and application thereof, amino acid mutation is performed on an amino acid sequence of excavated formate dehydrogenase ClFDH, such as SEQ ID NO.1, and the amino acid mutation site is at least one of the 52nd site, the 87th site, the 111th site, the 124 site, the 177th site and the 243th site. The obtained recombinant strain containing the mutant is subjected to double-enzyme coupling with a mannitol dehydrogenase strain containing AsMDH plasmids, so that the efficiency of producing D-mannitol by taking D-fructose as a raw material is remarkably improved. The catalytic conversion rate of a recombinant strain double-enzyme system containing the L124F mutant reaches 92.3% within 10 h and is obviously superior to the conversion rate before modification, and potential is provided for industrial development of mannitol.

This invention provides a method for producing allicinol from recombinant *Escherichia coli* using glycerol and glucose as substrates. Using *E. coli* K-12 as the substrate host, a pathway for the synthesis of allicinol from glycerol and glucose is constructed by expressing genes fucA, yqaB, aldO, and rdh, and knocking out the glucose-specific phosphotransferase system IIBC component gene ptsG. Subsequently, genes mzwf, mgnd, alsE, and a6PP are introduced, while genes pfkA, gnd, and edd are knocked out to regulate the flux of the glycolysis and pentose phosphate pathways, thereby directing more carbon sources to the allicinol synthesis pathway. Finally, the formate dehydrogenase gene fdh and the glycerol kinase mutant gene glpK22 are introduced, and the methylglyoxal synthase gene mgsA is knocked out to construct a cofactor NADH cycle system and optimize the allicinol synthesis pathway, thus enabling *E. coli* to efficiently produce allicinol from glycerol and glucose through fermentation.