Abstract: Disclosed are a gene mining method combining functional sequence and structure simulation, an NADH-preferring phosphinothricin dehydrogenase mutant and an application thereof. The gene mining method comprises the following steps: (1) analyzing a characteristic sequence which an NADH-type glutamate dehydrogenase should have; (2) searching a gene library based on the characteristic sequence; (3) performing clustering analysis and protein structure simulation on genes obtained by the searching; (4) selecting genes that feature high gene aggregation and a protein structure similar to that of the known phosphinothricin dehydrogenase as candidate genes. A wild-type phosphinothricin dehydrogenase with an amino acid sequence as set forth in SEQ ID No.

Abstract: Disclosed are a machine learning gene mining method and a phosphinothricin dehydrogenase mutant for amino translocation. The phosphinothricin dehydrogenase mutant for amino translocation is obtained by mutation of a wild-type phosphinothricin dehydrogenase with an amino acid sequence as shown in SEQ ID No.2 at one of the following sites: (1) E263D-K134R-H96A-R290V; (2) E263D-K134R-H96A; (3) E263D-K134R; (4) E263D; (5) E263N; (6) E263C; and (7) E263G. The present invention utilizes the site-saturation mutagenesis technology to mutate a phosphinothricin dehydrogenase gene as shown in SEQ ID No. 1, finds that the 263rd, 134th, 290th and 290th positions are the key sites affecting enzyme activity and stereoselectivity, and obtains a mutant with enzyme activity and ee value much higher than those of the parent phosphinothricin dehydrogenase.

Abstract: The present invention discloses a method for preparing L-glufosinate ammonium powder. The method includes the following steps: (1) obtaining a transformation solution for performing biocatalytic transformation to prepare L-glufosinate ammonium, and filtering out bacteria to obtain a filtrate; (2) detecting the amount of ammonium sulfate in the filtrate obtained in step (1), adding calcium hydroxide or calcium oxide to react with the ammonium sulfate to produce calcium sulfate precipitates, and filtering out the calcium sulfate precipitates to obtain a filtrate; (3) detecting the amount of glufosinate ammonium in the filtrate obtained in step (2), adding zinc salt, adjusting pH to 5.5-6.8 to produce glufosinate ammonium zinc salt precipitates, and filtering and collecting the glufosinate ammonium zinc salt precipitates; (4) adding a solvent to dissolve the glufosinate ammonium zinc salt precipitates collected in step (3), and adjusting pH of a solution to 2-2.

Abstract: The present invention discloses a method for preparing L-glufosinate ammonium by biological enzymatic de-racemization, a glufosinate ammonium dehydrogenase mutant and a use thereof. The method for preparing L-glufosinate ammonium by biological enzymatic de-racemization includes catalyzing D,L-glufosinate ammonium as a raw material by a multi-enzyme catalysis system to obtain L-glufosinate ammonium. The enzyme catalysis system includes D-amino acid oxidase for catalyzing D-glufosinate ammonium in the D,L-glufosinate ammonium to 2-carbonyl-4-[hydroxy(methyl)phosphonyl]butanoic acid, and a glufosinate ammonium dehydrogenase mutant for catalytically reducing 2-carbonyl-4-[hydroxy(methyl)phosphonyl]butanoic acid to L-glufosinate ammonium. The glufosinate ammonium dehydrogenase mutant is obtained by mutation of glufosinate-ammonium dehydrogenase in wild fungi Thiopseudomonas denitrificans at a mutation site of V377S.

Abstract: A cofactor self-sufficient Escherichia coli and its construction method and application in the synthesis of L-glufosinate are provided. The present invention expresses a NADH kinase and key enzymes of the cofactor synthesis pathway in E. coli, and knocks out the genes of enzymes that catabolizes cofactor, and with the addition of co-metabolic intermediates during cell incubation, the intracellular NADP(H) concentration is increased by at least 50% and the catalytic activity of glutamate dehydrogenase by 2-fold, resulting in a significant increase in the spatiotemporal yield of the-glufosinate synthesis reaction.

Abstract: 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.

Abstract: The present invention discloses an amino acid dehydrogenase mutant and application thereof in synthesizing L-glufosinate-ammonium, the amino acid dehydrogenase mutant is obtained by a single mutation or a multi-site mutation of the amino acid at position 95, 108, 172, 303 of the amino acid sequence shown in SEQ ID No. 2. The amino acid dehydrogenase mutant DyGDH-F95I-A108T-R172P-R303H prepared by the present invention has a specific enzyme activity that is 33 times higher than that of the original Aldo-keto reductase, and the concentration of the largest substrate, 2-carbonyl-4-(hydroxymethylphosphinyl)-butyric acid reaches 500 mM, the amino acid dehydrogenase mutant has more industrial application prospects.

Abstract: A method for asymmetrically preparing L-phosphinothricin by oxidation-reduction reaction through biological multienzyme coupling, where D,L-phosphinothricin as a raw material is catalyzed by an enzyme catalysis system to obtain L-phosphinothricin, wherein the enzyme catalysis system comprises a D-amino acid oxidase mutant for catalyzing D-phosphinothricin in D,L-phosphinothricin into 2-carbonyl-4-[hydroxy(methyl)phosphono]butyric acid and a transaminase for catalytic reduction of the 2-carbonyl-4-[hydroxy(methyl) phosphono]butyric acid into L-phosphinothricin; the D-amino acid oxidase mutant is obtained by mutation of D-amino acid oxidase in wild strain Rhodotorula taiwanensis at one of the following four sites: (1) M213S; (2) M213S-N54V-F58E; (3) M213S-N54V-F58E-D207A; (4) M213S-N54V-F58E-D207A-S60T.

Abstract: Disclosed are a machine learning gene mining method and a phosphinothricin dehydrogenase mutant for amino translocation. The phosphinothricin dehydrogenase mutant for amino translocation is obtained by mutation of a wild-type phosphinothricin dehydrogenase with an amino acid sequence as shown in SEQ ID No.2 at one of the following sites: (1) E263D-K134R-H96A-R290V; (2) E263D-K134R-H96A; (3) E263D-K134R; (4) E263D; (5) E263N; (6) E263C; and (7) E263G. The present invention utilizes the site-saturation mutagenesis technology to mutate a phosphinothricin dehydrogenase gene as shown in SEQ ID No. 1, finds that the 263rd, 134th, 290th and 290th positions are the key sites affecting enzyme activity and stereoselectivity, and obtains a mutant with enzyme activity and ee value much higher than those of the parent phosphinothricin dehydrogenase.

Abstract: Disclosed are a gene mining method combining functional sequence and structure simulation, an NADH-preferring phosphinothricin dehydrogenase mutant and an application thereof. The gene mining method comprises the following steps: (1) analyzing a characteristic sequence which an NADH-type glutamate dehydrogenase should have; (2) searching a gene library based on the characteristic sequence; (3) performing clustering analysis and protein structure simulation on genes obtained by the searching; (4) selecting genes that feature high gene aggregation and a protein structure similar to that of the known phosphinothricin dehydrogenase as candidate genes. A wild-type phosphinothricin dehydrogenase with an amino acid sequence as set forth in SEQ ID No.

Abstract: The present invention provides a glufosinate-ammonium dehydrogenase mutant and application in synthesis of L-glufosinate-ammonium thereof, the method uses 2-carbonyl-4-[(hydroxy)(methyl)phosphinoyl]-butyric acid or its salts as a substrate and the glufosinate-ammonium dehydrogenase or cells containing the glufosinate-ammonium dehydrogenase as a biocatalyst to carry out reductive amination, thereby obtaining L-glufosinate-ammonium. The method has features of high conversion rate of raw materials, high yield, easy separation and purification of the product, and high chiral purity; compared with other catalytic processes, the method in the present invention has features of relatively simple process and a conversion rate of raw materials up to 100%.

Abstract: The present invention discloses an amino acid dehydrogenase mutant and application thereof in synthesizing L-glufosinate-ammonium, the amino acid dehydrogenase mutant is obtained by a single mutation or a multi-site mutation of the amino acid at position 95, 108, 172, 303 of the amino acid sequence shown in SEQ ID No.2. The amino acid dehydrogenase mutant DyGDH-F95I-A108T-R172P-R303H prepared by the present invention has a specific enzyme activity that is 33 times higher than that of the original Aldo-keto reductase, and the concentration of the largest substrate, 2-carbonyl-4-(hydroxymethylphosphinyl)-butyric acid reaches 500 mM, the amino acid dehydrogenase mutant has more industrial application prospects.