Zhejiang University

Jianmiao Xu

Yuguo Zheng

Yaping Xue

Feng Cheng

Shuping Zou

Heng Li

Qinghua Li

Zhentao Jiang

Jumou Li

Liuyu Wang

Dongyang Wu

Jie Mao

Jiamin Zhang

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 2-2.5 to produce L-glufosinate ammonium precipitates; and (5) recrystallizing the L-glufosinate ammonium precipitates obtained in step (4) to obtain purified L-glufosinate ammonium powder. The method provided by the present invention is simple to operate and low in cost, and has a better industrialization prospect.

Preparation method for L-glufosinate-ammonium powder

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 strainat one of the following sites: (1) M213S-N54V-F58E; (2) M213S-N54V-F58E-D207A; (3) M213S-N54V-F58E-D207A-S60T. According to the present invention, the D-amino acid oxidase mutant provides better catalytic efficiency, and when racemic D,L-phosphinothricin is used as a substrate for catalytic reaction, the conversion rate is much higher than that of the wild type enzyme, and the PPO yield is also greatly improved.

Method for asymmetrically preparing L-phosphinothricin by oxidation-reduction reaction through biological multi-enzyme coupling

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. 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.

Phosphinothricin dehydrogenase mutant, genetically engineered bacterium and one-pot multi-enzyme synchronous directed evolution method

A cofactor self-sufficientand 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, 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 glufosinate dehydrogenase by 2-fold, resulting in a significant increase in the spatiotemporal yield of the glufosinate synthesis reaction.

Cofactor self-sufficientand construction method and application thereof

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%.

Method for the production of glufosinate-ammonium

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.2 derived from Lysinibacillus composti is obtained through the gene mining, and then mutated, and an NADH-preferring phosphinothricin dehydrogenase mutant is screened out, which has a mutation site selected from one of the following: (1) A144G-V375F-M91A; (2) A144G-V345A-M91A; (3) A144G. This mutant enzyme can be used for catalytic reaction with an inexpensive coenzyme NAD.

Gene mining method combining functional sequence and structure simulation, NADH-preferring phosphinothricin dehydrogenase mutant and application thereof

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.

Machine learning gene mining method and phosphinothricin dehydrogenase mutant for amino translocation

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. Using the amino acid dehydrogenase mutant to produce L-glufosinate-ammonium, the reaction time is significantly shortened, the general process takes 20 hours, and the reaction time of the present invention only requires 120 minutes, which shows that the amino acid dehydrogenase mutant has a good industrial application prospect.

Growing community of inventors

Zhejiang, China

Jianmiao Xu