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 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.

Methods for preparing crystalline L-glufosinate ammonium monohydrate are disclosed. The methods include forming a mixture comprising water, a water-miscible organic solvent, ammonium hydroxide, and a glufosinate starting material containing L-glufosinate ammonium and D-glufosinate ammonium. L-Glufosinate ammonium monohydrate is crystallized and separated from the mixture, providing L-glufosinate ammonium monohydrate Form B. Compositions and methods employing the crystalline L-glufosinate ammonium monohydrate are also described.

Provided are a D-amino acid oxidase and use thereof in the preparation of L-phosphinothricin or an intermediate thereof. Provided is a D-amino acid oxidase having an amino acid sequence comprising an amino acid residue difference as compared to SEQ ID NO: 1, the amino acid residue difference being selected from one or a plurality of: K29G/H/I/N/Q/W/Y/C/L; V42C/D/E/H/Y; E195N/Y/Q; C234L; and V326W. The activity and/or thermal stability of the D-amino acid oxidase is not lower than that of a D-amino acid oxidase having an amino acid sequence as set forth in SEQ ID NO: 1. Provided is a D-amino acid oxidase with higher thermal stability. The operating temperature range of the enzyme is expanded while the activity of the enzyme is improved. The enzyme can have a prolonged service life when used at a relatively low temperature, and can have an improved catalytic efficiency when used at a relatively high temperature.

A D-amino acid oxidase mutant with significantly improved catalytic performance, a gene encoding the gene, a vector containing the gene, a genetically engineered bacterium, and the application of the said mutant in the microbial catalytic preparation of L-ammonium glufosinate. The D-amino acid oxidase mutant was obtained from the amino acid shown in SEQ ID NO.1 by single-point mutation or multi-point combined mutation. The beneficial effects are mainly reflected in the following: the D-amino acid oxidase mutant with improved enzyme activity and thermal stability can be used in the microbial catalysed preparation of L-ammonium glufosinate, which is conducive to industrial production and has a better application prospect.

Disclosed is a method for preparing (S)-1,2,3,4-tetrahydroisoquinoline-1-carboxylic acid and derivatives thereof, comprising: taking a racemate of a compound represented by Formula (I) or a racemate of a salt of the compound represented by Formula (I) as a substrate, and making a R-isomer of the compound represented by Formula (I) in the substrate react under the catalysis of oxidative dehydrogenase to generate imino acid represented by formula (II); and converting the imino acid represented by Formula (II) into an S-isomer of the compound represented by Formula (I) in the presence of pipecolic acid reductase and a coenzyme capable of supplying hydrogen anions. The process has mild reaction conditions, strong stereoselectivity, high reaction efficiency, and high conversion rate. ##STR00001##

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. The glufosinate ammonium dehydrogenase mutant in the present invention has better catalytic efficiency. When racemic D, L-glufosinate ammonium is used as a substrate for a catalytic reaction, the conversion rate is much higher than the conversion rate of a wild-type enzyme, and the yield of 2-carbonyl-4-[hydroxy(methyl)phosphonyl]butanoic acid (PPO for short) is also greatly improved.