US 11,939,599 B2
Gene mining method combining functional sequence and structure simulation, NADH-preferring phosphinothricin dehydrogenase mutant and application thereof
Yaping Xue, Hangzhou (CN); Feng Cheng, Hangzhou (CN); Jiamin Zhang, Hangzhou (CN); Shuping Zou, Hangzhou (CN); Jianmiao Xu, Hangzhou (CN); and Yuguo Zheng, Hangzhou (CN)
Assigned to ZHEJIANG UNIVERSITY OF TECHNOLOGY, Hangzhou (CN)
Filed by ZHEJIANG UNIVERSITY OF TECHNOLOGY, Hangzhou (CN)
Filed on Dec. 30, 2021, as Appl. No. 17/566,373.
Prior Publication US 2022/0204949 A1, Jun. 30, 2022
Int. Cl. C12N 9/00 (2006.01); C12N 9/04 (2006.01); C12N 9/06 (2006.01); C12P 13/04 (2006.01); G16B 30/10 (2019.01)
CPC C12N 9/0016 (2013.01) [C12N 9/0006 (2013.01); C12P 13/04 (2013.01); G16B 30/10 (2019.02); C12Y 104/01002 (2013.01); C12Y 104/01003 (2013.01); C12Y 104/01004 (2013.01)] 6 Claims
 
1. A method for preparing L-phosphinothricin, characterized in that, 2-carbonyl-4-(hydroxymethylphosphono)-butyric acid as a substrate is allowed to react as catalyzed by a catalyst in the presence of an inorganic amino donor, a coenzyme regeneration and circulation material and a corresponding co-substrate to obtain L-phosphinothricin;
the catalyst is one of the following:
(1) an NADH phosphinothricin dehydrogenase mutant;
(2) a recombinant bacterium capable of expressing the NADH phosphinothricin dehydrogenase mutant or a crude enzyme liquid obtained by lysis of the recombinant bacterium;
wherein the NADH phosphinothricin dehydrogenase mutant is obtained by mutating a wild-type phosphinothricin dehydrogenase obtained from Lysinibacillus composti, wherein the NADH phosphinothricin dehydrogenase mutant has the amino acid sequence as set forth in SEQ ID No.2, excepting that the NADH-preferring phosphinothricin dehydrogenase mutant has a mutation site selected from one of the following:
(1) A144G-V375F-M91A;
(2) A144G-V345A-M91A.