Recombinant glufosinate-ammonium dehydrogenase, genetically engineered bacterium and application of recombinant glufosinate-ammonium dehydrogenase in preparation of L-glufosinate-ammonium

Abstract

The invention discloses recombinant glufosinate-ammonium dehydrogenase, a genetically engineered bacterium and application of recombinant glufosinate-ammonium dehydrogenase in preparation of L-glufosinate-ammonium. The amino acid sequence of the recombinant glufosinate-ammonium dehydrogenase is shown as SEQ ID NO. 2. According to the invention, a gene library of the recombinant glufosinate-ammonium dehydrogenase is constructed through a staggered extension pcr gene rearrangement technology, and the recombinant glufosinate-ammonium dehydrogenase with high enzyme activity, catalytic performanceand stereoselectivity is screened from the gene library; the activity of the recombinant glufosinate-ammonium dehydrogenase is respectively improved by 31% and 35% in comparison with the activity of aprevious mutant PPTDHE3-A164G and the activity of a previous mutant PPTDHE0-V375S; and finally, 108 g / L of 2-carbonyl-4-(hydroxymethyl phosphine oxide)-butyric acid is completely catalyzed to producethe L-glufosinate-ammonium, and only 20min is needed (transaminase generally needs 40h), wherein the ee value is larger than 99.5%.

Description

technical field [0001] The invention relates to the field of biochemical technology, in particular to recombinant glufosinate-ammonium dehydrogenase, genetically engineered bacteria and their application in preparing L-glufosinate-ammonium. Background technique [0002] The chemical name of glufosinate-ammonium is 4-[hydroxy (methyl) phosphono]-DL-homoalanine, which is the second most resistant herbicide in genetically modified crops in the world. After the merger, it is now owned by Bayer) to develop and produce, also known as glufosinate ammonium salt, Basta, Buster, etc., which are phosphonic acid herbicides, and non-selective (killing) contact herbicides are glutamine synthetase inhibitors. [0003] Glufosinate-ammonium has two optical isomers, namely L-glufosinate-ammonium and D-glufosinate-ammonium. But only the L-type has physiological activity, and is easy to decompose in the soil. It is less toxic to humans and animals, has a wide weeding spectrum, and has little d...

AI Technical Summary

Problems solved by technology

But utilize transaminase to prepare L-glufosinate-ammonium and there are two big defects, and one is that raw material PPO can not be completely converted into L-PPT, and the conversion rate is the highest only 90%; More than 4 times the equivalent of L-glutamic acid is needed as the amino donor, and the excess glutamic acid brings great trouble to the separation of L-glufosinate-ammonium

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