Enhancement of α-ketoisovalerate production by relieving the product inhibition of L-amino acid deaminase from Proteus mirabilis  

作　　者：Shanshan Pei Xiaobo Ruan Jia Liu Wei Song Xiulai Chen Qiuling Luo Liming Liu Jing Wu 

机构地区：[1]School of Pharmaceutical Science,Jiangnan University,Wuxi 214122,China [2]Key Laboratory of Industrial Biotechnology,Ministry of Education,Jiangnan University,Wuxi 214122,China [3]State Key Laboratory of Food Science and Technology,Jiangnan University,Wuxi 214122,China [4]National Engineering Laboratory for Cereal Fermentation Technology,Jiangnan University,Wuxi 214122,China

出　　处：《Chinese Journal of Chemical Engineering》2020年第8期2190-2199,共10页中国化学工程学报（英文版）

基　　金：financially supported by the national first-class discipline program of Light Industry Technology and Engineering(LITE201820);the Key Technologies Research and Development Program of Jiangsu Province(BE2018623)。

摘　　要：L-Amino acid deaminase(LAAD) is a key enzyme in the deamination of L-valine(L-val) to produce α-ketoisovalerate(KIV). However, the product inhibition of LAAD is a major hindrance to industrial KIV production.In the present study, a combination strategy of modification of flexible loop regions around the product binding site and the avoidance of dramatic change of main-chain dynamics was reported to reduce the product inhibition.The four mutant PM-LAAD^(M4)(PM-LAAD^(S98A/T105A/S106A/L341A)) achieved a 6.2-fold higher catalytic efficiency and an almost 6.7-fold reduction in product inhibition than the wild-type enzyme. Docking experiments suggested that weakened interactions between the product and enzyme, and the flexibility of the "lid" structure relieved LAAD product inhibition. Finally, the whole-cell biocatalyst PM-LAAD^(M4) has been applied to KIV production,the titer and conversion rate of KIV from L-val were 98.5 g·L^-1 and 99.2% at a 3-L scale, respectively. These results demonstrate that the newly engineered catalyst can significantly reduce the product inhibition, that making KIV a prospective product by bioconversion method, and also provide the understanding of the mechanism of the relieved product inhibition of PM-LAAD.

关 键 词：Product inhibition L-Amino acid deaminases α-Ketoisovalerate Bio-catalysis Protein engineering 

分 类 号：TQ426.97[化学工程]