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

Abstract

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.