A High-Specific-Activity L-aspartate-α-Decarboxylase from Bacillus aryabhattai Gel-09 and Site-Directed Mutation to Improve Its Substrate Tolerance.

Abstract

L-aspartate-α-decarboxylase (ADC) can recognize L-aspartic acid specifically and catalyze the decarboxylation of L-aspartic acid to β-alanine. In this study, a novel L-aspartate-α-decarboxylase (BaADC) with high specific activity from Bacillus aryabhattai Gel-09 was heterologously expressed and characterized. It exhibited optimal enzyme activity at pH 5.5 and 75°C, and its specific activity was 33.9U/mg. To improve the substrate tolerance of BaADC, site-directed mutation was used to construct variants. The optimal variant BaADC_I88M exhibited higher pH stability and thermostability, with 1.2-fold increase in catalytic efficiency. Moreover, through the fed-batch method, the conversion of L-aspartic acid to β-alanine catalyzed by BaADC_I88M reached 98.6% (128.67g/L) at 12h, which was 1.42-fold that of the wild-type enzyme. The mechanism of improved substrate tolerance was interpreted by molecular dynamics simulation and structural analysis, which revealed that the local conformational change in the active pocket could promote correct protonation. These results suggested that BaADC and its variant are potential candidates for use in the industrial production of β-alanine.