Abstract
L-aspartate-alpha-decarboxylase (ADC) catalyzes the decarboxylation of L-aspartate to produce β-alanine, which is the decisive step in the biosynthesis of β-alanine. However, the low catalytic stability and efficiency of ADC limit its industrial applications. In this study, a variant of ADC from Bacillus subtilis were used as a starting point for engineering. After constructing a random mutagenesis library by error-prone PCR, followed by high-throughput screening,four substitutions (S7 N, K63 N, A99T, and K113R) were identified. By screening saturation mutagenesis libraries on these positions and computational analysis, two recombined variants N3(S7 N/K63 N/I88 M/A99E/K113R/I126*) and Y1(S7Y/K63 N/I88 M/A99E/K113R/I126*) with improved performance were obtained. Compared to the wild type, the catalytic efficiency and catalytic stability of the best two variants were enhanced up to 95 %(variant N3) and up to 89 %(variant Y1), respectively. In addition, Y1 exhibited 3.37 times improved half-life and 2-fold improved total turnover number. Hydrophilicity analysis and molecular dynamics (MD) simulation revealed that the increased hydrophilicity and steric hindrance of key amino acid residues would affect the catalytic activity and stability. The improved catalytic performance of the variants could be attributed to their enhanced binding capacity to the substrate within the active pocket and the alleviation of mechanism-based inactivation.