Computational Redesign of Aspartase from Escherichia coli and Its Catalytic Synthesis of β-Alanine

Aspartase from Escherichia coli (AspA) catalyzes the direct conversion of acrylic acid to β-alanine; however, its substrate specificity and low catalytic efficiency limit its broader application. We engineered an AspA mutant capable of efficiently catalyzing the amination of acrylic acid for β-alanine synthesis, using Rosetta Enzyme Design to computationally redesign the Cβ-binding region of the acrylic acid binding site in AspA. Based on energy scores, structural configurations, and hydrogen bonding networks, 51 candidate variants with penalty scores below 30 were selected for mutant construction and performance testing; >70% of these variants exhibited enhanced catalytic activity in acrylic acid’s hydrogen amination. Four mutants achieved over 3-fold improved activity. The optimal mutant, M1 (T190I-M324I-K327L-N329C), demonstrated a 7.3-fold increased specific enzyme activity and a 13.0-fold improved kcat/Km compared with the wild type. Conformational changes in the S-loop and enhanced hydrophobic interactions near the active site contributed significantly to M1’s enhanced activity. Upon reaction optimization, the conversion of β-alanine synthesis using M1 in whole-cell catalysis increased from 5% with the wild type to 90% with M1. This study provides a reference for the biocatalytic synthesis of β-alanine, significantly enhancing the conversion of acrylic acid and demonstrating the enzyme’s potential for industrial applications.