Glu56Ser mutation improves the enzymatic activity and catalytic stability of Bacillus subtilis L-aspartate α-decarboxylase for an efficient β-alanine production

L-aspartate alpha-decarboxylase (ADC) is responsible for the decarboxylation of L-aspartate to form beta-alanine in the bacterial biosynthetic pathway. However, the low activity and stability of ADC are the primary factors restricting beta-alanine biosynthesis. In this study, to improve the ADC activity and thermostability and to attenuate the degree of its inactivation in the reaction, we expressed and characterized the Bacillus substilis ADC (BsADC) in Escherichia coli and rationally designed several site-directed mutations adjacent to its catalytic site. The Glu56Ser (E56S) mutant showed a 1.6-fold higher activity than the wild-type enzyme. This mutant retained approximately 65% activity when incubated at 70 degrees C for 12 h. More importantly, the E56S mutant showed an approximately 1.4-fold increased residual activity compared with the wild type during 2 h reaction at 37 degrees C, suggesting that the E56S mutation attenuated the mechanism-based inactivation of the enzyme. The mutant enzyme also catalyzed the beta-alanine synthesis with a product yield of 215.3 g per liter culture, which is the highest reported yield to date. This work proposes a protein engineering strategy that improves the enzymatic activity and thermostability and reduces the degree of enzyme inactivation in the reaction, thereby improving the efficiency of beta-alanine biosynthesis.