Amino acid residues adjacent to the catalytic cavity of tetramer L-asparaginase II contribute significantly to its catalytic efficiency and thermostability.

L-Asparaginase (L-asparagine amidohydrolase, EC 3.5.1.1) catalyzes the hydrolysis of L-asparagine to L-aspartic acid and ammonia. It can be used to reduce the formation of acrylamide, which is carcinogenic to humans in foods, via removal of the precursor, asparagine, from the primary ingredients. However, low activity and poor thermostability of L-asparaginase restrict its application in food industry. In this study, we successfully improved thermostability and catalytic efficiency of L-asparaginase II (BsAII) from Bacillus subtilis B11-06 by site-directed mutagenesis. According to sequences alignment and homologous modeling, residues G107, T109 and S166 which were adjacent to the catalytic cavity were selected and substituted by Asp, Gln/Ser and Ala, respectively, to construct mutants G107D, T109Q, T109S and S166A. The BsAII mutant of G107D (G107Dansz) displayed superior performance in thermal tolerance and higher activity than the wild-type enzyme (towards L-asparagine). Comparative analysis of hydrogen bond interactions, surface electrostatic potential and structure of substrate binding pocket between G107Danszand BsAII indicated that the substitution of G107, which was adjacent to catalytic cavity with Asp, resulted in small conformational changes and surface electrostatic potential redistribution and contributed to the improved protein stability and catalytic efficiency.