Improving the Activity and Stability of L-Asparaginase from Bacillus subtilis by Site-Directed Mutagenesis

Abstract:

L-Asparaginase catalyzes the hydrolysis of L-asparagine to L-aspartic acid and ammonia，which can hydrolyze free L-Asn to aspartic acid and decrease acrylamide formation during food processingd. In order to meet the high requirements and complicated procedures of food processing，the activity and stability of L-Asparaginase need to beimproved. In this study，we successfully improved the enzyme activity and stability of L-asparaginase （BsAII） from Bacillus subtilis B11-06 by site-directed mutagenesis. Five residues of BsAII were selected and used to construct the mutants by sequence alignment and homology modeling. The enzyme activity assayshowed that the enzyme activity of S299Nansz and P348Aaansz were increased by 28% and 32%，respectively，as compared to the BsAII. The thermal stability and pH stability of P348Aansz were significantly improved compared to that of BsAII. This study showed that residues 299 and 348 in amino acid sequence of BsAII had a great influence on the catalytic action，which provided a basis for the study of the catalytic mechanism of the enzyme，and extended the application in food industry.