Improving the Activity and Thermal Stability of Pyrococcus yayanosii CH1 L-Asparaginase by Site-Directed Mutagenesis

Abstract:

L-asparaginase II (EC 3.5.1.1), which can convert L-asparagine, the precursor of carcinogenic acrylamide, to L-aspartic acid, is attracting much attention in food industry. The use of L-asparaginase II can reduce the formation of acrylamide during the pretreatment of foods. However, the application of this enzyme is limited due to variant requirements of commercial use, especially for its activity and thermostability. In this study, by sequence aligning and homology modeling,three mutants of PyAsnase gene from the thermophilus Pyrococcus yayanosii CH1was constructed and over-expressed in Bacillus subtilis. According to the results, the specific activities of the mutants E22K and R111L were increased by 37.3% and 31.1%, respectively, and the half-life of the mutant obtained by the mutant M92A was extended by 30 min at 85 ℃. This work provides references for studying the interrelationship between function and structure of L-asparaginase, and also develops the application of L-asparaginase in food industry.