Construction of L-Asparaginase Stable Mutation for the Application in Food Acrylamide Mitigation

Acrylamide, a II A carcinogen, widely exists in fried and baked foods. L-asparaginase can inhibit acrylamide formation in foods, and enzymatic stability is the key to its application. In this study, the Escherichia coli L-asparaginase (ECA) stable variant, D60W/L211R/L310R, was obtained with molecular dynamics (MD) simulation, saturation mutation, and combinatorial mutation, the half-life of which increased to 110 min from 60 min at 50 degrees C. Furthermore, the working temperature (maintaining the activity above 80%) of mutation expanded from 31 degrees C-43 degrees C to 35 degrees C-55 degrees C, and the relative activity of mutation increased to 82% from 65% at a pH range of 6-10. On treating 60 U/mL and 100 U/g flour L-asparaginase stable mutant (D60W/L211R/L310R) under uncontrolled temperature and pH, the acrylamide content of potato chips and bread was reduced by 66.9% and 51.7%, which was 27% and 49.9% higher than that of the wild type, respectively. These results demonstrated that the mutation could be of great potential to reduce food acrylamide formation in practical applications.