The additive mutational effects from surface charge engineering: A compromise between enzyme activity, thermostability and ionic liquid tolerance

Abstract

Surface charge engineering is a powerful tool that could alter protein properties (e.g. activity, thermostability and resistance to ionic liquid, IL), providing an increasingly rich mutation library. In the present study, two quadruple mutants of Bacillus subtilis lipase A (BsLA) designed as mutagenesis studies for improved IL-tolerance (BsLA4M1) and thermostability (BsLA4M2) are proceeded. A combined octuple mutant (BsLA8M) is then generated via incorporation of the above eight mutations, demonstrating significant increase in resistance to irreversible thermal denaturation as well as ILs ([BMIM][Cl]), relative to the parent enzyme. Results also indicate that, less positively charged quadruple mutant BsLA4M2 yields significant enhancement of activity (170% of parent enzyme); whereas highly negatively charged mutant BsLA8M has a clear additive effect on the enzyme stability in the IL. The combined octuplet mutant BsLA8M with a net charge of −5 also displays extremely high resistance against irreversible thermal inactivation (T(50) increased from 51 to 97 ºC) and excellent thermal refolding. This work elucidates the interplays between enzyme activity, thermostability and resistance to harsh IL as the additive properties of the mutants from surface charge engineering.