Protein Engineering of Lipase A from Candida Antarctica to Improve Esterification of Tertiary Alcohols.

Chiral tertiary alcohols are important organic compounds in science as well as in industry. However, their preparation in enantiomerically pure form is still a challenge due to their complex structure and steric hindrances compared with primary and secondary alcohols, so kinetic resolution could be an attractive approach. Lipase A from Candida antarctica (CAL-A) has been shown to catalyze the enantioselective esterification of various tertiary alcohols with excellent enantioselectivity but low activity. Here we report a mutagenesis study by rational design to improve CAL-A activity against tertiary alcohols. Single mutants of CAL-A were selected, expressed, immobilized and screened for esterification of the tertiary alcohol 1,2,3,4-tetrahydronaphthalen-1-ol. A double mutant V278S+S429G showed a 1.5-fold higher reaction rate than that of the wild type CAL-A, while maintaining excellent enantioselectivity.