Improving the enantioselectivity of an esterase toward (S)-ketoprofen ethyl ester through protein engineering

Abstract

• Biochemical characteristics of Est25 were studied in various temperature, solvent and pH. • Mutated esterase (L255W) based on the modeled esterase structure has higher selectivity to (S)-ketoprofen ethyl ester than wild-type. • The addition of methanol or ethanol (as a solvent) led to drastic increases in the enantioselectivity of each enzyme. Esterases, enzymes that hydrolyze ester bonds, have been utilized to synthesize optically pure compounds like (S)-ketoprofen, which can be produced by the esterase-catalyzed hydrolysis of rac -ketoprofen ethyl ester. We previously reported a novel esterase, Est25, which was expressed in active form in Escherichia coli , and showed high hydrolyzing activities for various substrates. However, the enzyme did not show any preference for either of rac -ketoprofen ethyl ester. In this study, to improve the enantioselectivity of Est25, we used a protein engineering approach that involves building a homology model of Est25, library generation based on the model, screening for esterase activity, and screening for specificity toward (S)-ketoprofen ethyl ester. One variant, L255W was isolated showing significantly higher enantioselectivity than the wild-type enzyme. Enantioselectivity was further improved by adding one of the polar organic solvents, namely, ethanol and methanol. However, the mutant enzyme showed lower thermostability, higher sensitivity to pH, and lower resistance to polar solvents than Est25. The L255W mutation improves substrate specificity, but, at the same time, sacrifices structural stability. The approach used in this study is precise enough to engineer the substrate specificity of esterase, and general enough to be adapted to engineer other kinds of enzymes with a minimum amount of modifications.