Engineering of Candida antarctica Lipase B for Enhanced Perhydrolysis Activity by Modulating the Potential Water Channel

Abstract Although Candida antarctica lipase B (CAL‐B) naturally hydrolyzes an ester compounds, it can catalyze a variety of promiscuous reactions, such as perhydrolysis, aldol reaction, and Michael‐type reactions. Perhydrolysis is similar to hydrolysis but uses hydrogen peroxide instead of water molecules. It was recently proposed that water molecules access the active site of CAL‐B through a water channel. We hypothesized that hydrogen peroxide may also use the channel to enter the active site, and that the access of hydrogen peroxide is one of the key factors for perhydrolysis activity. We investigated the effects of mutations at two residues in the water channel of CAL‐B on the perhydrolysis activity. The residues, Pro280 and Ala281, are located at the constricted area of the water channel. We found that the substitution of Ala281 to a small and polar amino acid, such as serine or threonine, resulted in a noticeable increase in hydrolysis and perhydrolysis activity. The activity of the variant enzymes was up to three times and twice higher for the activities of hydrolysis and perhydrolysis, respectively, than those of CAL‐B. In contrast, the substitution of the Pro280 residue with a small and polar amino acid decreased both hydrolysis and perhydrolysis activities.