Substrate-binding Site Engineering of Candida antarctica Lipase B to Improve Selectivity for Synthesis of 1-monoacyl-sn-glycerols

Lipases are extensively used for regiospecific esterification of polyols with fatty acids. However, side reactions generating byproducts limited the enzymes for industrial applications. Here, we have engineered the substrate-binding site of Candida antarctica lipase B (CALB) to improve selectivity for monoacylation of glycerol using medium chain fatty acids ( e.g. , nonanoic acid) as acyl donors. The enzyme engineering was based on the substrate-binding region of a lipase from Penicillium camemberti ( i.e. , Lipase G), which showed very high selectivity for monoacylation of glycerol with medium chain fatty acids ( e.g. , decanoic acid) but low reaction rates. One of the CALB variants (e.g., CALBA282E/I285F), which was designed to have a narrow substrate binding region, has exhibited ca. 2-fold greater selectivity for the synthesis of 1-monoacyl-sn-glycerol with n-nonanoic acid. The double mutant allowed the production of 1-nonanoyl-glycerol to a concentration of 2.27 M in glycerol to a reaction rate of 1.0 M/h. This study will contribute to the use of lipases for regiospecific esterification of polyols with carboxylic acids.