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.