Altering the scissile fatty acid binding site of Candida antarctica lipase A by protein engineering for the selective hydrolysis of medium chain fatty acids

摘要

Candida antarctica lipase A (CAL-A) is the first representative of a new subclass of lipases because of its unique cap domain. The acyl-binding tunnel - having a short alternative binding region - is mainly formed by this domain. In order to create CAL-A variants with a high specificity for medium chain length (MCL) fatty acids (C6-C12), we used rational protein design to block the primary acyl-binding tunnel of CAL-A at position G237, which is near the junction to the alternative binding pocket. By closing the junction to the main tunnel, CAL-A variants (G237A/L/V/Y) have been created, which are highly specific for medium chain fatty acids (MCFAs) as determined by chain length profiles with p-nitrophenyl esters and triacylglycerides. Especially the CAL-A variants G237L/V/Y, in which the junction to the primary tunnel is completely closed, show a distinct preference for the hydrolysis of hexanoate esters. Hydrolytic activity for substrates with a chain length >C6 is suppressed extensively in mutants G237L/V/Y. Therefore, these highly MCL specific CAL-A variants may represent interesting biocatalysts for the production of MCL-derived esters for the food, flavor, and fragrance industry.