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

Abstract

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. Practical application: Since medium chain fatty acids (MCFAs: C6-C10) and their corresponding triacylglycerides (MCTs) provide quick access to energy and have been considered to be less implicated in the accumulation of body fat than long chain fatty acids, they represent interesting food additives. As functional oils, they are part of weight loss diets or are used in clinical nutrition. In the food industry MCTs are also utilized as storage stabilizing agents in cooking products, as release agents in food processing or as flavor diluent. Another interesting field of application of MCFA derived compounds, especially of C6 esters and alcohols, is as ingredients in flavors and fragrances. The CAL-A variants described in this study can be used for the biocatalytic synthesis of these compounds. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.