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.

Modulation of the thermostability and substrate specificity of Candida rugosa lipase1 by altering the acyl-binding residue Gly414 at the α-helix-connecting bend

“…Analogous results were reported by Schmitt et al [15]. Recently, Brundiek et al [49] reported that mutant G237A/L/V/Y altered the substrate specificity of Candida antarctica lipase A by interrupting 19 the acyl-binding tunnel. Zhou et al [50] reported that M148W substitution of meta-cleavage product hydrolase could interrupt the tunnel T2.…”

Balancing the Stability–Activity Trade‐Off by Fine‐Tuning Dehalogenase Access Tunnels

“…It has previously been shown that modifying the size, physico‐chemical properties and dynamics of access tunnels by protein engineering can change the catalytic activity, substrate specificity, enantioselectivity and stability of HLDs8, 33, 54–56 and many other enzymes with buried active sites57 such as cytochrome P450s,58–63 β‐glucosidases,64 lipases,15, 65–69 esterases70 and epoxide hydrolases 47. 71, 72…”

Modification of fatty acid selectivity of Candida antarctica lipase A by error-prone PCR

“…Interestingly, the unique construction of the acyl tunnel of lipase A determines a perfect fit for long-chain fatty acids and also affects the selectivity towards trans -fatty acids (Brundiek et al 2012). …”