Engineering the substrate-binding domain of an esterase enhances its hydrolytic activity toward fatty acid esters

The poor solubility and dispersibility of fatty acids in aqueous reaction media may limit the catalytic activity of fatty acid transformation enzymes. Therefore, we studied a novel method to increase the catalytic activity of an esterase by introducing a presumed substrate-binding domain. The primary structure of an esterase from Pseudomonas fluorescens WI SIK (PFEI) is similar to that of an esterase in P. fluorescens DSM 50106 (PFEII) but not Bacillus subtilis DSM 402 (BS2). However, the reaction kinetics for the formation of octylacetate and a ricinoleic acid-derived ester (3) were more similar to the kinetics in BS2. For instance, the k_cat value of PFEI with 3 was similar to that of BS2, which was approximately 12-fold lower than the k_cat value of PFEII. Furthermore, fusion of PFEI to the N-terminal hydrophobic domain of PFEII led to a substantial increase (an approximate 6-fold increase in the k_cat value) in its hydrolytic activity of 3. These results indicate that the N-terminal domain of PFEII, which is assumed to be involved in anchoring the enzyme in the membrane, interacts with fatty acid-like substrates, resulting in an improved enzymatic activity. Therefore, we conclude that the membrane-anchoring domains can be used to increase the catalytic activity of fatty acid transformation enzymes.