Conversion of a Monascus ruber esterase into a lipase by disrupting a salt bridge

Abstract

Cold-active lipases have emerged as an important class of biocatalysts for chemical and food industries due to their high efficiency at low temperature and long-chain substrate preference. In an effort to explore the feasibility of converting a cold-active esterase from Monascus ruber (Lip10) into a cold-active lipase, an Y264F variant in which the salt bridge between K243 and Y264 was disrupted has been constructed and characterized. The interfacial kinetic parameter, K_m^app for pNP-laurate (C12) and pNP-palmitate (C16), of Lip10 esterase was 4.2 and 5.7 times higher than those of the Y264F variant, respectively. Substrate specificity of the Y264F variant changed from shot-chain length substrate to medium- and long-chain length substrates, indicating that the Y264F variant turned into a lipase. Meanwhile, the Y264F variant displayed 48.6% maximum activity at 4 °C and 3.2 kcal/mol activation energy in the range of 5–30 °C, suggesting that it was still cold-active. Based on analysis of the structure-function relationships, it suggests that the shape of substrate channel controlled by the conserved salt bridge was very important for the substrate specificity. This study provides a way to alter the substrate preference of the Lip10 esterase as well as new insight into the structural basis of esterase substrate specificity.