Production of octyl butyrate using psychrophilic mutant lipase from Croceibacter atlanticus LipCA lipase developed by a molecular evolution technique

Lipases are used to synthesize a variety of industrially useful compounds. Among them, psychrophilic lipase can be used to synthesize thermo-labile compounds at low temperatures. In this study, random mutagenesis was introduced into Antarctic Croceibacter atlanticus lipase gene using error-prone PCR, resulting in changes in its protein sequence. Through two rounds of mutagenesis and screening, we found that a mutant R1 showed an enhanced activity at low temperatures. Mutant R1 had five mutations (F43L, S48G, S49G, D141K, and K297R) and higher k_cat/K_M value than the wild type (WT) at 10 °C. We immobilized this enzyme on methacrylate divinylbenzene resin and used it to synthesize octyl butyrate, a flavor compound. The esterification reaction proceeded even at 10 °C. Mutant R1 synthesized the ester compound faster than the WT. To determine which amino acids were responsible for the increase of activity, site-directed mutagenesis was performed to introduce five back mutations into mutant R1. Three back mutants (L43F, G48S, G49S) showed significant decreases of activity at low temperatures, indicating that these amino acids were closely related to the increase in activity. This psychrophilic mutant R1 is expected to be used in low-temperature enzyme conversion reactions in the food industry.