Xing_2021_Enzyme.Microb.Technol_149_109849

A lipase from Malassizia globose, named SMG1, is highly desirable for industrial application due to its substrate specificity towards mono- and diacylglycerol. To improve its thermostability, we constructed a mutant library using an error-prone polymerase chain reaction, which was screened for both initial and residual enzymatic activity. Selected mutants were further studied using purified proteins for their kinetic thermostability at 45 degC, T(50) (the temperature at which the enzyme loses half of its activity), and the optimal reaction temperature. Results showed that the majority of mutations with improved thermostability were on the protein surface. D245N and L270P showed the most significant thermostability enhancement with an approximately 3 degC increase in T(50) compared to wild-type (WT). In addition, combining these two mutations resulted in an increase of T(50) by 5 degreesC. Also, the optimal reaction temperatures of L270P and this double mutant are 10 degC higher than WT. The double mutant showed an approximately 100-fold increase in half-life at 45 degC and higher enzymatic activities at 30 degC and above compared to WT. High-temperature unfolding molecular dynamics simulation suggested that the double mutant stabilized a flexible loop in the catalytic pocket.