Enhancing the thermostability of a mono- and diacylglycerol lipase from Malassizia globose by stabilizing a flexible loop in the catalytic pocket

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 degrees C, T50 (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 degrees C increase in T50 compared to wild-type (WT). In addition, combining these two mutations resulted in an increase of T50 by 5 degrees C. Also, the optimal reaction temperatures of L270P and this double mutant are 10 degrees C higher than WT. The double mutant showed an approximately 100-fold increase in half-life at 45 degrees C and higher enzymatic activities at 30 degrees C and above compared to WT. High-temperature unfolding molecular dynamics simulation suggested that the double mutant stabilized a flexible loop in the catalytic pocket.

Automated summary

A lipase from Malassizia globose, named SMG1, was mutated to improve thermostability, with mutations on the protein surface resulting in significant enhancements. Specific mutations, such as D245N and L270P, showed the most significant increase in T50, with the double mutant exhibiting a 5-degree Celsius increase. The double mutant displayed a 100-fold increase in half-life at 45 degrees Celsius and higher enzymatic activities at temperatures above 30 degrees Celsius compared to the wild-type.