Rational engineering of phospholipase C from Bacillus cereus HSL3 for simultaneous thermostability and activity improvement

As an essential enzyme for phospholipid degradation, phospholipase C (PLC) has been used for enzymatic degumming of vegetable oils and production of valuable phospholipid derivatives. In this study, rational engineering based on B-factor analysis and molecular dynamic simulation analysis were employed to rationally identify mutation candidates and a PLC double mutant F96R/Q153P was designed from Bacillus cereus HSL3. Compared to the wild-type PLC, F96R/Q153P exhibited significantly improved thermal properties, including higher temperature optima and better thermal stability. It showed the highest optimal reaction temperature (90 °C) reported so far. F96R/Q153P displayed 4.94 times kcat and 2.37 times kcat/Km as much as the wild-type, as well as improved substrate adaptability. Structural insights revealed that the mutations caused reduced proportion of random coil and constraint of certain loop fluctuations. These results demonstrated the great potential of knowledge-based rational design for improving the catalytic characteristics of industrial enzymes in the enzymatic degumming process.