Computational-Aided Mutagenesis of Lipase CRL1 with Promoted Stability and Activity for Biosynthesis of Vitamin E Succinate.

Vitamin E succinate (VES) is a particularly potent form of vitamin E derivatives in daily supplements. The promising biocatalyst crude lipase CRL (Candida rugosa lipase) could produce vitamin E succinate but suffers from low yield due to limited thermostability. This study integrated computational-aided virtual mutagenesis and combinatorial mutations of lipase CRL1. A combinatorial variant 6M (K85P/V86W/T108V/T132W/F344I/V454L) synchronously enhanced thermostability and pH stability, and half-lives were 322.7 min (60 °C) and 201.2 min (70 °C), representing a 6.35-fold and 7.31-fold increase compared to WT, respectively. 6M's lipase activity was 2 times that of WT, and the catalytic efficiency (kcat/Km) showed a 6.8-fold improvement over that of WT. Mutant 6M could generate vitamin E succinate with an improved yield of 77.8% (60 °C, 12 h). The synergistically functional modifications of the lipase pave the way for the mutated lipase to become a highly promising and robust biocatalyst for industrial applications.