Modification to increase the thermostability and catalytic efficiency of a-L-rhamnosidase from Bacteroides thetaiotaomicron and high-level expression

The alpha-L-rhamnosidase BtRha from Bacteroides thetaiotao VPI-5482 is a specific enzyme that selectively hydrolyzes the alpha-1,2 glycosidic bond between rhamnose and rhamnose, allowing the bioconversion of epimedin C to icariin. In this study, BtRha was molecularly modified using B-factor-saturation mutagenesis strategy and the introduction of disulfide bonds, resulting in a mutant with significantly improved catalytic efficiency, S592C, and two thermally stable mutants, E39W and E39W-S592C. The results showed that the half-lives of E39W and E39WS592C at 55 C were 10.4 and 9.4-fold higher, respectively, than that of the original enzyme, The mutant S592C showed a 63.3% reduction in Km value and a 163.6% increase in catalytic efficiency (kcat/Km value), which improved the ability to hydrolyze epimedin C to icariin effectively. In addition, high-level expression of alpha-L-rhamnosidase mutant S592C was established. With 0.1 mM IPTG as an inducer, induction temperature of 32 C, induction pH of 7.0 and induction OD600 of 50, the maximum activity of mutant S592C reached 182.0 U/ mL in terrific broth medium after 22 h. This is the highest enzyme activity of alpha-L-rhamnosidase which can convert epimedin C to icariin to date. All the results provide a specific and cost-effective alpha-L-rhamnosidase mutant, which will raise its potential interest for the food and pharmaceutical applications.