The α-L-rhamnosidase BtRha from Bacteroides thetaiotao VPI-5482 is a specific enzyme that selectively hydrolyzes the α-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 E39W-S592C 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 α-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 α-L-rhamnosidase which can convert epimedin C to icariin to date. All the results provide a specific and cost-effective α-L-rhamnosidase mutant, which will raise its potential interest for the food and pharmaceutical applications.
Keywords: B-factor-saturation mutagenesis; Disulfide bonds; High-level expression; Icariin; α-L-rhamnosidase.
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