Screening Of Highly Active Glycosidase For Rare Ginsenosides Production

Rare ginsenoside compound K(C-K) has attracted a great deal of attention due to its unique anti-tumor and other medical effect. The content of ginsenoside C-K is very low in nature, which limits its scientific research and clinical applications. Rare saponins C-K can be biotransformed from other major saponin.The aim of this study is to engineer β-glycosidase (LacS) from Sulfolobus solfataricus which has been reported as a candidate for ginsenoside biotransformation for improved activities to meet the needs of industral application.Frist, a mutant library was established by error-prone PCR, and screen for the best hit by high-throughput screening method. The mutants with improved activities were purified and characterized. LacS catalyzes major ginsenoside Rbl to C-K via hydrolyzing3glycosides from Rbl, which established a high-throughput screening method by measuring ruducing sugars by3,5-dinitrosalicylic acid method.The improved mutant (Gly218Val) showed2.5times activity of transforming major ginsenoside Rbl into rare ginsenoside C-K, compared with that of wild type. The optimum reaction temperature of LacS mutant decreased from85"C (the optimal temperatur of wild type) to70℃.3.2mg/ml ginsenoside Rb1was transformed into ginsenoside C-K with a corresponding molar conversion ratio of97%in McBuffer (pH5.5) at70℃, which makes the rare ginsenoside C-K a potential industrial biocatalyst.Single substitution of Gly218position of LacS mutant greatly changed characteristics of enzyme. We propose that this substitution influences the three-dimensional structure of LacS, which results in the changing of capability of substrate capturing and binding.