Production of steviol from steviol glucosides using β-glycosidase from Sulfolobus solfataricus

⿢Thermostable β-glycosidase of S. solfataricus was expressed via lactose induction.⿢Kinetic parameters of SSbgly for lactose, oNPG, and pNPG were determined.⿢The SSbgly hydrolyzed stevioside to produce steviol with a yield of 99.2%.⿢Response Surface Method was used to determine optimum conditions of steviol production. Steviol is a diterpene isolated from the plant Stevia rebaudiana that has a potential role as an antihyperglycemic agent by stimulating insulin secretion from pancreatic beta cells and also has significant potential to diminish the renal clearance of anionic drugs and their metabolites. In this study, the lacS gene, which encodes a thermostable β-glycosidase (SSbgly) enzyme from the extremely thermoacidophillic archaeon Sulfolobus solfataricus, was cloned and expressed in E. coli Rossetta BL21(DE3)pLyS using lactose as an inducer. Through fermentation, SSbgly was expressed as a 61kDa protein with activity of 24.3U/mg and the OD600 of 23 was reached after 18h induction with 10mM lactose. Purified protein was obtained by Ni-Sepharose chromatography with a yield of 92.3%. SSbgly hydrolyzed steviol glycosides to produce steviol with a yield of 99.2%. The optimum conditions for steviol production were 50U/ml SSbgly and 90mg/ml Ste at 75°C as determined by the response surface method.