Isolation And Purification Of Stevioside Invertase And Microbial Transformation Of Steviol

Stevia is a natural, non-nutritive high-intensity sweetener, and its main ingredients include stevioside (SS), rebaudioside A(RA) and rebaudioside C (RC). SS is restricted in extensive application as natural edulcorator because of the bitter taste. RA has sweet quality, but low content. Rubusoside(RS), the main sweet component in the leaves of Rubus suavissimus, has low yield because of the limitation of geographical conditions. This work mainly isolated and purified enzymes that biotransformed stevioside to rubusoside by Chryseobacterium, and the properties of enzymatic were also studied.Ammonium sulfate fractionation, Phenyl-Sepharose CL-4B hydrophobic interaction chromatography and DEAE-Sepharose Fast Flow ion exchange chromatography were selected to separate and purify this specific enzyme.30%-80%of ammonium sulfate precipitation was used to precipitate and concentrate the protein. The target protein was adsorded when adapting0.05M, pH7.0, containing10%(NFI4)2SO4phosphate buffer on Phenyl-Sepharose CL-4B hydrophobic interaction chromatography, and only eluted by water. When adapting pH6.0,0.02M phosphate buffer, the target protein could be adsorded on DEAE-Sepharose Fast Flow ion exchange chromatography, and eluted by0.1M salt concentration. Telative molecular weights of the target protein were approximately20-3Okd,40-60kd and40-60kd by SDS-PAGE. Nano LC-MS/MS sequencing results showed that the transformation of the enzyme may be has oxidation reduction characteristic.The specific enzyme attained its maximum activity in the pH7.0at45℃. The suitable range of pH was6.0-7.0, and temperature was30℃-45℃. It was activated by Na+, Fe2+, and inhibited by Ca2+, Mg2+, Zn2+. It was aslo activated by10%ethanol,1%-5%methanol,10%-20%ethyl acetate, and the relatively activity was about141%,239%, and159%, respectively. The enzyme activity was inhibited by the butanol and acetone. Specific chemical modifiers were used to explore the functional groups of the enzyme. The result showed disulfide linkage and metal ions were maybe the necessary for the activity, but serine was not the activity of the enzyme groups.Steviol, the aglycone of stevioside, has the ent-kauranoid skeleton structure that could provide skeleton for new drugs. Based on the physical characterization and phylogenetic analysis by16S rRNA gene sequence analysis and phylogenetic tree construction of the strain. The strain XJ was identified as Microbacterium barkeri (CCTCCM2011182). The relative elimination rates of substrates were in the order of stevioside> rebaudioside A> rebaudioside C. Rubusoside was the intermediate production in the process. After isolation and purification, the purify of steviol was about90%and the yield was81.6%.