Enzymatic Modification And Microbial Transformation Of Steviol Glycosides

Steviol glycoside is a natural,non-nutritive high-intensity sweetener,and its main ingredients include stevioside(Stv)and rebaudioside A(RA).The content of Stv is highest in the steviol glycoside with bitter aftertaste.Rubusoside(RS)is a natural sweetener with high quality and has low yield.Stv would be glycosylated or deglycosylated by cyclodextrin glucanotransferase(CGTase)or microbiological fementation to produce glycosylated Stv or RS with better taste quality.In this work.strains would glycosylated or deglycosylated of Stv were screened,and the conversion method and conditions were studied.CGTase of strain CGT7 screening from soil presented specific glycosy lated activity on Stv,and the new product was identified as mono-glucosylated stevioside(Stv-Glu)with better taste quality by HPLC and LC-MS/MS.Based on the physical characterization and phylogenetic analysis by 16S rDNA gene sequence,the strain CGT7 was identified as Bacillus(Bacillus sp.CGT7).The highest enzyme activity was obtained with corn starch and soybean meal powder as carbon and nitrogen source respectively,culture conditions of 30?,pH8.5.The product concentration of 0.61 mg/mL was obtained with starch as glycosyl donors,reaction conditions of 45 ?,pH 7.0,mass ratio of 1.5:1.0(steviol glycoside:cornstarch)?steviol giycoside 1%.The Stv of stevia extract would be glycosylated specifically by CGTase from strain CGT7 to produce Stv-Glu.The highest conversion of Stv of 40.2%was obtained with reaction conditions of 45? starch 5%,pH 7.0.volume ratio of 10:7.5(enzyme liquid:stevia extract),for 10h.The strain CGT7 can not conver RA in the fermentation process and can conver Stv specifically with the final conversion of 33.9%.CGTase were separated and purified.20%-60%of(NH4)2SO4 precipitation was used to precipitate and concentrate the protein.The target protein was precipitated.SDS-PAGE electrophoresis results show that the relatively single band of target protein was obtained by DEAE Sepharose TM ion exchange chromatography with NaCI-Trish-HCl buffer solution(20mmol/L,pH8.3)gradient elution and Sephadex G-75 filtration chromatography with Trish-HCl buffer solution(20mmol/L,pH8.3)elution.The molecular of the CGTase weight was 37 kD.The optimum reaction pH of the CGTase was 5.5,and it was stable in the range of pH 5.0-9.5.Its optimum reaction temperature was 50? and the enzyme activity keep more than 80%after 2h incubated in 60?.Fusarium sp.was screened out with the highest conversion of Stv and can not convert RA.The new product was identified as RS by HPLC and LC-MS/MS.The galactooligosaccharide,lactobiose,IPTG can promote the conversion of Stv.The enzyme transforming Stv was a endoenzyme.The high galactosidase activity was detected at intracellular location in the middle and later periods of the fermentation.The results showed that galactosidase was a key enzyme in the process of conversion of Stv.The conversion of Stv of 91.8%was obtained with glucose 0.8%,steviol glycoside 4%,for 70h.Polyamide and macroporous resin were used in the purification process of RS.Macroporous resins D302 has the highest adsorptive capacity.According to the conclusion of purification process,polyamide is more advantageous to the purification of RS.The RS with purity of 95.2%and RA with purity of 64.5%were obtained with the process of deionized water and 10%ethanol solution elution,rotary evaporation and methanol recrystallization.