Synthesis Of Glycosides By ?-galactosidase And Cyclodextrin Glycosyltransferase

Natural extracts are important source of bioactive compounds.Rhodiolae grows under severe environment in alpine and axenic areas with high altitude,and has strong vitality and special adaptability.The extract of Rhodiolae has been commercially used to improve energy,hypoxia tolerance and so on.In recent years,it has been found that the active ingredient of salidroside(p-hydroxypHenylethyl-O-?-D-glucoside)has a wide range of biological activities,including antioxidant,anti-inflammatory,heart and neuroprotective effects.Notably,one analog with a galactose moiety attached to the tyrosol,namely tyrosol galactoside,was found to exhibit better neuroprotection activity than the salidroside containing a glucose moiety but the current enzymatic yield of the compound is rather low.Enzymatic synthesis of glycosides is characterized by stereo-and regioselectivity.It can catalyze the synthesis of specific glycoside products in the one-step,through a simple step and in a sustainable and environment-friendly.Glycosidase and Non-Leloir glycosyltransferases catalyze glycosylation reaction in a simple way.Their substrate is inexpensive,and the enzyme source is broad and readily available.Thus they are widely used to catalyze the glycosylation reaction.?-galactosidase(EC 3.2.1.23),a class of important glycosidases capable of breaking or introducing galactose residuces,have attracted particular interest in the applications.Some ?-galactosidase capable of synthesizing glycosidic bonds have been industrially used to produce galacto-oligosaccharides.The synthesis activity also has been extended to synthesize a variety of glycoside compounds.Cyclodextrin glycosyltransferase(EC 2.4.1.19)is a kind of Non-Leloir glycosyltransferase,which catalyzes the hydrolysis of?(1?4)-glucosidic bond.It can hydrolysis starch as substrate to produce glucotose,diglucotose,triglucotose,tetraglucotose,pentaglucotose,?-/?-/?-cyclodextrin.It also catalyze glycoside synthesis reaction with starch as saccharide donor.In this paper,tyrosol galactoside was synthesized by ?-galactosidase derived from Enterobacter cloacae B5 in a high yield,which laid a foundation for the large-scale production of this compound.In addition,a variety of new salidroside derivatives were synthesized by cyclodextrin glycosyltransferase from Bacillus subtilis.Those compounds might have potential applications drug screening.The P-galactoside gene bgaB5 from Enterobacter cloacae B5(GenBank accession No.DQ266449)had been inserted into the pET-15b vector and successfully expressed in Eschericha coil BL21(DE3)in the previous work.In this paper,the recombinant enzyme was expressed and purified for transglycosylation reaction.The lactose was used as the glycosyl donor and the tyrosol was used as the glycosyl acceptor.The reaction was detected by TLC and HPLC,respectively.The results showed that three novel spots(designated as P1,P2,P3)appeared signals below the spot of tyrosol by TLC which loaded the reaction mixture catalyzed by the recombinant BgaB5.The effect of substrate concentration,pH,temperature and reaction time on this reaction by BgaB5 were investigated.The optimal conditions for the synthesis of tyrosol derivatives by BgaB5 were 250 mM tyrosol and 1000 mM lactose at pH 7.5 and 5 min incubation at 50?.Under these conditions,the yield of total tyrosol derivatives reached a maximum of 50.0%,consisting of 39.4%of PI and 10.6%of P2 and P3 products.The glycosylated products were purified by Bio-gel P2 and preparative TLC.The MSresults showed that the molecular weight of P1 was 300,indicating a monosaccharide derivative.The molecular weight of P2 and P3 were 462,indicatingdisaccharide derivatives.NMR spectroscopy analysis further identified these compounds to bep-hydroxyphenethyl P-D-galactopyranoside,p-hydroxyphenethyl ?-D-galactopyranosyl-(1?3)-p-D-galactopyranoside,and p-hydroxyphenethyl ?-D-galactopyranosyl-(1?6)-(3-D-galactopyranoside,respectively.Apart from tyrosol monogalactoside,two novel tyrosol digalactosides were synthesis by BgaB5 for the first time.?-Cyclodextrin glycosyltransferase(?-CGTase)from Bacillus subtilis was purified by ammonium sulphate precipitation,DEAE anion exchange chromatography,Source 15Q anion exchange chromatography and Sephadex G-200 gel filtration chromatography for the first time.?-CGTase was a homodimer protein,and its subunit molecular weight was about 70.4 kDa.The optimum pH and temperature of ?-CGTase was 7.5 and 55?,respectively.The enzyme was stable in the range of pH6.5?8.5,and has high activity and stability at 50??60?.Zn2+,Ca2+and Na+had a slight effect on the activity of ?-CGTase,while Ni+and NH4+had inhibitory effects.The ?-CGTase catalyzed the glycosylation of esculin,resveratrol,salidroside,and sterioside when using soluble starch as donor.using B.subtilis ?-CGTase were for the first time used for glycosylation of salidroside with high efficiency.The reaction was detected by TLC and HPLC,respectively.The results showed that six novel spots(designated as S1,S2,S3,S4,S5,S6)appeared signals below the spot of salidroside by TLC which loaded the reaction mixture catalyzed by the ?-CGTase.The effect of salidroside,starch,reaction pH,reaction temperatuure and reaction time on this reaction by ?-CGTase were investigated.The optimal conditions for the synthesis of salidroside derivatives by ?-CGTase were 50 mM salidroside and 100 g/L starch at pH 7.5 and 2 hours incubation at 55?.Under these conditions,the yield of total salidroside derivatives reached a maximum of 85.9%,consisting of 34.5%of S1,and 21.6%of S2,and 14.8%of S3;and 7.5%of S4,and 4.7%of S5,and 2.8%of S6.The glycosylated products were concentrated by Bio-gel P2,HPLC,preparative TLC.The MS results showed that the molecular weight of S1 was 462;the molecular weight of S2 was 624;the molecular weight of S3 was 786;the molecular weight of S4 was 948;the molecular weight of S5 was 1110;the molecular weight of S6 was 1272.NMR spectroscopy analysis the structure of Slconfirmed it to be p-hydroxyphenethyl ?-D-glucoopyranosyl-(1?4)-?-D-glucoopyranoside.Based on the characteristic synthesitic property of the ?-CGTase and combined with the MS and NMR data,the other salidroside derivatives S2 to S6 were considered to be salidroside derivatives containing?-(1?4)-glucoopyranosyl linked disaccharide,taisaccharide,tetrasaccharide,pentasaccharide and ?? exasaccharide,respectively.