Study On Synthesis Of Glycosides Using β-Glucosidase In Non-aqueous Medium

Glycosides have a wide range of physiological and pharmacological activity, but its low content in the natural products made it can not meet demand. So mainly through the callus suspension culture and chemical synthesis to obtain them at present. The callus culture cycle is too long, and chemical synthesis, which requires multi-step protection and deprotection, has not high enough pure products. Enzymatic synthesis of glycosides has obvious advantages:mild reaction conditions, high purity and avoid cumbersome protection and to protect the steps of chemical synthesis; at the same time, glycoside receptor is cheap and easy to get, low cost. Biocatalytic Synthesis of glycosides has recently become research hot.As a glycoside hydrolase,β-glucosidase posseses the catalytic activity of glycosidic bond hydrolysis, meanwhile it owns the catalytic activity of glycosyltransferases. It can be used for synthesis of oligosaccharides, alkyl glycosides and aromatic glycosides, as well as amino acids, peptides, antibiotics, glycosylation etc. As the products of glucoside in the aqueous phase is easy to hydrolysis, significantly reduced the effect of the catalytic synthesis, relative to the water phase, the non-aqueous reaction media for enzymatic synthesis of glycosides has obvious advantages, through the effective inhibition of the product's hydrolysis to increase the conversion. The purposes of this paper, is to build reaction systems of enzyme-catalyzed glycoside synthesis in non-aqueous medium, usingβ-glucosidase from microorganism in organic solvents and ionic liquids, which is suitable to synthesis glycosides. Optimize the catalytic conditions in order to improve the yield of salidroside. It lays the foundation for the study of enzyme catalytic mechanisms and enzyme-catalyzed industry in the future.Main contents are as follows:1. Solid State Fermentation medium components and culture conditions were optimized to improve enzyme production capacity. The optimum fermentation medium:bran 8 g, corn cob 2 g, peptone 0.3%, salicin 0.05%, water lOmL, natural pH. The crude enzyme obtain from the fermentation extracts by ammonium sulfate fractionation, dialysis and freeze-dried, the enzyme specific activity rise from 59.94 U/mg to 164.57 U/mg, and purification was about 2.75.2. Construction of the organic solvent reaction system, and reaction conditions were optimized, The catalytic behaviors ofβ-glucosidase and polyethylene glycol (PEG)-modifiedβ-glucosidase in non-aqueous medium were investigated by HPLC. The results show that, PEG-β-glucosidase complexes has more tolerance, higher catalytic activity in organic solvent than the free enzyme, and the best conditions of the synthesis is:in hexane reaction system, substrate tyrosol of 30 mol/L, D-glucose and tyrosol molar ratio of 1:1, at 50℃for 50h, the yield of 59.2%. In order to verify the scope of the reaction system, hydroquinone glycoside was also synthesised.3. In ionic liquid reduction system, [bmim]PF6was the optimal reaction medium, and different reaction conditions were optimized in it. A yield of 88.5%(product content of 61.93 mmol/L) was obtained under the conditions of water content of 2%, temperature of 50℃, tyrosol content of 70 mmol/L, D-glucose/tyrosol molar ratio of 2, and reaction time of 24 h. The results suggest that both the modification with PEG and using ionic liquid [bmim]PF6 as reaction medium have obvious promoting effects on the yield of enzymatic.