| Glycosylation modification of bioactive compounds,not only increase its biological activity,reduce its toxicity,but also enhance the transhipment of drug and gene,and meantime can enhance its liposolubility and improve its efficacy.As a result,glycosylation modification has become today's hot spots in the field of biomedical research.So far,the industrialized production of the glycosylation modifica- tion, in particular the synthesis of Alkyl glycosides mainly relies on chemical methods.However,chemical products generally contain various isomers and mixtures,and also involve a number of steps for protection and deprotection. Enzymatic synthesis of glycosides,can not only avoid the complex reaction steps for protection and deprotection with high stereoselectivity,but also obtain products with high purity and high yield in mild reaction conditions, friendly to the environment.So enzymatic synthesis of glycosides in the industrial application is more and more be interested.Purpose of the study:(1) To explore the feasibility of enzymatic synthesis of VC glycosides,VE glycosides,salidroside and other glycosides with biological activity.(2) To establish the reaction system to synthesize VC glycosides,VE glycosides,salidroside and other glycosides.Methods and Results:(1) Four strains with highβ-glucosidase activity were isolated from the soil under the apple trees and pear trees.Strain Aspergillus oryzae produces the highest amount ofβ-glucosidase among the four strains,and its activity reached 107.9 U/mL.The results showed that the liquid medium with salicylaldehyde glycosides and solid medium were both optimal medium for producingβ-glucosidase.(2) The effects of temperature,pH,reaction time,organic solvents and metal ions on the activity of crudeβ-glucosidase derived from Aspergillus oryzae was researched and the optimal condition was obtained;β-glucosidase was purified by salting-out,dialysis desalination and freeze-drying,and the enzyme activity reached 127.6 U/mg,purifiing factor was 2.39.(3) The enzymatic synthesis of salidroside in non-aqueous medium relying on reaction of reversed hydrolysis withβ-glucosidase as a catalyst was explored.In this paper,the effects of different organic solvents,reaction time,temperature,pH and concentration of substrates on the synthesis of salidroside was investigated and the reaction conditions were optimized.The salidroside conversion rate reached 18.1%under the optimal conditions, equivalent to 7.39 g/L,much higher than the catalytic production of overall cells of Aspergillus oryzae(0.7 g/L).(4) Synthesis of a new VC glucoside:2-O-β-D-glucopyranosyl-L-ascorbic acid(AA2βG) isolated from a popular traditional Chinese food(Lyciun fruit) was explored,with crudeβ-glucosidase derived from Aspergillus oryzae as a catalyst and with cellobiose as a glucose donor.The new VC glucoside AA2βG has better oxidative stability than VC itself,and has substantial prospects in the beauty and skin care applications.Besides, methods of separation and purification of AA2βG,and its testing conditions with HPLC in the existing conditions were also explored.(5) Based on the synthesis AA2βG by microorganism,we studied the basic metabolism of VC in vivo,as well as the impacts of concentration of substrates,pH and reaction time on the synthesis of AA2βG.The results showed that the optimal conditions were as follows:ascorbic acid 7g/L, D-glucose 5g/L,reaction time 48 h,pH 7.Under the optimal conditions,the maximum amount of AA2βG reached 0.23 g/L.We concluded that,the intracellular accumulation of the substrate to a certain concentration can cause toxity to cells,and the cells will detoxify through the formation of ascorbic acid glycosides 2-O-β-D-glucopyranosyl-L-ascorbic acid.
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