Biosynthesis Of Phenyl Allyl β-D-Glucoside With Magnetic Cross-linked β-Glucosidase Aggregates

In this paper, magnetic cross-linked β-glucosidase aggregates (M-CLGAs) were prepared and were used for the catalytic synthesis of phenyl allyl P-D-glucoside, an active compound extracted from Rhodiola. The enzymatic synthesis of phenyl allyl P-D-glucoside has some characteristics, such as simple reaction process, mild conditions, high stereoselectivity and environment friendly, and so on. Therefore, the enzymatic catalytic biosynthesis of phenyl allyl β-D-glucoside by β-glucosidase is of great value in industrial application.M-CLGAs are a kind of immobilized enzyme with some advantages, such as high recovery of enzyme activity in preparing, simple preparation process, easy recovery and stability. Orthogonal test results showed that the optimum conditions for preparation were:P-glucosidase 2 mg, sedimentation agent volume 1 mL, magnetic ferriferrous oxide particles 6 mg, glutaraldehyde concentration 15 mmol/L, and cross linking time 2 h. The specific activity of magnetic cross-linked P-glucosidase aggregates prepared under this condition was up to 6.44 U/mg, which means 80.5% of the enzyme activity was recovered. SEM showed magnetic cross-linked P-glucosidase aggregates were spherical particles with good monodispersity and at a diameter of about 50 μm. Compared with the free enzyme, cross-linked magnetic β-glucosidase aggregates possessed improved thermal stability and pH stability, good operating stability, strong resistance to magnetic fields, but increased Km value.Furthermore, the M-CLGAs catalyzed direct glycosylation reaction to synthesize phenyl allyl P-D-glucoside in non-aqueous phase system was studied, and the effect of the combination of non-aqueous systems, substrate concentration, ionic liquids, water activity and other factors on glycosylation reaction were investigated, and the product structure was identified and characterized. The results showed that in non-aqueous system cinnamyl alcohol and D-glucose could be synthesized to from phenyl allyl P-D-glucoside by direct glycosylation catalyzed by M-CLGAs. Under the optimal conditions, that is, the reaction system 1,4-dioxane/citrate-phosphate buffer/ionic liquid (C4Mim·PF6) (8:2:1), the molar ratio of the substrate cinnamyl alcohol and D-glucose 4:1, namely cinnamyl alcohol and D-glucose concentration of 298 g/L and 100 g/L, pH 6,50℃,250 rpm,15 U/mL, after 60 h the product concentration was 8.037 g/L and the activity recovery of M-CLGAs reached 58.2%. The product was identified by NMR and LC-MS as phenyl allyl P-D-glucoside.Moreover, the preliminary study on transglycosylation reaction catalyzed by M-CLGAs to synthesize phenyl allyl β-D-glucoside was also conducted in this article. The effects of different substrates of glycoside, organic solvent, buffer, substrate concentration on the synthesis of phenyl allyl β-D-glucoside by transglycosylation were investigated. Under the optimum reaction system, which was cinnamyl alcohol and pNPG with molar concentration ratio of 1:1, reacting for 30 h in t-butanol and citric acid-phosphate buffer solution at a volume ratio of 9:1, the final phenyl allyl P-D-glucoside yield would be 10.77 g/L, which was greater than the yield of the product obtained by direct glycosylation reaction. And in the reaction, the activity recovery of M-CLGAs could reach 87.5%.