Research On Enzymatic Transformation Of Flavonoid Glycosides From Ginkgo Bilobal

Ginkgo biloba is one of the oldest and rarest plants on earth.Flavonoids are important active components in ginkgo biloba leaves.In this paper,two strains with high production ofβ-glucosidase were isolated from fermented fermented soybean food in guizhou province,and ginkgo flavonoid glycosides with high biological activity were prepared by hydrolyzing ginkgo flavonoid glycosides with free enzymes.β-glucosidase was purified by acetone precipitation and reverse micelle extraction.Secondly,the enzymatic properties of purified-glucosidase were studied in order to lay a foundation for the industrial application of the enzyme.Finally,the bioinformatics method was used to study the molecular docking of several major glycosides in ginkgo flavonoids with-glucosidase at the active sites,and the reasons for the high substrate selectivity of-glucosidase on ginkgo flavonoids and the functions of related proteins were analyzed.Firstly,two strains with high enzyme activity against ginkgo flavonoids were screened by the plate color method of gardenosine-glutamate sodium with ginkgo flavonoids extract as the sole carbon source.Subsequently,microscopical examination,molecular biological identification of 16S rRNA and physiological and biochemical identification were performed on the two strains.The newly identified strain GUXN01 was Bacillus subtilis,and the newly identified strain GUYZ13 was Bacillus amyloliquefaciens.Secondly,by using acetone precipitation and reversedmicelle method of combining the purification beta-glycosidase enzymes,obtained the optimum parameters of acetone precipitation for:strain GUYZ13 when hydrolyzed acetone and volume ratio of 1:3.8,-18℃under precipitation 0.5 h ShiChunHua effect best,under the condition of the beta-glycosidase enzymes enzyme activity recovery of 63.3%plus or minus 1.21%,purified ratio was 4.7+/-0.05;Strain GUXN01 when hydrolyzed acetone and volume ratio of 1:5,-18℃under precipitation 0.5h ShiChunHua effect best,under the condition of the beta-glycosidase enzymes enzyme activity recovery was 63.2+/-1.05%,purification ratio was 2.69+/-0.05.Acetone precipitation method using reverse micelle extraction of beta-glycosidase enzymes,the optimal reversedmicelle strains GUYZ13 conditions for water pH value of 7,0.05 mol/L NaCl salt ion concentration,CTAB concentration as the tendency for 25 mmol/L,cosolvent is hexanol/isooctane ratio of 2:8,reaction temperature of 30℃,the reaction time of30 min;Strain GUXN01 optimal reversedmicelle conditions for water pH value of 8,0.05 mol/L NaCl salt ion concentration,CTAB concentration as the tendency for 25mmol/L,cosolvent is hexanol/isooctane ratio of 2:8,reaction temperature of 30℃,reaction time for 30 min.Under the best condition,the total enzyme activity recovery ofβ-glucosidase produced by GUYZ13 strain was 41.7%after reverse micellar extraction of acetone precipitation,and the total purification multiple was 7.78 times.The total enzyme activity rate of-glucosidase produced by strain GUXN01 was45.3%,and the total purification rate was 4.37 times.Sds-page characterization of the purified enzyme solution showed that this method could purify the target enzyme to electrophoresis.Third,the enzymatic properties of purified-glucosidase were studied.GUXN01β-glucosidase hydrolyzed gardenoside,rutin and ginkgo flavonoids showed high enzyme activity and strong substrate specificity.The enzyme activities of gardenoside,rutin and gentianediose were 2.364 U/g,2.613 U/g and 3.543U/g,respectively.With ginkgo flavonoids as the substrate,the Km value of the enzyme produced by GUXN01 was 0.010mmol/L and Vmax was 7.812 mmol/L（l.min）.β-glucosidase produced by GUYZ13 has hydrolytic activity on oligosaccharides and glucoside substrates,such as salicylate,gentianediose and amygdalin.The Km value of the enzyme produced by GUYZ13 was 0.038 mmol/L and Vmax was 7.99 mmol/（l.min）.The study also found that GUYZ13,beta glucose from the GUXN01 glycosidase with the increase of temperature,enzyme activity increases,then the two strains of bacteria are best at 50℃enzyme activity.Under the condition of pH value of 5.0,the temperature of 30℃,strain GUYZ13 and the activity of enzymes from the strain GUXN01 is stable,almost no loss of enzyme activity,greater than 65℃,the enzyme activity are falling sharply.Ca⁺²,Mn²⁺and Mg²⁺significantly increased the enzyme activity of the two strains and activated them.The inhibition and activation ofβ-glucosidase by Na⁺and K⁺were not significant,but the activity of the enzyme was significantly inhibited by Fe³⁺.Finally,by the methods of bioinformatics,the selection of the main components of the several kinds of ginkgo flavone glycosides,using molecular docking method,illustrates the ginkgo flavonoid glycosides from molecular level with the beta glycosidase enzymes loci interaction mode,explains the beta glycosidase enzymes from the mechanism of ginkgo biloba extract has the cause of the high substrate specificity and the related function of the protein.The interaction mechanism between substrate and target enzyme-glucosidase was clarified.The specificity and affinity of-glucosidase to different substrates were mainly determined by the structure of the enzyme molecule,especially the structure of its active center and the structure of the substrate molecule.