Process Development And Optimization On Glycoside Hydrolase-catalyzed Hydrolysis Of Flavonoid Glycosides

Flavonoid glycosides such as rutin,hesperidin and naringin are a group of biologically active polypHenolic compounds widely distributed in plants,which aglycones are connected with a disaccharide by glycosidic linkages.Chemical or enzymatic hydrolysis of flavonoid glycosides can form biologically active flavonoid aglycone or aglycone monosaccharide.Chemical hydrolysis of flavonoid glycosides requires the use of strong acid and has no selectivity on the types of the glycosidic bonds.In comparison with chemical hydrolysis,enzymatic hydrolysis of flavonoid glycosides acts on specific type of glycosidic linkage under mild conditions.Here,extracellular ?-L-1,2-rhamnosidase and ?-L-1,6-rhamnosidase from Aspergillus niger WZ001 was used as biocatalysts in the hydrolysis of rutin,naringin and hesperidin in order to obtain the corresponding aglycone quercetin,naringenin and hesperetin.?-L-1,6-rhamnosidase from Lactobacillus plantarum WZ011 was cloned from its genomic DNA,over-expressed in the E.coli and applied on the hydrolysis of hesperidin to give hesperetin monosaccharide.In addition,the over-expression of ?-L-1,6-rhamnosidase from Lactobacillus plantarum WZ011 was explored in Pichia pastoris.The main findings are listed as follows:1.The conditions of ?-L-1,6-rhamnosidase production in the submerged fermentation of Aspergillus niger WZ001 were optimized.The results showed that when the mixture of 0.1 %(w/v)rutin and 1 %(w/v)naringin was used as inducer,the maximal activity of ?-L-1,6-rhamnosidase and ?-L-1,2 rhamnosidase reached up to 1260 U/ml after 60 h fermentation and 1489 U/ml after 84 h fermentation,respectively.The 10 %(w/v)substrate was subjected to hydrolysis in 25 %(v/v)DMSO as a co-solvent at the optimum temperature of 50 ? and the optimal pH of 5,in which the amount of enzyme immobilized in sodium alginate was 2 %(w/v).Rutin and naringin were completely hydrolyzed after 10 h and 6 h,respectively,while the conversion of hesperidin 14 h was aournd 60 %.After seven rounds of reactions,the remaining activity was 60 % of the initial activity.The product aglycone was recovered through the adsorption of the resin D4020 in a single time and the recovery was around 94%.The product was separated and purified silica gel thin layer chromatograpHy,giving 99 % purity of quercetin,hesperetin or naringenin.2.The gene encoding ?-L-1,6-rhamnosidase from Lactobacillus plantarum WZ011 was cloned from its genomic DNA and over-expressed in E.coli with the specific activity of 127 U/mg.As a bifunctional enzyme,recombinant ?-L-1,6-rhamnosidase exhibited the activity of ?-D-glucosidase and catalyzed the complete hydrolysis of hesperidin to hesperetin.When 30 %(v/v)DMSO was used as a cosolvent in the hydrolysis of hesperidin,the activity ?-D-glucosidase was inhibited and the hydrolyzed product was hesperetin monosaccharide.The hydrolysis of hesperidin with the optimum substrate concentration of 3 %(w/v)was conducted at the optimum pH 5 and the optimum temperature 55 ? for 10 h,and the hydrolyzed product hesperetin monosaccharide was recovered through the adsorption of the resion D4020 for three times,giving the recovery of 91 %.The recovered product was isolated and purified by silica gel thin layer chromatograpHy and its purity was greater than 99 %.3.To construct the yeast strain over-expressing ?-L-1,6-rhamnosidase,the gene encoding ?-L-1,6-rhamnosidase from Lactobacillus plantarum WZ011 was cloned onto the plasmid pPICZ?A,and the resulting recombinant plasmid was transformed into the host strain P.pastoris GS115.The genetically engineered P.pastoris GS115 was induced,and the SDS-PAGE analysis was used to determine if the gene encoding ?-L-1,6-rhamnosidase from Lactobacillus plantarum WZ011 was expressed.The SDS-PAGE analyses showed the the targeted protein band but its activity was not approved by the hydrolysis of hesperidin.