Study On The Specificity Of Hydrolyzed Quercetin Glycoside Substrate By β - Glucosidase From Roots

Flavonoids are a class of secondary metabolites in plants and have may play a dietary role in reducing the risk from chronic diseases such as cardiovascular disease and cancer. However, flavonoids are generally not found as free aglycones, but rather as complex conjugates with sugar residues and the aglycones are likely to have a greater biological effect than the glycoside, so the abilities for converting flavonoids glycosides into the aglycones and the substrate specificity of β-glucosidases on flavonoids have been extensively studied.The β-glucosidase (Tm-BglA) from Thermotoga maritima which belong to glycoside hydrolases family 1 can efficiently hydrolyzed flavonoid glycosides to increase the flavonoid aglycones which are likely to have a greater biological effect than the glycoside. In this study, based on the multiple sequence alignment of the Tm-BglA sequence with sequences of oligosaccharide, flavonoid and isoflavonoid hydrolyzing GH1 enzymes and analysis of the BglA structural model, we designed three substitutions at three positions close to the +2 subsites (F221L, N223L, and G224T) including single point mutations, double point mutations and three mutations by inverse PCR method. Here seven mutant recombinant plasmids were constructed and transformed into the E. coli host to expression. The seven mutants have been purified, characterized and compared to the wild-type enzyme. The results show that the three positions changing have not much impact on the stability of the optimal conditions of the enzyme and also have notable impact for the kinetic parameters of the artificial substrate pNPG. However, the mutations all lead to the reducing of the activity of hydrolytic activity of soy isoflavones and the G224T mutation decreased most significantly. Meanwhile, mutations have different effects on the hydrolytic activity of the quercetin-4’-O-glucose which class to flavonoid. The mutation which including F221L point mutation has no effect the hydrolytic activity of the quercetin-4’-O-glucose compared to the wild-type enzyme activity and the mutation containing N223L point mutation almost have no hydrolytic activity of the quercetin-4’-O-glucose. However, the mutation which including G224T point mutation showed a significantly increase in quercetin-3,4’-O-glucose’s and quercetin-4’-O-glucose’s conversion 28.6% and 35.5%, respectively, decrease in Km (1.24) and displayed an increase in Kcat (2.4) for the hydrolytic activity of the quercetin-4’-O-glucose. This results show that the F221,N223,G224 are extremely important sites on the enzyme’s substrate specificity and G224 looks more prominent. These results provide important theoretical basis for study of the beta-glucosidase substrate specificity, and also has important application value for the biotransformation of flavonoids.