Improve The Substrate Specificity Of ?-glucosidase Hydrolyzing Plant Polyphenol Glycosides

Plant polyphenols,also named vegetable tannin,that 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,plant polyphenols are generally not found as free aglycones,but rather as complex conjugates with sugar residues and the aglycones are likely to have a higher biological effect than the glycoside,so the abilities for converting polyphenols glycosides into the aglycones and the substrate specificity of ?-glucosidases on flavonoids have been extensively studied.Thus deglycosylated can make the polyphenols glycosides play its function.Enzymatic deglycosylation is the most economical and environmentally method,and ?-glucosidase is widely used now.However,because of the specificity inconsistent to aglycone of-glucosidase,the way to improve the ability of deglycosylating plant polyphenols is focused on the role of research now.The p-glucosidase(Tm-BglA)from Thermotoga maritima which belong to glycoside hydrolases family 1.In early experiments,-glucosidase was found that it can hydrolysis flavonoid glycosides to aglycone,and the activity of the hydrolysis of ?-glucosidase for different flavonoids substrate are varied.By site-directed mutagenesis,we found the original 224rd amino acids were replaced from glycine to threonine,and this change can significantly improve the hydrolysis efficiency to quercetin-4'-O-glucose.This program studies the key amino acids of Tm-BglA in hydrolysizing of glycosides.We can determine the mutation site by docking of Tm-BglA and molecular cellotetraose,and sequence alignmed of Tm-BglA and other GH1-glycosidase.So,here choose W168 located on ?-4 folding,N223,G224 stocked on ?-5 folding and N246 located on ?-5,that all close to the aglycone binding site subsites as site-directed mutagenesis.Using inverse-PCR,here was constructed single mutant of W168Y,W168S,N223T,N223S,G224F,N246S and double mutant N223S/G224T.Here mutants recombinant plasmids were constructed and transformed into the E.coli host to expression.The mutants have been purified,characterized and compared to the wild-type enzyme.The results show that the four 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,to study the position of the amino acid of Tm-BglA.After the success of mutants recombinant plasmids constructed and transformed into the E.coli,here was determinated enzymatic properties and hydrolytic activity of plant polyphenols of mutations after expressed and purificated enzyme,compared with Tm-BglA,combining the results of molecular docking,we can evaluate the ability of the mutant enzyme.The results showed that 168,223,224 and 246 point mutations didn't affect the normal expression of Tm-BglA.These mutations also didn't affect the overall structure of Tm-BglA.There was no change on optimal conditions of Tm-BglA,but had an impact on the kinetic parameters of pNPG.The hydrolysis activity of soy isoflavones of each mutations enzyme was all reduced compared with the wild-type enzyme,and the hydrolytic activity of quercetin glycosides,polydatin,arctiin was different varing with the enzyme,too.Including N223,G224 point mutations in these mutant enzymes,have a higher hydrolytic activity on quercetin glycosides,polydatin,arctiin.The rate ofN223S/G224T hydrolyzed quercetin glycosides was increased 30%to 55%higher than Tm-BglA,which reacted time is 10 minutes.The rate of N223T hydrolyzed polydatin was increased 65.0%higher than Tm-BglA,which reacted time is 10 minutes.And G224F increased 28.1%when substrate is arctiin.This shows that the two amino acid sites in the hydrolysis of Tm-BglA played a key role in hydrolyzing plant polyphenols.The results ca improve the study of key amino acid of ?-glucosidase.Combining molecular docking on the experimental results are analyzed and discussed,analysis of the interaction between quercetin-4'-O-glucose and Tm-BglA,calculated free energy of binding of both-10.87 kcal/mol,223 amino acids by asparagine mutation of serine,and threonine 224 mutation,the spatial distance enzyme and substrate closer;the Tm-BglA and polydatin docking result,the calculated free energy of a combination of both-8.11kcal/mol,from the 223 amino acid aspartic acid is mutated to threonine,224 amino acids after mutation of phenylalanine,the spatial distance was shortened,it may be that the mutant enzymes are able to improve its efficiency for hydrolysis polydatin the reason;and by the results of Tm-BglA docking with arctiin found,Tm-BglA combined with arctiin free energy-5.49kcal/mol,the 223 amino acid mutated from aspartic acid,threonine,after the 224 amino acid is mutated to phenylalanine,the spatial distance was shortened,this may be the reason for the mutant enzymes is possible to improve the efficiency of hydrolysis for arctiin.