Molecule Evolution Of Glycoside Hydrolase Family 1 And Family 6 From Scytalidium Thermophilium

Cellulose is a renewable resourece which is rich in content in nature.It plays an important role in solving energy shortage and resource depletion.However cellulose is abundant in nature,the utilization of it is very low,and resulting in a lot of waste.Cellulase is a kind of enzyme,which play a very important role in degrading of cellulose.Cellulase can degrade cellulose into glucose.Scytalidium thermophilum is a thermophilic fungus that produces thermostable enzymes and is one of the powerful decomposers of crystalline cellulose.In this study,the exoglucanase gene cbhk and ?-glucosidase gene bgk from Scytalidium thermophilum were cloned and expressed in P.pastoris GS115.The expression products,recombinase CBHK and BGK,were purification by affinity chromography.SDS-PAGE anaylysis showed that they had a mass weight of approximate 72 k Da and 55 k Da,respectively.The molecular weight of CBHK was 72 k Da,which was greater than the theoretical molecular weight of 50 k Da,which may be caused by glycosylation.The molecular weight of?-glucosidase BGK was determined by SDS-PAGE.The size of 55 k Da is basically the same as the theoretical molecular weight.Improving the enzyme activity play a critical role in enchancing the hydrolysis of cellulose.Based on the structure of Scytalidium thermophilum ?-glucosidase(Hi BG),12 amino acids in the intrance channel of active site were mutated in this study.Based on homology modeling,the site directed mutagenesis of four site from Scytalidium thermophilum exoglucanase was carried out,and the gene was expressed in P.pastoris GS115.Mutants W163H?E437D and Q459 T showed decreased activity by 50%?40% and 35%,respectively,compared to the wild type exoglucanase.The enzyme activity of W397 H almost be fully lost.Compared to wild type ?-glucosidase(WT),the activity of all mutations were reduced.The activity of mutations A260 N,F348G,Y179 F and F180 H decreased by 20%,20%,30% and 30%;mutations D237 S,L173Q enzyme activity decreased by 55% and 60%.The enzyme activity of W168 H,N335F and W349 G almost be fully lost.?-glucosidase is the key enzyme in the hydrolysis of cellulose.For most cellulolytic systems ?-glucosidase is the rate-limiting factor,however,most ?-glucosidase are feedback inhibited by the glucose product,which restricts their application.Remarkably,some?-glucosidase of the glycoside hydrolase(GH)1 family are tolerant to glucose,but the molecular mechanism of glucose tolerance is still elusive.In this study,12 amino acid residues of W168,L173,F348,W349,C169,F180,D237,Y179,A260,H307,N335 and E437 in the substrate entrance region were mutated and expressed in Pichia pastoris GS115.Mutation L173 Q losed glucose tolerance,while mutation Y179 F losed glucose tolerance when at high glucose concentrations.This study suggested that the substrate entrance residues can effect enzyme activity and glucose tolerance,and the specificity of catalytic channel structural may be involved in the mechanism of glucose tolerance.