Cloning,Expression And Molecular Modification Of Glycosidehydrolase Family 12 Gene From Scytalidium Thermophilium

Cellulose is the main component of the plant cell wall,and it is the most abundant renewable resource on the planet.Under the circumstances of the current energy crisis and environmental pressure in the world,how to more effectively exert the function of microbial cellulase to decompose and transform the huge cellulose stored in nature into energy is of great significance for solving resource and environmental problems,especially for the sustainable development of human society,site-directed mutagenesis technology has become an important technology for improving cellulase activity.Scytalidium thermophilum is a thermophilic fungus that produces thermostable enzymes and is one of the powerful decomposers of crystalline cellulose.The study aimed to search for amino acid sites that affect the activity of the Glycoside Hydrolase 12 family?GH12?of Scytalidium thermoohilium,to study the catalytic mechanism of GH12,and to lay a foundation for the search for the remaining amino acid sites with improved enzyme activity and improved enzymatic properties.In this study,the gene steg1 from GH12 of Scytalidium thermoohilium was cloned and the full-length cDNA was 711bp,encoding 237 amino acids.The target gene was transformed into Pichia pastoris to induce expression and purification of the protein.The results of SDS-PAGE analysis showed that the molecular weight of STEG1 protein was 26kDa,which was basically consistent with the theoretical value of 27kDa.The study found that the catalytic domain of the three-dimensional structure of STEG1 is a flute-shaped active channel consisting of 18 amino acids,including F220,P146,and Y145.,I144,D72,W137,W37,I147,N35,M135,V74,E133,D116,M171,N168,F118,Y77,W128.Based on structural analysis,it was found that protein forms different structures in the process of degrading cellulose.Among them,during the reaction with various carbohydrates,hydrogen bonds are more easily formed between some amino acid sites located on the active channel and glucosyl groups,so these amino acids are more likely to bind with the substrate,and the hydrogen bonds formed between-COOH and N in the main chain of Y145 and-OH of glucosyl group are in close proximity.Therefore,six mutation sites?N35,W37,Y77,W128,Y145,and N168?located in the catalytical active channel were selected for site-directed mutagenesis,and eight mutants?N35Q,W37H,Y77F,W128S,Y145F,Y145A,Y145W,N168Q?were obtained,which have different changes in the enzymatic properties.The results showed that compared with wild-type STEG1,the activity of all mutant enzymes decreased,and the thermal stability of the mutant enzymes Y77F and N35Q was improved,among which the K_m and k_catat values of Y77F,W128S,and Y145A decreased;the K_m and k_catat values of N35Q increased;in addition,The K_m values of W37H,Y145F,Y145W and N168Q increased,and the k_catat values decreased.The optimum pH of STEG1 and the mutants was 5.0,both remained unchanged.The optimum temperature of Y77F and N35Q was reduced to 50?,and the optimum temperature of STEG1 and W37H,W128S,Y145A,Y145F,Y145,N168Q was55°C.The results showed that the selected six amino acids play an important role in the degradation of cellulose by GH12.