Cloning,Expression And Molecular Modification Of Glycoside Hydrolase Family 7 Genes From Thermophilic Fungi

As a renewable resource with the most sugar content,cellulose has great significance in solving energy shortage problem.Studies have found that degradation of cellulose by cellulase is an environmentally friendly and efficient method.In the process of the degradation of cellulose,cellulase with high activity and thermal stability is the key to improve the efficiency of the degradation.Cellulase produced by thermophilic fungi has high activity and thermal stability.In this study,an exoglucanase cbh1 gene of glycoside hydrolase family 7 from Myceliophthora thermophila and an endoglucanase eg2 gene of glycoside hydrolase family 7 from Chaetomium thermophilum were cloned,expressed in Pichia pastoris.The expressed CBH1 and EG2 were purified using nickel affinity chromatography and detected by SDS-PAGE electrophoresis.The theoretical molecular mass of CBH1 was 54 kDa,whereas the electrophoresis band showed a protein of 72 kDa,speculating that the protein may be modified with glycosylation.The electrophoresis band of EG2 showed that the protein was 45 kDa,which was consistent with the theoretical molecular mass(45 kDa).In order to obtain CBH1 and EG2 with higher activity,molecular modification of CBH1 and EG2 was carried out.Using homology modeling,three-dimensional structure of CBH1 and EG2 was predicted.The three-dimensional structure shows CBH1 has a loop structure,which is located at the top of the active site tunnel at catalytic centre and consists of 9 amino acids,namely G245-Y253.The loop structure was deleted to obtain deletion mutant CBH1-DM.Based on the three-dimensional structures,we mutated four conservative aromatic amino acid residues W38,W40,W372 and W381 in the substrate binding path of CBH1 and five nonconservative amino acid residues A141,L145,M195,I207 and M342 within the active site 6 ? in the active channel of EG2 by site-directed mutagenesis,to form mutant enzymes W38 Y,W38F,W40 Y,W40F,W372 Y,W372F,W381 Y and W381 F for CBH1 and A141S,L145 T,M195L,I207 V and M342 F for EG2.The enzymatic properties of wild enzymes and mutant enzymes were determined.The experimental results show that this study has successfully obtained two mutants with increased enzyme activity,namely CBH1-DM and W38 Y.The specific activity of CBH1-DMwas 2.42 U/mg,which is 2.1 times that of wild enzyme CBH1(1.17 U/mg),the specific activity of mutant W38 Y was 1.3 U/mg,which is 1.1 times that of CBH1.The enzyme activity of the remaining mutant enzymes decreased in varying degrees.The optimum temperature of CBH1 and CBH1-DM were 50 ?,and the optimum temperature of the remaining mutant enzymes was 60 ?.The optimum pH of CBH1 and mutant enzymes was5.0.After treatment at 70 ? for 1 hour,CBH1 only had 35% of enzyme activity,while CBH1-DM had 65% of enzyme activity,showing that the thermal stability of CBH1-DM was significantly improved.The thermal stability of other mutant enzymes was reduced in varying degrees.Compared with the wild enzyme EG2,activity of its mutant enzymes was decreased to varying degrees.The optimum temperature and optimum pH of the wild enzyme EG2 and the mutant enzymes was 50 ? and 5.0,respectively.After treatment at 80 ? for 1 hour,EG2 had 35% of activity,while M195 L had 65% of enzyme activity,showing that the thermal stability of M195 L was improved.The thermal stability of other mutant enzymes was reduced in varying degrees.