Construction Of Yeast Engineering Strain With High Expression And Thermalstability Cellulase Fusion

It’s well known that cellulose is the most abundant organic compound on earth,widely spreading as one of the cheapest renewal resources, but most of it can’t be used directly and becomes cellulose waste. Although cellulases have been used as important industrial enzymes, more work should be done to improve enzyme activity, thermostability, and pH stability to meet the industry need. Thermophilic microbes are main sources of cellulases with high thermo stability. Cellulases from the thermophilic fungi have been reported to be stable and highly active at high temperature, and thermophilic fungi can produce multiple forms of the cellulase components. Studies show that glycoside hydrolase family 61 can be greatly improved cellulase activity.In this study, the genes eg1 and 61 f have been cloned from Thermoascus aurantiacus var.levisporus and the two genes are fused to create a gene hybrid named eg1-61 f by fusion PCR.The genes 61 f and cbh1 cloned from C. thermophilum have been fused to create a gene hybrid named 61f-cbh1 by fusion PCR. After cloned into the victor pPIC9 k, eg1-61 f and 61f-cbh1 were expressed in the yeast Pichia pastoris. The strains named EG1-61 F and 61F-CBH1 with a high expression efficiency under induction of methanol were obtained through screening and the expressed products possesses an excellent thermostability. Further analyses showed the molecular mass of the enzyme EG1-61 F is 59 kDa, and the optimum pH and temperature of the recombinant enzyme activity are 5.0 and 60 oCespectively. The recombinant enzyme remains 83% of its original activity after 60 min at 80 oC, and the half-life was 50 min at 90 oC and remains 56%of its original activity. The activity of the recombinant enzyme can remain stable in pH between 4.0 and 6.0. Compared with the original enzymes 61 f and eg1(the activity of 61 f is very weak, the performance of EG1 is endonuclease activity), the fused enzyme activity is about 2.1times higher than that of EG1.Further analyses showed that the molecular mass of the enzyme 61F-CBH1 is 102 kDa, and the optimum pH and temperature of the recombinant enzyme activity are 6.0 and 60 oC respectively. The recombinant enzyme remains 96%of its original activity after 60 min at 60 oC, and 93%of its original activity at 70℃. Compared with the original enzymes 61 f and cbh1(the activity of 61 f is very weak, the performance of cbh1 is exoglucanases activity),the fused enzyme activity is about 1.5 times higher than that of cbh1.