Cloning a novel endo-1,4-β-d-glucanase gene from Trichoderma virens and heterologous expression in E. coli

Rong Zeng, Qiao Hu, Xiao-Yan Yin, Hao Huang, Jia-Bao Yan, Zhi-Wei Gong, Zhong-Hua Yang

Endo-1,4-β-D-glucanase (EG), as a key constituent of cellulase taking the responsibility of cutting β-1,4 glycosidic bonds, plays the essential role in the process of degrading cellulose by cellulase. Cloning and expressing the EG gene is important to the cellulase research and application. In this work, a novel EG gene was cloned from Trichoderma virens ZY-01, which was a cellulase secreting microbe isolated by our laboratory. The DNA sequence showed that the length of the cloned EG is 1069 bp, which had 95.2% similarity to the EG IV from T. viride AS 3.3711. Further, the expression vector pET-32a-EG was constructed and was successfully heterologously expressed in Escherichia coli. The expression product was purified with Ni(2+) affinity chromatography and its enzymatic properties were investigated. The SDS-PAGE showed the target protein is 39 kDa, which is consistent with the translated result from the DNA sequence. The kinetic parameter for the expression product was Km = 13.71 mg/mL and Vmax=0.51 μmol/min·mL. The optimal reaction pH and temperature was pH = 7.0 and T = 40 °C, which is similar to the native EG produced by Trichoderma virens ZY-01. It provides the foundation for the endo-1,4-β-D-glucanase further evolution and application.