Abstract
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 Ni2+ 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s13568-016-0282-0) contains supplementary material, which is available to authorized users.
Highlights
Cellulose, as a kind of renewable bioresource, is the most abundant biomass in nature
Cloning and sequence analysis of EG gene from T. virens ZY‐01 The total RNA was extracted from the T. virens ZY-01 spores with RNAprep Pure Plant Kit after lysis the spores in liquid nitrogen
The RNA sample was detected with agarose gel electrophoresis, and the results indicated that the total RNA was pure and intact
Summary
As a kind of renewable bioresource, is the most abundant biomass in nature. The output of cellulose and hemicellulose is over 75 billion ton each year (Fang and Xia 2015a; Yücel and Aksu 2015). Hydrolysis of cellulose and hemicellulose to fermentable sugars is an economical and promising route for cellulose biomass utilization. Cellulase plays the key role in the route of cellulose utilization with biological technology (Fang and Xia 2015a). It is helpful to solve energy crisis, food shortage and environmental pollution. Endo-1,4-β-d-glucanase (or endoglucanase, EG) is the major constituent of cellulase, which catalyzes the hydrolysis of the 1,4-β-d-glycosidic linkages in cellulose, hemicellulose, lichenin, and cereal β-d-glucans
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