Molecular Cloning of Cellulase Genes from Trichoderma harzianum

Cellulases are attractive source for utilization of the agro-industrial waste materials. Exoglucanase (EC; 3.2.1.91), Endoglucanase (EC. 3.2.1.4) and β- glucosidase (EC. 3.2.1.21) were isolated from Trichoderma harzianum (E-58 strain). The fungus was grown on Vogel's medium with different carbon sources. Maximal production of the enzymes was achieved at 28 °C, pH 5.5 under continuous shaking at 120 rpm for 5 days. Glucose repressed the synthesis of the enzymes whereas carboxymethylcellulose (CMC) produced the enzymes in substantial amounts. Maximum activity of cellulases (exoglucanase, endoglucanase and β-glucosidase) were found to be 2.764, 14.4 and 0.629IU mL-1, respectively. Corresponding genes for cellulases were isolated with the help of RTPCR. RNA was isolated from mycelia of T. harzianum grown on CMC and xylan. First strand of cDNA was synthesized using oligo dT (18) primer and subjected to PCR with specific primers. The amplified products were purified through agarose gel electrophoresis and ligated into SmaI site of pUC18. The plasmids containing exg, egl and bgl genes were transformed into E. coli for further characterization.