Molecular Cloning of Cellulase Genes from Trichoderma harzianum

Abstract

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 βglucosidase (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, 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. INTRODUCTION Despite the fact that the world community is no longer pre-occupied with fossil fuel shortage, there is still a considerable research and development directed towards understanding and commercializing enzymatic hydrolysis of cellulose [1]. Cellulose is the most abundant organic polymer in this planet and is an important renewable energy source along with sugars and starches [2]. Energy production from cellulosic raw material involves its hydrolysis into glucose. Highly ordered cellulose substrates are converted into soluble sugars only when exoglucanase, endoglucanase and β-glucosidase are present in solution simultaneously in right proportion. A number of fungal species are known for the production of cellulases such as Aspergillus niger, Sporotrichum spp, Chaetomium thermophile, Trichoderma species etc [3]. Gene cloning is recently being employed for studying the structure and function of a number of enzymes and proteins and their over expression. This strategy has been found very efficient as compared to other traditional methods. Present study was designed to layout strategy for enhanced production of cellulases. In this paper we have reported the isolation and cloning of cellulase genes (exg ,egl and bgl) from T.harzianum (E-58 strain). MATERIALS AND METHODS Growth Conditions and Enzyme Assay Trichoderma harzianum (E58) was grown at 28C with shaking at 120 rpm in Vogel's medium (0.5% Trisodium citrate, 0.5% KH2PO4.0.2% NH4NO3, 0.4% (NH4)2SO4, 0.02%