Purification, Characterization, Gene Cloning And Expression Of Thermostable Cellulases From Thermophilic Fungi

Cellulose is by far the most abundant carbohydrate available on earth, and can be converted into glucose by cellulases.Cellulases have been attracted increasing attention due to their application potential in the field such as bioconversions of cellulose waste, textile industry ,paper and pulp industries, deetergent industry protoplasts generation from yeast and filametous fungi and so on. However, there have several limits, for example, difficult degradation ,high cost and environment pollution, on hydrolysis cellulose by chemical methods. In contrast, biodegradation process not only increase utilization ratio but also gain greater benefit.Cellulose may be degradated by many kinds of micrograms, such as fungi, bacteria and so on. In the process of degradation, there is an important enzyme named 1,4-β-D-glucan cellobiohydrolase, which belongs to cellulase family. The cellobiohydrolase hydrolyse cellulose chains by removing cellobiose either from the nonreducing ends, which results in rapid release of reducing sugars but little change in polymer length.β-glucosidase is another cellulase, which can cleave cellobiose to glucose.Cellulases from the themophilic fungi have reported to be stable at high temperature and like mescophilic fungi, the thermophilic fungi, produce multiple forms of the cellalase components.Chaetomium thermophilum CT2 and Talaromyces ermersonii are both thermophilic fungi, can thrive at temperature between 45 and 50℃,while most fungi will die above 40℃. C.thermophilum CT2 and Talaromyces ermersonii are shown to produce themostable enzymes such as protease, glucomyces, lipoxygenase, cellulases, xylanases and so on. Cellulases from the fungi have been studied. In the present study, two new extracellular exoglucanase from C. thermophilum CT2 and two new extracellular exoglucanase from Talaromyces ermersonii are produced when they were grown in minimal liquid medium containing Avicel as the only carbon sourse. The enzymes were purified from the culture filtrate of the strain by fractional ammonium sulphate precipitation, DEAE-Sepharose chromatography and Sephacryl S-100 chromatography. The purified enzymes were shown to be homogeneous by SDS-PAGE. The molecular weight of two exoglucanase from C. thermophilum CT2 were estimated to be 66.3kDa and 180.3 kDa. a newβ-glucosidase from C. thermophilum CT2 were estimated to be 118kDa. The molecular weight of two exoglucanase from Talaromyces ermersonii were estimated to be 70kDa and 72.2 kDa. Then characterization of the purified enzymes were carried out.Degenerate primers based on existed cellobiohydrolases present in the databases. A 1356-length cDNA fragment encoding the CBH gene was obtained through RT-PCR. The RACE-PCR was manipulated to generate full-length cDNA clones. The gene has been registered in GenBank with accession number DQ085790. Then partial genomic DNA of CBH gene was cloned and the length was 1507bp with three introns. The gene has been registered in GenBank with accession number EF222284. The cbh3 gene and expression vector pPIC9K were both digested with EcoRⅠand NotⅠ, then ligationed in vitro to construct the expression plasmid pPIC9K/cbh. The expression plasmid was transformed to Pichia pastoris GS115 competent cell after linearized with restriction enzyme SacI. The recombinant Pichia pastoris GS-CBH was got and the expressed cellobiohydrolase was characterized .