Purification, Characterization And Gene Cloning Of Cellulase From Thermomyces Lanuginosus

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 potential application in the field such as bioconversions of cellulose waste, textile industry, paper and pulp industry, detergent industry ,protoplasts generation from yeast and filamentous fungi and so on. However, there are several limits in hydrolysis cellulose by chemical methods, such as high cost degradation and environment pollution. In contrast, the biodegradation process could not only increase utilization ratio but also gain greater benefit. Cellulase from the themophilic fungi have been reported to be stable at high temperature and like mescophilic fungi, the thermophilic fungi, produce multiple forms of the cellulase components.Many organisms are capable of producing cellulase whom were screened and isolated. But commercial cellulase from mesophilic organisms are limited on agricultural and industrial application due to their high cost, low thermostability, shorter shelf-life and lower activity. It is implied that research on thermostable cellulase is of importance in industrial exploitation. Thermomyces lanuginosus is a thermophilic fungus with higher growth temperature, and is shown to produce thermostable protease, glucomylase, lipoxygenase and lipase. However, cellulase from the genus Thermomyces lanuginosus has not yet been studied. In the present study, an extracellular cellulase from Thermomyces lanuginosus was produced when Thermomyces lanuginosus was grown in minimal liquid medium containing Avicel as the only carbon source. The enzyme was purified from the culture filtrate of the strain by fractional ammonium sulphate precipitation, DEAE-Sepharose chromatography and Pheny1-Sepharose chromatography. The two purified enzymes were shown to be homogeneous by SDS-PAGE, and the molecular weights were estimated to be 46.6kDa to 47kDa (cellobiohydrolase) and 118kDa to 120kDa (β-glucosidase), as identified by 12.5% SDS-PAGE and gel filtration correspondingly.The cellobiohydrolase exhibited optimum catalytic activity at pH 5.0 and 60℃. The half life time of the enzyme at 70℃was 35min. Different metal ions showed different effects on the cellobiohydrolase activity. Ag+, NH4+, Zn²⁺ cause obvious inhibition, while Na+, Mn²⁺, K+ cause some inhibition on it, and Ca²⁺ , Ba²⁺, Mg²⁺ enhanced the enzyme activity. The Vmax and Km values of cellobiohydrolase on Avicel were 22.12 mg/mL.min and 9.56 mg/mL respectively.Theβ-glucosidase exhibited optimum catalytic activity at pH 5.0 and 60℃. The half life time of the enzyme at 70℃was15 min. Different metal ions showed different effects on theβ-glucosidase activity. Na+, Mn²⁺, K+ enhanced the enzyme activity, and Ag+, NH4+, Zn²⁺ cause obvious inhibition, while Ca²⁺ , Ba²⁺, Mg²⁺ cause some inhibition on it. The Vmax and Km values ofβ-glucosidase on Avicel were 11.94 mg/ml.min and 1.98 mg/ml respectively.To clone cellulase gene from Thermomyces lanuginosus, degenerate primers were designed based on the conserved amino acid sequences of cellobiohydrolase reported from 12 different kinds of fungi . The cDNA fragment of Thermomyces lanuginosus cellobiohydrolase was obtained through RT-PCR. The RACE was used to generate full-length cDNA of the cellobiohydrolase. The full length of cbh gene is 1710bp which encoding 457 amino acids. The signal sequence of deduced cbh amino acid sequence was analysed by SignalP-NN and SignalP-HNN on the Signal.P server. The result is that the cbh gene has a signal site (amino acids 1-17) with the sequence of MYQRALLFSFFLAAARA, showing the protein was a kind of secreted protein.