Purification And Properties Of β-glucosidase From Chaetomium Thermophilum And Cloning Of The Encoding Gene

β-glucosidases (β-D-glucoside glucohydrolases; EC 3.2.1.21) occur ubiquitously in plants, mammals and microorganisms. β-glucosidases have been the subject of much recent research due to the key role these enzymes play in biological processes and potential biotechnological applications. Microbial β-glucosidases are mainly applied to the hydrolyzation of cellulose and the flavor improvement industry.Chaetomium thermophilum, a kind of the thermophilic fungi, is a metatrophic fungus. It can be thrive at the temperature between 45℃ and 50℃ while most fungi will die above 40 ℃ soonly. Only a few reports have been published about β-glucosidases from thermophilic fungi. Chu Chunxue et al. have purified one β-glucosidase from C. thermophilum, and the molecular weight of the enzyme is 80kDa.In the first part of the article, β-glucosidase from culture supernatant of C. thermophilum was purified to homogeneity, by using ammonium sulfate fraction, DEAE-sepharose fast flow chromatography, phenyl-sepharose fast flow chromatography and sephacryl S-100 chromatography, and its properties were studied. The molecular mass of the enzyme was about from 118kDa to 120kDa, as identified by 10% SDS-PAGE and gel filtration correspondingly. Its optimum pH value and temperature were 4.0～5.0 and 70℃ respectively. It was stable in pH 4.0～11.0 and under 60℃. Different metal ions showed different effects on the β-glucosidase activity. Ca²⁺ and Ba²⁺ enhanced its activity, while Zn²⁺, Cu²⁺, Al³⁺, Ag⁺ and Hg²⁺ caused obvious inhibition.In the second part of the article, primers were designed based on the conserved amino acid sequences of 8 different kinds of fungus and a 1872bp cDNA fragment bgl encoding the gene of C. thermophilum β-glucosidase was obtained through RT-PCR and 3'-RACE. The cDNA of bgl contained a region of 1770bp encoding 589 amino acids, terminal codon TGA and polyA.