[Purification and characterization of a cellobiohydrolase from the thermophilic fungus Chaetomium thermophilus CT2].
Chaetomium thermophilus CT2 was a cellulolytic fungus. It was a widely-existing saprophyte, which grower rapidly in soil. The cellulases synthesized by C. thermophilus CT2 was overall, consisting of three principal types of enzymes. The cellobiohydrolase was one of these three cellulases, which was associated with the endo-beta-1,4-glucanase and beta-glucosidase activities. C. thermophilus CT2 produced cellobiohydrolase available at 50 degrees C, when grown on ferment liquid substrate, containing 1% Avicel, 0.14% (NH4 )2SO4, 0.2% KH2PO4, 0.03% CaC2 x 2H2O, 0.03% MnSO4 x 7H2O, 0.1% peptone, 0.05% yeast extract, 0.1% Tween 80 and trace element solution at 1mL/L, containing 18mmol/L FeSO4 x 7H2O, 6.6mmol/L MnSO4, 4.8mmol/L ZnSO4 x 7H2O and 15mmol/L COCl2. A cellobiohydrolase was purified to homogeneity by an inexpensive and straightforward method for extraction of the enzyme involving fractional ammonium sulphate precipitation, ion-exchange chromatography on DEAE-Sepharose Fast Flow, gel filtration on Sephacryl S-100 and ion-exchange chromatography on Q Sepharose Fast Flow. The molecular weight of the enzyme was estimated to be 66.3kDa by 12.5% SDS-PAGE and was to be 67.1kDa by gel filtration on Sephacryl S-100 respectively. Kinetic studies of the purified cellobiohydrolase of C. thermophilus CT2 showed that the Km for p-NPC (p-trophenylbeta-d-cellobioside) was 0.956mmol/L as determined from a Lineweaver-Bark plot. Optimum enzyme activity was at 65 degrees C and pH5.0. It was thermostable at 60 degrees C and remained 20% activity after 20min at 80 degrees C. The half life time of the enzyme at 70 degrees C was 1h. It indicated that the cellobiohydrolase possessed of excellent acid stability and thermostable property. The properties of the cellobiohydrolase make it possible to be good material in scientific researches of protein thermostable mechanism and good model for designing and constructing a new type protein in industry. The enzyme may also provide instructive insight on the diversity and mechanism of cellulose degradation by C. thermophilus CT2. As a thermophilic fungus C. thermophilus CT2 is an attractive potential source of cellulases. It indicates that C. thermophilus CT2 may be a new excellent industrialized fungus for producing cellulases through molecule biology means.
Li
Ya-ling
YL
Department of Environmental Biology, Shandong Agricultural University, Tai'an 271018, China. yalingli@163.com
Li
Duo-chuan
DC
Teng
Fang-chao
FC
D043366:Cellulose 1,4-beta-Cellobiosidase / Q000737:chemistry* / Q000302:isolation & purification*; D002597:Chaetomium / Q000201:enzymology*; D005656:Fungal Proteins / Q000737:chemistry / Q000302:isolation & purification
D005656:Fungal Proteins; D043366:Cellulose 1,4-beta-Cellobiosidase