Identification and purification of the main components of cellulases from a mutant strain of Trichoderma viride T 100-14.
A new mutant strain of fungus Trichoderma viride T 100-14 was cultivated on 1% microcrystalline cellulose (Avicel) for 120h and the resulting culture filtrate was prepared for protein identification and purification. To identify the predominant catalytic components, cellulases were separated by an adapted two-dimensional electrophoresis technique. The apparent major spots were identified by high performance liquid chromatography electrospray ionization mass (HPLC-ESI-MS). Seven of the components were previously known, i.e., the endoglucanases Cel7B (EG I), Cel12A (EG III), Cel61A (EG IV), the cellobiohydrolases Cel7A (CBH I), Cel6A (CBH II), Cel6B (CBH IIb) and the beta-glucosidase. The seven major components in the fermentation broth of T. viride T 100-14 probably constitute the essential enzymes for crystalline cellulose hydrolysis and they were further purified to electrophoretic homogeneity by a series of chromatography column. Hydrolysis studies of the purified elements revealed that three of the cellulases were classified as cellobiohydrolases due to their main activities on p-nitrophenyl-beta-d-cellobioside (pNPC). Three of the cellulases, with the abilities of hydrolyzing both carboxymethyl-cellulose (CMC) and Avicel indicate their endoglucanase activities. It deserved noting that the beta-glucosidase from the T 100-14 displayed an extremely high activity on p-nitrophenyl-beta-D-glycopyranoside (pNPG), which suggested it was a good candidate for the conversion of cellobiose to glucose.
Zhou
Jin
J
State Key Laboratory of Bioreactor Engineering, National Research Center for Biotechnology (Shanghai), East China University of Science and Technology, Shanghai 200237, PR China.
Wang
Yong-Hong
YH
Chu
Ju
J
Zhuang
Ying-Ping
YP
Zhang
Si-Liang
SL
Yin
Peng
P
D044602:Cellulases / Q000302:isolation & purification*; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D009154:Mutation; D021241:Spectrometry, Mass, Electrospray Ionization; D014242:Trichoderma / Q000201:enzymology* / Q000235:genetics
D044602:Cellulases