Isolation and purification of an endoglucanase from Trichoderma reesei M(7)

Crude cellulolytic enzyme preparation was isolated from the culture supernatant of Trichoderma reesei M(7) by ammonium sulfate precipitation (80% saturation). An endo-1,4-beta-D-glucanase (EC 3.2.1.4) from the multicomponent cellulolytic complex of Trichoderma reesi M(7) was purified for further investigations and biotechnological applications. The endoglucanase active fraction F-II-III-II was isolated in a molecular homogeneous form by a simple three-step chromatographic procedure on Sephadex G-75, DEAE Sephadex A-50 and PBE 94 columns. The homogeneity of the purified enzyme was assessed by gel filtration chromatography on Superose 12 column, SDS-PAGE and analytical isoelectric focusing. The optimum pH and temperature values were estimated to be 5.0 and 60 degrees C (for the crude cellulolytic preparation) and 6.0 and 60 degrees C (for the purified endoglucanase), respectively. The homogenous protein correspond to a relative molecular mass and pI value of 51 kDa and 5.82, respectively. The predominant products of the enzymatic hydrolysis of cellulosic substrates by crude enzyme preparation were identified as glucose, xylose and cellobiose. The biotechnological potential of the purified endoglucanase preparation will be investigated further.