Purification And Characterization Of Cellulases From The Fungus Trichoderma Viride

Four endoglucanases (EG1, EG2, EG3 and EG4) were isolated and purified from leavening of Trichoderma viride M1 by a combination of DEAE-650C anionic chromatography, CM-650M cationic chromatography, Sephacryl S-200 gel chromatography and Octyl Sepharose CL-4B hydrophobic chromatography. Four endoglucanases displayed one single band on SDS-PAGE respectively.It indicates that better separation and purification was achieved, the specific activity of EG1, EG2, EG3 and EG4 with carboxymethyl cellulose was 41.83 U/mg, 139.96 U/mg,117.72 U/mg and 126.50U/mg respectively, and the recovery coefficient of EG1, EG2, EG3 and EG4 with carboxymethyl cellulose was 8.36%, 11.31%,14.72% and 24.31%, and the specific activity of those four endoglucanases increased 7.04-fold,23.56-fold,19.82-fold and 21.30-fold, respectively.The enzymatic characterizations of obtained endoglucanases were studied. For EG1, EG2, EG3 and EG4, their relative molecular weight were 25.4 kDa,28.8 kDa, 56.3kDa and 60.5 kDa, respectively, and the optimum temperture were 50℃,45℃,55℃ and 60℃ respectively, while those endoglucanases had their optimum activity at pH 6.0,5.0,4.0 and 5.0. The values of Km for the four endoglucanases which calculated from Lineweaver-Burk plots were 9.51mg/mL,5.06 mg/mL,4.16 mg/mL and 4.86 mg/mL.Temperature and pH stability studies show that the EG1, EG3 and EG4 had the relatively good stability during 30℃～50℃, but the stability decreased when the temperature exceeds 60℃.In terms of the EG2, it lost its activity quickly when the temperature upon 40℃.The cellulases were relatively stable within pH 4.0～6.0, it was also found that the EG3 had the poor pH stability.Several metal ions on endoglucanases activity have been studied, the activity of EG1, EG2 and EG3 were stimulated by 5mmol/L Ca2+, and inhibited by 5mmol/L Zn2+, Mg2+ and K+, but the activity of EG4 was stimulated by 5mmol/L Zn2+, Mg2+ and K+. Analysis of substrate specificity learned that four endoglucanases preferred CMC of lower degree of polymerization; furthermore, EG2 also had activity on Xylan and EG4 had the highest ability to hydrolyze Avicel and cellulose powder. Both of EG1 and EG4 showed affinity for Avicel indicating the presence of the cellulose binding domain (CBD),while the EG2 and EG3 were found to lack CBD which were capable of degrading only the soluble forms of cellulose.