Purification And Characterization Of Cellulases From The Fungus Trichoderma Reesei

Using ultrafiltration, cationic (CM-Sepharose FF) and anionic (DEAE-Sepharose FF) chromatography and size exclusion (Sephadex G-100), six cellulolytic enzymes were purified from the culture broth of Trichoderma reesei : three endoglucanases (EGⅠ,EGⅡ,EGⅢ), two cellobiohydrolases (CBHⅠ,CBHⅡ) and aβ-gulcosidase (GB). The six cellulases displayed one single band on SDS-PAGE respectively. The specific activity of EGⅠ,EGⅡand EGⅢwith carboxymethyl cellulose was estimated to be 176.35 IU·mg^-1,153.96 IU·mg^-1,64.22 IU·mg-1 respectively. For CBHⅠand CBHⅡ, the specific activity was 16.86 IU·mg-1,4.82 IU·mg-1 respectively with microcellulose. The specific activity of theβ-gulcosidase with salicin was 31.00 IU·mg^-1.Basic enzymatic characteristic of obtained cellulases were studied. The relative molecular weight of EGⅠ, EGⅡ, EGⅢ, CBHⅠ, CBHⅡand GB was 50 kD, 46 kD, 25 kD, 65 kD, 58.1 kD and 75kD, respectively. Kinetics method showed the Km of EGⅠ,EGⅡ,EGⅢfor carboxymethyl cellulose was 6.70 mg/mL, 8.46 mg/mL, 13.22 mg/mL respectively. For CBHⅠand CBHⅡ, the Km with microcellulose was 1.37 mg/mL and 3.46 mg/mL respectively. The Km ofβ-gulcosidase with salicin was 2.20 mg/mL. For EGⅠ, EGⅡ, EGⅢ, CBHⅠ, CBHⅡand GB, the optimum temperture was 55℃, 50℃, 55℃, 60℃, 45℃and 60℃respectively, while their optimum pH at 5.6, 4.4, 5.0, 5.6, 5.0 and 5.0.Besides, the cellulases had the relatively good stability under 30℃, while in the temperture upon 80℃, they lost their activity quickly. The cellulases were relatively stable within pH 5.0～6.2. EGⅠ和CBHⅠhad the stronger stability, which had strong activity after being kept at pH 6.8 for 5.0h.