里氏木霉纤维素酶系的分离及其酶学性质

Purification and Characterization of Cellulase from Trichoderma reesei

采用两级超滤、DEAE-Sepharose FF阴离子交换层析、CM-Sepharose FF阳离子交换层析和Seph-adex G-100凝胶渗透层析等分级纯化步骤,从里氏木霉纤维素酶系中分离纯化得到电泳纯的3个内切葡聚糖酶组分EGⅠ、EGⅡ和EGⅢ,2个外切葡聚糖酶组分CBHⅠ和CBHⅡ和1个β-葡萄糖苷酶组分,它们对各自底物的比活力分别为176.35、153.96、64.22、16.86、4.82、31.00 IU/mg,米氏常数分别为6.70、8.46、13.22、1.37、3.46、2.20 mg/mL。同一类酶组分的米氏常数Km越大,转换数Kcat越小。分离纯化所得EGⅠ、EGⅡ、EGⅢ、CBHⅠ、CBHⅡ和GB等酶组分的分子量分别为50、46、25、65、58、75 kDa。

The purification steps, e.g. a two-step ultrafiltration, cationic （CM-Sepharose FF） and anionic （DEAE-Sepharose FF） chromatography and size exclusion （Sephadex G- 100） chromatography and the six homogeneous cellulase etc, were used to purify three endoglucanases （EG Ⅰ , EGⅡ and EGⅢ ）, two cellohiohydrolases （CBH Ⅰ and CBH Ⅱ ） and a β-glucosidase （GB） according to SDS--PAGE analysis from the culture broth of Trichoderma reesei. The specific activities of EG Ⅰ , EG Ⅱ and EG m with carhoxymethyl celluloses were estimated to he 176. 35, 153.96, 64.22 IU/mg, respectively. For CBH Ⅰ and CBH Ⅱ , their specific activities with microcelluloses were 16.86, 4. 82 IU/mg, respectively. The GB with salicin was 31.00 IU/mg. Their Km were 6.70, 8.46, 13.22, 1.37, 3.46, 2.20 mg/mL, respectively. The greater Km of the purified enzyme component of the same class is, the smaller their turnover is. Their molecular weights of EG Ⅰ , EGⅡ , EGⅢ, CBH Ⅰ , CBHⅡ and GB were 50, 46, 25, 65, 58, 75kDa, respectively.