【摘要】 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。更多还原

【Abstract】 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(EGⅠ,EGⅡ and EGⅢ),two cellobiohydrolases(CBHⅠand CBHⅡ) and a β—glucosidase(GB) according to SDS-PAGE analysis from the culture broth of Trichoderma reesei.The specific activities of EGⅠ,EGⅡ and EGⅢ with carboxymethyl celluloses were estimated to be 176.35,153.96,64.22 IU/mg,respectively.For CBHⅠand CBHⅡ,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 EGⅠ,EGⅡ,EGⅢ,CBHⅠ,CBHⅡ and GB were 50,46,25,65,58,75 kDa,respectively.更多还原