绿色木霉内切葡聚糖酶的分离纯化及酶学性质研究

Purification and Characterization of Endoglucanases from Trichoderma viride

采用DEAE-650C弱阴离子交换层析、CM-650M弱阳离子交换层析、Sephacryl S-200凝胶层析和Octyl Sepharose CL-4B疏水层析等分离纯化技术,从绿色木霉M1发酵液中分离纯化得到4个电泳纯的内切葡聚糖酶组分（EG1、EG2、EG3和EG4）,比活力分别为41.83,139.96,117.72,126.50 U/mg,分子质量分别为25.4,28.8,56.3,60.5 ku,最适反应温度分别为50,45,55,60℃,最适反应p H分别为6.0,5.0,4.0,5.0,米氏常数Km值分别为9.51,5.06,4.16,4.86 mg/mL。组分EG1、EG3和EG4在30-50℃范围较稳定,组分EG1、EG2和EG4在p H4.0-6.0范围稳定性较好,而组分EG3只在p H5.0的条件下较稳定。Zn^2＋、Mg^2＋和K^＋对组分EG4有激活作用,对组分EG1、EG2和EG3有抑制作用,Ca^2＋对组分EG1、EG2和EG3有激活作用。

Four endoglucanases（EG1, EG2, EG3 and EG4） were isolated and purified to electrophoretic homogeneity from leavening of Trichoderma viride M1 by a combination of DEAE-650 C anion-exchange chromatography, CM-650 M cationic-exchange chromatography, Sephacryl S-200 gel chromatography and Octyl Sepharose CL-4B hydrophobic chromatography. The specific activity of EG1, EG2, EG3 and EG4 with carboxymethyl cellulose were 41.83, 139.96, 117.72 and 126.50 U/mg respectively. For EG1, EG2, EG3 and EG4, the relative molecular weight were 25.4, 28.8, 56.3,60.5 ku, respectively. And the optimum temperture were 50, 45, 55, 60 ℃ respectively, while those endoglucanases had their optimum activity at p H 6.0, 5.0, 4.0 and 5.0. The values of Kmfor the four endoglucanases were 9.51, 5.06,4.16, 4.86 mg/mL severally. The EG1, EG3 and EG4 had the relatively good stability during 30-50℃. The EG1, EG2 and EG4 were relatively stable within the p H range of 4.0-6.0. The activity of EG1, EG2 and EG3 were stimulated by5mmol/L Ca^2＋, and inhibited by 5 mmol/L Zn2＋, Mg2＋and K＋, but the activity of EG4 was stimulated by 5 mmol/L Zn^2＋,Mg^2＋and K^＋.