黄绿木霉纤维素酶提纯及其性质研究  

Purification and characteristics of celluloses from trichoderma aureoviride

作　　者：宋金柱[1] 杨谦[1] 陈中祥[1] 张晶[1] 

机构地区：[1]哈尔滨工业大学生命科学与工程系,哈尔滨150001

出　　处：《哈尔滨工业大学学报》2006年第11期1923-1926,共4页Journal of Harbin Institute of Technology

基　　金：国家自然科学基金资助项目(50178021)

摘　　要：通过(NH4)2SO4分级沉淀、Sephadex G-100分子筛层析和DEAE Sephadex A-50离子交换层析等步骤,分离纯化出木霉T2纤维素酶系中达到电泳纯的3种内切葡聚糖酶(EGⅠ、EGⅡ、EGⅢ)和2种β-葡萄糖苷酶(BGⅠ、BGⅡ).通过SDS-PAGE和IEF电泳测得5个酶组分的相对分子质量分别为62.3 ku、71.9 ku、52.6 ku、85.3 ku和78.3 ku,等电点分别为5.4、4.8、5.0、5.6和5.8.此酶系均为糖蛋白,含糖量分别为17.7%、11.7%、15.6%、17.8%、19.3%.5个酶组分均属酸性纤维素酶,最适pH都是5.0;3种内切葡聚糖酶的最适温度皆为60℃,2种β-葡萄糖苷酶的最适温度都是55℃.Mn2+、Fe2+、Zn2+对5种纤维素酶都有一定的激活作用,尤其是Mn2+对内切葡聚糖酶的激活作用极为显著;Mg2+、Cu2+、脲对5种纤维素酶都具有不同程度的抑制作用.酶学动力学分析表明5种纤维素酶组分的Km值分别为0.093、0.083 8、0.079 0、0.085 1和0.077 8 mg/mL,Kcat值为101.7、119.6、11.4、111.4和162.1 s-1.内切葡聚糖酶对底物CMC-Na亲和力的大小与酶的催化效率之间并无相关性,BG对底物水杨苷亲和力的大小与酶的催化效率之间有一定的相关性.β-葡萄糖苷酶有较高的底物专一性,只对水杨苷有活力;内切葡聚糖酶在对羧甲基纤维素钠有活力的同时,对滤纸和秸秆粉也有微弱的活力.Three endoglucanases and two β -glucosidases from the fungus were separated and purified to electrophoretic homogeneous by （NH4 ）2SO4 gradient precipitation, Sephadex G- 100 gel chromatography and DEAE Sephadex A -50 ion exchange chromatography. They were designated to be EG Ⅰ, EG Ⅱ , EG In, BG Ⅰ and BG Ⅱ. Five components had molecular weights of 62. 3 ku, 71.9 ku, 52.6 ku, 85.3 ku and 78.3 ku by SDS - PAGE, respectively, and IEF showed their isoelectric point were 5.4, 4. 8, 5.0, 5.6, and 5.8, respectively. All of them had maxium reactive activities at pH 5. The optimum temperature of endoglucanases is 60 ℃, and the optimum temperature of β - glucosidases is 55 ℃. The activities of all components were stimulated by Mn^2＋ , Fe^2＋ , Zn^2＋ , and especially, EG were mostly activated by Mn^2＋ , while the activities of all components were inhibited by Mg^2＋ , Cu^2＋ , carbamide. The kinetics showed the Km of five components were 0.093 mg/ml, 0. 083 8 mg/ml, 0. 079 0 mg/ml, 0. 085 1 mg/ml and 0.077 8 mg/ml, respectively. Kcat were 101.7 s^-1 ,119. 6 s^-1, 11.4 s^-1, 111.4 s^-1 and 162. 1 s^-1, respectively. There was no relativies between the catalytic efficiency of endoglucanases and its Km, however, there was correlation between the catalytic efficiency of β - glucosidases and its Km. Substrate specificity of the five components measurement indicated that BG Ⅰ and BG Ⅱ could only active Salicin, yet EG Ⅰ , EG Ⅱ and EG Ⅲ could hydrolyse not only CMC -Na,but also filter paper and straw powder faintly.

关 键 词：木霉T2 纤维素酶 提纯 性质 

分 类 号：Q539.3[生物学—生物化学]