纤维素酶系中内切β-葡聚糖苷酶的分离纯化及酶学性质的研究
摘要
利用本实验室筛选的绿色木霉MJ1固体发酵产物，采用硫酸铵分级沉淀、SephadexG-100凝胶柱层析、DEAE FF弱阴离子交换柱层析(快速蛋白液相色谱系统(FPLC))一系列分离纯化技术分离纯化出一单一组分一内切β-葡聚糖苷酶，经SDS-PAGE电泳分析表明该内切酶的相对分子量约为64.7KD。
分离纯化出的内切β-葡聚糖苷酶的比活力提高了28.6倍，回收率为19.7％。酶学试验研究表明：该酶的最适反应温度53℃，最适pH为4.2，Lineweaver-Burk法求得动力学参数，Km和Vmax分别为1.230×10~(-2)g／mL、2.396×10~(-2)mg／(mL·min)。
在一系列假设基础上提出绿色木霉产纤维素酶水解CMC-Na的反应机理，对“三状态”动力学模型进行改进，建立了pH值对纤维素酶活力影响的动力学模型，并推导出反应速率表达式。L-B作图法求得木霉产纤维素酶水解CMC-Na在不同pH的动力学参数：Km′和Vmax′，试验得到最佳初始pH值与模型计算得的最佳pH值结果相符合，从而模型得以验证。
An endo-β-glucanase was isolated and purified from the leavening of Trichoderma viride called after MJ1 by means of ammonium sulfate and Sephadex G-100 chromatography, and further fractionated by FPLC (AKTA UPC-900) respectively.
The specific activity of the endoglucanase from MJ1 increased 28.6-fold, and coefficient of recovery was 19.7%. The molecular weight was 64.7KD determined by SDS-PAGE. The optimal reaction temperature of the endoglucanase was 53℃, and the optimal pH was 4.2. The values of Km and Vmax calculated from Lineweaver-Burk plots were 1.230×10~(-2)g/mL and 2.396×10~(-2) mg/(mL · min) respectively.
The reaction mechanism of carboxymethyl cellulose sodium(CMC-Na) hydrolyzed by cellulase from Trichoderma viride was brought forward on a series of assumption.The kinetic model was established by the influence of pH on endo-β-glucanase activity and the expression of reaction rate was educed, improving upon the "three-state" enzyme catalysis kinetic model. By changing pH of the buffer solution, a series of Km'and Vmax' of CMC-Na hydrolyzed by cellulase of Trichoderma viride were calculated from the Lineweaver-Burk plot. The optimal initial pH value gained by the kinetic model was consistent with data got from experiment. Consequently the kinetic model was validated.
分离纯化出的内切β-葡聚糖苷酶的比活力提高了28.6倍，回收率为19.7％。酶学试验研究表明：该酶的最适反应温度53℃，最适pH为4.2，Lineweaver-Burk法求得动力学参数，Km和Vmax分别为1.230×10~(-2)g／mL、2.396×10~(-2)mg／(mL·min)。
在一系列假设基础上提出绿色木霉产纤维素酶水解CMC-Na的反应机理，对“三状态”动力学模型进行改进，建立了pH值对纤维素酶活力影响的动力学模型，并推导出反应速率表达式。L-B作图法求得木霉产纤维素酶水解CMC-Na在不同pH的动力学参数：Km′和Vmax′，试验得到最佳初始pH值与模型计算得的最佳pH值结果相符合，从而模型得以验证。
An endo-β-glucanase was isolated and purified from the leavening of Trichoderma viride called after MJ1 by means of ammonium sulfate and Sephadex G-100 chromatography, and further fractionated by FPLC (AKTA UPC-900) respectively.
The specific activity of the endoglucanase from MJ1 increased 28.6-fold, and coefficient of recovery was 19.7%. The molecular weight was 64.7KD determined by SDS-PAGE. The optimal reaction temperature of the endoglucanase was 53℃, and the optimal pH was 4.2. The values of Km and Vmax calculated from Lineweaver-Burk plots were 1.230×10~(-2)g/mL and 2.396×10~(-2) mg/(mL · min) respectively.
The reaction mechanism of carboxymethyl cellulose sodium(CMC-Na) hydrolyzed by cellulase from Trichoderma viride was brought forward on a series of assumption.The kinetic model was established by the influence of pH on endo-β-glucanase activity and the expression of reaction rate was educed, improving upon the "three-state" enzyme catalysis kinetic model. By changing pH of the buffer solution, a series of Km'and Vmax' of CMC-Na hydrolyzed by cellulase of Trichoderma viride were calculated from the Lineweaver-Burk plot. The optimal initial pH value gained by the kinetic model was consistent with data got from experiment. Consequently the kinetic model was validated.
引文
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[9] 李廷生，赵江，王平诸．果胶酶与纤维素酶复合制剂的研制．郑州工程学院学报．2000，21(4)：38～40
[10] 葛文中，李楠．绿色木霉应用的研究进展．黑龙江八一农垦大学学报．2005，17(2)：76
[11] 冯镖．工业纤维素酶的测定和比较．无锡轻工业学院学报．1994，13(4)：323～330
[12] 张树政主编．酶制剂工业．北京：科学出版社，1998：595
[13] 陈洪章．纤维素生物技术．北京：化学工业出版社，2005，50～52
[14] 汪维云等．纤维素科学及纤维素酶的研究进展．江苏理工大学学报．1998，19(3)：2027
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