黑曲霉Ⅲ与绿色木霉Ⅰ混合发酵产纤维素酶的分离纯化及酶学性质研究

摘要

Cellulase produced by mixed fermentation of Apsergillus niger Ⅲ and Trichoderma viride Ⅰ was isolated and purified and its enzymatic properties were studied. Five elution peaks were obtained through ammonium sulfate salting-out and Sephadex G-100 column chromatography, namely peakl -5. Peak 2 contained endoglucanase and exoglucanase, among them, endoglucanase activity was higher. Peak 3 contained compar- atively full enzyme species including endoglucanase and exoglucanase and glucosidase. Peak 5 contained endoglucanase and exoglucanase. And there was no cellulase activity for peak 1 and peak 4. By DEAE FF weak anion exchange, a kind of glycosidase enzymes was purified from peak 2, and its molecular weight was 61.5kD by SDS-PAGE cataphoresis. Within the range of pH 5.0 -6.0, the initial activity of CMCase of peak 2 maintained 70 %-80 %, and the optimum pH was 5.0. Higher enzyme activity appeared at 30-50 ℃, the optimal temperature was at 50 ℃, and enzyme activity rapidly decreased when temperature was higher than 60 ℃.%对黑曲霉Ⅲ与绿色木霉Ⅰ混合发酵所产纤维素酶进行了分离纯化,并对其酶学性质进行了研究。通过硫酸铵分级沉淀、Sephadex G-100凝胶柱层析,得到5个洗脱峰,其中峰2含内切葡聚糖苷酶和外切葡聚糖苷酶,且内切葡聚糖苷酶活最高;峰3含内切酶、外切酶及β-葡萄糖苷酶,酶系较全面;峰5含内切酶和外切酶;峰1和峰4没有纤维素酶活性。采用DEAF FF弱阴离子交换柱层析对峰2进行分离纯化,从中分离纯化得到1种内切葡聚糖苷酶组分,经SDS-PAGE电泳分析,其分子量为61.5 KD。酶学性质研究结果表明,CMC酶活在pH4.0～6.0的条件下,可保持初始酶活的70%～80%,最适酶反应pH值为5.0;温度在30～50℃范围内,酶活较高,最适酶反应温度为50℃,若超过60℃,酶活迅速下降。