【作者】 支晓鹏；

【导师】 龙敏南；

【作者基本信息】 厦门大学 ， 微生物学， 2007， 硕士

【摘要】 本实验室筛选到一株产纤维素酶和木聚糖酶菌株,青霉(Penicillium sp.) T24-2。液态发酵培养96h后,发酵液离心去除菌体和残渣,粗酶液采用(NH4)2S04分级沉淀、Sephadex G-25层析、Sephadex G-75凝胶过滤层析、Sephadex G-200凝胶过滤层析、DEAE-Sepharose Fast Flow离子交换层析等步骤,分离纯化出青霉T24-2中达到电泳纯的β-葡萄糖苷酶和2种木聚糖酶XYN-1,XYN-2。XYN-1被纯化了3.8倍,比活由37.5 U/mg上升到144.2 U/mg;XYN-2被纯化了5.4倍,比活由37.5U/mg上升到202.5U/mg;β-葡萄糖苷酶则被纯化20.6倍,比活由26.5 U/mg上升到546.7 U/mg。通过SDS-PAGE电泳测得木聚糖酶和β-葡萄糖苷酶酶组分亚基的相对分子量分别为43.7kD,20.7kD和140.0kD。木聚糖酶XYN-1和XYN-2的最适反应pH分别为pH5.5,pH4.5,最适反应温度分别为70℃和50℃,最稳定的pH分别为pH4和pH5,两种木聚糖酶均在30℃-40℃下处理30 min后比较稳定。β-萄糖苷酶的最适反应pH为pH3.5,最适反应温度为70℃,最稳定的pH为pH4.5,在30℃～50℃之间比较稳定。在反应体系金属离子和化合物的终浓度均为5mmol/L条件下,ZnSO4和MgCl2对木聚糖酶XYN-1酶活有激活作用,而MgCl2则对XYN-2酶活有一定的抑制作用。MnSO4对木聚糖酶XYN-1酶活有强烈的抑制作用,但是对于XYN-2, MnSO4却起到了激活作用。SDS对两种木聚糖酶都有强烈的抑制作用。NiCl2和ZnSO4对β-葡萄糖苷酶酶活有激活作用,而FeSO4,MgCl2,MnSO4则对β-葡萄糖苷酶有较强的抑制作用。实验过程中发现,对稻草粉不进行预处理和进行预处理两种情况下,青霉T24-2菌株液态发酵能够产生更高浓度的胞外蛋白。另外,该菌株能以尿素为氮源表达CMC酶活和木聚糖酶酶活,是一个优良的纤维素酶和木聚糖酶高产菌株,这是该菌株比较突出的两个特点,应在今后对其进一步的深入研究。本研究对青霉T24-2菌株所产3种胞外酶进行分离纯化并对其酶学性质进行了研究,为利用该菌株构建产纤维素酶和半纤维素酶的基因工程菌株打下基础。更多还原

【Abstract】 Penicillium sp. T24-2 is a strain which exhibited high cellulase activity and xylanase activity. After culturing the strain in liquid medium for 96h, two xylanases and oneβ-glucosidase from the fermented liquor were separated and purified.The xylanase XYN-1 was purified 3.8 folds with a specific activity of 144.2 U/mg after four step purification of (NH4)2SO4 precipitation, Sephadex G-25, DE32 and Sephadex G-200 chromatography. The xylanase XYN-2 was purified 5.4 folds with a specific activity of 202.5 U/mg after purified by (NH4)2SO4 precipitation, Sephadex G-25, Ultrafiltration and Sephadex G-50 chromatography. Theβ-glucosidase was purified 20.6 folds with a specific activity of 546.7 U/mg by (NH4)2SO4 precipitation, Sephadex G-75 chromatography and DEAE-Sepharose Fast Flow chromatography.The molecular weight of XYN-1, XYN-2 andβ-glucosidase were determined as 40.7kD, 20.7kD and 140kD respectively, by SDS-PAGE.The affection of pH and temperature on the activity of enzyme were investigated. The maximum activities were obtained at pH5.5 for XYN-1, pH4.5 for XYN-2, pH3.5 forβ-glucosidase.The optimunm reaction temperature for XYN-1 and XYN-2 were 70℃and 50℃, respectively. The optimum reaction temperature forβ-glucosidase was 70℃. The two xylanases andβ-glucosidase showed good stability in acidic condition. All the threee enzymes were stalbe below 40℃.Several chemicals on xylanases andβ-glucosidase activity have been studied. The activity of XYN-1 was stimulated by ZnSO4 and MgCl2, but inhibited by MnSO4.The activity of XYN-2 was stimulated by MnSO4, but inhibited by MgCl2.Both XYN-1 and XYN-2 are inhibited severely by SDS. The activity ofβ-glucosidase was stimulated by NiCl2 and ZnSO4, but inhibited by FeSO4, MgCl2 and MnSO4. It is amazing that SDS has no obvious negative effects on the activity ofβ-glucosidase.Penicillium sp. T24-2 could produce cellulase using unpretreated rice straw as carbon source and using urea as nitrogen source. It will greatly reduce the cost in the large amount production of cellulase and xylanase. It suggests a good application in the future production of cellulose and xylanase.更多还原