Generoso W C, Malagó W, Pereira N, Henrique-Silva F
Laboratório de Biologia Molecular, Departamento de Genética e Evolução, Universidade Federal de São Carlos, São Carlos, SP, Brasil.
Genet Mol Res. 2012 May 21;11(2):1544-57. doi: 10.4238/2012.May.21.11.
Filamentous fungi from the genus Trichoderma have been widely investigated due to their considerable production of important biotechnological enzymes. Previous studies have demonstrated that the T. harzianum strain IOC-3844 has a high degree of cellulolytic activity. After excluding the native signal peptide, the open reading frame of the T. harzianum endoglucanase III enzyme was cloned in the expression vector pPICZαA, enabling protein secretion to the culture medium. The recombinant plasmid was used to transform Pichia pastoris. Recombinant expression in the selected clone yielded 300 mg pure enzyme per liter of induced medium. The recombinant enzyme proved to be active in a qualitative analysis using Congo red. A quantitative assay, using dinitrosalicylic acid, revealed a high degree of activity at pH 5.5 and around 48°C. This information contributes to our understanding of the cellulolytic repertory of T. harzianum and the determination of a set of enzymes that can be incorporated into mixes for second-generation ethanol production.
由于木霉属丝状真菌能大量产生重要的生物技术酶，因此已对其进行了广泛研究。先前的研究表明，哈茨木霉IOC - 3844菌株具有高度的纤维素分解活性。去除天然信号肽后，将哈茨木霉内切葡聚糖酶III的开放阅读框克隆到表达载体pPICZαA中，使蛋白质能够分泌到培养基中。重组质粒用于转化巴斯德毕赤酵母。在选定的克隆中进行重组表达，每升诱导培养基可产生300毫克纯酶。在使用刚果红的定性分析中，重组酶被证明具有活性。使用二硝基水杨酸进行的定量测定表明，该酶在pH 5.5和48°C左右具有高度活性。这些信息有助于我们了解哈茨木霉的纤维素分解酶库，并确定一组可用于第二代乙醇生产混合物的酶。
