Recombinant expression and characterization of an endoglucanase III (cel12a) from Trichoderma harzianum (Hypocreaceae) in the yeast Pichia pastons

• Published in: Genetics and molecular research 2012-01, Vol.11 (2), p.1544-1557
• Main Authors: Generoso, W C, Malago, W Jr, Pereira, N Jr, Henrique-Silva, F
• Format: Article
• Language: English
• Subjects: Cell culture; Endoglucanase; Enzymes; Ethanol; Expression vectors; Fungi; Hypocreaceae; Open reading frames; pH effects; Pichia; Plasmids; Secretion; Signal peptides
• ISSN: 1676-5680; 1676-5680
• 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 pPICZ alpha A, enabling protein secretion to the culture medium. The recombinant plasmid was used to transform Pichia pastons. 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 degree 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.