Recombinant expression and characterization of an endoglucanase III  (cel12a) from  Trichoderma harzianum (Hypocreaceae)  in the yeast Pichia pastoris.
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.
Laboratório de Biologia Molecular, Departamento de Genética e Evolução, Universidade Federal de São Carlos, São Carlos, SP, Brasil.
W C
WC
Generoso
W
W
Malagó
N
N
Pereira
F
F
Henrique-Silva
D002480:Cellulase / Q000235:genetics / Q000378:metabolism*; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014242:Trichoderma / Q000201:enzymology*
D011994:Recombinant Proteins; D002480:Cellulase