The
endoglucanase II of Trichoderma reesei is considered the most effective
enzyme for biofinishing cotton fabrics and biostoning denim garments. However, the commercially available preparation of
endoglucanase II is usually mixed with other
cellulase components, especially
endoglucanase I, resulting in hydrolysis and
weight loss of garments during biofinishing and biostoning. We thus isolated the
endoglucanase II gene from T. reesei to express this in Pichia pastoris, under the control of a
methanol-inducible AOX1 promoter, to avoid the presence of other
cellulase components. A highly expressible Mut(+) transformant was selected and its expression in BMMH medium was found most suitable for the production of large amounts of the
recombinant protein. Recombinant
endoglucanase II was purified to electrophoretic homogeneity, and functionally characterized by activity staining. The specific activity of recombinant
endoglucanase II was found to be 220.57 EU/mg of
protein. Purified recombinant
endoglucanase II was estimated to have a molecular mass of 52.8 kDa. The increase in molecular mass was likely due to hyperglycosylation. Hyperglycosylation of recombinant
endoglucanase II secreted by P. pastoris did not change the temperature or pH optima as compared to the native
protein, but did result in increased thermostability. Kinetic analysis showed that recombinant
endoglucanase was most active against amorphous
cellulose, such as
carboxymethyl cellulose, for which it also had a high affinity.