Functional characterisation of cellobiohydrolase I (Cbh1) from Trichoderma virens UKM1 expressed in Aspergillus niger

Cellobiohydrolases catalyze the processive hydrolysis of cellulose into cellobiose. Here, a Trichoderma virens cDNA predicted to encode for cellobiohydrolase (cbhI) was cloned and expressed heterologously in Aspergillus niger. The cbh1 gene has an open reading frame of 1518 bp, encoding for a putative protein of 505 amino acid residues with a calculated molecular mass of approximately 54 kDa. The predicted CbhI amino acid sequence has a fungal type carbohydrate binding module separated from a catalytic domain by a threonine rich linker region and showed high sequence homology with glycoside hydrolase family 7 proteins. The partially purified enzyme has an optimum pH of 4.0 with stability ranging from pH 3.0 to 6.0 and an optimum temperature of 60 degrees C. The partially purified CbhI has a specific activity of 4.195 Umg(-1) and a low K-m value of 1.88 mM when p-nitrophenyl-beta-D-cellobioside (pNPC) is used as the substrate. The catalytic efficiency (k(cat)/K-m) was 5.68 x 10(-4) mM(-1) s(-1), which is comparable to the CbhI enzymes from Trichoderma viridae and Phanaerochaete chrysosporiwn. CbhI also showed activity towards complex substrates such as Avicel (0.011 Umg(-1)), which could be useful in complex biomass degradation. Interestingly, CbhI also exhibited a relatively high inhibition constant (K-i) for cellobiose with a value of 8.65 mM, making this enzyme more resistant to end-product inhibition compared to other fungal cellobiohydrolases.