Purification, cloning and characterisation of two forms of thermostable and highly active cellobiohydrolase I (Cel7A) produced by the industrial strain of

Two forms of cellobiohydrolase I (CBH I, Cel7A) were purified from the culture ultrafiltrate of a mutant strain of the fungus Chrysosporium lucknowense, an industrial producer of cellulases and hemicellulases. The enzymes had different molecular masses (52 and 65 kDa, SDS-PAGE data) but the same pI (4.5). Peptide sequencing showed that a single gene encodes both proteins. Both enzymes displayed maximum activity at pH 5.0-5.5; they had similar specific activities against soluble substrates. However, the 65 kDa CBH I was much more efficient in hydrolysis of Avicel and cotton cellulose, and its adsorption ability on Avicel was notably higher in comparison to the 52 kDa enzyme. Using the in-gel tryptic digestion followed by MALDI-TOF mass-spectrometry, it was shown that the 52 kDa enzyme represents the core catalytic module of the intact 65 kDa CBH I without a cellulose-binding module and major part of glycosylated linker. Both enzymes were stable at 50°C for 24 h. At higher temperature, the 65 kDa enzyme showed better thermostability: it retained >90% of activity after 7 h at 60°C and 50% of activity after 3 h at 65°C. The intact CBH I is also notably more thermostable than the Trichoderma reesei CBH I (by 6°C, the data of differential scanning microcalorimetry study). The cbh1 gene was cloned and then the amino acid sequence of Cel7A was deduced from the gene sequence. The enzyme had high degree of similarity (up to 74%) to family 7 cellobiohydrolases and lower degree of similarity (up to 41%) to family 7 endoglucanases. © 2004 Elsevier Inc. All rights reserved.