Abstract
Cel5A of Thermotoga maritima, a 37 kDa cellulase of the family GH5, was expressed in partially soluble state in E. coli. However, the truncated version tCel5A1, produced by removing ten residues from the C-terminal of Cel5A, was expressed in a completely soluble form. tCel5A1 showed 7.3- fold increased specific activity against carboxy methyl cellulose while the increase in activities against regenerated amorphous cellulose and Avicel were 21 and 16 fold, respectively. tCel5A1 is stable at 60 degrees C for more than 2hr and it showed temperature and pH optima 70 degrees C and 6.0, respectively, under the assay conditions used. These characteristics are similar to those of the native enzyme. As expected, CD spectral analysis showed that C-terminal truncation has little effect on the secondary structure of the molecule. tCel5A1 showed higher binding to pretreated rice straw (84%) as compared to the native form (46%). Molecular modelling analysis of tCel5A1 showed that the removal of C-terminal residues exposed the active site residues Glu253, Trp286, and Phe292, which are located in the catalytic cavity close to the C-terminus. Making these residues more accessible to the substrate would result in increased activity. The ratio of 10.01 between the soluble to the insoluble reducing groups produced from RAC on treatment with tCel5A1, and the presence of cellobiose as the major end product in the hydrolysate showed that tCel5A1 is a processive cellulase. Although other processive cellulases belonging to the family GH5, mainly of the fungal origin, have been reported, but tCel5A1, to our knowledge, is the first processive cellulase from an extreme thermophile reported so far.