The non-catalytic, family 11 carbohydrate binding module (CtCBM11) belonging to a bifunctional cellulosomal cellulase from Clostridium thermocellum was hyperexpressed in E. coli and functionally characterized. Affi nity fi electrophoresis of CtCBM11 on nondenaturing PAGE containing cellulosic polysaccharides showed binding with β-glucan, lichenan, hydroxyethyl cellulose and carboxymethyl cellulose. In order to elucidate the involvement of conserved aromatic residues Tyr 22, Trp 65 and Tyr 129 in the polysaccharide binding, site-directed mutagenesis was carried out and the residues were changed to alanine. The results of affi nity electrophoresis and binding adsorption fi isotherms showed that of the three mutants Y22A, W65A and Y129A of CtCBM11, two mutants Y22A and Y129A showed no or reduced binding affi nity with polysaccharides. fi These results showed that tyrosine residue 22 and 129 are involved in the polysaccharide binding. These residues are present in the putative binding cleft and play a critical role in the recognition of all the ligands recognized by the protein.
The recombinant enzyme lichenase of size 30 kDa was over-expressed using E. coli cells and purifi ed by immobilized metal ion affi nity chromatography (IMAC) and size exclusion chromatography. The enzyme displayed high activity towards lichenan and β-glucan. The enzyme showed no activity towards carboxymethyl cellulose, laminarin, galactomannan or glucomannan. Surprisingly, affi nity-gel electrophoresis on native-PAGE showed that the enzyme binds only glucomannan and not lichenan or β-glucan or other manno-confi gured substrates. The enzyme was thermally stable between the temperatures 60°C and 70°C. Presence of Cu 2+ ions at a concentration of 5 mM enhanced enzyme activity by 10% but higher concentrations of Cu 2+ (>25 mM) showed a sharp fall in the enzyme activity. Heavy metal ions Ni 2+ , Co 2+ and Zn 2+ did not affect the activity of the enzyme at low concentrations (0-10 mM) but at higher concentrations (>10 mM), caused a decrease in the enzyme activity. The crystals of lichenase were produced and the 3-dimensional structure of native form of enzyme was previously solved at 1.50 Å. Lichenase displayed (ß/α) 8 -fold a common fold among many glycoside hydrolase families. A cleft was identifi ed that represented the probable location of active site.
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