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Functional characterization and mutation analysis of family 11, Carbohydrate-Binding Module (CtCBM11) of cellulosomal bifunctional cellulase from Clostridium thermocellum

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Abstract

The non-catalytic, family 11 carbohydrate binding module (CtCBM11) belonging to a bifunctional cellulosomal cellulase from Clostridium thermocellum was hyper-expressed in E. coli and functionally characterized. Affinity 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 affinity electrophoresis and binding adsorption isotherms showed that of the three mutants Y22A, W65A and Y129A of CtCBM11, two mutants Y22A and Y129A showed no or reduced binding affinity with polysaccharides. 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.

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Authors and Affiliations

  1. Department of Biotechnology, Indian Institute of Technology Guwahati, North Guwahati, 781 039, Assam, India

    S. Bharali, R. K. Purama, A. Majumder & A. Goyal

  2. CIISA-Faculdade de Medicina Veterinária, Rua Prof. Cid dos Santos, 1300 477, Lisbon, Portugal

    C. M. G. A. Fontes

Authors
  1. S. Bharali
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  2. R. K. Purama
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  3. A. Majumder
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  4. C. M. G. A. Fontes
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  5. A. Goyal
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Correspondence to A. Goyal.

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Bharali, S., Purama, R.K., Majumder, A. et al. Functional characterization and mutation analysis of family 11, Carbohydrate-Binding Module (CtCBM11) of cellulosomal bifunctional cellulase from Clostridium thermocellum . Indian J Microbiol 47, 109–118 (2007). https://doi.org/10.1007/s12088-007-0023-9

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  • DOI: https://doi.org/10.1007/s12088-007-0023-9

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