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Biochemistry (Moscow)

, Volume 78, Issue 11, pp 1272–1279 | Cite as

Functional and structural characterization of family 6 carbohydrate-binding module (CtCBM6A) of Clostridium thermocellum α-L-arabinofuranosidase

  • S. Ahmed
  • A. S. Luís
  • J. L. A. Brás
  • C. M. G. A. Fontes
  • A. GoyalEmail author
Article

Abstract

The gene encoding the family 6 carbohydrate-binding module (CtCBM6A) from Clostridium thermocellum, cloned in pET-21a(+) expression vector, was overexpressed using Escherichia coli BL-21(DE3) cells and purified by immobilized metal-ion affinity chromatography. SDS-PAGE analysis of the recombinant CtCBM6A showed molecular size of approximately 15 kDa. Ligand-binding analysis of CtCBM6A with rye arabinoxylan and oat spelt xylan by affinity gel electrophoresis showed low affinity for these ligands (K a of 40 and 26 liter/g, respectively), and analysis by fluorescence spectroscopy (K a of 33 and 15 liter/g, respectively) corroborated lower binding affinity with the above soluble ligands. However, CtCBM6A displayed significantly higher ligand-binding affinity with insoluble wheat arabinoxylan with equilibrium association constant K a of 230 M−1 and binding capacity (N 0) of 11 μmole/g. The protein melting curve of CtCBM6A displayed a peak shift from 53 to 58°C in the presence of Ca2+, indicating that Ca2+ imparts thermal stability to the CtCBM6A structure. Homology modeling of CtCBM6A revealed a characteristic β-sandwich core structure. The Ramachandran plot of CtCBM6A showed 89% of the residues in the most favorable region, 10% in additionally favored region, and 1% in generously allowed region, indicating that CtCBM6A has a stable conformation.

Key words

CtCBM6A insoluble wheat arabinoxylan oat spelt xylan homology modeling affinity gel electrophoresis 

Abbreviations

CtCBM6A

family 6 carbohydrate-binding module of type A from Clostridium thermocellum

MSA

multiple sequence alignment

WAXI

wheat arabinoxylan insoluble

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Copyright information

© Pleiades Publishing, Ltd. 2013

Authors and Affiliations

  • S. Ahmed
    • 1
  • A. S. Luís
    • 2
  • J. L. A. Brás
    • 2
  • C. M. G. A. Fontes
    • 2
  • A. Goyal
    • 1
    Email author
  1. 1.Department of BiotechnologyIndian Institute of Technology GuwahatiGuwahatiIndia
  2. 2.CIISA-Faculdade de Medicina VeterinariaLisbonPortugal

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