Functional and Structural Characterization of Family 6 Carbohydrate-Binding Module (CtCBM6A) of Clostridium thermocellum α-L-Arabinofuranosidase

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S. Ahmed¹, A. S. Luís², J. L. A. Brás², C. M. G. A. Fontes², and A. Goyal^1*

¹Department of Biotechnology, Indian Institute of Technology Guwahati, Guwahati-781039, Assam, India; fax: (361) 258-2249; E-mail: arungoyl@iitg.ernet.in

²CIISA-Faculdade de Medicina Veterinaria, Avenida da Universidade Técnica, 1300-477 Lisbon, Portugal

^* To whom correspondence should be addressed.

Received May 21, 2013; Revision received July 16, 2013

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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 (N₀) of 11 µmole/g. The protein melting curve of CtCBM6A displayed a peak shift from 53 to 58ºC 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.

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KEY WORDS: CtCBM6A, insoluble wheat arabinoxylan, oat spelt xylan, homology modeling, affinity gel electrophoresis

DOI: 10.1134/S0006297913110072

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