Mannan specific family 35 carbohydrate-binding module (CtCBM35) of Clostridium thermocellum: structure analysis and ligand binding |
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Authors: | Arabinda Ghosh Anil Kumar Verma Ana Sofia Luis Joana Luis Armada Bras Carlos Mendes Fontes Arun Goyal |
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Affiliation: | 1. Department of Biotechnology, Indian Institute of Technology Guwahati, Guwahati, 781039, Assam, India 2. CIISA — Faculdade de Medicina Veterinaria, Avenida da Universidade Técnica, 1300-477, Lisboa, Portugal
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Abstract: | The three-dimensional model of the CtCBM35 (Cthe 2811), i.e. the family 35 carbohydrate binding module (CBM) from the Clostridium thermocellum family 26 glycoside hydrolase (GH) β-mannanase, generated by Modeller9v8 displayed predominance of β-sheets arranged as β-sandwich fold. Multiple sequence alignment of CtCBM35 with other CBM35s showed a conserved signature sequence motif Trp-Gly-Tyr, which is probably a specific determinant for mannan binding. Cloned CtCBM35 from Clostridium thermocellum ATCC 27405 was a homogenous, soluble 16 kDa protein. Ligand binding analysis of CtCBM35 by affinity electrophoresis displayed higher binding affinity against konjac glucomannan (K a = 2.5 × 105 M?1) than carob galactomannan (K a = 1.4 × 105 M?1). The presence of Ca2+ ions imparted slightly higher binding affinity of CtCBM35 against carob galactomannan and konjac glucomannan than without Ca2+ ion additive. However, CtCBM35 exhibited a low ligand-binding affinity K a = 2.5 × 10?5 M?1 with insoluble ivory nut mannan. Ligand binding study by fluorescence spectroscopy showed K a against konjac glucomannan and carob galactomannan, 2.4 × 105 M?1 and 1.44 × 105 M?1, and ΔG of binding ?27.0 and ?25.0 kJ/mol, respectively, substantiating the findings of affinity electrophoresis. Ca2+ ions escalated the thermostability of CtCBM35 and its melting temperature was shifted to 70°C from initial 55°C. Therefore thermostable CtCBM35 targets more β-(1,4)-manno-configured ligands from plant cell wall hemicellulosic reservoir. Thus a non-catalytic CtCBM35 of multienzyme cellulosomal enzymes may gain interest in the biofuel and food industry in the form of released sugars by targeting plant cell wall polysaccharides. |
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