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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Abbreviations
- BSA:
-
bovine serum albumin
- CAZy:
-
Carbohydrate-Active enZyme database
- CBM:
-
carbohydrate-binding module
- CtCBM35 (i.e. Cthe_2811):
-
CBM35 from the Clostridium thermocellum GH26 β-mannanase
- EDTA:
-
ethylenediaminetetraacetic acid
- GH:
-
glycoside hydrolase
- PDB:
-
Protein Data Bank
- RMSD:
-
root mean square deviation
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Based on a contribution presented at the International Conference on Advances in Biotechnology & Bioinformatics (ICABB-2013), November 25–27, 2013, Pune, Maharashtra, India. Electronic supplementary material. The online version of this article (DOI:10.2478/s11756-014-0444-y) contains supplementary material, which is available to authorized users.
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Ghosh, A., Verma, A.K., Luis, A.S. et al. Mannan specific family 35 carbohydrate-binding module (CtCBM35) of Clostridium thermocellum: structure analysis and ligand binding. Biologia 69, 1271–1282 (2014). https://doi.org/10.2478/s11756-014-0444-y
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DOI: https://doi.org/10.2478/s11756-014-0444-y