Mannan specific family 35 carbohydrate-binding module (CtCBM35) of Clostridium thermocellum: structure analysis and ligand binding

作者：Ghosh Arabinda; Verma Anil Kumar; Luis Ana Sofia; Armada Bras Joana Luis; Fontes Carlos Mendes; Goyal Arun^*

来源：Biologia, 2014, 69(10): 1271-1282.

DOI：10.2478/s11756-014-0444-y

• homology modeling
• affinity electrophoresis
• fluorescence spectra
• protein melting

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) beta-mannanase, generated by Modeller9v8 displayed predominance of beta-sheets arranged as beta-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 x 10(5) M-1) than carob galactomannan (K-a (=) 1.4 x 10(5) 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 x 10(5) M-1 with insoluble ivory nut mannan. Ligand binding study by fluorescence spectroscopy showed Ka against konjac glucomannan and carob galactomannan, 2.4 x 10(5) M-1 and 1.4 x 10(5) M-1, and Delta G 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 degrees C from initial 55 degrees C. Therefore thermostable CtCBM35 targets more beta-(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.

• 出版日期2014-10