Deciphering ligand specificity of a Clostridium thermocellum family 35 carbohydrate binding module (CtCBM35) for gluco- and galacto- substituted mannans and its calcium induced stability.

Arabinda Ghosh,Ana Sofia Luís,Neeta Pathaw,Carlos M G A Fontes,Nikhil K Chrungoo,Joana L A Brás,Arun Goyal

doi:10.1371/journal.pone.0080415

Arabinda Ghosh, Ana Sofia Luís + Show 5 more

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https://doi.org/10.1371/journal.pone.0080415

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Abstract

This study investigated the role of CBM35 from Clostridium thermocellum (CtCBM35) in polysaccharide recognition. CtCBM35 was cloned into pET28a (+) vector with an engineered His6 tag and expressed in Escherichia coli BL21 (DE3) cells. A homogenous 15 kDa protein was purified by immobilized metal ion chromatography (IMAC). Ligand binding analysis of CtCBM35 was carried out by affinity electrophoresis using various soluble ligands. CtCBM35 showed a manno-configured ligand specific binding displaying significant association with konjac glucomannan (K a = 14.3×104 M−1), carob galactomannan (K a = 12.4×104 M−1) and negligible association (K a = 12 µM−1) with insoluble mannan. Binding of CtCBM35 with polysaccharides which was calcium dependent exhibited two fold higher association in presence of 10 mM Ca2+ ion with konjac glucomannan (K a = 41×104 M−1) and carob galactomannan (K a = 30×104 M−1). The polysaccharide binding was further investigated by fluorescence spectrophotometric studies. On binding with carob galactomannan and konjac glucomannan the conformation of CtCBM35 changed significantly with regular 21 nm peak shifts towards lower quantum yield. The degree of association (K a) with konjac glucomannan and carob galactomannan, 14.3×104 M−1 and 11.4×104 M−1, respectively, corroborated the findings from affinity electrophoresis. The association of CtCBM35with konjac glucomannan led to higher free energy of binding (ΔG) −25 kJ mole−1 as compared to carob galactomannan (ΔG) −22 kJ mole−1. On binding CtCBM35 with konjac glucomannan and carob galactomannan the hydrodynamic radius (RH) as analysed by dynamic light scattering (DLS) study, increased to 8 nm and 6 nm, respectively, from 4.25 nm in absence of ligand. The presence of 10 mM Ca2+ ions imparted stiffer orientation of CtCBM35 particles with increased RH of 4.52 nm. Due to such stiffer orientation CtCBM35 became more thermostable and its melting temperature was shifted to 70°C from initial 50°C.

Highlights

Hydrolytic enzymes and their enhanced polysaccharide specificity often improve by appended non catalytic carbohydrate binding module either at their N or C terminal ends
Expression and purification of CtCBM35 The open reading frame (ORF) region encoding CtCBM35 was amplified by polymerase chain reaction and successfully ligated to pET28a (+) expression vector and transformed E. coli DH5a cells
The recombinant CtCBM35 was purified by Ni2+-NTA (Immobilized metal ion chromatography) and the elution was accomplished with 300 mM Imidazole

Summary

Introduction

Hydrolytic enzymes and their enhanced polysaccharide specificity often improve by appended non catalytic carbohydrate binding module either at their N or C terminal ends. Polysaccharide recognition, binding and enhanced catalysis of hydrolytic enzymes truly facilitates by non catalytic modular carbohydrate binding modules. 35 carbohydrate binding module is often appended to glycoside hydrolase family 26 (GH26) and GH5 mannanases [2,3,4], xylanases (GH30) [5] which significantly alter the polysaccharide specificity for plant cell wall polysaccharides such as galactomannan, glucomannan, mannan and glucouronoxylan. CBM35 usually accommodates the polysaccharides utilizing a planer surface of aromatic side chains which interact with the flat chains of manno-configured carbohydrate residues. This form of conformation is known as type B module [5]. Substantial rise in effective enzyme catalysis process may induce by this carbohydrate binding module will meet the requirement for carbohydrate fermentation to most demanding biofuel [11]

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