ORIGINAL ARTICLE The family 6 carbohydrate-binding module ( CtCBM6B) of Clostridium thermocellum alpha-L-arabinofuranosidase binds xylans and thermally stabilized by Ca 2 ions SHADAB AHMED 1 , ANA SOFIA LUIS 2 , JOANA L. A. BRÁS 2 , CARLOS M. G. A. FONTES 2 & ARUN GOYAL 1 1 Department of Biotechnology, Indian Institute of Technology Guwahati, Guwahati, Assam, India and 2 CIISA-Faculdade de MedicinaVeterinaria, Avenida da Universidade Técnica, Lisbon, Portugal Abstract The gene encoding CtCBM6B of Clostridium thermocellum α-L-arabinofuranosidase ( Ct43Ara f) was cloned in pET-21a( +) vector, over-expressed using Escherichia coli BL-21(DE3) cells and purified by immobilized metal-ion affinity chromatog- raphy (IMAC). The recombinant CtCBM6B showed a molecular size close to 15 kDa by SDS-PAGE analysis, which was close to the expected size of 14.74 kDa. The ligand-binding affinity of CtCBM6B was assessed against ligands for which the catalytic enzyme, Ct43Ara f showed maximum activity. The affinity-gel electrophoresis of CtCBM6B with rye arab- inoxylan showed lower equilibrium association constant ( K a, 4.0% C -1 ), whereas, it exhibited higher affinity ( K a , 19.6% C -1 ) with oat spelt xylan. The ligand-binding analysis of CtCBM6B by fluorescence spectroscopy also revealed similar results with low K a (3.26% C -1 ) with rye arabinoxylan and higher affinity for oat spelt xylan ( K a , 17.9% C -1 ) which was corroborated by greater blue-shift in case of oat spelt xylan binding. The CtCBM6B binding with insoluble wheat arabinoxylan by adsorption isotherm analysis showed significant binding affinity as reflected by the equilibrium association constant ( K a ), 9.4  10 3 M -1 . The qualitative analysis by SDS-PAGE also corroborated the CtCBM6B binding with insoluble wheat arabinoxylan. The protein-melting curve of CtCBM6B displayed the peak shift from 53 °C to 59 °C in the presence of Ca 2 + ions indicating that Ca 2 + ions impart thermal stability to the CtCBM6B structure. Keywords: Clostridium thermocellum, CtCBM6B, pET-21a( +), rye arabinoxylan, oat spelt xylan, affinity gel electrophoresis Correspondence: Professor Arun Goyal, Department of Biotechnology, Indian Institute of Technology Guwahati, Guwahati- 781 039, Assam, India. Tel: +(361) 258 2208 (O) Fax: +(361) 258 2249, 269 0762. E-mail: arungoyl@iitg.ernet.in. URL: http://www.iitg.ernet.in/biotech/A.Goyal.html. http://www. biomedexperts.com/Profile.bme/486220/Arun_Goyal (Received 12 May 2013; revised 2 June 2013; accepted 7 June 2013) Introduction Carbohydrate-binding modules (CBMs) are non- catalytic components usually found associated with the carbohydrate-active enzymes glycoside hydrolases (GHs) (Fierobe et al. 2002; Fontes & Gilbert 2010). Their primary function is to assist in targeting the appended catalytic modules to the ligand and con- centrating them on the surface of the ligand. Similar to GHs, the CBMs are also categorized into different families (http://www.cazy.org/CBM.html). The affin- ity-gel electrophoresis has been the most popular method for determining CBM-ligand-binding affin- ity for the last decade (Takeo 2000; Tomme et al. 2000). There are several CBM families that display diversity in ligand-binding affinity (Ficko-Blean et al. 2012), which is mainly because of the flexibility in the recognition of sugar ring configuration, type of linkage, and degree of polymerization. The family 6 CBMs (CBM6s) have been previously demon- strated to bind with amorphous cellulose, xylans, and a few modules also bind to β-1,3-glucan, β-1,3- 1,4-glucan, and β-1,4-glucan (http://www.cazy.org/ CBM6.html). CBM6s are found in a range of GHs displaying activity against xylan, mannan, β-glucans, arabinoxylans, and arabinans. Several complemen- tary biochemical studies have been performed in past demonstrating the ability of CBM6s to interact with diverse carbohydrate targets (Abbott et al. 2009; Abbott & Boraston 2012). Because of the ligand diversity and continually expanding knowledge Biocatalysis and Biotransformation, 2013; 31(4): 217–225 ISSN 1024-2422 print/ISSN 1029-2446 online © 2013 Informa UK, Ltd. DOI: 10.3109/10242422.2013.828047 This paper was delivered at the 10th Carbohydrate Bioengineering Meeting, Prague 21–24th April 2013. Biocatal Biotransformation Downloaded from informahealthcare.com by 117.226.20.164 on 10/03/13 For personal use only.