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The family 6 carbohydrate-binding module (CtCBM6B) of Clostridium thermocellum alpha-L-arabinofuranosidase binds xylans and thermally stabilized by Ca2+ ions

2013-08Journal article Restricted access
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dc.contributor.authorAhmed, Shadab
dc.contributor.authorLuis, Ana Sofia
dc.contributor.authorBrás, Joana L. A.
dc.contributor.authorFontes, Carlos M. G. A.
dc.contributor.authorGoyal, Arun
dc.date.accessioned2014-09-02T15:54:48Z
dc.date.available2014-09-02T15:54:48Z
dc.date.issued2013-08
dc.descriptionArticles in International Journalspor
dc.description.abstractThe gene encoding CtCBM6B of Clostridium thermocellum α-L-arabinofuranosidase (Ct43Araf) was cloned in pET-21a(+) vector, over-expressed using Escherichia coli BL-21(DE3) cells and purified by immobilized metal-ion affinity chromatography (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, Ct43Araf showed maximum activity. The affinity-gel electrophoresis of CtCBM6B with rye arabinoxylan showed lower equilibrium association constant (Ka, 4.0% C− 1), whereas, it exhibited higher affinity (Ka, 19.6% C− 1) with oat spelt xylan. The ligand-binding analysis of CtCBM6B by fluorescence spectroscopy also revealed similar results with low Ka (3.26% C− 1) with rye arabinoxylan and higher affinity for oat spelt xylan (Ka, 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 (Ka), 9.4 × 103 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 Ca2+ ions indicating that Ca2+ ions impart thermal stability to the CtCBM6B structure.por
dc.identifier.citationAhmed, S., Luis, A.S., Brás, J.L.A, Fontes, C.M.G.A., Goyal, A. (2013). The family 6 carbohydrate-binding module (CtCBM6B) of Clostridium thermocellum alpha-L-arabinofuranosidase binds xylans and thermally stabilized by Ca2+ ions. Biocatalysis and Biotransformation, 31(4), 217-225. doi:10.3109/10242422.2013.828047por
dc.identifier.doi10.3109/10242422.2013.828047
dc.identifier.issn1024-2422
dc.identifier.urihttp://hdl.handle.net/10400.5/7060
dc.language.isoengpor
dc.peerreviewedyespor
dc.subjectClostridium thermocellumpor
dc.subjectCtCBM6Bpor
dc.subjectpET-21a(+)por
dc.subjectrye arabinoxylanpor
dc.subjectoat spelt xylanpor
dc.subjectaffinity gel electrophoresispor
dc.titleThe family 6 carbohydrate-binding module (CtCBM6B) of Clostridium thermocellum alpha-L-arabinofuranosidase binds xylans and thermally stabilized by Ca2+ ionspor
dc.typejournal article
dspace.entity.typePublication
oaire.citation.endPage225por
oaire.citation.startPage217por
oaire.citation.titleBiocatalysis and Biotransformationpor
oaire.citation.volume31(4)por
rcaap.rightsrestrictedAccesspor
rcaap.typearticlepor

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