title: Expression, purification and characterization of a functional carbohydrate-binding module from Streptomyces sp. SirexAA-E.
abstract: Streptomyces sp. SirexAA-E (ActE) has been identified as a highly cellulolytic actinobacterium capable of deconstructing lignocellulosic biomass. SirexAA-E CAZymes most frequently contain a carbohydrate-binding module from family 2a (CBM2a). The DNA encoding the CBM2a from gene locus SACTE_0237, the most abundantly expressed cellulase from SirexAA-E, was cloned into an Escherichia coli expression vector and expressed as a C-terminal fusion protein to GFP. The GFP-CBM2a fusion protein was purified from insoluble inclusion bodies and refolded. The solubilized protein was separated by size-exclusion chromatography into high molecular weight GFP-CBM2a multimers and monomeric GFP-CBM2a. Only the monomeric CBM2a protein was found to have high relative affinity (partition coefficient of 0.62±0.04L/g) to cellulose. Binding of monomeric CBM2a prepared in this manner exhibits fully reversible, high affinity binding to cellulose.
authors.affiliation: Department of Biochemistry, University of Wisconsin, Madison, WI 53706, United States.
authors.forename: Sungsoo
authors.initials: S
authors.lastname: Lim
authors.affiliation: Department of Biochemistry, University of Wisconsin, Madison, WI 53706, United States; DOE Great Lakes Bioenergy Research Center (GLBRC), Madison, WI 53703, United States; Department of Chemical Engineering and Materials Science, Michigan State University, East Lansing, MI 48824, United States. Electronic address: chundawa@egr.msu.edu.
authors.forename: Shishir P S
authors.initials: SP
authors.lastname: Chundawat
authors.affiliation: Department of Biochemistry, University of Wisconsin, Madison, WI 53706, United States; DOE Great Lakes Bioenergy Research Center (GLBRC), Madison, WI 53703, United States. Electronic address: bgfox@biochem.wisc.edu.
authors.forename: Brian G
authors.initials: BG
authors.lastname: Fox
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D002482:Cellulose / Q000378:metabolism; D002846:Chromatography, Affinity; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D007700:Kinetics; D008958:Models, Molecular; D011485:Protein Binding; D011956:Receptors, Cell Surface / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D013302:Streptomyces / Q000737:chemistry / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D011956:Receptors, Cell Surface; C055213:saccharide-binding proteins; D002482:Cellulose
keywords: Biofuels; Carbohydrate-binding module (CBM); Cellulose; Protein refolding; Streptomyces sp. SirexAA-E