Expression, purification and characterization of a functional carbohydrate-binding module from Streptomyces sp SirexAA-E

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 carbohydratebinding 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 colt 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 +/- 0.04 L/g) to cellulose. Binding of monomeric CBM2a prepared in this manner exhibits fully reversible, high affinity binding to cellulose. (C) 2014 Elsevier Inc. All rights reserved.