The celB gene of Caldicellulosiruptor saccharolyticus was cloned and expressed in Escherichia coil to create a recombinant biocatalyst for hydrolyzing lignocellulosic biomass at high temperature. The GH5 domain of CelB hydrolyzed 4-nitrophenyl-beta-D-cellobioside and carboxymethyl cellulose with optimum activity at pH 4.7-5.5 and 80 degrees C. The recombinant GH5 and CBM3-GH5 constructs were both stable at 80 degrees C with half-lives of 23 h and 39 h, respectively, and retained > 94% activity after 48 h at 70 degrees C. Enzymatic hydrolysis of corn stover and cellulose pretreated with the ionic liquid 1-ethyl-3-methylimidazolium acetate showed that GH5 and CBM3-GH5 primarily produce cellobiose, with product yields for CBM3-GH5 being 1.2- to 2-fold higher than those for GH5. Confocal microscopy of bound protein on cellulose confirmed tighter binding of CBM3-GH5 to cellulose than GH5, indicating that the enhancement of enzymatic activity on solid substrates may be due to the substrate binding activity of CBM3 domain. Published by Elsevier Ltd.