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iv INTRODUCTION 1 LITERATURE REVIEW 4 History of acetone–butanol fermentations 4 Carbohydrate chemistry and glycoside hydrolases 4 Cellulolytic organisms and cellulosomes 5 The Clostridium acetobutylicum cellulosome 9 Glycoside hydrolase family 44 10 GH44 putative proteins and their source organisms 14 GH44 produced proteins and their source organisms 15 CLONING EXPERIMENTS 21 Domains of CAC0915 and preliminary design 21 Codon–bias adjustment of CAC0915 22 PCRJet synthesis of CAC–1 26 Motivation for the isolation of PfamB 26 Comparison of expected amino acid sequences 28 PCR amplification of PfamB fragments 28 Ligation and transformation into pGEM–T Easy in E. coli Novablue 32 Digestions, ligation and transformation into pET–22b in E. coli BL21 (DE3) 37 PROTEIN PRODUCTION AND PURIFICATION 41 Initial production and screening of C–His recombinant protein 41 Selection of cell lysis buffer based on protein solubility 43 Temperature stability qualitatively tested by Congo Red staining 44 Ni–NTA chromatography validation 45 Bulk production fermentation and processing 47 Purification by chromatography through affinity to a Ni–NTA column 50

Cloning, expression, purification, and characterization of a glycoside hydrolase family 44 cellulase from Clostridium acetobutylicum in Escherichia coli