| Glycoside hydrolase family 44 (GH44) remains one of the smallest and least studied families of cellulases. The cellulosome of Clostridium acetobutylicum ATCC 824 contains the gene CAC0915, which codes for a putative extracellular GH44 catalytic domain with a C-terminal type I dockerin domain. The GH44 catalytic domain was synthesized with codon modifications, ligated into the expression vector pET--22b, and transformed into Escherichia coli BL21 (DE3). Expression during cell growth was induced by isopropyl-beta-D-thiogalactopyranoside and the recombinant protein, produced as a fusion protein containing a C-terminal His6-tag, and isolated from the harvested cells' cytoplasm after sonication. The enzyme has a mass of approximately 57 kDa and was present in the soluble fraction from the cell lysate, which was subsequently purified by affinity and gel filtration chromatography. The enzyme degrades CMC and xylan and is most active on those substrates at the pH values of 4 and 6, with specific activities of 7.8 and 5.9 imol reducing sugar/mg protein-min, respectively. This enzyme is active at 50°C but appears to be unstable at that temperature without the presence of substrate. Further characterization is pending. This enzyme is not active on crystalline cellulose. |
