Expression and Characterization of CMCax Having β-1,4-Endoglucanase Activity from Acetobacter xylinum

다국어 초록 (Multilingual Abstract)

The CMCax gene from Acetobacter xylinum ATCC 23769 was cloned and expressed in E. coli. With this gene, three gene products - mature CMCax containing signal peptide (pre-CMCax), and a glutathione-S-transferase(GST)-CMCax fusion enzyme - were expressed. CMCax and pre-CMCax are aggregated to multimeric forms which showed high CMC hydrolysis activity, whereas GST-CMCax was less aggregated and showed lower activity, indicating that oligomerization of CMCax contributes to the cellulose hydrolysis activity to achieve greater efficiency. The enzyme was identified to be an β-1,4-endoglucanase, which catalyzes the cleavage of internal β-1,4-glycosidic bounds of cellulose. The reaction products, cellobiose and cellotriose, from cellopentaose as a substrate, were identified by HPLC. Substrate specificity of cellotetraose by this enzyme was poor, and the reaction products consisted of glucose, cellobiose, and cellotriose in a very low yield. These results suggested that cellopentaose might be the oligosaccharide substrate consisting of the lowest number of glucose. The optimum pH of CMCax and pre-CMCax was about 4.5, whereas that of GST-CMCax was rather broad at pH 4.5-8. The physiological significance of cellulose-hydrolyzing enzyme, CMCax, having such low β-1,4-endoglucanase activity and low optimum pH in cellulose-producing A. xylinum is not clearly known yet, but it seems to be closely related to the production of cellulose.