Construction of Escherichia coli BL21/A-53 producing histidine-tagged carboxymethylcellulase and comparison of its characteristics with CMCase without histidine-tag.

To enhance recovery yield of carboxymethylcellulase (CMCase), E. coli BL21/A-53 producing the histidine-tagged CMCase was constructed in this study. The recovery yield of the histidine-tagged CMCase using the His-tag affinity chromatography was 39.8%. The predicted molecular weight of the histidine-tagged CMCase was determined as 56,260 Da. Its K_m and V_max were 9.3 g l⁻¹ and 76.3 g l⁻¹·min⁻¹, respectively. The histidine-tagged CMCase hydrolyzed avicel, carboxymethylcellulose (CMC), filter paper, pullulan, xylan, but there was no detectable activity on cellobiose, p-Nitrophenyl-β-D-glucopyranoside (pNPG). The optimal temperature and pH for the enzymatic reaction of the histidine-tagged CMCase was 50 °C and 5.0. The histidine-tagged CMCase was enhanced by CoCl₂ until the concentration of 100 mM, but inhibited by EDTA, HgCl₂, MnCl₂, NiCl₂, and RbCl₂. The characteristics of the histidine-tagged CMCase produced by E. coli BL21/A-53 were compared with those of CMCase without the histidine-tag of Bacillus subtilis subsp. subtilis A-53. The little changed characteristics of the histidine-tagged CMCase compared to the CMCase without a His-tag seemed to be the conformational change in the structure due to a His-tag. [ABSTRACT FROM AUTHOR]