To enhance purification yield of the carboxymethylcellulase (CMCase) of P. aquimaris LBH-10, E. coli BL21/LBH-10 was constructed to produce the six histidine-tagged CMCase (CMCase with a His-tag). The purification yield of the CMCase with a His-tag produced by E. coli BL21/LBH-10 was 44.4%. The molecular weight of the CMCase with a His-tag was determined as 56 kDa. Its Km and Vmax were 7.4 g/L and 70.9 g/L min, respectively. The CMCase with a His-tag hydrolyzed avicel, carboxymethylcellulose (CMC), filter paper, pullulan, and xylan but did not hydrolyze cellobiose and p-nitrophenyl-β-d-glucopyranoside. The optimal temperature for reaction was 50 °C and more than 75% of its original activity was maintained at broad temperatures ranging from 20 to 70 °C after 24 h. The optimal pH was 4.0 and more than 60% of its original activity was maintained at pH ranging from 4.0 to 7.0. The activity of the CMCase with a His-tag was enhanced by CoCl2, KCl, PbCl2, RbCl2, and SrCl2 until the concentration of 100 mM, but inhibited by EDTA, HgCl2, MnCl2, and NiCl2. The characteristics of the CMCase with a His-tag produced by E. coli BL21/LBH-10 were little different from the CMCase without a His-tag, which seemed to resulted from the conformational change in the structure due to a His-tag. The purification yield of the CMCase with a His-tag using affinity chromatography from the cell broth after cell breakdown was proven to be more economic than that from the supernatant with its low concentration of cellulase.
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This paper was financially supported by Dong-A University Research Fund.
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The authors declare that they have no conflict of interest.
This article does not contain any studies with human participants performed by any of authors.
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