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Molecular Biology Reports

, Volume 46, Issue 2, pp 1973–1983 | Cite as

Enhanced purification of histidine-tagged carboxymethylcellulase produced by Escherichia coli BL21/LBH-10 and comparison of its characteristics with carboxymethylcellulase without histidine-tag

  • Duk-Un Kang
  • Yong-Suk Lee
  • Jin-Woo LeeEmail author
Original Article
  • 101 Downloads

Abstract

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.

Keywords

Affinity chromatography Carboxymethylcellulase Characterization Escherichia coli BL21 Purification 

Notes

Acknowledgements

This paper was financially supported by Dong-A University Research Fund.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

This article does not contain any studies with human participants performed by any of authors.

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Copyright information

© Springer Nature B.V. 2019

Authors and Affiliations

  1. 1.Department of Applied Biology, Graduate SchoolDong-A UniversityBusanKorea
  2. 2.Department of BiotechnologyDong-A UniversityBusanKorea

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