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Oriented Immobilization and Characterization of a Poly-Lysine-Tagged Cephalosporin C Acylase on Glyoxyl Agarose Support

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Abstract

Cephalosporin C acylase (CCA), an important industrial enzyme for the production of 7-aminocephalosporanic acid, has very limited and scattered surface lysine residues. A mutant of cephalosporin C acylase (mCCA) has been designed to fuse a poly-lysine tag to the C-terminal of the β-subunit, which is far away from the active site. The free mCCA showed a near equal specific activity with the wild-type CCA, while a much higher activity recovery was obtained for the mCCA than its wild-type counterpart after immobilization on glyoxyl agarose support (73.3 versus 53.3 %). The mCCA’s oriented immobilization enables it to obtain a higher substrate affinity and even a higher thermal stability than the wild-type enzyme. The improvement of stability might be attributed to the multipoint covalent attachment by the oriented enzyme immobilization via the adhered poly-lysine tag, which prevents the dissociation of the β-subunit of CCA from the support.

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Acknowledgments

This work was supported by the National Science Foundation of China (21276023; 21476025), Beijing Natural Science foundation (2143041), and the Fundamental Research Funds for the Central Universities of China (FRF-BR-11-002A; FRF-SD-12-007A). The authors thank North China Pharmaceutical Co., Ltd. for the generous supply of CPC and 7-ACA.

Conflict of Interest

The authors have no conflict of interest to declare.

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Authors and Affiliations

  1. School of Chemistry and Biological Engineering, University of Science and Technology Beijing, Beijing, 100083, China

    Hui Luo, Huan Zhao & Qixin Wang

  2. School of Civil and Environmental Engineering, University of Science and Technology Beijing, Beijing, 100083, China

    Yanhong Chang

  3. Key Laboratory of Educational Ministry for High Efficient Mining and Safety in Metal Mine, University of Science and Technology Beijing, Beijing, 100083, China

    Yanhong Chang

  4. Department of Chemical Engineering, Tsinghua University, Beijing, 100084, China

    Huimin Yu & Zhongyao Shen

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  1. Hui Luo
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  2. Huan Zhao
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  3. Yanhong Chang
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  4. Qixin Wang
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  5. Huimin Yu
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  6. Zhongyao Shen
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Correspondence to Hui Luo.

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Luo, H., Zhao, H., Chang, Y. et al. Oriented Immobilization and Characterization of a Poly-Lysine-Tagged Cephalosporin C Acylase on Glyoxyl Agarose Support. Appl Biochem Biotechnol 175, 2114–2123 (2015). https://doi.org/10.1007/s12010-014-1411-3

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  • DOI: https://doi.org/10.1007/s12010-014-1411-3

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