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Engineering of a CPC acylase using a facile pH indicator assay

  • Biocatalysis
  • Published:
Journal of Industrial Microbiology & Biotechnology

Abstract

Cephalosporin C (CPC) acylase is important for the one-step production of 7-aminocephalosporanic acid (7-ACA), a key intermediate for cephalosporin antibiotics. However, its application is hampered by the low activity, substrate inhibition, and product inhibition. In this study, two rounds of combinatorial active-site saturation testing (CASTing) were carried out on the CPC acylase acyII from Pseudomonas SE83, and one mutant H57βA/H70βY with no substrate inhibition was obtained. For further engineering to reduce the product inhibition, a quick pH indicator assay was developed, allowing for real-time monitoring of the product inhibition in the presence of added 7-ACA. The utility of the assay was demonstrated by screening six libraries of site-directed saturation mutagenesis libraries of H57βA/H70βY. A new mutant H57βA/H70βY/I176βN was obtained, which showed a k cat 3.26-fold and a K IP 3.08-fold that of the wild type, respectively. Given the commercial value of the enzyme, both this pH indicator assay and the triple mutant should be useful for further engineering of the enzyme to increase the specific activity and to decrease the product inhibition.

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Acknowledgments

This work was supported by grants from the National Basic Research Program of China (2009CB724704 and 2013CB733902), and a grant from the National High-Tech Program of China (2012AA022205).

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Author notes

  1. Xiangdong Huo

    Present address: Institute of Microbiology, Xinjiang Academy of Agricultural Sciences, 403 Nanchang Road, Urumqi, 830091, Xinjiang, China

  2. Yu Qian

    Present address: China Huanqiu Contracting and Engineering Corporation, One Chuangda Second Road, Beijing, 100029, China

Authors and Affiliations

  1. Department of Chemical Engineering, National Engineering Laboratory for Industrial Enzymes, Tsinghua University, One Tsinghua Garden Road, Beijing, 100084, China

    Yingzhou Xiao, Xiangdong Huo, Yu Qian, Yan Zhang, Pingkai Ouyang & Zhanglin Lin

  2. School of Life Sciences, Tsinghua University, One Tsinghua Garden Road, Beijing, 100084, China

    Guoqiang Chen

  3. College of Biotechnology and Pharmaceutical Engineering, Nanjing University of Technology, 30 South Puzhu Road, Nanjing, 211816, Jiangsu, China

    Pingkai Ouyang

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  1. Yingzhou Xiao
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  4. Yan Zhang
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Correspondence to Pingkai Ouyang or Zhanglin Lin.

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Xiao, Y., Huo, X., Qian, Y. et al. Engineering of a CPC acylase using a facile pH indicator assay. J Ind Microbiol Biotechnol 41, 1617–1625 (2014). https://doi.org/10.1007/s10295-014-1501-9

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  • DOI: https://doi.org/10.1007/s10295-014-1501-9

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