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Gene cloning and enzymatic characterization of an endoprotease Endo-Pro-Aspergillus niger

  • Biotechnology Methods
  • Published:
Journal of Industrial Microbiology & Biotechnology

Abstract

A novel endoprotease Endo-Pro-Aspergillus niger (endoprotease EPR) was first successfully expressed at high level in the methylotrophic yeast Pichia pastoris and the purification procedure was established. The endoprotease EPR is 95 % identity with proline specific endopeptidase from A. niger CBS513.88 (EMBL; AX458699), while sharing low identity with those from other microorganisms. The purified endoprotease EPR was a monomer of 60 kDa. Furthermore, the peptide mass fingerprinting (PMF) analysis confirmed that the purified protein was an endoprotease Endo-Pro-Aspergillus niger. A three-dimensional model revealed that the active site of the enzyme was located in Ser(179)-Asp(458)-His(491), based on template 3n2zB with sequence identity of 17.6 %. The optimum pH and temperature of the endoprotease EPR were pH 4–5 and 35 °C, and the stabilities were pH 3–7 and 15–60 °C, respectively. Furthermore, the endoprotease EPR had the ability to digest peptides with the C-terminal of proline as well as alanine, and was also capable of hydrolyzing larger peptides. The properties of the endoprotease EPR made it a highly promising candidate for future application in the field of brewing and food process.

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Acknowledgments

Financial support from the National High Technology Research and Development Program of China (863 Program) (No. 2012AA022207 and 2008AA10Z304), the Fundamental Research Funds for the Central Universities (JUSRP11014), and the Ministry of Education, R.P. China, and from NSFC (20802027) is greatly appreciated.

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  1. State Key Laboratory of Food Science and Technology, The Key Laboratory of Industrial Biotechnology of Ministry of Education, School of Biotechnology, Jiangnan University, 1800 Lihu Ave, Wuxi 214122, Jiangsu, People’s Republic of China

    Chao Kang, Xiao-Wei Yu & Yan Xu

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  1. Chao Kang
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  2. Xiao-Wei Yu
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  3. Yan Xu
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Correspondence to Xiao-Wei Yu or Yan Xu.

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Kang, C., Yu, XW. & Xu, Y. Gene cloning and enzymatic characterization of an endoprotease Endo-Pro-Aspergillus niger . J Ind Microbiol Biotechnol 40, 855–864 (2013). https://doi.org/10.1007/s10295-013-1284-4

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  • DOI: https://doi.org/10.1007/s10295-013-1284-4

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