Biotechnology Letters

, Volume 35, Issue 12, pp 2121–2127

Glycosylation analysis of recombinant neutral protease I from Aspergillus oryzae expressed in Pichia pastoris

  • Da Lei
  • Yang Xu
  • Qinghua He
  • Yifeng Pan
  • Bo Chen
  • Liang Xiong
  • Yanping Li
Original Research Paper

DOI: 10.1007/s10529-013-1314-3

Cite this article as:
Lei, D., Xu, Y., He, Q. et al. Biotechnol Lett (2013) 35: 2121. doi:10.1007/s10529-013-1314-3

Abstract

Neutral protease I from Aspergillus oryzae 3.042 was expressed in Pichia pastoris and its N-glycosylation properties were analyzed. After purification by nickel-affinity chromatography column, the recombinant neutral protease (rNPI) was confirmed to be N-glycosylated by periodicacid/Schiff’s base staining and Endo H digestion. Moreover, the deglycosylated protein’s molecular weight decreased to 43.3 kDa from 54.5 kDa analyzed by SDS-PAGE and MALDI–TOF–MS, and the hyperglycosylation extent was 21 %. The N-glycosylation site of rNPI was analyzed by nano LC–MS/MS after digesting by trypsin and Glu-C, and the unique potential site Asn41 of mature peptide was found to be glycosylated. Homology modeling of the 3D structure of rNPI indicated that the attached N-glycans hardly affected neutral protease’s activity due to the great distance away from the active site of the enzyme.

Keywords

Aspergillus oryzae N-glycosylation Nano LC–MS/MS Pichia pastoris Protease Recombinant neutral protease I 

Copyright information

© Springer Science+Business Media Dordrecht 2013

Authors and Affiliations

  • Da Lei
    • 1
  • Yang Xu
    • 1
    • 2
  • Qinghua He
    • 1
    • 2
  • Yifeng Pan
    • 1
  • Bo Chen
    • 1
  • Liang Xiong
    • 1
  • Yanping Li
    • 1
    • 2
  1. 1.State Key Laboratory of Food Science and TechnologyNanchang UniversityNanchangChina
  2. 2.Jiangxi-OAI Joint Research InstituteNanchang UniversityNanchangChina

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