Applied Biochemistry and Biotechnology

, Volume 170, Issue 4, pp 934–950

Zingipain, a Ginger Protease with Acetylcholinesterase Inhibitory Activity

  • Porlin Rungsaeng
  • Polkit Sangvanich
  • Aphichart Karnchanatat

DOI: 10.1007/s12010-013-0243-x

Cite this article as:
Rungsaeng, P., Sangvanich, P. & Karnchanatat, A. Appl Biochem Biotechnol (2013) 170: 934. doi:10.1007/s12010-013-0243-x


In order to search for new acetylcholinesterase inhibitors (AChEIs), 15 Zingiberaceae plants were tested for AChEI activity in rhizome extracts. The crude homogenate and ammonium sulfate cut fraction of Zingiber officinale contained a significant AChEI activity. Eighty percent saturation ammonium sulfate precipitation and diethylaminoethyl cellulose ion exchange chromatography (unbound fraction) enriched the protein to a single band on nondenaturing and reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis (approximately 33.5 kDa). Gelatin-degrading zymography showed that the AChEI-containing band also contained cysteine protease activity. The AChEI activity was largely stable between −20 and 60 °C (at least over 120 min) and over a broad pH range (2–12). The AChEI activity was stimulated strongly by Mn2+ and Cu2+ at 1–10 mM and weakly by Ca2+, Fe2+, Mg2+, and Zn2+ at 1 mM, but was inhibited at 10 mM. In contrast, Hg2+ and ethylenediaminetetraacetic acid were very and moderately strongly inhibitory, respectively. In-gel tryptic digestion with liquid chromatography–tandem mass spectroscopy resolution revealed two heterogeneous peptides, a 16-amino-acid-long fragment with 100 % similarity to zingipain-1, which is a cysteine protease from Z. officinale, and a 9-amino-acid-long fragment that was 100 % identical to actinidin Act 2a, suggesting that the preparation was heterogeneous. AChEI exhibited noncompetitive inhibition of AChE for the hydrolysis of acetylthiocholine iodide with a Ki value of 9.31 mg/ml.


Acetylcholinesterase inhibitorGinger proteaseZingipain

Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  • Porlin Rungsaeng
    • 1
  • Polkit Sangvanich
    • 2
  • Aphichart Karnchanatat
    • 3
  1. 1.Program in Biotechnology, Faculty of ScienceChulalongkorn UniversityBangkokThailand
  2. 2.Department of Chemistry, Faculty of ScienceChulalongkorn UniversityBangkokThailand
  3. 3.Institute of Biotechnology and Genetic EngineeringChulalongkorn UniversityBangkokThailand