Journal of Protein Chemistry

, Volume 20, Issue 1, pp 49–58

Kinetic and Structural Properties of Two Isoforms of Trypsin Isolated from the Viscera of Japanese Anchovy, Engraulis japonicus

  • Md. Nazmul Ahsan
  • Shugo Watabe

DOI: 10.1023/A:1011005104727

Cite this article as:
Ahsan, M.N. & Watabe, S. J Protein Chem (2001) 20: 49. doi:10.1023/A:1011005104727


Two isoforms of anchovy trypsin (aT-I and aT-II) were purified from the visceral extracts by (NH4)2SO4 fractionation followed by affinity chromatography, gel filtration, and ion-exchange chromatography. The homogeneity of the purified preparation was evidenced by both native- and SDS-PAGE, and further by gelatin zymography. Identities of aT-I and aT-II as trypsins were established by N-terminal amino acid sequencing, which matched exactly to the corresponding stretches of their respective amino acid sequences obtained by molecular cloning [Ahsan et al. (2000), Marine Biotechnol., in press]. Both isoforms were completely inhibited by serine protease inhibitors as well as by specific trypsin inhibitors. The purified anchovy trypsins showed considerably higher catalytic efficiencies (kcat/Km) than bovine trypsin as measured toward benzoyl-arginine p-nitroanilide (BAPA) and benzoyl-arginine ethyl ester (BAEE) at 25°C; in particular, aT-II was 35 times more efficient than its mammalian counterpart against BAPA. This was due mainly to a dramatic decrease of Km values for anchovy trypsins, which are indicative of an evolutionary response toward increased substrate binding at suboptimal temperatures in the marine environment.

Anchovy trypsin isoform characterization S1 pocket 

Copyright information

© Plenum Publishing Corporation 2001

Authors and Affiliations

  • Md. Nazmul Ahsan
    • 1
  • Shugo Watabe
    • 1
  1. 1.Laboratory of Aquatic Molecular Biology and Biotechnology, Graduate School of Agricultural and Life SciencesUniversity of TokyoTokyoJapan

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