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.

AnchovytrypsinisoformcharacterizationS1 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