Table of Contents
Journal of Amino Acids
Volume 2011 (2011), Article ID 912382, 8 pages
Research Article

Simple Preparation of Pacific Cod Trypsin for Enzymatic Peptide Synthesis

1Faculty of Pharmaceutical Sciences, Health Sciences University of Hokkaido, Ishikari-Tobetsu, Hokkaido 061-0293, Japan
2Laboratory of Marine Products and Food Science, Research Faculty of Fisheries Sciences, Hokkaido University, Hakodate, Hokkaido 041-8611, Japan
3Department of Food Science and Technology, Faculty of Technology and Community Development, Thaksin University, Phattalung Campus, Phattalung 93110, Thailand
4Faculty of Agro-Industry, King Mongkut's Institute of Technology Ladkrabang, Choakhunthaharn Building, Choakhunthaharn Road, Ladkrabang, Bangkok 10520, Thailand
5Department of Food Technology, Faculty of Agro-Industry, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand
6Faculty of Food Science and Biotechnology, Pukyong National University, Busan 608-737, Republic of Korea

Received 24 February 2011; Revised 19 June 2011; Accepted 16 July 2011

Academic Editor: Nabil Miled

Copyright © 2011 Tomoyoshi Fuchise et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


Trypsin from the pyloric caeca of Pacific cod (Gadus macrocephalus) was easily prepared by affinity chromatography on Benzamidine Sepharose 6B and gel filtration on Superdex 75. Pacific cod trypsin was composed of three isozymes, and their molecular masses were estimated 23,756.34 Da, 23,939.62 Da, and 24,114.81 Da by desorption/ionization time-of-flight mass spectroscopy (MALDI/TOF-MS) and their isoelectric points (pIs) were approximately 5.1, 6.0, and 6.2, respectively. The isolated Pacific cod trypsin showed high similarity to other frigid-zone fish trypsins. The kinetic behavior of tryptic hydrolysis toward N-p-tosyl-L-arginine methyl ester hydrochloride (TAME), N-benzoyl-L-arginine p-nitroanilide hydrochloride (BAPA), and p-amidinophenyl ester were also analyzed. In addition, the cod trypsin-catalyzed dipeptide synthesis was investigated using twelve series of “inverse subdtrates” that is p- and m-isomer of amidinophenyl, guanidinophenyl, (amidinomethyl)phenyl, (guanidinomethyl)phenyl, and four position isomers of guanidinonaphtyl esters derived from N-(tert-butoxycarbonyl)amino acid as acyl donor components. They were found to couple with an acyl acceptor such as L-alanine p-nitroanilide to produce dipeptide in the presence of the trypsin. All inverse substrates tested in this study undergo less enantioselective coupling reaction. The p-guanidinophenyl ester was most practical substrate in twelve series tested. The enzymatic hydrolysis of the resulting products was negligible.