Simple Preparation of Pacific Cod Trypsin for Enzymatic Peptide Synthesis

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. 1. Introduction Proteinases from cold-water fish are of interest to us owing to their greater proteolytic activity towards native protein substrates and lower activation energy for catalysis compared with proteinases from mammalian or microbial sources [1]. Also, their survival in cold water required adaptation of their proteinase activity to low temperatures of their habitats. Proteinases from cold-adapted fish thus often have higher enzymatic activity at low temperatures than those from warm-blooded animals [2, 3]. High activity of these fish proteinases at low temperatures may be interesting for several industrial applications, such as in certain food processing operations that require low processing temperatures. Furthermore, proteinases from cold adapted fish inactivated at relatively low temperatures making such enzymes potentially useful in food applications where easy enzyme denaturation is desirable [4]. One of the main digestive proteinases, which are detected in pyloric caeca and intestine of fish, is trypsin (EC 3.4.21.4). Trypsin is a member of a large family of