Mackerel trypsin purified from defatted viscera by supercritical carbon dioxide.

Byung-Soo Chun,Soottawat Benjakul,Hideki Kishimura,Sappasith Klomklao,Sitthipong Nalinanon

doi:10.4061/2011/728082

Abstract

Viscera of mackerel (Scomber sp.) were defatted by supercritical carbon dioxide (SCO2) treatment. Trypsin (SC-T) was then extracted from the defatted powder and purified by a series of chromatographies including Sephacryl S-200 and Sephadex G-50. The purified SC-T was nearly homogeneous on SDS-PAGE, and its molecular weight was estimated as approximately 24,000 Da. N-terminal twenty amino acids sequence of SC-T was IVGGYECTAHSQPHQVSLNS. The specific trypsin inhibitors, soybean trypsin inhibitor and TLCK, strongly inhibited the activities of SC-T. The pH and temperature optimums of SC-T were at around pH 8.0 and 60°C, respectively, using N α-p-tosyl-L-arginine methyl ester as a substrate. The SC-T was unstable below pH 5.0 and above 40°C, and it was stabilized by calcium ion. These enzymatic characteristics of SC-T were the same as those of other fish trypsins, especially spotted mackerel (S. borealis) trypsin, purified from viscera defatted by acetone. Therefore, we concluded that the SCO2 defatting process is useful as a substitute for organic solvent defatting process.

Highlights

Fish viscera are one of the sources of digestive enzymes that may have some unique properties of fascinate with both basic research and industrial applications
With the aim of utilization of fish trypsin for food industry, we purified a trypsin (SC-T) from the mackerel viscera powder treated by Supercritical CO2 (SCO2) defatting process and compared its enzymatic properties with those of other fish trypsins purified from the viscera defatted by acetone
Nα-p-Tosyl-Larginine methyl ester hydrochloride (TAME) and Ethylenediaminetetraacetic acid (EDTA) were obtained from Wako Pure Chemicals (Osaka, Japan). 1-(L-trans-epoxysuccinylleucylamino)-4-guanidinobutane (E-64), soybean trypsin inhibitor, N-p-tosyl-L-lysine chloromethyl ketone (TLCK), and pepstatin A were purchased from Sigma Chemical Co

Summary

Introduction

Fish viscera are one of the sources of digestive enzymes that may have some unique properties of fascinate with both basic research and industrial applications. Their survival in waters required adaptation of their enzyme activity to low temperatures of their habitats. The strong positive correlation between the habitat temperature of marine fish and the thermostability of its trypsin has been demonstrated [3,4,5,6,7,8,9,10,11]. High activity at low temperatures and instability against heat, low pH, and autolysis of fish proteinases are interesting for some industrial applications [12]. Pacific cod and Atlantic cod trypsins were utilized as catalyst of enzymatic peptide synthesis [9, 15]

Methods

Results

Conclusion