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20 ประจำป
2553
- 12 -
Purification and Characteristics of Trypsins from Cold-Zone Fish, Pacific Cod
(
Gadus macrocephalus
) and Saffron Cod (
Eleginus gracilis
)
Tomoyoshi Fuchise
1
, Hideki Kishimura
2
, Haruo Sekizaki
1
, Yoshiyuki Nonami
2
, Gaku Kanno
2
,
Sappasith Klomklao
3
, Soottawat Benjakul
4
and Byung-Soo Chun
5
Abstract
Trypsins from the pyloric ceca of Pacific cod (
Gadus macrocephalus
) (GM-T) and saffron
cod (
Eleginus gracilis
) (EG-T) were purified by gel filtration on Sephacryl S-200 and Sephadex
G-50. The final enzyme preparations were nearly homogeneous on SDS–PAGE and the molecular
weights of both enzymes were estimated to be approximately 24 kDa by SDS–PAGE. The specific
trypsin inhibitors, soybean trypsin inhibitor and TLCK, strongly inhibited the activities of GM-T and
EG-T. The optimum pH and optimum temperature of both trypsins were around pH 8.0 and 50
o
C,
respectively, using N
α
-p-tosyl-L-arginine methyl ester as substrate. The GM-T and EG-T were
unstable above 30
o
C and belowpH5.0, and theywere stabilized by calcium ion. The N-terminal amino
acid sequences of GM-T (IVGGYECTRHSQAHQVSLNS) and EG-T (IVGGYECPRHSQAHQVS-
LNS) were found. The percentage of hydrophobic amino acid in the N-terminal 20 amino acids
sequences of these cold-zone fish trypsins was lower (28%) than those of temperate-zone fish
trypsins (34%), tropical-zone fish trypsins (37%) and mammalian trypsins (34%). Whereas the
content of charged amino acids in the GM-T and EG-T was relatively higher than those of trypsins
from temperate-zone fish, tropical-zone fish and mammals. Moreover, the GM-T catalyzed
synthesis of N
α
-(tert-butoxycarbonyl)-L-alanyl-L-alanine-p-nitroanilide (N
α
-Boc-L-Ala-
L-Ala-pNA) has been studied by using N
α
-(tert-butoxycarbonyl)-L-alanine-p-guanidinophenyl
ester [N
α
-Boc-L-Ala- OpGu (inverse substrate)] as acyl donor and L-alanine-p-nitroanilide
(L-Ala-pNA) as acyl acceptor, respectively.
Keywords:
Pacific cod,
Gadus macrocephalus
, Saffron cod,
Eleginus gracilis
, Pyloric ceca,
Trypsin, Thermostability, Peptide synthesis
1
Faculty of Pharmaceutical Sciences, Health Sciences University of Hokkaido, Ishikari-Tobetsu, Hokkaido
061-0293, Japan
2
Laboratory of Marine Products and Food Science, Research Faculty of Fisheries Sciences, Hokkaido University,
Hakodate, 041-8611, Hokkaido Japan
3
Department of Food Science and Technology, Faculty of Technology and Community Development, Thaksin
University, Phatthalung Campus, Phatthalung 93110, Thailand
4
Department of Food Technology, Faculty of Agro-Industry, Prince of Songkla University, Hat Yai, Songkhla
90112, Thailand
5
Faculty of Food Science and Biotechnology, Pukyong National University, Busan 608-737, Korea
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