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งที่
20 ประจำป
2553
- 11 -
Biochemical Properties of Two Isoforms of Trypsin Purified from the Intestine
of Skipjack Tuna (
Katsuwonus pelamis
)
Sappasith Klomklao
1*
,Hideki Kishimura
2
, Yoshiyuki Nonami
2
and Soottawat Benjakul
3
Abstract
Two trypsins (A and B) from the intestine of skipjack tuna (
Katsuwonus pelamis
) were
purified by Sephacryl S-200, Sephadex G-50 and DEAE-cellulose with a 177- and 257-fold increase
in specific activity and 23% and 21% recovery for trypsin A and B, respectively. Purified trypsins
revealed a single band on native-PAGE. The molecular weights of both trypsins were 24 kDa
as estimated by size exclusion chromatography and SDS–PAGE. Trypsin A and B exhibited the
maximal activity at 55 °C and 60 °C, respectively, and had the same optimal pH at 9.0. Both
trypsins were stable up to 50 °C and in the pH range from 6.0 to 11.0. Both trypsin A and B
were stabilized by calcium ion. Activity of both trypsins continuously decreased with increasing
NaCl concentration (0–30%) and were inhibited by the specific trypsin inhibitors – soybean
trypsin inhibitor and
N-p
-tosyl-l-lysine chloromethyl ketone. Apparent
K
m
and
K
cat
of trypsin
A and B were 0.22–0.31 mM and 69.5–82.5 S
-1
, respectively. The N-terminal amino acid
sequences of the first 20 amino acids of trypsin A and B were IVGGYECQAHSQPPQVSLNA
and IVGGYECQAHSQPPQVSLNS, respectively.
Keywords:
Trypsin, Serine proteinase, Tuna, Purification, Isolation, Viscera, N-terminal
amino acid sequence
1
Department of Food Science and Technology, Faculty of Technology and Community Development, Thaksin
University, Phatthalung Campus, Phatthalung 93110, Thailand
*
Corresponding author: Sappasith Klomklao, Fax: +66 7469 3996; e-mail:
2
Laboratory of Marine Products and Food Science, Research Faculty of Fisheries Sciences, Hokkaido University,
Hakodate, Hokkaido 041-8611, Japan
3
Department of Food Technology, Faculty of Agro-Industry, Prince of Songkla University, Hat Yai, Songkhla
90112, Thailand
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