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20 ประจำป
2553
- 9 -
Trypsin from the Pyloric Ceca of Pectoral Rattail (
Coryphaenoides pectoralis
):
Purification and Characterization.
Sappasith Klomklao
1*
, Hideki Kishimura
2
and Soottawat Benjakul
3
Abstract
Trypsin from the pyloric ceca of pectoral rattail (
Coryphaenoides pectoralis
) was purified
and characterized. Purification was carried out by ammonium sulfate precipitation, followed
by column chromatographies on Sephacryl S-200, DEAE-cellulose and Sephadex G-50. The
enzyme was purified 89-fold with a yield of 2.2%. Purified trypsin had an apparent molecular
weight of 24 kDa when analyzed using SDS-PAGE and size exclusion chromatography. Optimal
profiles of pH and temperature of the enzyme were 8.5 and 45
o
C, respectively, using N
α
-p
-tosyl-L-
argininemethylesterhydrochlorideasasubstrate.ItwasstableinawidepHrangeof6-11butunstableata
temperature greater than 40
o
C. Trypsin was stabilized by calcium ion. The activity of purified trypsin
was effectively inhibited by soybean trypsin inhibitor and TLCK and was partially inhibited by
EDTA. Activity continuously decreased with increasing NaCl concentration (0-30%). The kinetic
trypsin constants K
m
and K
cat
were 0.15 mM and 210 s
-1
, respectively, while the catalytic efficiency
(K
cat
/K
m
) was 1400 s
-1
mM
-1
. The N-terminal amino acid sequence of trypsin was determined to
be 12 residues (IVGGYECQEHSQ), and the sequence showed high homology to other fish trypsins.
Keywords:
Trypsin, Purification, Isolation, Pyloric ceca, 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, 041-8611, Hokkaido Japan
3
Department of Food Technology, Faculty of Agro-Industry, Prince of Songkla University, Hat Yai, Songkhla
90112, Thailand
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