Trypsin from the pyloric ceca of pectoral rattail (Coryphaenoides pectoralis): purification and characterization.
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 degrees C, respectively, using N(alpha)-p-tosyl-l-arginine methyl ester hydrochloride as a substrate. It was stable in a wide pH range of 6-11 but unstable at a temperature greater than 40 degrees 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.
Klomklao
Sappasith
S
Department of Food Science and Technology, Faculty of Technology and Community Development, Thaksin University, Phattalung Campus, Phattalung, 93110, Thailand. sappasith@tsu.ac.th
Kishimura
Hideki
H
Benjakul
Soottawat
S
D000595:Amino Acid Sequence; D000818:Animals; D004795:Enzyme Stability; D029941:Fish Proteins / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D048388:Gadiformes / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D011708:Pylorus / Q000737:chemistry / Q000201:enzymology*; D016415:Sequence Alignment; D013379:Substrate Specificity; D014357:Trypsin / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism
D029941:Fish Proteins; D014357:Trypsin