Trypsin from the pyloric caeca of bluefish (Pomatomus saltatrix).
Trypsin was purified from the pyloric caeca of bluefish (Pomatomus saltatrix) by ammonium sulfate precipitation, acetone precipitation and soybean trypsin inhibitor-Sepharose 4B affinity chromatography. Bluefish trypsin migrated as a single band using both sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and native-PAGE and had a molecular mass of 28 kDa. The optima pH and temperature for the hydrolysis of benzoyl-dl-arginine-p-nitroanilide (BAPNA) were 9.5 and 55 degrees C, respectively. The enzyme was stable over a broad pH range (7 to 12), but was unstable at acidic pH, and at temperatures greater than 40 degrees C. The enzyme was inhibited by specific trypsin inhibitors: soybean trypsin inhibitor (SBTI), N-p-tosyl-l-lysine chloromethyl ketone (TLCK) and the serine protease inhibitor phenylmethyl sulfonylfluoride (PMSF). CaCl2 partially protected trypsin against activity loss at 40 degrees C, but NaCl (0 to 30%) decreased the activity in a concentration dependent manner. The N-terminal amino acid sequence of trypsin was determined as IVGGYECKPKSAPVQVSLNL and was highly homologous to other known vertebrate trypsins.
Klomklao
Sappasith
S
Department of Food Science and Technology, Faculty of Technology and Community Development, Thaksin University, Phattalung Campus, Phattalung, 93110, Thailand.
Benjakul
Soottawat
S
Visessanguan
Wonnop
W
Kishimura
Hideki
H
Simpson
Benjamin K
BK
D020816:Amino Acid Motifs; D000818:Animals; D002122:Calcium Chloride / Q000494:pharmacology; D002432:Cecum / Q000201:enzymology*; D016384:Consensus Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability / Q000187:drug effects; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D010473:Perciformes; D011708:Pylorus / Q000201:enzymology*; D012965:Sodium Chloride / Q000494:pharmacology; D013696:Temperature; D013816:Thermodynamics; D014357:Trypsin / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D014361:Trypsin Inhibitors / Q000494:pharmacology
D014361:Trypsin Inhibitors; D012965:Sodium Chloride; D014357:Trypsin; D002122:Calcium Chloride