Purification and Characterization of Trypsin from the Spleen of Tongol Tuna (Thunnus tonggol)

Trypsin from tongol tuna (Thunnus tonggol) spleen was purified to 402-fold by ammonium sulfate precipitation, followed by a series of chromatographic separations. The molecular mass of trypsin was estimated to be 24 kDa by size-exclusion chromatography and sodium dodecyl sulfate−polyacrylamide gel e...

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Bibliographic Details
Published in:Journal of agricultural and food chemistry 2006-07, Vol.54 (15), p.5617-5622
Main Authors: Klomklao, Sappasith, Benjakul, Soottawat, Visessanguan, Wonnop, Kishimura, Hideki, Simpson, Benjamin K
Format: Article
Language:English
Subjects:
Amino Acid Sequence
amino acid sequences
Animals
Biological and medical sciences
enzyme activity
Enzyme Stability
fish waste
Food industries
Fundamental and applied biological sciences. Psychology
Hydrogen-Ion Concentration
Kinetics
Molecular Sequence Data
Molecular Weight
proteolysis
purification
spleen
Spleen - enzymology
Temperature
Thunnus
Thunnus tonggol
trypsin
Trypsin - chemistry
Trypsin - isolation & purification
Trypsin - metabolism
Trypsin Inhibitors - pharmacology
Tuna
waste utilization
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Summary:Trypsin from tongol tuna (Thunnus tonggol) spleen was purified to 402-fold by ammonium sulfate precipitation, followed by a series of chromatographic separations. The molecular mass of trypsin was estimated to be 24 kDa by size-exclusion chromatography and sodium dodecyl sulfate−polyacrylamide gel electrophoresis (SDS−PAGE). Trypsin appearing as a single band on native PAGE showed the maximal activity at pH 8.5 and 65 °C. It was stable in a wide pH range of 6−11 but unstable at the temperatures greater than 50 °C. The enzyme required calcium ion for thermal stability. The activity was strongly inhibited by 1.0 g/L soybean trypsin inhibitor and 5 mM TLCK and partially inhibited by 2 mM ethylenediaminetetraacetic acid. Activity was lowered with an increasing NaCl concentration (0−30%). The enzyme had a K m for N α-p-tosyl-l-arginine methyl ester hydrochloride of 0.25 mM and a K cat of 200 s-1. The N-terminal amino acid sequence of trypsin was determined as IVGGYECQAHSQPHQVSLNA and was very homologous to other trypsins. Keywords: Trypsin; proteinase; tuna; viscera; purification, N-terminal amino acid sequence
ISSN:0021-8561
1520-5118
DOI:10.1021/jf060699d