Trypsins from yellowfin tuna (Thunnus albacores) spleen: purification and characterization.
Two anionic trypsins (A and B) were purified to homogeneity from yellowfin tuna (Thunnus albacores) spleen by a series of column chromatographies including Sephacryl S-200, Sephadex G-50 and DEAE-cellulose. Purity was increased to 70.6- and 91.5-fold with approximately 2.8% and 15.6% yield for trypsin A and B, respectively. The apparent molecular weight of both trypsins was estimated to be 24 kDa by size exclusion chromatography and SDS-PAGE. Both trypsin A and B appeared as a single band on native-PAGE. Trypsin A and B exhibited the maximal activity at 55 and 65 degrees C, respectively, and had the same optimal pH at 8.5 using TAME as a substrate. Both trypsins were stable to heat treatment up to 50 degrees C and in the pH range of 6.0 to 11.0. Both trypsin A and B were stabilized by calcium ion. The activities were inhibited effectively by soybean trypsin inhibitor, TLCK and partially inhibited by EDTA, but were not inhibited by E-64, N-ethylmaleimide, iodoacetic acid, TPCK and pepstatin A. Activity of both trypsins continuously decreased with increasing NaCl concentration (0-30%). Apparent Km and Kcat of trypsin A and B for TAME were 0.2-0.33 mM and 66.7-80 S(-1), respectively. The N-terminal amino acid sequences of trypsin A, IVGGYECQAHSQPHQVSLNA, and trypsin B, IVGGYECQAHSQPPQVSLNA, indicated the high homology between both enzymes.
Klomklao
Sappasith
S
Department of Food Technology, Faculty of Agro-Industry, Prince of Songkla University, Hat Yai, Songkhla, 90112, Thailand.
Benjakul
Soottawat
S
Visessanguan
Wonnop
W
Kishimura
Hideki
H
Simpson
Benjamin K
BK
Saeki
Hiroki
H
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002848:Chromatography, DEAE-Cellulose; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry; D017385:Sequence Homology; D013154:Spleen / Q000201:enzymology*; D014357:Trypsin / Q000145:classification / Q000302:isolation & purification* / Q000378:metabolism; D014361:Trypsin Inhibitors / Q000494:pharmacology; D014413:Tuna
D000596:Amino Acids; D010446:Peptide Fragments; D014361:Trypsin Inhibitors; D014357:Trypsin