Purification and characterization of three trypsin isoforms from viscera of sardinelle (Sardinella aurita).
Three trypsin isoforms A, B and C were purified to homogeneity from the viscera of sardinelle (Sardinella aurita). Purification was achieved by ammonium sulfate precipitation (20-70% (w/v)), Sephadex G-100 gel filtration and Mono Q-Sepharose anion-exchange chromatography. The molecular weights of these purified enzymes were estimated to be 28.8 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Based on the native PAGE and casein-zymography, each purified trypsin appeared as a single band. Trypsins A and C exhibited the maximal activity at 55°C, while trypsin B at 50°C. All isoforms showed the same optimal pH (pH 9.0) using Nα-benzoyl-DL: -arginine-p-nitroanilide (BAPNA) as a substrate. The three trypsins were stable at temperatures below 40°C and over a broad pH range (7.0-11.0). The activities of the three isoforms were strongly inhibited by soybean trypsin inhibitor and phenylmethylsulfonyl fluoride, a serine protease inhibitor, and partially inhibited by ethylenediaminetetraacetic acid, a metalloenzyme inhibitor. Kinetic constants of trypsins A, B and C for BAPNA were evaluated at 25°C and pH 9.0. The values of K (m) and k (cat) were 0.125, 0.083 and 0.10 mM, and 2.24, 1.21 and 5.76 s(-1), respectively. The N-terminal sequences of the first 10 amino acids were "I V G G Y E C Q K Y" for trypsin A and "I V G G Y E A Q S Y" for trypsins B and C. These sequences showed highly homology to other fish trypsins.
Ben Khaled
Hayet
H
Laboratoire de Génie Enzymatique et de Microbiologie, Ecole Nationale d'Ingénieurs de Sfax, Route Soukra Km 3.5, Sfax, Tunisia.
Jellouli
Kemel
K
Souissi
Nabil
N
Ghorbel
Sofiane
S
Barkia
Ahmed
A
Nasri
Moncef
M
D000595:Amino Acid Sequence; D000818:Animals; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D005399:Fishes / Q000502:physiology*; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D008670:Metals / Q000494:pharmacology; D008969:Molecular Sequence Data; D016415:Sequence Alignment; D012965:Sodium Chloride / Q000494:pharmacology; D013696:Temperature; D014357:Trypsin / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D014781:Viscera / Q000201:enzymology*
D004791:Enzyme Inhibitors; D007527:Isoenzymes; D008670:Metals; D012965:Sodium Chloride; D014357:Trypsin