title: Trypsin from viscera of vermiculated sailfin catfish, Pterygoplichthys disjunctivus, Weber, 1991: its purification and characterization.
abstract: Pterygoplichthys disjunctivus viscera trypsin was purified by fractionation with ammonium sulphate, gel filtration, affinity and ion exchange chromatography (DEAE-Sepharose). Trypsin molecular weight was approximately 27.5kDa according to SDS-PAGE, shown a single band in zymography. It exhibited maximal activity at pH 9.5 and 40°C, using N-benzoyl-dl-arginine-p-nitroanilide (BAPNA) as substrate. Enzyme was effectively inhibited by phenyl methyl sulphonyl fluoride (PMSF) (100%), N-α-p-tosyl-l-lysine chloromethyl ketone (TLCK) (85.4%), benzamidine (80.2%), and soybean trypsin inhibitor (75.6%) and partially inhibited by N-tosyl-l-phenylalanine chloromethyl ketone (TPCK) (10.3%), ethylendiaminetetraacetic acid (EDTA) (8.7%) and pepstatin A (1.2%). Enzyme activity was slightly affected by metal ions (Fe(2+)>Hg(2+)>Mn(2+)>K(+)>Mg(2+)>Li(+)>Cu(2+)). Trypsin activity decreased continuously as NaCl concentration increased (0-30%). Km and kcat values were 0.13mM and 1.46s(-1), respectively. Results suggest the enzyme have a potential application where room processing temperatures (25-35°C) or high salt (30%) concentration are needed, such as in fish sauce production.
authors.affiliation: Centro de Investigación en Alimentación y Desarrollo (CIAD), Carretera a La Victoria Km. 0.6, Apdo. Postal 1735, Hermosillo, Sonora 83000, Mexico.
authors.forename: Ana Gloria
authors.initials: AG
authors.lastname: Villalba-Villalba
authors.forename: Juan Carlos
authors.initials: JC
authors.lastname: Ramírez-Suárez
authors.forename: Elisa Miriam
authors.initials: EM
authors.lastname: Valenzuela-Soto
authors.forename: Guillermina García
authors.initials: GG
authors.lastname: Sánchez
authors.forename: Gisela Carvallo
authors.initials: GC
authors.lastname: Ruiz
authors.forename: Ramón
authors.initials: R
authors.lastname: Pacheco-Aguilar
mesh_terms: D000818:Animals; D002397:Catfishes / Q000378:metabolism*; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D029941:Fish Proteins / Q000737:chemistry* / Q000302:isolation & purification*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D014357:Trypsin / Q000737:chemistry* / Q000302:isolation & purification*; D014781:Viscera / Q000737:chemistry / Q000201:enzymology*
chemical_list: D029941:Fish Proteins; D014357:Trypsin