title: Purification and characterization of chymotrypsin from viscera of vermiculated sailfin catfish, Pterygoplichthys disjunctivus, Weber, 1991.
abstract: Pterygoplichthys disjunctivus viscera chymotrypsin was purified by fractionation with ammonium sulfate (30-70 % saturation), gel filtration, affinity, and ion exchange chromatography. Chymotrypsin molecular weight was approximately 29 kDa according to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), shown a single band in zymogram. Electrofocusing study suggested being an anionic enzyme (pI ≈ 3.9), exhibiting maximal activity at pH 9 and 50 °C, using Suc-Ala-Ala-Pro-Phe-p-nitroanilide (SAAPNA) as substrate. Enzyme was effectively inhibited by phenyl methyl sulfonyl fluoride (PMSF) (99 %), and N-tosyl-L-phenylalanine chloromethyl ketone (TPCK) (94 %). Enzyme activity was affected by the following ions in decreasing order: Hg(2+), Fe(2+), Cu(2+), Li(1+), Mg(2+), K(1+), Mn(2+), while Ca(2+) had no effect. Chymotrypsin activity decreased continuously as NaCl concentration increased (from 0 to 30 %). K m and V max values were 0.72 ± 1.4 mM and 1.15 ± 0.06 μmol/min/mg of protein, respectively (SAAPNA as substrate). Results suggest the enzyme has a potential application where low processing temperatures are needed, such as in fish sauce production.
authors: Centro de Investigación en Alimentación y Desarrollo, Carretera a La Victoria Km. 0.6. Apdo. Postal 1735, 83304 Hermosillo, Sonora, Mexico.
authors: Ana Gloria
authors: AG
authors: Villalba-Villalba
authors: Juan Carlos
authors: JC
authors: Ramírez-Suárez
authors: Ramón
authors: R
authors: Pacheco-Aguilar
authors: Elisa Miriam
authors: EM
authors: Valenzuela-Soto
authors: María Elena
authors: ME
authors: Lugo-Sánchez
authors: Ciria Guadalupe
authors: CG
authors: Figueroa-Soto
mesh_terms: D000818:Animals; D002397:Catfishes / Q000378:metabolism*; D005591:Chemical Fractionation; D002918:Chymotrypsin / Q000302:isolation & purification* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel / Q000662:veterinary; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D019216:Metals, Heavy / Q000378:metabolism; D008800:Mexico; D012965:Sodium Chloride / Q000378:metabolism; D013696:Temperature; D014781:Viscera / Q000737:chemistry*
chemical_list: D019216:Metals, Heavy; D012965:Sodium Chloride; D002918:Chymotrypsin