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Purification and characterization of chymotrypsin from viscera of vermiculated sailfin catfish, Pterygoplichthys disjunctivus, Weber, 1991

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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: Hg2+, Fe2+, Cu2+, Li1+, Mg2+, K1+, Mn2+, while Ca2+ 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.

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Acknowledgments

This study was supported by the Fondo Mixto CONACYT-Gobierno del Estado de Michoacán under the project “Desarrollo Tecnológico para el Aprovechamiento e Industrialización del Pez Diablo en la Región del Bajo Balsas en Michoacán, FOMIX # 37147.”

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  1. Centro de Investigación en Alimentación y Desarrollo (CIAD), Carretera a La Victoria Km. 0.6. Apdo. Postal 1735, 83304, Hermosillo, Sonora, Mexico

    Ana Gloria Villalba-Villalba, Juan Carlos Ramírez-Suárez, Ramón Pacheco-Aguilar, Elisa Miriam Valenzuela-Soto, María Elena Lugo-Sánchez & Ciria Guadalupe Figueroa-Soto

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  1. Ana Gloria Villalba-Villalba
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  2. Juan Carlos Ramírez-Suárez
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  3. Ramón Pacheco-Aguilar
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  4. Elisa Miriam Valenzuela-Soto
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  5. María Elena Lugo-Sánchez
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  6. Ciria Guadalupe Figueroa-Soto
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Correspondence to Juan Carlos Ramírez-Suárez.

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Villalba-Villalba, A.G., Ramírez-Suárez, J.C., Pacheco-Aguilar, R. et al. Purification and characterization of chymotrypsin from viscera of vermiculated sailfin catfish, Pterygoplichthys disjunctivus, Weber, 1991. Fish Physiol Biochem 39, 121–130 (2013). https://doi.org/10.1007/s10695-012-9684-3

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  • DOI: https://doi.org/10.1007/s10695-012-9684-3

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