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Fish Physiology and Biochemistry

, Volume 39, Issue 2, pp 121–130 | Cite as

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

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

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.

Keywords

Chymotrypsin Enzyme purification Pterygoplichthys disjunctivus Viscera 

Notes

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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Copyright information

© Springer Science+Business Media B.V. 2012

Authors and Affiliations

  • Ana Gloria Villalba-Villalba
    • 1
  • Juan Carlos Ramírez-Suárez
    • 1
  • Ramón Pacheco-Aguilar
    • 1
  • Elisa Miriam Valenzuela-Soto
    • 1
  • María Elena Lugo-Sánchez
    • 1
  • Ciria Guadalupe Figueroa-Soto
    • 1
  1. 1.Centro de Investigación en Alimentación y Desarrollo (CIAD)HermosilloMexico

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