Purification of hydroperoxide lyase from tomato fruits

Authors

  • Purvika Shah Department of Biochemistry and Microbiology Rutgers University, New Brunswick, NJ
  • Theodore, Jr. Chase Department of Biochemistry and Microbiology Rutgers University, New Brunswick, NJ

Abstract

Tomato fruit hydroperoxide lyase was initially purified using the procedure set forth in Fauconnier et al. (1997) for tomato leaves. The enzyme, hydroperoxide lyase (HPOOH lyase) was difficult to purify; for this reason, several changes to the procedure of Fauconnier et al. were explored. Tomatoes at different stages of ripening were extracted, purified, and assayed to determine the stage with the highest activity. Storage of the tomato fruit for future extractions and storage of the enzyme for future analysis and purification was tested. Two types of assays, one involving disappearance of absorbance and the other involving appearance of absorbance, were also tested.


It was shown that polyethylene glycol (PEG), used to precipitate leaf enzyme, could not precipitate fruit enzyme. Ammonium sulfate was later used to precipitate the fruit enzyme, but the activity significantly decreased. Centrifugation steps were eliminated to allow more time for further purification steps and to preserve enzymatic activity, which often decreased during each centrifugation step. From among the tomatoes in various stages of ripeness, green tomatoes showed the highest activity. These tomatoes could not be obtained after frost began and so they were lyophilized and also stored frozen. However, assays showed that the enzyme was not active when fruit were stored in the freezer or lyophilized. The assay involving appearance of NADH absorbance was tested, but activity was not observed. Instead, the assay involving disappearance of absorbance of unsaturated fatty acid hydroperoxides was used to monitor enzymatic activity. The behavior in purification of tomato fruit HPOOH lyase appeared, from our data, to be different from tomato leaf HPOOH lyase. We have not been able to sufficiently obtain a purified enzyme due to difficulties in precipitating the enzyme.

Downloads

Download data is not yet available.

Author Biography

Purvika Shah, Department of Biochemistry and Microbiology Rutgers University, New Brunswick, NJ

Rutgers Undergraduate Research Fellow

Downloads

Published

1999-09-01

How to Cite

Shah, P., & Chase, T. J. (1999). Purification of hydroperoxide lyase from tomato fruits. The Rutger Scholar, 1. Retrieved from https://rutgersscholar.libraries.rutgers.edu/index.php/scholar/article/view/2

Issue

Section

Articles