An improved method to characterize crude lipoxygenase extract from wheat germ

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Abstract

Introduction

Spectrophotometric assay is commonly applied in measuring activity of lipoxygenase (LOX). But the accuracy of this method is greatly affected by transparence of the substrate solution and the purity of enzyme solution; it is especially difficult to directly measure the activity of LOX in crude enzyme solution in the condition of pH value lower than 7.0.

Objectives

To provide a convenient, efficient, accurate and reproducible assay of LOX activity from wheat germ (WG), an improved method to characterize crude lipoxygenase extracted from wheat germ was established.

Methods

First the stability of substrate at different pH values was studied. Then a transparent substrate solution was obtained by mixed solution of ‘absolute ethanol – linoleic acid – Tween 20′. When determining LOX activity, we took substrate solution mixed with completely inactivated enzyme solution as the blank, which effectively eliminated the assay error and the complicated purifying process. Detection wavelength was 234 nm at 25 °C. In order to study the enzyme characteristics of crude LOX from WG, we used this modified method to determine the optimum pH and temperature of LOX. The kinetic parameters including Michaelis constant (Km) and the maximum rate (Vmax) were determined as well.

Results

The optimum conditions of assaying LOX activity of WG were: a mixed solution of 2.0 mL 0.1 M phosphate buffered solution (pH = 6.5); 200 μL substrate solution [the concentration of linoleic acid and Tween 20 are 1.25–2.53 mM and 0.08–0.12% (w/v)]; and the temperature of 25 °C. When LOX activity was determined at the optimum condition, Km value and Vmax of the crude LOX extract were 2.64 mM and 256.41 unit per mg protein per min, respectively.

Conclusion

The method is convenient, efficient, accurate and reproducible for the determination of LOX activity in WG.

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