Colorimetric Enzymatic Assay of L-Malic Acid Using Dehydrogenase from Baker’s Yeast

Maristela F.S. Peres¹*, Cecilia Laluce² and Edwil A.L. Gattás<sup>1

1</sup>School of Pharmaceutical Science, São Paulo State University – UNESP, Department of Food and Nutrition, Rodovia Araraquara-Jaú, Km 1, BR-14801-902, SP, Brazil
²Department of Biochemistry and Chemical Technology, Institute of Chemistry, UNESP, R. Prof. Francisco Degni s/n C.P. 355, BR-14801-970, Araraquara, SP, Brazil

Article history:
Received July 17, 2006
Accepted September 23, 2007

Key words:
colorimetric assay of L-malic acid, fruit juices, wine, malate dehydrogenase (MDH), baker’s yeast

Summary:
A colorimetric method has been developed and optimized to measure L-malic acid in samples of fruit juices and wine. This method is based on oxidation of the analyte, catalyzed by malate dehydrogenase (MDH) from dry baker’s yeast, and in combination with the reduction of a tetrazolium salt (MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide). In the present study, the method exhibited sensitivity in the range of 500–4000 μM of L-malic acid in the reaction cuvette, with the lower detection limit of 6.7·10^–2 g/L, the upper limit of 53.6·10^–2 g/L and a maximum standard deviation of only 2.5 % for the analyzed samples. The MDH activity from baker’s yeast was also optimized, the enzyme showed a high stability at pH=8.0–9.0 and the activity was maintained completely at temperatures up to 40 °C for 1 hour. The results show that the colorimetric method using enzymatic preparations from dry baker’s yeast is a simple and low-cost method with possibility of wide application.

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