Available online at www.sciencedirect.com Journal of Molecular Catalysis B: Enzymatic 52–53 (2008) 140–145 Kinetic properties of glycerophosphate oxidase isolated from dry baker’s yeast Luciana Amade Camargo a , Maria Henriques Lourenc ¸o Ribeiro b , Maristela de Freitas Sanches Peres a , Edwil Aparecida de Lucca Gatt´ as a,∗ a School of Pharmaceutical Science, S˜ ao Paulo State University-UNESP, Department of Food and Nutrition, Rodovia Araraquara-Ja´ u, Km 1, 14801 902 S ˜ ao Paulo, Brazil b Faculdade de Farm´ acia, i-Med (CECF) Research Institute for Medicines and Pharmaceutical Sciences, University of Lisbon, Av. Prof. Gama Pinto, 1649-003 Lisbon, Portugal Available online 19 November 2007 Abstract The glycerophosphate oxidase is a flavoprotein responsible for the catalysis of the oxidation of the glycerophosphate to dihydroxyacetone phosphate, through the reduction of the oxygen to hydrogen peroxide. The glycerophosphate oxidase from baker’s yeast was specific for l-- glycerol phosphate. It was estimated by monitoring the consumption of oxygen with an oxygraph. An increase of 32% in consumption of oxygen was obtained when the enzyme was concentrated 16-fold. The assay of enzyme was determined by the peroxidase chromogen method followed at 500nm. The procedure for the standardization of the activity of the glycerophosphate oxidase from baker’s yeast was accomplished, and the pH and temperature stability showed that the enzyme presented a high stability at pH 8.0, and the thermal stability was maintained up to 60 ◦ C during 1 h. Such method allowed quantifying in the range 92–230 mM of glycerol phosphate, an important intermediate metabolite from lipid biosynthesis and glycolytic routes. © 2007 Elsevier B.V. All rights reserved. Keywords: Glycerophosphate oxidase; Kinetic properties; Baker’s yeast 1. Introduction Glycerophosphate oxidase (sn-glycerol-3-phosphate: oxy- gen 2-oxidoreductase, EC 1.1.3.21, GPO) is an oxido-reductase isolated from different microorganisms such as lactic acid bac- teria [1–5] and yeast [6]. Evidence for the involvement of this enzyme in lactic acid bacteria, was shown through the lack of the membrane-associated electron transport chain found in E. coli and in eukaryotic mitochondria [7,8]. The enzyme differs markedly from the l--glycerophosphate dehydrogenases iso- lated from both mitochondria [9] and from E. coli [10,11], in fact that two of these membrane-bound enzymes contain non- heme iron as well as FAD; furthermore, these enzymes do not catalyze the reduction of oxygen. The animal and yeast oxidases are mitochondrial origin and catalyze transfer of electrons from l--glycerol phosphate via the respiratory chain, and the bacte- rial enzymes (E. coli, for example) are bound to the membrane ∗ Corresponding author. Tel.: +55 16 3301 6929; fax: +55 16 3301 6920. E-mail address: gattas@fcfar.unesp.br (E.A.d.L. Gatt´ as). and do not utilize molecular oxygen as an electron acceptor. Whereas the enzymes isolated from the lactic acid bacteria are soluble enzymes, which do utilize molecular oxygen [2]. This enzyme catalyzes the oxidation of -glycerol phosphate to dihydroxyacetone phosphate with the concomitant reduc- tion of oxygen to hydrogen peroxide. Glycerophosphate oxidase from the cells of the mutant strain A. viridans was isolated and purified and its properties were established [5]. GPO produc- tion is dependent on the time and on the composition of culture medium [5]. Commercial dry yeast can be used as a source of glycerophosphate oxidase for enzymatic assay. Most studies on these oxidative enzymes have utilized crude extracts, however, the assays that use partially purified preparations have a quite low cost and are viable for different samples. GPO has practical application in several coupled systems for the quantitative determination of magnesium [12], glycerol phosphate [4], triacylglycerol [13–16], glycerol [17,18], phos- phatidic acid [19] and other phospholipids. It can also be used for the measurement of enzymatic activities (glycerol kinase [20] and similar enzymes coupled to other enzymes and chromogen reagent) in blood serum or other biological materials [3,5,21]. 1381-1177/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.molcatb.2007.11.011