Determination of the ratio of d- and l-amino acids in brewing by an immobilised amino acid oxidase enzyme reactor coupled to amperometric detection

To determine the quantity of free amino acids, the d- and l-forms separately, is an important task in modern nutritional studies. The aim of our present work was to develop rapid, routine methods for fast determination of the different forms of free amino acids. We utilized two enzymes (l-amino acid oxidase, d-amino acid oxidase) with broad specificity. In our home-made reactors, the enzymes were immobilized in a thin-layer Plexi-cell on natural protein membrane. The enzyme-cell was built into a FIA system and the hydrogen peroxide generated during the enzymatic reaction was determined by an amperometric detector. The electrode potential was fixed at +100 mV. The parameters for the biochemical and electrochemical reactions were optimized in each case. The optimal pH value for measuring l- and d-amino acids was found ca. 8.8 and 9.5, respectively. The LAO reactor could be used for more than 900 measurements, while the DAO reactor for about 1000 measurements. The working concentration range was between 0.1–3 and 0.2–3 mM, respectively. The same standard solution (l- and d-Methionine, 1 mM) was injected 25 times sequentially and the standard deviations were 2 and 2.7%, respectively. After determining the optimal parameters, the specificity of the immobilized enzyme preparations towards different amino acids and in samples from different stages of brewing was investigated.