微生物与生化技术杂志

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Medium/High Throughput D-Amino Acid Oxidase Colorimetric Method for Determination of D-Amino Acids. Application for Amino Acid Racemases

Armand Berneman, Marcelo Alves-Ferreira, Nicolas Coatnoan, Nathalie Chamond and Paola Minoprio

Free amino acids detection and quantification in samples still calls for elaborated, powerful and usually high cost and sometimes time-consuming technologies such as gas chromatography, HPLC or highly selective and sensitive column-switching techniques. Here, we undertook to develop a simple, one-step, microtiter plate method to detect and/or quantify most D-amino acids in solutions based on the enzyme D-amino acid oxidase and the generation of stoechiometric amounts of hydrogen peroxide. Inclusion of horseradish peroxidase and OPD allows the determination of D- amino acid concentration through the absorbance of the reaction products as compared to serial dilutions of equimolar amounts of L/D amino acid standards. The methodology is easily adapted for a medium/high throughput analysis of amino acid racemase activities.