A High-Throughput Screening Assay for Amino Acid Decarboxylase Activity

作者：Medici Rosario; de Maria Pablo Dominguez; Otten Linda G; Straathof Adrie J J^*

来源：Advanced Synthesis & Catalysis, 2011, 353(13): 2369-2376.

DOI：10.1002/adsc.201100386

• amino acid decarboxylation
• amino acids
• enzyme catalysis
• fluorescence
• high-throughput screening

The development of sensitive and easy-to-apply high-throughput screening methods is a common need in modern biocatalysis. With these powerful analytical tools in hands, chemists can easily assess enzyme libraries to identify either novel biocatalysts or improved mutants. Within biocatalysis, amino acid decarboxylases are gaining an increased importance, with several diverse applications ranging from the synthesis of bio-commodities to medical applications (e. g., synthesis of enzyme inhibitors at the level of L-DOPA decarboxylase). Herein, an efficient and simple analytical method for high-throughput screening of amino acid decarboxylase activity is reported. The method is valid for the discrimination of a broad range of amino acid/amine pairs such as L-tyrosine/tyramine, L-DOPA/dopamine, 5-hydroxy-L-tryptophan/serotonin, L-histidine/histamine, L-serine/ethanolamine, L-tryptophan/tryptamine, L-glutamic acid/GABA, and L-alanine/ethylamine. It has proven its versatility by using pure substrates, mixtures, or enzymatic reactions, both coming either from commercial enzymes or derived from cell-free (crude) extracts. The limit of detection was 13 mM for ethanolamine in the presence of 50 mM L-serine, while z' values were in the range 0.75-0.93, indicating the suitability for high-throughput screening.

• 出版日期2011-9