A chromogenic substrate for solid-phase detection of phospholipase A2 |
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| Authors: | Chisato Eba Aoi Okano Hideo Nakano Yugo Iwasaki |
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| Institution: | Laboratory of Molecular Biotechnology, Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan |
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| Abstract: | A method for solid-phase detection of phospholipase A2 (PLA2) was developed. The method uses 1-octanoyloxynaphthalene-3-sulfonic acid, which was found to be a good substrate of PLA2. The substrate is hydrolyzed by PLA2 into 1-naphthol-3-sulfonic acid, which is spontaneously coupled with coexisting diazonium salt to form a red-purple azo dye. Streptomyces and bovine pancreatic PLA2 spotted on a nitrocellulose membrane could be detected by this method with considerable sensitivity. In addition, colonies of recombinant Escherichia coli producing bacterial PLA2 were distinguishable from those producing an inactive mutant PLA2, facilitating high-throughput screening in directed evolution of the enzyme. |
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| Keywords: | Phospholipase A2 Solid-phase detection Diazo coupling Artificial substrate High-throughput screening |
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