Purified luciferase from luciola cruciata

A purified luciferase from Luciola cruciata characterized as follows:

• (a) action:
• said purified luciferase is derived from Luciola cruciata and catalyzes the oxidation of luciferin by an oxygen molecule, as shown by the enzymatic reaction formula:
• Luciferen+ATP+O.sub.2 .fwdarw.Oxyluciferin+AMP+pyrophosphoric acid+CO.sub.2 +light;
• (b) optimum pH, and pH range for stability:
• the optimum pH is 8.0 to 9.5, and the pH range for stability is 6.5 to 9.0;
• and
• (c) substrate specificity:
• the luciferase does not act on ADP, CTP, UTP and GTP;
• said purified luciferase having the purity of luciferase purified from L. cruciata by:
• dissolving the precipitate formed at 30 to 60% ammonium sulfate saturation from crude L. cruciata luciferase enzyme solution in a solution prepared by adding ethylenediaminetetraacetate to 1mM and ammonium sulfate to 10% to 25mM tris(hydroxy)aminomethane-hydrochloric acid buffer, pH=7.8;
• subjecting said dissolved precipitate solution to gel filtration chromatography and recovering the active fraction;
• dialyzing said active fraction against a buffer solution prepared by adding 0.1 M sodium chloride and 10% (V/V) ethylene glycol to a 10 mM sodium hydrogen phosphate-sodium dihydrogenphosphate solution;
• adsorbing said dialyzed material on a hydroxyapatite column equilibrated with 10 mM phosphate buffer;
• and
• collecting the fraction having luciferase activity eluted from said hydroxyapatite column by a linear gradient from 10 to 100 mM phosphate buffer, pH=7.5.