Purified luciferase from luciola lateralis

A purified luciferase from Luciola lateralis characterized as follows:

• (a) action:
• said luciferase is a purified enzyme which catalyzes the oxidation of luciferin by an oxygen molecule, as shown by the enzymatic reaction formula:
• luciferin+ATP+O.sub.2 .fwdarw.oxyluciferin+AMP+pyrophosphoric acid+CO.sub.2 +light
• (b) substrate specificity:
• said luciferase does not act on ADP, CTP, UTP and GTP;
• (c) optimum pH, and pH range for luciferase stability:
• the optimum pH is 7.5 to 9.5 when luciferin is used as a substrate, and the stable pH range is 60 to 10.5;
• (d) optimum temperature range for activity:
• 0.degree. to 50.degree. C.;
• (e) conditions of inactivation by pH, temperature;
• at pH of 5.0 or lower and 12.0 or higher, said luciferase is completely inactivated when it is exposed to such a condition for four hours,
• at pH 7.8, said luciferase is completely inactivated by heat treatment at a temperature of 55.degree. C. for 15 minutes;
• (f) thermal stability:
• after being treated at a temperature of 40.degree. C. for 15 minutes, said luciferase has a residual enzymatic activity of 78.2%;
• even after being treated at a temperature of 50.degree. C. for 8 minutes, said luciferase has a residual enzyme activity of 6%.
• said purified luciferase having the purity of luciferase purified from L. lateralis by:
• dissolving the precipitate formed at 30 to 60% ammonium sulfate saturation from crude L. lateralis luciferase enzyme solution in a solution prepared by adding ethylenediaminetetraacetate to 1 mM and ammonium sulfate to 10% to 25 mM 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;
• absorbing said dialyzed fraction 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.