Purified luciferase from luciola cruciata
First Claim
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1. 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;
space="preserve" listing-type="equation">Luciferen+ATP+O.sub.2 →
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.
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Abstract
Disclosed is a purified luciferase and a method for making it. The luciferase is obtained from Luciola cruciata. The luciferase has a pH range for stabililty of 6.5-9.0 and a optimum pH range of 8.0-9.5. The enzyme does not act on ADP, CTP, UTP and GTP.
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1 Claim
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1. A purified luciferase from Luciola cruciata characterized as follows:
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(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;
space="preserve" listing-type="equation">Luciferen+ATP+O.sub.2 →
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.
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Specification