Purified luciferase from luciola cruciata

• US 5,182,202 A
• Filed: 08/05/1991
• Issued: 01/26/1993
• Est. Priority Date: 11/30/1987
• Status: Expired due to Fees

• Current Assignee: Kikkoman Corporation
• Original Assignee: Kikkoman Corporation
• Inventors: Kajiyama, Naoki, Masuda, Tsutomu, Nakano, Eiichi, Tatsumi, Hiroki
• Primary Examiner(s): Naff, David M.
• Assistant Examiner(s): MELLER, MICHAEL V
• Application Number:
  US07/742,477
• Time in Patent Office: 540 Days
• Field of Search: 435/189
• US Class Current: 435/189
• CPC Class Codes: C12N 9/0069 acting on single donors with incorporation of molecular oxygen, i.e. oxygenases (1.13)
• CPC Class Codes: C12Y 113/12007 Photinus-luciferin 4-monooxygenase (ATP-hydrolysing) (1.13.12.7), i.e. firefly-luciferase

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.

Claims

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.