Purified luciferase from luciola lateralis

• US 5,352,598 A
• Filed: 08/29/1991
• Issued: 10/04/1994
• Est. Priority Date: 04/12/1988
• Status: Expired due to Fees

• Current Assignee: Kikkoman Corporation
• Original Assignee: Kikkoman Corporation
• Inventors: Masuda, Tsutomu, Kajiyama, Naoki, Nakano, Eiichi, Tatsumi, Hiroki
• Primary Examiner(s): Robinson, Douglas W.
• Assistant Examiner(s): Meller, Michael V.
• Application Number:
  US07/759,814
• Time in Patent Office: 1,132 Days
• Field of Search: 435/189, 435/243, 435/815, 435/816
• 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
• CPC Class Codes: Y10S 435/815 by sorption
• CPC Class Codes: Y10S 435/816 by solubility

Abstract

A purified luciferase from Luciola lateralis is disclosed. The enzyme is characterized by having properties including: an optimum pH range of 7.5 to 9.5, an optimum temperature range of 0° C. to 50° C., and that the enzyme does not act on ADP, CTP, UTP, and GTP. The enzyme is purified by using a process which includes: gel filtration chromatography, hydroxyapatite column chromatography, and a tris(hydroxy)aminomethane-hydro-chloric acid buffer.

Claims

1. 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+O₂ →

oxyluciferin+AMP+pyrophosphoric acid+CO₂ +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°

to 50°

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°

C. for 15 minutes;

(f) thermal stability;

after being treated at a temperature of 40°

C. for 15 minutes, said luciferase has a residual enzymatic activity of 78.2%;

even after being treated at a temperature of 50°

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.