Sarcosine oxidase and process for producing the same

Details Sarcosine oxidase and process for producing the same Sarcosine oxidase and process for producing the same

1999-12-09

2001-05-08

Nashed, Nashaat T. (Department: 1652)

Chemistry: molecular biology and microbiology

Enzyme , proenzyme; compositions thereof; process for...

Oxidoreductase

C435S252330, C435S320100

Reexamination Certificate

active

06228626

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to sarcosine oxidase which shows a high reactivity under neutral conditions, and to a process for producing the sarcosine oxidase.
BACKGROUND OF THE INVENTION
Sarcosine oxidase is an enzyme which catalyzes a reaction to oxidatively hydrolyze sarcosine to produce glycine, formaldehyde, and hydrogen peroxide. This enzyme can be used for determining creatinine or creatine levels in human sera or urine samples in combination with creatininase or creatinase and therefore is useful as a diagnostic enzyme for a variety of diseases such as hepatic disease.
Conventional sarcosine oxidases have drawbacks such as drastically decreased reactivity under neutral to weakly acidic conditions. Accordingly, they have generally been used under weakly alkaline conditions. See JP-B-1-34035, JP-A-5-115281, JP-A-8-238087, JP-A-6-113840, and JP-A-4-94688. However, when reacted with sera at the optimum pH range, those sarcosine oxidases react well with the substrate but are readily affected by bilirubin, causing errors in assay.
On the contrary, if sarcosine oxidase can be reacted with sera at a pH of approximately 6.5 it will not be affected by bilirubin and thus will not cause such errors. Accordingly, there has always been a need for a sarcosine oxidase which can be used under neutral to weakly acidic conditions.
Moreover, use of such enzyme with an increased reactivity under weakly alkaline conditions (i.e., lower Km value) will be very economical since a smaller amount of the enzyme can provide a sufficient reaction when used as a diagnostic enzyme under normal conditions.
An object of the invention is to provide sarcosine oxidase having a high reactivity under neutral conditions.
Another object of the invention is to provide a process for producing the sarcosine oxidase.
SUMMARY OF THE INVENTION
The present invention is based on a finding that a genetic mutation of a sarcosine oxidase gene derived from Bacillus sp. NS-129 (as disclosed in JP-B-6-65303) gave a mutant sarcosine oxidase which can not only show a higher reactivity (i.e., smaller Km value) than that of a wild-type sarcosine oxidase under weakly alkaline conditions but also keep a relatively higher reactivity under neutral conditions (pH7.0).
Thus, the present invention relates to a sarcosine oxidase which has the following physico-chemical properties:
(a) action: oxidatively hydrolyzing 1 mole sarcosine to give 1 mole glycine, 1 mole formaldehyde, and 1 mole hydrogen peroxide;
(b) substrate specificity: specific for sarcosine;
(c) optimum pH: 7.0-8.0;
(d) stable pH range: 7.0-9.5;
(e) suitable temperature range for action: 50° C.;
(f) thermostability: 55° C. or less; and
(g) molecular weight: 44,000 daltons (when estimated roughly from the amino acid sequence of wild-type).
In an embodiment of the invention, the sarcosine oxidase is obtainable from
E. coli
JM109 (pSO12 EH) (Accession No. FERM BP-6597) or a variant derived therefrom.
Further, the present invention relates to a process for producing sarcosine oxidase, comprising the steps of culturing a microorganism having an ability to produce the sarcosine oxidase and collecting the sarcosine oxidase from the culture.
In an embodiments of the invention, the microorganism is
E. coli
JM 109-pSO12 EH, FERM BP-6597, or a variant derived therefrom.
The term “variant” used herein means a mutant strain from
E. coli
JM109-pSO12EH, FERM BP-6597, which is capable of producing a sarcosine oxidase with the above-described physico-chemical properties.
This specification includes part or all of the contents disclosed in the specification and/or drawings of Japanese Patent Application No. 10-354482, which is a priority-application of the present application.


REFERENCES:
patent: 1-034035 (1989-07-01), None
patent: 4-094688 (1992-03-01), None
patent: 5-115281 (1993-05-01), None
patent: 6-113840 (1994-04-01), None
patent: 6-065303 (1994-08-01), None
patent: 8-238087 (1996-09-01), None
Madaras et al. Miniaturized biosensors employing electropolymerized permselective films andtheir use for creatine assays in human serum. Anal Chem. Nov. 1, 1996, vol. 68, pp. 3832-3239.*
Nishiya et al. Alteration of substrate specificity and optimum pH of sarcosine oxidase by random and site-directed mutagenesis. Applied and Environmental Microbiology. 1994, vol. 60, pp. 4213-4215.*
Suzuki et al. Cloning, sequencing, and overexpression inEscherichia coliof a sarcosine oxidase-encoding gene linked to theBacillus creatinasegene. Journal of Fermentation and Bioengineering. 1994, vol. 77, pp. 231-234.

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