Creatine amidinohydrolase and process for producing the same

Details Creatine amidinohydrolase and process for producing the same Creatine amidinohydrolase and process for producing the same

2001-05-24

2004-03-02

Patterson, Jr., Charles L. (Department: 1652)

Chemistry: molecular biology and microbiology

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

Hydrolase

Reexamination Certificate

active

06699700

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to a creatine amidinohydrolase having an optimum pH in the weakly acidic range, and a method for producing the same.
BACKGROUND OF THE INVENTION
Creatine amidinohydrolase is an enzyme which has a catalytic action of hydrolyzing creatine to generate sarcosine and urea. Creatine amidinohydrolase can be used for measuring the amount of creatine existing in human serum or urine, and so this enzyme can be used as a diagnostic agent for various diseases including renal diseases.
Previously, the optimum pH of creatine amidinohydrolase derived from Alcaligenes (which is described in Japanese Patent No. 2788174; Japanese Patent Application Laid-Open Nos. 7-265074 (1995), 9-215494 (1997), and 10-174585 (1998)) has been reported to be from 7.0 to 9.0. So, in a case where the creatine amidinohydrolase is reacted with serum in the optimum pH range, there are disadvantages such that the creatine amidinohydrolase is so susceptible to bilirubin that a measurement error may occur at the measurement of creatinine, although the reaction with substrate is good.
In contrast, when the reaction with serum is performed around pH6.5, the reaction can effectively be performed without the influence of bilirubin and the measurement error does not occur. And so, a creatine amidinohydrolase having an optimum pH of about 6.5 has been required.
In this situation, the object of the present invention is to provide a creatine amidinohydrolase having an optimum pH in the weakly acidic range, and a method for producing the same.
SUMMARY OF THE INVENTION
After thorough studies to achieve the above object, the present inventors have found a creatine amidinohydrolase having an optimum pH in the weakly acidic range can be obtained by making a modification of a creatine amidinohydrolase gene derived from Alcaligenes (Japanese Patent Application Laid-Open No. 8-89255 (1996)).
Therefore, in one aspect of the present invention, a creatine amidinohydrolase having the following physicochemical properties is provided:
(a) hydrolyzing 1 mole of creatine to generate 1 mole of sarcosine and 1 mole of urea;
(b) having a substrate specificity to creatine;
(c) having an optimum pH ranging from 6.0 to 7.0, particularly pH of about 6.5;
(d) having a stable pH ranging from 4.0 to 11.0;
(e) having an optimum temperature ranging from 50 to 55° C.; and
(f) having a molecular weight of approx. 92,000 daltons (as measured by gel filtration).
In one embodiment, the enzyme of the present invention is a mutant of a creatine amidinohydrolase derived from Alcaligenes. Specifically, the enzyme of the present invention has an amino acid sequence comprising a mutation(s) relative to the amino acid sequence shown in SEQ ID NO: 1. More specifically, the enzyme of the present invention is obtainable from an
Escherichia coli
strain, FERM BP-6580. The term “mutation” as used herein means a deletion, substitution, addition or insertion of at least one amino acid.
In another aspect of the present invention, a method for producing a creatine amidinohydrolase is provided, that comprises: culturing a microorganism having an ability to produce the creatine amidinohydrolase with the above properties; and collecting the creatine amidinohydrolase from the obtained culture.
In one embodiment of the present invention, the microorganism is an
Escherichia coli
strain, FERM BP-6580, or a strain derived from said
Escherichia coli
strain. The term “strain derived from” as used herein means a bacterial strain, which can be isolated from the parent strain treated by a natural mutation or an artificial mutation, having an ability to produce a creatine amidinohydrolase with the above properties.
In addition, the present specification includes part or all of the contents as disclosed in the specification and/or drawings of Japanese Patent Application No. 10-334252 (filed on Nov. 25, 1998), which is a priority document of the present application.


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