Enzyme

Details Enzyme Enzyme

2001-10-11

2004-09-28

Saucier, Sandra E. (Department: 1651)

Chemistry: molecular biology and microbiology

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

Hydrolase

C435S023000, C435S212000, C435S219000

Reexamination Certificate

active

06797503

ABSTRACT:

TECHNICAL FIELD
The present invention relates to an enzyme.
BACKGROUND ART
Proteases are used in various industrial fields. For example, proteases are used for determining a glycated protein, e.g., glycated albumin, in serum, which can serve as a significant index for the diagnosis, treatment, etc. of diabetes.
Such determination of the glycated protein utilizing the protease can be carried out, for example, by degrading the glycated protein with the protease, reacting the resultant degradation product with a fructosyl amino acid oxidase (hereinafter, referred to as “FAOD”), and then determining oxygen consumption or hydrogen peroxide generation to find the amount of the glycated protein. Examples of the protease include those disclosed in JP 5(1993)-192193 A and JP 7(1995)-289253 A.
The above-mentioned protease pre-treatment of the glycated protein is conducted because FAOD and the like easily act on a glycated amino acid and a glycated peptide whereas they hardly act on the glycated protein itself. Particularly, since the glycated site of glycated hemoglobin (hereinafter, referred to as “HbA1c”) is the N-terminal amino acid residue of the &bgr;-chain, there has been a demand for a protease capable of treating HbA1c so that FAOD easily can act on this site of the HbA1c.
DISCLOSURE OF THE INVENTION
Therefore, it is an object of the present invention to provide a novel enzyme capable of treating a glycated protein and a glycated peptide so that FAOD easily can act thereon.
First, among various FAODs, the inventors of the present invention have studied the mechanism of the action of the FAOD that acts on a glycated protein, glycated peptide, glycated amino acid, etc. in which a sugar is bound to an &agr;-amino group. From this study, the inventors have found that such FAOD easily acts on the glycated amino acid in which a sugar is bound to an &agr;-amino group whereas it hardly acts on the above glycated protein and glycated peptide. Based on this finding, the inventors have isolated various bacteria from nature, cultured them, and studied the enzymes produced by them. As a result, the inventors have succeeded in isolating the bacteria producing a novel enzyme capable of releasing an amino acid having a glycated &agr;-amino group (&agr;-Glycated Amino acid: hereinafter, referred to as “&agr;-GA”) from the glycated protein or glycated peptide in which sugar is bound to an &agr;-amino group (an N-terminal amino group), thereby establishing the present invention. The novel enzyme (&agr;-Glycated Amino acid Releasing Enzyme: hereinafter, referred to as “&agr;-GARE”) according to the present invention can release &agr;-GA, for example, from the above glycated protein or glycated peptide. Hence, the determination of HbA1c using the FAOD that easily acts on &agr;-GA can be made practical in clinical tests etc. by the use of this novel enzyme. The novel enzyme according to the present invention can be utilized not only for the determination of HbA1c but also in various application fields, e.g., for the determination of other glycated proteins. Furthermore, in addition to the catalytic functions of releasing &agr;-GA, &agr;-GARE of the invention may have other catalytic functions, e.g., the function of cleaving other peptide bonds. Examples of novel bacterial strains isolated by the present inventors include bacterial strains of the genus Corynebacterium and the genus Pseudomonas. However, it is to be noted that &agr;-GARE according to the present invention is not limited to those derived from the strains of these genera.
The glycated amino acid released by &agr;-GARE is not specifically limited as long as it has a glycated &agr;-amino group. However, since the N-terminal valine residue is glycated in HbA1c as described above, the glycated amino acid released by &agr;-GARE preferably is a glycated valine (hereinafter, referred to as “&agr;-GV”).
Examples of &agr;-GARE according to the present invention include the following two types.
The first &agr;-GARE (hereinafter, referred to as “&agr;-GARE-1”) is derived from the bacterial strain of the genus Corynebacterium and most preferably from
Corynebacterium ureolyticum
KDK1002
. Corynebacterium ureolyticum
KDK1002 was isolated from the soil novelly by the present inventors.
Corynebacterium ureolyticum
KDK1002 has been deposited with the National Institute of Bioscience and Human Technology (NIBH), Agency of Industrial Science and Technology, Ministry of International Trade and Industry (1-3, Higashi 1-chome, Tsukuba-shi, Ibaraki 305-0046, JAPAN) under the Accession Number FERM P-17135 since Jan. 11, 1999. Bacteriological properties of this strain are as shown below.
(Morphological Characteristics)
This strain is a non-motile, rod-shaped bacterium (bacillus) of 0.8×1.2 &mgr;m.
(Culture Characteristics)
When cultured in an agar medium according to the usual method, the strain forms a colony that is circular in form and low convex in elevation. The colony is cream-colored.
(Physiological Characteristics)
Gram's stain:
positive
Oxygen requirement:
facultative anaerobic
Nitrate reduction:
−
Pyrazinamidase:
+
Pyrrolidonylarylamidase:
−
Alkaline phosphatase:
−
&bgr;-glucuronidase:
−
&bgr;-galactosidase:
−
&agr;-glucosidase:
−
N-acetyl-&bgr;-glucosaminase:
−
Esculin (glucosidase):
−
Urease:
+
Gelatin degradation:
−
&bgr;-hemolysis:
−
Carbohydrate fermentability
Glucose:
−
Ribose:
−
Xylose:
−
Mannitol:
−
Maltose:
−
Lactose:
−
Sucrose:
−
Glycogen:
−
The second &agr;-GARE (hereinafter, referred to as “&agr;-GARE-2”) is derived from the bacterial strain of the genus Pseudomonas and most preferably from
Pseudomonas alcaligenes
KDK1001
. Pseudomonas alcaligenes
KDK1001 also was isolated from the soil novelly by the present inventors.
Pseudomonas alcaligenes
KDK1001 has been deposited with the National Institute of Bioscience and Human Technology (NIBH), Agency of Industrial Science and Technology, Ministry of International Trade and Industry (1-3, Higashi 1-chome, Tsukuba-shi, Ibaraki 305-0046, JAPAN) under the Accession Number FERM P-17133 since Jan. 11, 1999. Bacteriological properties of this strain are as shown below.
(Morphological Characteristics)
This strain is a rod-shaped bacterium (bacillus) of 0.3×1.5 &mgr;m, which is motile by polar flagellums.
(Culture Characteristics)
When cultured in an agar medium according to the usual method, the strain forms a colony that is circular in form, low convex in elevation, with a smooth surface. The colony initially is translucent and then turns to light yellow. When cultured in the Mac Conkey's culture medium, the strain grows, albeit weakly. Further, at a culture temperature of 40° C., the strain does not grow at all.
(Physiological Characteristics)
Gram's stain:
negative
Oxygen requirement:
aerobic
Nitrate reduction:
+
Indole production:
−
Glucose acidification:
−
Arginine dihydrolase:
−
Urease:
−
Esculin hydrolysis:
+
Gelatin hydrolysis:
−
&bgr;-galactosidase:
+
Gas generation from glucose:
−
Substrate utilization
Glucose:
+
L-arabinose:
+
D-mannose:
+
D-mannitol:
+
N-acetyl-D-gluconsamine:
−
Maltose:
+
Potassium gluconate:
+
n-capric acid:
+
Adipic acid:
−
DL-malic acid:
+
Sodium citrate:
−
Phenyl acetate:
−
A method of determining a glycated protein or a glycated peptide according to the present invention includes: degrading a glycated protein or a glycated peptide with an enzyme; causing a redox reaction between the resultant degradation product and FAOD; and determining the redox reaction so as to determine the glycated protein or the glycated peptide. In this method, the novel enzyme (&agr;-GARE) according to the present invention is used as the above enzyme. The type of the &agr;-GARE used in this method is decided appropriately depending on the type, concentration, etc. of the glycated protein or glycated peptide to be determined.

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