U.S. patent number RE39,352 [Application Number 10/807,228] was granted by the patent office on 2006-10-17 for creatine amidinohydrolase, production thereof and use thereof.
This patent grant is currently assigned to Toyo Boseki Kabushiki Kaisha. Invention is credited to Takashi Hattori, Yoshihisa Kawamura, Yoshiaki Nishiya, Atsushi Sogabe.
United States Patent |
RE39,352 |
Sogabe , et al. |
October 17, 2006 |
**Please see images for:
( Certificate of Correction ) ** |
Creatine amidinohydrolase, production thereof and use thereof
Abstract
A creatine amidinohydrolase having the following physicochemical
properties: Action: catalyzing the following reaction;
creatine+H.sub.2O.fwdarw.sarcosine+urea Optimum temperature: about
40-50.degree. C. Optimum pH: pH about 8.0-9.0 Heat stability: not
more than 50.degree. C. (pH 7.5, 30 min) Km value for creatine in a
coupling assay using a sarcosine oxidase and a peroxidase: about
3.5-10.00 mM Molecular weight: about 43,000 (SDS-PAGE) Isoelectric
point: .[.3.5.]. .Iadd.4.5.Iaddend., a method for producing said
enzyme, comprising culture of microorganism producing said enzyme,
a method for the determination of creatine or creatinine in a
sample using said enzyme, and a reagent therefor.
Inventors: |
Sogabe; Atsushi (Tsuruga,
JP), Hattori; Takashi (Tsuruga, JP),
Nishiya; Yoshiaki (Tsuruga, JP), Kawamura;
Yoshihisa (Tsuruga, JP) |
Assignee: |
Toyo Boseki Kabushiki Kaisha
(Osaka, JP)
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Family
ID: |
12165916 |
Appl.
No.: |
10/807,228 |
Filed: |
March 23, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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09940941 |
Aug 28, 2001 |
Re. 38687 |
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Reissue of: |
08799897 |
Feb 13, 1997 |
06080553 |
Jun 27, 2000 |
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Foreign Application Priority Data
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Feb 13, 1996 [JP] |
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8-025435 |
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Current U.S.
Class: |
435/227; 435/192;
435/440; 435/441; 435/320.1; 435/252.33; 435/252.3; 435/446;
435/829; 435/91.2; 536/23.2; 435/18 |
Current CPC
Class: |
C12Q
1/34 (20130101); G01N 33/52 (20130101); G01N
2333/978 (20130101); Y10S 435/829 (20130101) |
Current International
Class: |
C12N
9/78 (20060101); C07H 21/04 (20060101); C12N
1/21 (20060101); C12N 15/01 (20060101); C12Q
1/34 (20060101) |
Field of
Search: |
;435/227,440,441,446,91.2,18,192,252.33,320.1,829 ;536/23.2 |
References Cited
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Holz et al. |
3907644 |
September 1975 |
Mollering et al. |
4420562 |
December 1983 |
Ikuta et al. |
5451520 |
September 1995 |
Furukawa et al. |
5932466 |
August 1999 |
Furukawa et al. |
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Foreign Patent Documents
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62-091182 |
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Apr 1987 |
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JP |
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62 091182 |
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Apr 1987 |
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JP |
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07-265074 |
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Oct 1995 |
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JP |
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07 265074 |
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JP |
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07-274961 |
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JP |
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Sep 1998 |
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JP |
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Primary Examiner: Slobodyansky; Elizabeth
Attorney, Agent or Firm: Leydig, Voit & Mayer, Ltd.
Parent Case Text
.Iadd.CROSS-REFERENCE TO RELATED APPLICATIONS
More than one reissue application has been filed for the reissue of
U.S. Pat. No. 6,080,553. The reissue applications are the present
application and application Ser. No. 09/940,941, each of which is a
divisional reissue of U.S. Pat. No. 6,080,553. .Iaddend.
Claims
What is claimed is:
.[.1. A creatine amidinohydrolase having the following
physicochemical properties: Action: catalyzing the following
reaction; creatine+H.sub.2O.fwdarw.sarcosine+urea Optimum
temperature: about 40-50.degree. C. Optimum pH: pH about 8.0-9.0
K.sub.m value for creatine in a coupling assay using a sarcosine
oxidase and a peroxidase: 3.5-10.0 mM Molecular weight: about
43,000 (SDS-PAGE) Isoelectric point: about 3.5..].
.[.2. A creatine amidinohydrolase having the following
physicochemical properties: Action: catalyzing the following
reaction; creatine+H.sub.2O.fwdarw.sarcosine+urea Optimum
temperature: about 40-50.degree. C. Optimum pH: pH about 8.0-9.0
K.sub.m value for creatine in a coupling assay using a sarcosine
oxidase and a peroxidase: 4.5.+-.1.0 mM Molecular weight: about
43,000 (SDS-PAGE) Isoelectric point: about 3.5..].
.[.3. The creatine amidinohydrolase of claim 2, which is obtained
from Escherchia coli JM109 (pCRH273M2) (FERM BP-5375)..].
.[.4. A creatine amidinohydrolase having the following
physicochemical properties: Action: catalyzing the following
reaction; creatine+H.sub.2O.fwdarw.sarcosine+urea Optimum
temperature: about 40-50.degree. C. Optimum pH: pH about 8.0-9.0
K.sub.m value for creatine in a coupling assay using a sarcosine
oxidase and a peroxidase: 6.5.+-.1.0 mM Molecular weight: about
43,000 (SDS-PAGE) Isoelectric point: about 3.5..].
.[.5. The creatine amidinohydrolase of claim 4, which is obtained
from Escherchia coli JM109 (pCRH273M1) (FERM BP-5374)..].
.[.6. A creatine amidinohydrolase having the following
physicochemical properties: Action: catalyzing the following
reaction; creatine+H.sub.2O.fwdarw.sarcosine+urea Optimum
temperature: about 40-50.degree. C. Optimum pH: pH about 8.0-9.0
K.sub.m value for creatine in a coupling assay using a sarcosine
oxidase and a peroxidase: 9.0.+-.1.0 mM Molecular weight: about
43,000 (SDS-PAGE) Isoelectric point: about 3.5..].
.[.7. The creatine amidinohydrolase of claim 6, which is obtained
from Escherchia coli JM109 (pCRH273M3) (FERM BP-5376)..].
.[.8. A method for producing the creatine amidinohydrolase of claim
1, comprising culturing a microorganism producing said creatine
amidinohydrolase in a nutrient medium and recovering said creatine
amidinohydrolase from the resulting culture..].
.[.9. The method of claim 8, wherein said microorganism is selected
from the group consisting of Escherichia coli JM109 (pCRH273M1)
(FERM BP-5374), Escherichia coli JM109 (pCRH273M2) (FERM BP-5375)
and Escherichia coli JM109 (pCRH273M3) (FERM BP-5376)..].
.[.10. A reagent for determination of creatine in a sample,
comprising the creatine amidinohydrolase of claim 1, a sarcosine
oxidase and a composition for the detection of hydrogen
peroxide..].
.[.11. The reagent of claim 10, in which the composition for the
detection of hydrogen peroxide comprises an enzyme having a
peroxidase activity, a chromophore and a buffer..].
.[.12. The reagent of claim 11, in which the enzyme having the
peroxidase activity is selected from the group consisting of
peroxidase, haloperoxidase, bromoperoxidase, lactoperoxidase and
myeloperoxidase..].
.[.13. The reaction of claim 11, in which the chromophore comprises
a hydrogen receptor and a coupler..].
.[.14. The reagent of claim 13, in which the hydrogen receptor is
4-aminoantipyrine or a 3-methyl-2-benzothiazoline-hydrazine
derivative..].
.[.15. The reagent of claim 13, in which the coupler is an aniline
derivative or a phenol derivative..].
.[.16. A method for determining creatine in a sample, which
comprises measuring the absorbance of the pigment produced by the
reaction of the reagent of claim 10 with the sample..].
.[.17. A reagent for determination of creatinine in a sample,
comprising a creatinine amidohydrolase, the creatine
amidinohydrolase of claim 1, a sarcosine oxidase and a composition
for the detection of hydrogen peroxide..].
.[.18. The reagent of claim 17, in which the composition for the
detection of hydrogen peroxide comprises an enzyme having a
peroxidase activity, a chromophore and a buffer..].
.[.19. The reagent of claim 18, in which the enzyme having the
peroxidase activity is selected from the group consisting of
peroxidase, haloperoxidase, bromoperoxidase, lactoperoxidase and
myeloperoxidase..].
.[.20. The reagent of claim 18, in which the chromophore comprises
a hydrogen receptor and a coupler..].
.[.21. The reagent of claim 20, in which the hydrogen receptor is
4-aminoantipyrine or a 3-methyl-2-benzothiazoline-hydrazine
derivative..].
.[.22. The reagent of claim 20, in which the coupler is an aniline
derivative or a phenol derivative..].
.[.23. A method for determining creatinine in a sample, which
comprises measuring the absorbance of the pigment produced by the
reaction of the reagent of claim 17 with the sample..].
.Iadd.24. A method of preparing a creatine amidinohydrolase
comprising: (i) mutating (a) the nucleic acid sequence of SEQ ID
NO:2 or (b) a nucleic acid sequence encoding the amino acid
sequence of SEQ ID NO:1 to provide mutant nucleic acid sequences,
(ii) determining Km values for creatine of proteins encoded by the
mutant nucleic acid sequences in a coupling assay using a sarcosine
oxidase and a peroxidase, (iii) selecting and isolating a desired
mutant nucleic acid sequence that encodes a creatine
amidinohydrolase having the following physicochemical properties:
Action: catalyzing the following reaction:
creatine+H.sub.2O.fwdarw.sarcosine+urea Km values for creatine in a
coupling assay using a sarcosine oxidase and a peroxidase: 3.5-10.0
mM, Molecular weight: about 43,000(SDS-PAGE) Isoelectric point:
about 4.5 Optimum temperature: about 40-50.degree. C. (at pH of
about 7.5) Optimum pH: about 8.0-9.0(at a temperature of about
37.degree. C.) (iv) expressing the desired mutant nucleic acid
sequence in a host to produce creatine amidinohydrolase, and (v)
harvesting the produced creatine amidinohydrolase. .Iaddend.
.Iadd.25. The method of claim 24, wherein the sarcosine oxidase is
originated from the genus Arthrobacter, Corynebacterium,
Alcaligenes, Pseudomonas, Micrococcus, or Bacillus. .Iaddend.
.Iadd.26. A method of preparing a creatine amidinohydrolase
comprising: (i) selecting (a) a nucleic acid sequence of SEQ ID
NO:2 or (b) a nucleic acid sequence encoding the amino acid
sequence of SEQ ID NO:1 to provide a source nucleic acid sequence,
(ii) mutating the source nucleic acid sequence to provide mutant
nucleic acid sequences that encode mutant creatine
amidinohydrolases, (iii) selecting a mutant nucleic acid sequence
that encodes a creatine amidinohydrolase which has a reduced Km
value as compared to the Km value of creatine amidinohydrolase
encoded by the source nucleic acid sequence by: (A) determining a
first activity of creatine amidinohydrolase encoded by the source
nucleic acid sequence with a first concentration of creatine and a
second activity of creatine amidinohydrolase encoded by the source
nucleic acid sequence with a second concentration of creatine,
wherein the second concentration of creatine is less than the first
concentration of creatine, (B) determining a first activity of the
mutant creatine amidinohydrolase with the first concentration of
creatine and a second activity of the mutant creatine
amidinohydrolase with the second concentration of creatine, wherein
the second concentration of creatine is less than the first
concentration of creatine, (C) calculating a ratio of the second
activity of the creatine amidinohydrolase encoded by the source
nucleic acid sequence divided by the first activity of the creatine
amidinohydrolase encoded by the source nucleic acid sequence, (D)
calculating a ratio of the second activity of the mutant creatine
amidinohydrolase divided by the first activity of the mutant
creatine amidinohydrolase, (E) comparing the ratio calculated in
step (iii)(C) to the ratio calculated in step (iii)(D), wherein a
mutant creatine amidinohydrolase that has a reduced Km value as
compared to the Km value of creatine amidinohydrolase encoded by
the source nucleic acid sequence has a greater ratio than the ratio
for creatine amidinohydrolase encoded by the source nucleic acid,
(iv) selecting and isolating a desired mutant nucleic acid sequence
that encodes a creatine amidinohydrolase having the following
physicochemical properties: Action: catalyzing the following
reaction: creatine+H.sub.2O.fwdarw.sarcosine+urea Km values for
creatine in a coupling assay using a sarcosine oxidase and a
peroxidase: 3.5-10.0 mM, Molecular weight: about 43,000(SDS-PAGE)
Isoelectric point: about 4.5 Optimum temperature: about
40-50.degree. C. (at pH of about 7.5) Optimum pH: about 8.0-9.0(at
a temperature of about 37.degree. C.) (v) expressing the desired
mutant nucleic acid sequence in a host to produce creatine
amidinohydrolase; and (vi) harvesting the produced creatine
amidinohydrolase. .Iaddend.
Description
FIELD OF THE INVENTION
The present invention relates to a novel creatine amidinohydrolase,
specifically, a novel creatine amidinohydrolase having a very low
Km value for creatine, and a method for producing said enzyme. The
present invention also relates to a method for the determination of
creatine or creatinine in a sample by the use of said enzyme, and a
reagent therefor.
BACKGROUND OF THE INVENTION
A creatine and a creatinine are found in blood and urine. A quick
and accurate determination of their amounts is very important in
making diagnosis of the diseases such as uremia, chronic nephritis,
acute nephritis, giantism, tonic muscular dystrophy and the like.
For making diagnosis of these diseases, creatine and creatinine in
blood, as well as urine are frequently determined
quantitatively.
A creatine can be determined by allowing creatine aminohydrolase
and sarcosine oxidase to react on creatine in a sample and
determining the amount of the generated hydrogen peroxide by a
method for measuring hydrogen peroxide. A creatinine can be
determined by allowing creatinine amidohydrolase, creatine
amidinohydrolase and sarcosine oxidase to react on creatinine in a
sample and determining the generated hydrogen peroxide by a method
for measuring hydrogen peroxide.
The creatinine amidohydrolase, creatine amidinohydrolase and
sarcosine oxidase are widely found in the world of microorganisms,
have been industrially produced and used as reagents for clinical
tests.
Yet, the creatine amidinohydrolase produced from various known cell
lines show lower heat stability and greater Km value for creatine.
For example, an enzyme derived from the bacteria belonging to the
genus Bacillus (U.S. Pat. No. 4,420,562) is thermally stable only
at a temperature not more than 40.degree. C. An enzyme derived from
Pseudomonas putida has a smaller apparent Km value for creatine of
1.33 mM [Archives Biochemistry and Biophysics 177, 508-515 (1976)],
though the method for determining the activity is different and the
Km value for creatine determined by a coupling assay using
sarcosine oxidase and peroxidase widely used as reagents for
clinical tests, has been unknown. The enzymes derived from the
bacteria belonging to the genus Corynebacterium, Micrococcus,
Actinobacillus or Bacillus (Japanese Patent Examined Publication
No. 76915/1991) is thermally stable at a temperature not more than
50.degree. C., whereas Km value for creatine is as great as about
20 mM, and these enzymes are not suitable for use as reagents for
clinical tests.
In an attempt to resolve such problems, the present inventors
previously found that the bacteria belonging to the genus
Alcaligenes produced a creatine amidinohydrolase which was superior
in heat stability and had a relatively smaller Km value (Km value:
ca. 15.2) for creatine (Japanese Patent Unexamined Publication No.
63363/1994). Furthermore, they have established a technique for
isolating a creatine amidinohydrolase gene having a relatively
small Km value for creatine from said bacterial cell line and
producing said enzyme in a large amount using Gram negative
bacteria as a host (Japanese Patent Application No.
117283/1995).
Moreover, a creatine amidinohydrolase stable in a high pH range and
having a small Km value has been reported to be derived from the
same genus Alcaligenes cell line (U.S. Pat. No. 5,451,520).
Yet, these creatine amidinohydrolases still have greater Km values
as enzymes to be used as routine reagents for clinical tests, and a
creatine amidinohydrolase having smaller Km value has been
desired.
SUMMARY OF THE INVENTION
It is therefore an object of the present invention to produce a
novel creatine amidinohydrolase having a small Km value for
creatine to the extent sufficient for use as a general reagent for
clinical tests, preferably not more than about 15.0, and provide a
means for determining creatine or creatinine in a sample using said
enzyme.
The present invention is based on the successful provision of a
creatine amidinohydrolase gene which expresses a novel creatine
amidinohydrolase having a small Km value for creatine, by
introducing a mutation, by genetic engineering and protein
engineering, into a creatine amidinohydrolase gene derived from
conventionally known bacteria belonging to the genus Alcaligenes,
which is a known creatine amidinohydrolase having a rather small Km
value. The creatine amidinohydrolase of the present invention can
be produced in large amounts by culturing a microorganism capable
of expressing said gene in a nutrient medium.
The novel creatine amidinohydrolase of the present invention has a
very small Km value for creatine as compared to conventionally
known enzymes, and shows superior reactivity to creatine contained
in a trace amount in a sample. Thus, it is useful as a reagent for
determining creatine or creatinine with high sensitivity and high
precision.
Accordingly, the present invention provides a novel creatine
amidinohydrolase having the following physicochemical properties.
Action: catalyzing the following reaction:
creatine+H.sub.2O.fwdarw.sarcosine+urea Optimum temperature: ca.
40-50.degree. C. Optimum pH: ca. 8.0-9.0 Heat stability: stable at
not more than about 50.degree. C. (pH 7.5, 30 min) Km value
relative to creatine in a coupling assay using a sarcosine oxidase
and a peroxidase: ca. 3.5-10.0 mM Molecular weight: ca. 43,000
(SDS-PAGE) Isoelectric point: ca. .[.3.5.]. .Iadd.4.5 .Iaddend.
The present invention also provides a method for producing said
creatine amidinohydrolase, comprising culturing a microorganism
capable of producing a novel creatine amidinohydrolase having the
following physicochemical properties, in a nutrient medium, and
harvesting said creatine amidinohydrolase from the culture. Action:
catalyzing the following reaction:
creatine+H.sub.2O.fwdarw.sarcosine+urea Optimum temperature: ca.
40-50.degree. C. Optimum pH: ca. 8.0-9.0 Heat stability: stable at
not more than about 50.degree. C. (pH 7.5, 30 min) Km value
relative to creatine in a coupling assay using a sarcosine oxidase
and a peroxidase: ca. 3.5-10.0 mM Molecular weight: ca. 43,000
(SDS-PAGE) Isoelectric point: ca. .[.3.5.]. .Iadd.4.5 .Iaddend.
The present invention further provides a reagent for determining
creatine in a sample, comprising the above-said creatine
amidinohydrolase, sarcosine oxidase and a composition for detection
of hydrogen peroxide, and a method for determining creatine in a
sample by the use of said reagent.
The present invention further provides a reagent for determining
creatinine in a sample, comprising a creatinine amidohydrolase, the
above-mentioned creatine amidinohydrolase, sarcosine oxidase and a
composition for detection of hydrogen peroxide, and a method for
determining creatinine in a sample by the use of said reagent.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows a physical map of recombinant plasmid pCRH273.
FIG. 2 shows the time course determination results of creatinine in
a sample, by the use of the creatine amidinohydrolase of the
present invention and a wild creatine amidinohydrolase.
DETAILED DESCRIPTION OF THE INVENTION
One embodiment of the present invention is a novel creatine
amidinohydrolase having the following physicochemical properties.
Action: catalyzing the following reaction:
creatine+H.sub.2O.fwdarw.sarcosine+urea Optimum temperature: ca.
40-50.degree. C. Optimum pH: ca. 8.0-9.0 Heat stability: stable at
not more than about 50.degree. C. (pH 7.5, 30 min) Km value
relative to creatine in a coupling assay using a sarcosine oxidase
and a peroxidase: ca. 4.5.+-.1.0 mM Molecular weight: ca. 43,000
(SDS-PAGE) Isoelectric point: ca. .[.3.5.]. .Iadd.4.5 .Iaddend.
Another embodiment of the present invention is a novel creatine
amidinohydrolase having the following physicochemical properties.
Action: catalyzing the following reaction:
creatine+H.sub.2O.fwdarw.sarcosine+urea Optimum temperature: ca.
40-50.degree. C. Optimum pH: ca. 8.0-9.0 Heat stability: stable at
not more than about 50.degree. C. (pH 7.5, 30 min) Km value
relative to creatine in a coupling assay using a sarcosine oxidase
and a peroxidase: ca. 6.5.+-.1.0 mM Molecular weight: ca. 43,000
(SDS-PAGE) Isoelectric point: ca. .[.3.5.]. .Iadd.4.5 .Iaddend.
A still another embodiment of the present invention is a novel
creatine amidinohydrolase having the following physicochemical
properties. Action: catalyzing the following reaction:
creatine+H.sub.2O.fwdarw.sarcosine+urea Optimum temperature: ca.
40-50.degree. C. Optimum pH: ca. 8.0-9.0 Heat stability: stable at
not more than about 50.degree. C. (pH 7.5, 30 min) Km value
relative to creatine in a coupling assay using a sarcosine oxidase
and a peroxidase: ca. 9.0.+-.1.0 mM Molecular weight: ca. 43,000
(SDS-PAGE) Isoelectric point: ca. .[.3.5.]. .Iadd.4.5 .Iaddend.
One method for producing the creatine amidinohydrolase of the
present invention comprises mutation of a gene encoding a wild
creatine amidinohydrolase by genetic engineering and protein
engineering method, generating a mutant DNA encoding a novel
creatine amidinohydrolase having a smaller Km value for creatine
than the wild creatine amidinohydrolase, expressing said DNA in a
suitable host and harvesting the creatine amidinohydrolase thus
produced.
While the gene encoding a wild creatine amidinohydrolase which is
to be mutated is not particularly limited, in one embodiment of the
present invention, it is the creatine amidinohydrolase gene
depicted in the Sequence Listing.cndot.SEQ ID:No.2, which is
derived from Alcaligenes.cndot.faecalis TE3581 (FERM P-14237).
In another embodiment of the present invention, a novel creatine
amidinohydrolase having a smaller Km value for creatine than a wild
creatine amidinohydrolase is produced by mutating the gene encoding
the amino acid sequence depicted in the Sequence Listing.cndot.SEQ
ID No:1.
A wild creatine amidinohydrolase gene can be mutated by any known
method. For example, a wild creatine amidinohydrolase DNA or a
microorganism cells having said gene is brought into contact with a
mutagenic agent, or ultraviolet irradiation is applied, or a
protein engineering method is used such as PCR and site-directed
mutagenesis. Alternatively, an Escherichia coli susceptible to gene
mutation at high frequency due to defective gene repair mechanism
may be transformed with a wild creatine amidinohydrolase gene DNA
for mutation in vivo.
For example, Escherichia coli is transformed with the mutant
creatine amidinohydrolase gene obtained above and plated on a
creatine amidinohydrolase activity detection agar medium [J.
Ferment. Bioeng., Vol. 76 No. 2 77-81(1993)], and the colonies
showing clear color development are selected. The selected colonies
are inoculated to a nutritive medium (e.g., LB medium and
2.times.YT medium) and cultured overnight at 37.degree. C. The
cells are disrupted and a crude enzyme solution is extracted.
The method for disrupting the cells may be any known method, such
as physical rupture (e.g., ultrasonication and glass bead rupture),
as well as by the use of a lysozyme. This crude enzyme solution is
used to determine the creatine amidinohydrolase activity of two
kinds of activity determination reaction solutions having different
substrate concentrations. Comparison of the activity ratios of the
two with that obtained using a wild creatine amidinohydrolase leads
to the screening of the creatine amidinohydrolase having smaller Km
value.
The method for obtaining the purified creatine amidinohydrolase
from the cell line selected as above may be any known method, such
as the following.
After the cells obtained by culturing in a nutrient medium are
recovered, they are ruptured by an enzymatic or physical method and
extracted to give a crude enzyme solution. A creatine
amidinohydrolase fraction is recovered from the obtained crude
enzyme solution by ammonium sulfate precipitation. The enzyme
solution is subjected to desalting by Sephadex G-25 (Pharmacia
Biotech) gel filtration and the like.
After this operation, the resulting enzyme solution is separated
and purified by octyl Sepharose CL-6B (Pharmacia Biotech) column
chromatography to give a standard purified enzyme product. This
product is purified to the degree that it shows almost a single
band by SDS-PAGE.
The microorganism to be used in the present invention to produce
the novel creatine amidinohydrolase is exemplified by Escherichia
coli JM109 (pCRH273M1) (FERM BP-5374), Escherichia coli JM109
(pCRH273M2) (FERM BP-5375), Escherichia coli JM109 (pCRH273M3)
(FERM BP-5376) and the like.
The method for culturing these microorganisms and recovering the
creatine amidinohydrolase of the present invention from the
cultures thereof are not particularly limited, and conventional
methods can be applied.
The novel creatine amidinohydrolase obtained by the above-mentioned
production method of the present invention has the following
physicochemical properties. Action: catalyzing the following
reaction: creatine+H.sub.2O.fwdarw.sarcosine+urea Optimum
temperature: ca. 40-50.degree. C. Optimum pH: ca. 8.0-9.0 Heat
stability: stable at not more than about 50.degree. C. (pH 7.5, 30
min) Km value relative to creatine in a coupling assay using a
sarcosine oxidase and a peroxidase: ca. 3.5-10.0 mM Molecular
weight: ca. 43,000 (SDS-PAGE) Isoelectric point: ca. .[.3.5.].
.Iadd.4.5 .Iaddend.
The Km value in the present invention is the value relative to
creatine in a coupling assay using a sarcosine oxidase and a
peroxidase. While the conventional enzyme derived from Pseudomonas
putida has a small apparent Km value for creatine of 1.33 mM
[Archives Biochemistry and Biophysics 177, 508-515 (1976)], the
activity is determined by measuring the residual creatine in the
reaction mixture with .alpha.-naphthol and diacetyl, and the Km
value for creatine by a coupling assay using a sarcosine oxidase
and a peroxidase, which are widely used as reagents for clinical
tests, has been unknown.
The creatine amidinohydrolase of the present invention can be used
for the determination of creatine upon combination with a sarcosine
oxidase and a composition for detection of hydrogen peroxide.
Moreover, when creatinine amidohydrolase is concurrently used,
creatinine can be determined as well.
The determination method of the present invention utilizes the
following reactions. ##STR00001## ##STR00002##
When creatinine is determined, the following reaction is further
utilized. ##STR00003##
The quinonimine pigment produced is generally subjected to the
determination of absorbance at 500-650 nm wavelength. The method
for determining creatine is an end method or a rate method, though
the end method is generally used.
The inventive creatine amidinohydrolase having smaller Km value can
reduce the amount of the enzyme to be used in the test reagent for
creatine or creatinine determination to about 1/3-1/4 as compared
to the necessary amount of conventional enzymes, and achieves good
reactivity in the latter half of the reaction.
The reagent for determining creatine in a sample of the present
invention contains the above-mentioned creatine amidinohydrolase,
sarcosine oxidase, and a composition for detecting hydrogen
peroxide.
The reagent for determining creatinine in a sample of the present
invention contains a creatinine amidohydrolase, the above-mentioned
creatinine amidohydrolase, sarcosine oxidase, and a composition for
detecting hydrogen peroxide.
The sarcosine oxidase to be used for detecting creatine or
creatinine of the present invention can be obtained from the
microorganisms originated from the genera Arthrobacter,
Corynebacterium, Alcaligenes, Pseudomonas, Micrococcus, Bacillus
and the like, and some of them are commercially available.
The creatinine amidohydrolase can be obtained from the
microorganisms originated from the genera Pseudomonas,
Flavobacterium, Alcaligenes, Penicillium and the like, and some of
them are commercially available.
The composition for the detection of hydrogen peroxide contains an
enzyme having a peroxidase activity, chromophore and a buffer. The
enzyme having a peroxidase activity is exemplified by peroxidase,
haloperoxidase, bromoperoxidase, lactoperoxidase, myeloperoxidase
and the like. The chromophore comprises a hydrogen receptor and a
coupler. The hydrogen receptor may be any as long as it receives
hydrogen in the reaction with hydrogen peroxide, peroxidase and a
coupler, which is specifically exemplified by 4-aminoantipyrine,
3-methyl-2-benzothiazoline-hydrazine derivative and the like.
Examples of the coupler include aniline derivatives such as aniline
and N-ethyl-N-(2-hydroxy-3-sulfopropyl)-m-toluidine (TOOS), phenol
derivatives such as phenol and p-chlorophenol, and the like.
The reagent for the determination of creatine of the present
invention contains each ingredient in a preferable proportion of
creatine amidinohydrolase ca. 5-300 U/ml, sarcosine oxidase ca.
1-100 U/ml, peroxidase ca. 0.01-50 U/ml, hydrogen donor ca. 0.1-10
mM, and a coupler ca. 0.1-50 mM.
The reagent for the determination of creatinine of the present
invention contains each ingredient in a preferable proportion of
creatinine amidohydrolase ca. 10-300 U/ml, creatine
amidinohydrolase ca. 10-300 U/ml, sarcosine oxidase ca. 1-100 U/ml,
peroxidase ca. 0.01-50 U/ml, hydrogen donor ca. 0.1-10 mM, and a
coupler ca. 0.1-50 mM.
The reagent for the determination of creatine or creatinine of the
present invention is generally used with a buffer having a pH of
about 6-8. Examples of the buffer include phosphate buffer, Good
buffer, Tris buffer and the like.
Where necessary, ascorbate oxidase or catalase may be added to the
reagent of the present invention. Other compounds may be also added
to the reagent of the present invention for smooth enzyme reaction
and color development. Such compounds are, for example,
stabilizers, surfactants, excipients and the like.
EXAMPLES
The present invention is described in detail by way of the
following Examples.
In the Examples, the activity of creatine amidinohydrolase was
determined as follows. The enzyme activity in the present invention
is defined to be the enzyme amount capable of producing 1 .mu.mole
of sarcosine per min under the following conditions being one unit
(U).
TABLE-US-00001 Reaction mixture composition 0.3 H HEPES pH 7.6
0.005% 4-aminoantipyrins 0.015% phenol 1.8% creatine 6 U/ml
sarcosine oxidase 6 U/ml perioxidase
The above-mentioned reaction mixture (3 ml) is taken with a cuvette
(d=1 cm) and preliminarily heated to 37.degree. C. for about 3
minutes. An enzyme solution (0.1 ml) is added, and the mixture is
gently admixed. Using water as a control, changes in absorbance at
500 nm are recorded for 5 minutes using a spectrophotometer
controlled to 37.degree. C. Based on the linear portion of 2-5
minutes thereof, changes in absorbance per minute are determined
(.DELTA.OD) test.
The blank test is performed in the same manner as above except that
a solution (0.1 ml, 50 mM potassium phosphate buffer, pH 7.5) for
diluting the enzyme is used instead of the enzyme solution and
changes in absorbance per minute are determined (.DELTA.OD
blank).
The enzyme amount is calculated by inserting each measure into the
following formula. .DELTA. .DELTA. .DELTA. .times..times.
.times..times..times. ##EQU00001## wherein each constant denotes
the following: 13.3: millimolar absorbance coefficient
(cm.sup.2/.mu.M) under the above measurement conditions of
quinonimine pigment 1/2: coefficient indicating that the
quinonimine pigment formed from one molecule of hydrogen peroxide
generated in the enzyme reaction is 1/2 molecule 1.0: light path
length (cm) 0.1: amount of enzyme added (ml)
Reference Example 1
Isolation of chromosomal DNA
The chromosomal DNA of Alcaligenes.cndot.faecalis TE3581 was
isolated by the following method.
The cells (FERM P-14237) were shake-cultured overnight at
30.degree. C. in a nutrient broth (150 ml) and the cells were
collected by centrifugation (8000 rpm, 10 min). The cells were
suspended in a solution (5 ml) containing 10% sucrose, 50 mM
Tris-HCl (pH 8.0) and 50 mM EDTA, and a lysozyme solution (1 ml, 10
mg/ml) was added. The mixture was incubated at 37.degree. C. for 15
min. Then, 10% SDS solution (1 ml) was added. An equivalent amount
(1 ml) of a chloroform.cndot.phenol solution (1:1) was added to
this mixture. The mixture was stirred and separated into an aqueous
layer and a solvent layer by centrifugation at 10,000 rpm for 3
min. The aqueous layer was separated, and onto this aqueous layer
was gently layered a 2-fold amount of ethanol. The content was
slowly stirred with a glass rod to allow the DNA to wind around the
rod.
This DNA was dissolved in 10 mM Tris-HCl solution (pH 8.0,
hereinafter abbreviated as TE) containing 1 mM EDTA. This solution
was treated with an equivalent amount of chloroform.cndot.phenol
solution. The aqueous layer was separated by centrifugation, and a
2-fold amount of ethanol was added. The DNA was separated again by
the method described above and dissolved in 2 ml of TE.
Reference Example 2
Preparation of DNA fragment containing a gene encoding creatinine
amidinohydrolase and recombinant vector containing said DNA
fragment
The DNA (20 .mu.g) obtained in Reference Example 1 was partially
cleaved with restriction enzyme Sau3AI (Toyo Boseki Kabushiki
Kaisha) and 2-10 kbp fragments were recovered by sucrose density
gradient centrifugation. Meanwhile, pBluescript KS(+) cleaved with
restriction enzyme BamHI (Toyo Boseki Kabushiki Kaisha) was
dephosphorylated with bacterial alkaline phosphatase (Toyo Boseki
Kabushiki Kaisha). Then, the both DNAs were treated with T4DNA
ligase (1 unit, Toyo Boseki Kabushiki Kaisha) at 16.degree. C. for
12 hr to ligate the DNA. Escherichia coli JM109 competent cell
(Toyo Boseki Kabushiki Kaisha) was transformed with the ligated DNA
and plated onto a creatine amidinohydrolase activity detection agar
medium [0.5% yeast extract, 0.2% meat extract, 0.5% polypeptone,
0.1% NaCl, 0.1% KH.sub.2PO.sub.4, 0.05% MgSO.sub.4/7H.sub.2O, 1.15%
creatine, 10 U/ml sarcosine oxidase (Toyo Boseki Kabushiki Kaisha),
0.5 U/ml peroxidase (Toyo Boseki Kabushiki Kaisha), 0.01%
o-dianisidine, 50 .mu.g/ml ampicillin and 1.5% agar]. The activity
of creatine amidinohydrolase was detected using, as the indices,
the colonies grown in the above-mentioned medium and stained in
brown. The colonies (ca. 1.times.10.sup.5) of the transformant were
obtained per DNA 1 .mu.g used.
About 12,000 colonies were screened, and 6 colonies were found
stained in brown. These strains were cultured in LB liquid medium
(1% polypeptone, 0.5% yeast extract, 0.5% NaCl, 50 .mu.g/ml
ampicillin) and creatine amidinohydrolase activity was determined,
as a result of which creatine amidinohydrolase activity was
detected in every strain. The plasmid of the strain which showed
the highest creatine amidinohydrolase activity contained ca. 5 kbp
insert DNA fragment, and this plasmid was named pCRH17.
Then, the insert DNA of pCHR17 was cleaved with restriction enzymes
EcoRV (Toyo Boseki Kabushiki Kaisha) and Pst1 (Toyo Boseki
Kabushiki Kaisha), and ligated to pBluescript KS(+) cleaved with
said restriction enzymes to prepare pCRH173.
Example 1
Preparation of recombinant plasmid pCRH273 by mutating creatine
amidinohydrolase gene
The region of from .beta.-galactosidase structural gene derived
from the vector to the upstream region of the creatine
amidinohydrolase structural gene of the insert DNA was deleted from
the recombinant plasmid pCRH173 of Reference Example 2, using the
synthetic DNA depicted in SEQ ID No:3 and a commercially available
mutation introduction kit (Transformer.TM.; Clonetech) to prepare
recombinant plasmid pCRH173M. The detailed method for introducing
the mutation was given in the protocol attached to the kit.
The pCRH173M was cleaved with restriction enzyme EcoRI (Toyo Boseki
Kabushiki Kaisha) and self-ligated to prepare pCRH273 (FIG. 1).
Example 2
Selection of candidate cell lines producing the objective mutant
creatine amidinohydrolase
A commercially available Escherichia coli competent cell (E. coli
XLI-Red; Clonetech) was transformed with the pCRH273 prepared in
Example 1, and the entire amount thereof was inoculated to 3 ml of
LB liquid medium (1% polypeptone, 0.5% yeast extract, 1.0% NaCl)
containing ampicillin (50 .mu.g/ml; Nakarai Tesque), which was
followed by shake culture overnight at 37.degree. C. A plasmid was
recovered from the entire amount of this culture by a conventional
method. The commercially available Escherichia coli competent cell
(E. coli JM109, Toyo Boseki Kabushiki Kaisha) was transformed again
with this plasmid and plated onto a creatine amidinohydrolase
activity detection agar medium, which was then incubated overnight
at 37.degree. C. The cell lines which showed a strong expression of
the creatine amidinohydrolase activity, i.e., the strains which
showed a deep color development, were selected from the mutant
creatine amidinohydrolase library thus obtained.
Example 3
Screening of creatine amidinohydrolase-producing cell line having a
reduced Km value
The candidate cell lines selected in Example 2 were inoculated to 3
ml of TB medium (1.2% polypeptone, 2.4% yeast extract, 0.4%
glycerol, 0.0231% KH.sub.2PO.sub.4, 0.1254% K.sub.2HPO.sub.4)
containing ampicillin (200 .mu.g/ml) and shake-cultured overnight
at 37.degree. C. The cells were recovered from 1 ml of the culture
by centrifugation, and a crude enzyme solution was prepared
therefrom by rupture with glass beads. Using the crude enzyme
solution thus obtained and following the above-mentioned activity
determination method, creatine amidinohydrolase was determined.
Meanwhile, using an activity determination reagent having a 1/10
substrate concentration, the creatine amidinohydrolase activity was
determined in the same manner. The cell line wherein the ratio of
the two kinds of the activity measures (activity with 1/10
substrate concentration+activity obtained by conventional manner)
increased beyond that of a wild creatine amidinohydrolase was
selected as a mutant having a reduced Km value.
About 20,000 cell lines were screened by the above method, and
three mutant cell lines having a smaller Km value for creatine were
obtained, and the respective recombinant plasmids thereof were
named pCRH273M1 (FERM BP-5374), pCRH273M2 (FERM BP-5375) and
pCRH273M3 (FERM BP-5376).
Example 4
Preparation of creatine amidinohydrolase from Escherichia coli
JM109 (pCRH273M1)
TB medium (6 L) was dispensed to 10 L jar fermentors, and subjected
to autoclaving at 121.degree. C. for 15 min. After allowing them to
cool, 50 mg/ml ampicillin (Nakarai Tesque) and 200 mM IPTG (Nippon
Seika Corp.), which had been separately sterilized by filtration,
were added by 6 ml each. To this medium was added 60 ml of the
culture of Escherichia coli JM109 (pCRH273M1)(FERM BP-5374) after
previous shake culture at 30.degree. C. for 24 hr, which was
followed by aeration culture at 37.degree. C. for 24 hr. The
activity of creatine amidinohydrolase after the completion of the
culture was 8.7 U/ml.
The above-mentioned cells were collected by centrifugation, and
suspended in 50 mM phosphate buffer, pH 7.0.
The cells in this suspension were ruptured with a French press and
subjected to centrifugation to give a supernatant. The obtained
crude enzyme solution was subjected to ammonium sulfate
fractionation, desalting with Sephadex G-25 (Pharmacia Biotech) gel
filtration and purified by octyl Sepharose CL-6B (Pharmacia
Biotech) column chromatography to give a purified enzyme product.
The standard creatine amidinohydrolase product obtained by this
method showed a nearly single band by SDS-PAGE and had a specific
activity then of 18.4 U/mg protein.
Table 1 shows the purification performed so far. Table 2 shows
physicochemical properties of the creatine amidinohydrolase
obtained by the above methods.
TABLE-US-00002 TABLE 1 Purification of Cratine amidinohydrolase
from Escherichia coli JM109 (pCRH273M1) Total Activity Specific
activity Yield Step (U) (U/mg-protein) (%) French press rupture
52200 100.0 (NH.sub.4).sub.2SO.sub.4 precipitation - redissolution
49746 8.3 95.3 Sephadex G-25 46927 10.3 89.9 Octyl Sepharose CL-6B
33094 18.4 63.4
TABLE-US-00003 TABLE 2 Physiochemical properties of creation
amidinohydrolase purified from Escherichia coli JM109 (pCRH273M1)
Item Physicochemical properties Action creatine + H.sub.2O .fwdarw.
sarcosine + urea Optimal temperature ca. 40.degree. C.-50.degree.
C. Optimal pH ca. 8.0-9.0 Thermal stability ca. 50.degree. C. (50
mM potassium phosphate buffer, pH 7.5, 30 min treatment) pH
stability ca. 5-8 (40.degree. C., 18 hr preservation) Km value ca.
6.5 mM (creatine) Molecular weight ca. 43,000 (SDS-PAGE)
Isoelectric point ca. [3.5] 4.5 (Isoelectric focusing)
Example 5
Preparation of creatine amidinohydrolase from Escherichia coli
JM109 (pCRH273M2)
TB medium (6 L) was dispensed to 10 L jar fermentors, and subjected
to autoclaving at 121.degree. C. for 15 min. After allowing them to
cool, 50 mg/ml ampicillin (Nakarai Tesque) and 200 mM IPTG (Nippon
Seika Corp.), which had been separately sterilized by filtration,
were added by 6 ml each. To this medium was added 60 ml of the
culture of Escherichia coli JM109 (pCRH273M2)(FERM BP-5375) after
previous shake culture at 30.degree. C. for 24 hr, which was
followed by aeration culture at 37.degree. C. for 24 hr. The
activity of creatine amidinohydrolase after the completion of the
culture was 5.6 U/ml.
The above-mentioned cells were collected by centrifugation, and
suspended in 50 mM phosphate buffer, pH 7.0.
The cells in this suspension were ruptured with a French press and
subjected to centrifugation to give a supernatant. The obtained
crude enzyme solution was subjected to ammonium sulfate
fractionation, desalting with Sephadex G-25 (Pharmacia Biotech) gel
filtration and purified by octyl Sepharose CL-6B (Pharmacia
Biotech) column chromatography to give a purified enzyme product.
The standard creatine amidinohydrolase product obtained by this
method showed a nearly single band by SDS-PAGE and had a specific
activity then of 14.3 U/mg protein.
Table 3 shows the purification performed so far. Table 4 shows
physicochemical properties of the creatine amidinohydrolase
obtained by the above methods.
TABLE-US-00004 TABLE 3 Purification of creatine amidinohydrolase
from Escherichia coli JM109 (pCRH273M2) Total activity Specific
activity Yield Step (U) (U/mg-protein) (%) French press rupture
33600 100.0 (NH.sub.4).sub.2SO.sub.4 precipitation - redissolution
25636 7.2 76.3 Sephadex G-25 24326 9.8 72.4 Octyl Sepharose CL-6B
19689 14.3 58.6
TABLE-US-00005 TABLE 4 Phsicochemical properties of ceatine
amidinohydralase purified from Escherichia coli JM109 (pCRH273M2)
Item Physicochemical properties Action creatine + H.sub.2O .fwdarw.
sarcosine + urea Optimal temperature ca. 45.degree. C.-50.degree.
C. Optimal pH ca. 8.0-9.0 Thermal stability ca. 40.degree. C. (50
mM potassium phosphate buffer, pH 7.5, 30 min treatment) pH
stability ca. 5-8 (40.degree. C., 18 hr preservation) Km value ca.
4.5 mM (creatine) Molecular weight ca. 43,000 (SDS-PAGE)
Isoelectric point ca. [3.5] 4.5 (isoelectric focusing)
Example 6
Preparation of creatine amidinohydrolase from Escherichia coli
JM109 (pCRH273M3)
TB medium (6 L) was dispensed to 10 L jar fermentors, and subjected
to autoclaving at 121.degree. C. for 15 min. After allowing them to
cool, 50 mg/ml ampicillin (Nakarai Tesque) and 200 mM IPTG (Nippon
Seika Corp.) which had been separately sterilized by filtration
were added by 6 ml each. To this medium was added 60 ml of culture
of Escherichia coli JM109 (pCRH273M3)(FERM BP-5376) after previous
shake culture at 30.degree. C. for 24 hr, which was followed by
aeration culture at 37.degree. C. for 24 hr. The activity of
creatine amidinohydrolase after the completion of the culture was
8.3 U/ml.
The above-mentioned cells were collected by centrifugation, and
suspended in 50 mM phosphate buffer, pH 7.0.
The cells in this suspension were ruptured with a French press and
subjected to centrifugation to give a supernatant. The obtained
crude enzyme solution was subjected to ammonium sulfate
fractionation, desalting by Sephadex G-25 (Pharmacia Biotech) gel
filtration and purified by octyl Sepharose CL-6B (Pharmacia
Biotech) column chromatography to give a purified enzyme product.
The standard creatine amidinohydrolase product obtained by this
method showed a nearly single band by SDS-PAGE and had a specific
activity then of 14.8 U/mg protein.
Table 5 shows the purification performed so far. Table 6 shows
physicochemical properties of the creatine amidinohydrolase
obtained by the above methods.
TABLE-US-00006 TABLE 5 Purification of creatine amidinohydrolase
from Escherichia coli JM109 (pCRH273M3) Total activity Specific
activity Yield Step (U) (U/mg-protein) (%) French press rupture
49800 100.0 (NH.sub.4).sub.2SO.sub.4 precipitation - redissolution
43027 8.3 86.4 Sephadex G-25 39989 9.9 80.3 Octyl Sepharose CL-6B
32021 14.8 64.3
TABLE-US-00007 TABLE 6 Phsicochemical properties of ceatine
amidinohydralase purified from Escherichia coli JM109 (pCRH273M3)
Item Physicochemical properties Action creatine + H.sub.2O .fwdarw.
sarcosine + urea Optimal temperature ca. 40.degree. C.-45.degree.
C. Optimal pH ca. 8.0-9.0 Thermal stability ca. 40.degree. C. (50
mM potassium phosphate buffer, pH 7.5, 30 min treatment) pH
stability ca. 5-8 (40.degree. C., 18 hr preservation) Km value ca.
9.0 mM (creatine) Molecular weight ca. 43,000 (SDS-PAGE)
Isoelectric point ca. [3.5] 4.5 (isoelectric focusing)
The following Table 7 summarizes the Km values for creatine of the
novel creatine amidinohydrolases of the present invention and wild
creatine amidinohydrolase. As is evident from Table 7, the novel
creatine amidinohydrolases of the present invention had reduced Km
values as compared to the wild creatine amidinohydrolase.
TABLE-US-00008 TABLE 7 Enzyme Km value wild 15.2 mM pCRH273M1 6.5
mM pCRH273M2 4.5 mM pCRH273M3 9.0 mM
Example 7
Using the purified creatine amidinohydrolase prepared in Example 5
and wild creatine amidinohydrolase, a creatinine determination
reagent having the following composition was prepared, and the
amounts of the creatine amidinohydrolase necessary for giving a
creatinine determination reagent was compared.
TABLE-US-00009 creatine amidinohydrolase of Example 5 20, 40, 60
U/ml or wild cratine amidinohydrolase creatinine amidohydrolase 150
U/ml sarcosine oxidase 7 U/ml peroxidase 3 PU/ml MOPS buffer 0.1 M,
pH 8.0 Triton X-100 0.1% 4-aminoantipyrine 0.15 mM TOOS (aniline
derivative) 0.2 mM
The above-mentioned solution (3 ml) was added to a sample (60
.mu.l) containing creatinine (100 mg/dl) and changes in absorbance
were determined at 37.degree. C. at wavelength 546 nm. The time
course results are shown in FIG. 2. In the Figure, "Wild" shows a
wild creatine amidinohydrolase and "pCRH273M2" is the creatine
amidinohydrolase of the present invention.
As is evident from FIG. 2, when the determination was ended in 5
minutes, the creatine amidinohydrolase of the present invention
enabled determination with less enzyme amount (ca. 1/3 amount) as
compared to the wild creatine amidinohydrolase. It was also
confirmed that the reactivity during the latter half of the
determination, i.e., when the creatine in the sample decreased, was
fine.
SEQUENCE LISTINGS
1
SEQUENCE LISTING (1) GENERAL INFORMATION: (iii) NUMBER OF
SEQUENCES: 3 (2) INFORMATION FOR SEQ ID NO: 1: (i) SEQUENCE
CHARACTERISTICS: (A) LENGTH: 404 amino acids (B) TYPE: amino acid
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: <Unknown> (A)
DESCRIPTION: protein (vi) ORIGINAL SOURCE: (A) ORGANISM:
Alcaligenes faecalis (B) STRAIN: TE3581 (FERM P-14237) (ix)
FEATURE: (A) NAME/KEY: mat peptide (B) LOCATION: 1 to 404 (D) OTHER
INFORMATION: protein having creatine amidino- hydrolase activity
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: Met Thr Asp Asp Met Leu
His Val Met Lys Trp His Asn Gly Glu Lys 1 5 10 15 Asp Tyr Ser Pro
Phe Ser Asp Ala Glu Met Thr Arg Arg Gln Asn Asp 20 25 30 Val Arg
Gly Trp Met Ala Lys Asn Asn Val Asp Ala Ala Leu Phe Thr 35 40 45
Ser Tyr His Cys Ile Asn Tyr Tyr Ser Gly Trp Leu Tyr Cys Tyr Phe 50
55 60 Gly Arg Lys Tyr Gly Met Val Ile Asp His Asn Asn Ala Thr Thr
Ile 65 70 75 80 Ser Ala Gly Ile Asp Gly Gly Gln Pro Trp Arg Arg Ser
Phe Gly Asp 85 90 95 Asn Ile Thr Tyr Thr Asp Trp Arg Arg Asp Asn
Phe Tyr Arg Ala Val 100 105 110 Arg Gln Leu Thr Thr Gly Ala Lys Arg
Ile Gly Ile Glu Phe Asp His 115 120 125 Val Asn Leu Asp Phe Arg Arg
Gln Leu Glu Glu Ala Leu Pro Gly Val 130 135 140 Glu Phe Val Asp Ile
Ser Gln Pro Ser Met Trp Met Arg Thr Ile Lys 145 150 155 160 Ser Leu
Glu Glu Gln Lys Leu Ile Arg Glu Gly Ala Arg Val Cys Asp 165 170 175
Val Gly Gly Ala Ala Cys Ala Ala Ala Ile Lys Ala Gly Val Pro Glu 180
185 190 His Glu Val Ala Ile Ala Thr Thr Asn Ala Met Ile Arg Glu Ile
Ala 195 200 205 Lys Ser Phe Pro Phe Val Glu Leu Met Asp Thr Trp Thr
Trp Phe Gln 210 215 220 Ser Gly Ile Asn Thr Asp Gly Ala His Asn Pro
Val Thr Asn Arg Ile 225 230 235 240 Val Gln Ser Gly Asp Ile Leu Ser
Leu Asn Thr Phe Pro Met Ile Phe 245 250 255 Gly Tyr Tyr Thr Ala Leu
Glu Arg Thr Leu Phe Cys Asp His Val Asp 260 265 270 Asp Ala Ser Leu
Asp Ile Trp Glu Lys Asn Val Ala Val His Arg Arg 275 280 285 Gly Leu
Glu Leu Ile Lys Pro Gly Ala Arg Cys Lys Asp Ile Ala Ile 290 295 300
Glu Leu Asn Glu Met Tyr Arg Glu Trp Asp Leu Leu Lys Tyr Arg Ser 305
310 315 320 Phe Gly Tyr Gly His Ser Phe Gly Val Leu Cys His Tyr Tyr
Gly Arg 325 330 335 Glu Ala Gly Val Glu Leu Arg Glu Asp Ile Asp Thr
Glu Leu Lys Pro 340 345 350 Gly Met Val Val Ser Met Glu Pro Met Val
Met Leu Pro Glu Gly Met 355 360 365 Pro Gly Ala Gly Gly Tyr Arg Glu
His Asp Ile Leu Ile Val Gly Glu 370 375 380 Asp Gly Ala Glu Asn Ile
Thr Gly Phe Pro Phe Gly Pro Glu His Asn 385 390 395 400 Ile Ile Arg
Asn 404 (2) INFORMATION FOR SEQ ID NO: 2: (i) SEQUENCE
CHARACTERISTICS: (A) LENGTH: 1212 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE:
genomic DNA (vi) ORIGINAL SOURCE: (A) ORGANISM: Alcaligenes
faecalis (B) STRAIN: TE3581 (FERM P-14237) (ix) FEATURE: (A)
NAME/KEY: CDS (B) LOCATION: 1 to 1212 (xi) SEQUENCE DESCRIPTION:
SEQ ID NO: 2: ATG ACT GAC GAC ATG TTG CAC GTG ATG AAA TGG CAC AAC
GGC GAG AAA 48 Met Thr Asp Asp Met Leu His Val Met Lys Trp His Asn
Gly Glu Lys 1 5 10 15 GAT TAT TCG CCG TTT TCG GAT GCC GAG ATG ACC
CGC CGC CAA AAC GAC 96 Asp Tyr Ser Pro Phe Ser Asp Ala Glu Met Thr
Arg Arg Gln Asn Asp 20 25 30 GTT CGC GGC TGG ATG GCC AAG AAC AAT
GTC GAT GCG GCG CTG TTC ACC 144 Val Arg Gly Trp Met Ala Lys Asn Asn
Val Asp Ala Ala Leu Phe Thr 35 40 45 TCT TAT CAC TGC ATC AAC TAC
TAT TCC GGC TGG CTG TAC TGC TAT TTC 192 Ser Tyr His Cys Ile Asn Tyr
Tyr Ser Gly Trp Leu Tyr Cys Tyr Phe 50 55 60 GGA CGC AAG TAC GGC
ATG GTC ATC GAC CAC AAC AAC GCC ACG ACG ATT 240 Gly Arg Lys Tyr Gly
Met Val Ile Asp His Asn Asn Ala Thr Thr Ile 65 70 75 80 TCG GCC GGC
ATC GAC GGC GGC CAG CCC TGG CGC CGC AGC TTC GGC GAC 288 Ser Ala Gly
Ile Asp Gly Gly Gln Pro Trp Arg Arg Ser Phe Gly Asp 85 90 95 AAC
ATC ACC TAC ACC GAC TGG CGC CGC GAC AAT TTC TAT CGC GCC GTG 336 Asn
Ile Thr Tyr Thr Asp Trp Arg Arg Asp Asn Phe Tyr Arg Ala Val 100 105
110 CGC CAG CTG ACC ACG GGC GCC AAG CGC ATC GGC ATC GAG TTC GAC CAC
384 Arg Gln Leu Thr Thr Gly Ala Lys Arg Ile Gly Ile Glu Phe Asp His
115 120 125 GTC AAT CTC GAC TTC CGC CGC CAG CTC GAG GAA GCC CTA CCG
GGC GTC 432 Val Asn Leu Asp Phe Arg Arg Gln Leu Glu Glu Ala Leu Pro
Gly Val 130 135 140 GAG TTC GTC GAC ATC AGC CAG CCC TCG ATG TGG ATG
CGC ACC ATC AAG 480 Glu Phe Val Asp Ile Ser Gln Pro Ser Met Trp Met
Arg Thr Ile Lys 145 150 155 160 TCG CTC GAA GAG CAG AAG CTG ATC CGC
GAA GGC GCC CGC GTG TGT GAC 528 Ser Leu Glu Glu Gln Lys Leu Ile Arg
Glu Gly Ala Arg Val Cys Asp 165 170 175 GTC GGC GGC GCG GCC TGC GCG
GCT GCC ATC AAG GCC GGC GTG CCC GAG 576 Val Gly Gly Ala Ala Cys Ala
Ala Ala Ile Lys Ala Gly Val Pro Glu 180 185 190 CAT GAA GTG GCG ATC
GCC ACC ACC AAT GCG ATG ATC CGC GAG ATC GCC 624 His Glu Val Ala Ile
Ala Thr Thr Asn Ala Met Ile Arg Glu Ile Ala 195 200 205 AAA TCG TTC
CCC TTC GTG GAG CTG ATG GAC ACC TGG ACC TGG TTC CAG 672 Lys Ser Phe
Pro Phe Val Glu Leu Met Asp Thr Trp Thr Trp Phe Gln 210 215 220 TCG
GGC ATC AAC ACC GAC GGC GCG CAC AAT CCG GTC ACC AAC CGC ATC 720 Ser
Gly Ile Asn Thr Asp Gly Ala His Asn Pro Val Thr Asn Arg Ile 225 230
235 240 GTG CAA TCC GGC GAC ATC CTT TCG CTC AAC ACC TTC CCG ATG ATC
TTC 768 Val Gln Ser Gly Asp Ile Leu Ser Leu Asn Thr Phe Pro Met Ile
Phe 245 250 255 GGC TAC TAC ACC GCG CTG GAG CGC ACG CTG TTC TGC GAC
CAT GTC GAT 816 Gly Tyr Tyr Thr Ala Leu Glu Arg Thr Leu Phe Cys Asp
His Val Asp 260 265 270 GAC GCC AGC CTC GAC ATC TGG GAG AAG AAC GTG
GCC GTG CAT CGC CGC 864 Asp Ala Ser Leu Asp Ile Trp Glu Lys Asn Val
Ala Val His Arg Arg 275 280 285 GGG CTC GAG CTG ATC AAG CCG GGC GCG
CGC TGC AAG GAC ATC GCC ATC 912 Gly Leu Glu Leu Ile Lys Pro Gly Ala
Arg Cys Lys Asp Ile Ala Ile 290 295 300 GAG CTC AAC GAG ATG TAC CGC
GAG TGG GAC CTG CTG AAG TAC CGC TCC 960 Glu Leu Asn Glu Met Tyr Arg
Glu Trp Asp Leu Leu Lys Tyr Arg Ser 305 310 315 320 TTC GGC TAT GGC
CAC TCC TTC GGC GTG CTG TGC CAC TAC TAC GGT CGC 1008 Phe Gly Tyr
Gly His Ser Phe Gly Val Leu Cys His Tyr Tyr Gly Arg 325 330 335 GAG
GCC GGC GTG GAG CTG CGC GAG GAC ATC GAC ACC GAG CTG AAG CCC 1056
Glu Ala Gly Val Glu Leu Arg Glu Asp Ile Asp Thr Glu Leu Lys Pro 340
345 350 GGC ATG GTG GTC TCC ATG GAG CCG ATG GTG ATG CTG CCG GAG GGC
ATG 1104 Gly Met Val Val Ser Met Glu Pro Met Val Met Leu Pro Glu
Gly Met 355 360 365 CCC GGT GCC GGC GGC TAT CGC GAG CAC GAC ATC CTG
ATC GTC GGG GAG 1152 Pro Gly Ala Gly Gly Tyr Arg Glu His Asp Ile
Leu Ile Val Gly Glu 370 375 380 GAC GGT GCC GAG AAC ATC ACC GGC TTC
CCG TTC GGT CCG GAA CAC AAC 1200 Asp Gly Ala Glu Asn Ile Thr Gly
Phe Pro Phe Gly Pro Glu His Asn 385 390 395 400 ATC ATC CGC AAC
1212 Ile Ile Arg Asn 404 (2) INFORMATION FOR SEQ ID NO: 3: (i)
SEQUENCE CHARACTERISTICS: (A) LENGTH: 39 base pairs (B) TYPE:
nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)
MOLECULE TYPE: other nucleic acid (synthetic DNA) (xi) SEQUENCE
DESCRIPTION: SEQ ID NO: 3: CAACATGTCG TCAGTCATAT GTGTTTCCTG
TGTGAAATT 39
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