JP2005192538A - New creatinine amidinohydrolase, method for producing the same and use of the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a creatinine amidinohydrolase having a small Km value, preferably approximately ≤15.0 as a general clinical testing enzyme. <P>SOLUTION: This new creatinine amidinohydrolase having the following physicochemical properties is obtained by using a gene encoding the creatinine amidinohydrolase and derived from bacteria belonging to the genus Alcaligenes, introducing a mutation in the gene and by using a protein engineering method. As the creatine or creatinine determination reagent, the enzyme is used. The physicochemical properties are as follows: The activity is: Creatine + H<SB>2</SB>O → Sarcosine + Urea. Optimal temperature is approximately 40-50°C. Optimal pH is approximately 8-9. Thermal stability is approximately ≤50°C (at pH 7.5 for 30 min). Km value for the creatine is 3.0-10.0 mM in the case of performing an activity measurement by using sarcosineoxidase and peroxidase as coordinate enzymes. Molecular weight is approximately 43,000 (SDS-PAGE). Isoelectric point is approximately 4.5. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a novel creatine amidinohydrolase, a method for producing the same, and a reagent for quantification of creatine or creatinine using the enzyme.

Creatinine and creatine are found in the blood or urine, and the rapid and accurate detection and measurement of the amount diagnoses diseases such as uremia, chronic nephritis, acute nephritis, giantism, ankylosing myopathy Very important to. In order to make such a diagnosis, quantification of creatine and creatinine in blood or urine is frequently performed.
As a conventional method for quantifying creatine, creatine amidinohydrolase and sarcosine oxidase are allowed to act on creatine in a sample, and the generated hydrogen peroxide is measured by hydrogen peroxide measuring means to quantify creatine in the sample. In addition, there is a method in which creatinine amide hydrolase, creatine amidinohydrolase and sarcosine oxidase are allowed to act on creatinine, and the generated hydrogen peroxide is measured by a hydrogen peroxide measuring means to quantify creatinine in a sample.
On the other hand, creatinine amide hydrolase, creatine amidinohydrolase, and sarcosine oxidase are widely found in the microbial community, and are already industrially produced and used as clinical diagnostics.

However, creatine amidinohydrolase produced from various known microbial cells has low heat stability and a large Km value for creatine. For example, an enzyme derived from the genus Bacillus (Japanese Patent Publication No. 61-17465) has a low thermal stability of 40 ° C. or lower. Furthermore, the apparent Km value for Pseudomonas putida is as low as 1.33 mM for creatine (ArchivesBiochemistry and Biophysics 177, 508-515 (1976)), but the activity measurement method is different and is widely used in clinical diagnostics. The Km value for creatine in an activity measurement method using sarcosine oxidase and peroxidase as a conjugate enzyme was unknown. The enzymes derived from Corynebacterium, Micrococcus, Actinobacillus, and Bacillus (Japanese Patent Publication No. 3-76915) have a thermal stability of 50 ° C. or less, but the Km value for creatine was as large as about 20 mM. .
In order to solve the above problems, the present inventors have already demonstrated that bacteria belonging to the genus Alcaligenes are excellent in thermal stability and have a relatively small Km value for creatine (Km value: about 15.2). It has been found that amidinohydrolase is produced (Japanese Patent Application No. 6-63363). Furthermore, a creatine amidinohydrolase gene having a relatively low Km value for creatine was isolated from the strain, and a technique for producing the enzyme in large quantities using a gram-negative bacterium as a host was established (Japanese Patent Application No. 7-117283).
Further, a creatine amidinohydrolase that is stable in a high pH range and has a small Km value has been reported from the same Alkagenes bacterium (Japanese Patent Laid-Open No. 7-170979).

  However, these creatine amidinohydrolases have been demanded as creatine amidinohydrolase having a large Km value and a small Km value, preferably about 15.0 or less, as an enzyme that is a general clinical test drug.

  As a result of intensive studies to achieve the above object, the present inventors have created a novel creatine amidinohydrolase having a smaller Km value for creatine by a protein engineering technique using the creatine amidinohydrolase gene derived from the genus Alkagenes. Succeeded in doing.

  That is, the present invention is a novel creatine amidinohydrolase having the following physicochemical properties.

Action: Creatine + H ₂ O → Sarcosine + Urea Optimal temperature: about 40 ° C to 50 ° C
Optimum pH: about 8.0 to 9.0
Thermal stability: about 50 ° C. or less (pH 7.5, 30 minutes)
Km value for creatine when sarcosine oxidase and peroxidase are used as conjugate enzymes and the activity is measured: about 3.5 to 10.0 mM
Molecular weight: about 43,000 (SDS-PAGE)
Isoelectric point: about 4.5
The present invention also provides a novel creatine amidinohydrolase production method characterized by culturing a microorganism that produces a novel creatine amidinohydrolase having the following physicochemical properties in a nutrient medium and collecting the novel creatine amidinohydrolase: is there.

Action: Creatine + H ₂ O → Sarcosine + Urea Optimal temperature: about 40 ° C to 50 ° C
Optimum pH: about 8.0 to 9.0
Thermal stability: about 50 ° C. or less (pH 7.5, 30 minutes)
Km value for creatine when sarcosine oxidase and peroxidase are used as conjugate enzymes and the activity is measured: about 3.5 to 10.0 mM
Molecular weight: about 43,000 (SDS-PAGE)
Isoelectric point: about 4.5
The present invention is a reagent for quantifying creatine in a sample, comprising the creatine amidinohydrolase, sarcosine oxidase and a composition for detecting hydrogen peroxide.

  The present invention also provides a reagent for quantifying creatinine in a sample, comprising creatinine amide hydrolase, the creatine amidinohydrolase, sarcosine oxidase, and a composition for detecting hydrogen peroxide.

  According to the present invention, a novel creatine amidinohydrolase having a small Km value that is useful as an enzyme for diagnostic drugs can be created, and the creatine amidinohydrolase can be produced industrially in large quantities.

  One embodiment of the present invention is a novel creatine amidinohydrolase having the following physicochemical properties:

Action: Creatine + H ₂ O → Sarcosine + Urea Optimal temperature: about 40 ° C to 50 ° C
Optimum pH: about 8.0 to 9.0
Thermal stability: about 50 ° C. or less (pH 7.5, 30 minutes)
Km value for creatine when sarcosine oxidase and peroxidase are used as conjugate enzymes and the activity is measured: about 4.5 ± 1.0
Molecular weight: about 43,000 (SDS-PAGE)
Isoelectric point: about 4.5
Another embodiment is a novel creatine amidinohydrolase having the following physicochemical properties.

Action: Creatine + H ₂ O → Sarcosine + Urea Optimal temperature: about 40 ° C to 50 ° C
Optimum pH: about 8.0 to 9.0
Thermal stability: about 50 ° C. or less (pH 7.5, 30 minutes)
Km value for creatine when sarcosine oxidase and peroxidase are used as conjugate enzymes and the activity is measured: about 6.5 ± 1.0
Molecular weight: about 43,000 (SDS-PAGE)
Isoelectric point: about 4.5
Furthermore, another embodiment is a novel creatine amidinohydrolase having the following physicochemical properties:

Action: Creatine + H ₂ O → Sarcosine + Urea Optimal temperature: about 40 ° C to 50 ° C
Optimum pH: about 8.0 to 9.0
Thermal stability: about 50 ° C. or less (pH 7.5, 30 minutes)
Km value for creatine when sarcosine oxidase and peroxidase are used as conjugate enzymes and the activity is measured: about 9.0 ± 1.0
Molecular weight: about 43,000 (SDS-PAGE)
Isoelectric point: about 4.5
One of the methods for producing the creatine amidinohydrolase of the present invention is to introduce a mutation into a gene encoding wild type creatine amidinohydrolase, and to increase the Km value for creatine as compared with wild-type creatine amidinohydrolase by a protein engineering technique. This is a method for producing a reduced novel creatine amidinohydrolase.

The gene encoding wild-type creatine amidinohydrolase for introducing the mutation is not particularly limited, but in one embodiment of the present invention, Alcaligenes faecalis (Alcaligenes
faecalis) The creatine amidinohydrolase gene of SEQ ID NO: 2 derived from TE3581 (FERM P-14237) was used.

  Another embodiment of the present invention is a novel creatine having a reduced Km value for creatine as compared to wild-type creatine amidinohydrolase by introducing a mutation into the gene encoding the amino acid sequence described in SEQ ID NO: 1 Amidinohydrolase is produced.

  Any known method can be used for introducing a mutation into the wild-type creatine amidinohydrolase gene. For example, protein engineering techniques such as PCR and site-specific mutagenesis can be used, such as a method of contacting wild-type creatine amidinohydrolase gene DNA with a drug as a mutation source or a method using ultraviolet irradiation. In addition, since the gene repair mechanism is deficient, it is also possible to introduce mutations in vivo using E. coli, in which mutations occur frequently in genes.

After transforming the mutant creatine amidinohydrolase gene obtained as described above into, for example, E. coli, an agar medium for detecting creatine amidinohydrolase activity (J. Ferment. Bioeng., Vol. 76 No. 2 77-81 (1993) ) And select colonies with strong color development. The selected colonies are inoculated into a nutrient medium such as LB medium or 2 × YT medium, cultured at 37 ° C. overnight, and then disrupted to extract the crude enzyme solution.
Any known method may be used as a method for crushing bacterial cells. For example, a physical crushing method such as ultrasonic treatment or glass bead crushing or a method using a lytic enzyme such as lysozyme can be used. Using this crude enzyme solution, the creatine amidinohydrolase activity was measured with two types of activity measurement reaction solutions with different substrate concentrations, and the ratio of both was compared with that of wild-type creatine amidinohydrolase, thereby reducing the Km value for creatine. The resulting creatine amidinohydrolase can be screened.
As a method for obtaining purified creatine amidinohydrolase from a strain selected by the above method, any known method may be used, for example, the following method.
After collecting the cells obtained by culturing in a nutrient medium, the crude enzyme solution is obtained by crushing and extraction by enzymatic or physical crushing methods. A creatine amidinohydrolase fraction is recovered from the obtained crude enzyme extract by ammonium sulfate precipitation. This crude enzyme solution can be desalted by a method such as Sephadex G-25 (Pharmacia Biotech) gel filtration.
After this operation, separation and purification can be performed by octyl sepharose CL-6B (Pharmacia Biotech) column chromatography to obtain a purified enzyme preparation. This purified enzyme preparation is purified to such an extent that it shows an almost single band by SDS-PAGE.
Examples of microorganisms that produce a novel creatine amidinohydrolase used in the present invention include Escherichia coli JM109 (pCRH273M1) (FERM BP-5374), Escherichia coli JM109 (pCRH273M2) (FERM BP-5375), and Escherichia coli JM109. pCRH273M3) (FERM BP-5376).
The method for culturing these microorganisms and collecting the creatine amidinohydrolase of the present invention from the culture is not particularly limited, and follows a normal method.
The novel creatine amidinohydrolase obtained by the above production method of the present invention has the following physicochemical properties.

Action: Creatine + H ₂ O → Sarcosine + Urea Optimal temperature: about 40 ° C to 50 ° C
Optimum pH: about 8.0 to 9.0
Thermal stability: about 50 ° C. or less (pH 7.5, 30 minutes)
Km value for creatine when sarcosine oxidase and peroxidase are used as conjugate enzymes and the activity is measured: about 3.5 to 10.0 mM
Molecular weight: about 43,000 (SDS-PAGE)
Isoelectric point: about 4.5
The Km value in the present invention is a Km value for creatine when sarcosine oxidase and peroxidase are used as conjugate enzymes and activity is measured. The conventional enzyme derived from Pseudomonas putida has a small apparent Km value for creatine of 1.33 mM (ArchivesBiochemistry and Biophysics 177, 508-515).
(1976)), the activity measurement method is a method for quantifying the remaining creatine in the reaction solution with α-naphthol and diacetyl, and the activity using sarcosine oxidase and peroxidase, which are widely used in clinical test drugs, as a conjugate enzyme The Km value for creatine in the measurement method was unknown.

  The creatine amidinohydrolase of the present invention can be used for measuring creatine by combining sarcosine oxidase and a composition for detecting hydrogen peroxide. Creatinine can be quantified in the presence of creatinine amide hydrolase.

  The creatine quantification reagent in the sample of the present invention contains the creatine amidinohydrolase, sarcosine oxidase and the composition for detecting hydrogen peroxide.

  The reagent for quantifying creatinine in the sample of the present invention contains creatinine amide hydrolase, the above creatine amidinohydrolase, sarcosine oxidase, and a composition for detecting hydrogen peroxide.

  The sarcosine oxidase used for quantification of creatine or creatinine of the present invention can be obtained from microorganisms of origin such as Arthrobacter, Corynebacterium, Alkagenes, Pseudomonas, Micrococcus, Bacillus, etc. Some of these are already on the market.

  Creatinine amide hydrolase can be obtained from microorganisms of the genus Pseudomonas, Flavobacterium, Alkagenes, Penicillium, and some of these are already commercially available.

  The composition for detecting hydrogen peroxide includes an enzyme having peroxidase activity, a chromogen, and a buffer. Examples of the enzyme having peroxidase activity include peroxidase, haloperoxidase, bromoperoxidase, lactoperoxidase, myeloperoxidase and the like. The chromogen further comprises a hydrogen acceptor and a coupler. The hydrogen acceptor may be a compound that accepts hydrogen in the reaction with hydrogen peroxide, peroxidase, or a coupler, and examples thereof include 4-aminoantipyrine, 3-methyl-2-benzthiazoline-hydrazine derivatives, and the like. Examples of the coupler include aniline derivatives such as aniline and N-ethyl-N- (2-hydroxy-3-sulfopropyl) -m-toluidine (TOOS), and phenol derivatives such as phenol and p-chlorophenol.

  In the reagent for quantifying creatine of the present invention, preferred amounts of each component are about 5 to 300 U / ml of creatine amidinohydrolase, about 1 to 100 U / ml of sarcosine oxidase, about 0.01 to 50 U / ml of peroxidase, about 0 of hydrogen acceptor. 1 to 10 mM, coupler about 0.1 to 50 mM.

  In the reagent for quantifying creatinine of the present invention, preferable amounts of each component are about 10 to 300 U / ml of creatinine amide hydrolase, about 10 to 300 U / ml of creatine amidinohydrolase, about 1 to 100 U / ml of sarcosine oxidase, about 0.01 of peroxidase. ˜50 U / ml, hydrogen acceptor about 0.1-10 mM, coupler about 0.1-50 mM.

  The creatine or creatinine quantification reagent of the present invention is usually used together with a buffer solution having a pH of about 6-8. For example, phosphate buffer, Good buffer, Tris buffer and the like can be mentioned.

  Ascorbate oxidase or catalase may be added to the reagent of the present invention as necessary. Furthermore, other compounds may be added to the reagent of the present invention in order to smoothly perform an enzyme reaction or a color reaction. Examples of such compounds include stabilizers, surfactants, excipients and the like.

  The present invention utilizes the following measurement reaction.

  The produced quinoneimine dye is usually subjected to absorbance measurement at a wavelength of 500 to 650 nm. The creatine is quantified by the endo method and the rate method, but the endo method is generally used.

  The creatine amidinohydrolase having a small Km value according to the present invention can reduce the amount of enzyme used in a test reagent to about 1/3 to 1/4 in the quantification of creatine or creatinine as compared with conventional enzymes. And the reactivity in the second half of the reaction is improved.

  Hereinafter, the present invention will be specifically described by way of examples.

In the examples, the activity of creatine amidinohydrolase was measured as follows. In addition, the definition of the enzyme activity of this invention makes the amount of enzyme which produces | generates 1 micromol sarcosine in 1 minute on the following conditions as 1 unit (U).
Reaction mixture composition 0.3M HEPES pH 7.6
0.005% 4-aminoantipyrine 0.015% phenol 1.8% creatine 6 U / ml sarcosine oxidase 6 U / ml peroxidase 3 ml of the above reaction mixture is placed in a cuvette (d = 1 cm) and preheated at 37 ° C. for about 3 minutes. To do. Next, add 0.1 ml of the enzyme solution, mix gently, and record the change in absorbance at 500 nm for 5 minutes using a spectrophotometer controlled at 37 ° C. with water as a control. Absorbance change per unit is obtained (ΔOD test).

In the blind test, 0.1 ml of an enzyme diluent (50 mM potassium phosphate buffer, pH 7.5) is added instead of the enzyme solution, and the same operation as above is performed to determine the change in absorbance per minute (ΔOD blank). .
a formula

13.3: mmol extinction coefficient of quinoneimine dye under the above measurement conditions
(Cm ² / μM)
1/2: Coefficient of quinoneimine dye formed from one molecule of hydrogen peroxide generated by enzyme reaction being 1/2 molecule 1.0: Optical path length (cm)
0.1: Amount of added enzyme solution (ml)
Reference Example 1 Separation of Chromosomal DNA Chromosomal DNA of Alcaligenes faecalis TE3581 was isolated by the following method.

  The cells (FERM P-14237) were cultured with shaking in 150 ml of ordinary bouillon medium at 30 ° C. overnight, and then collected by centrifugation (8000 rpm, 10 minutes). Suspend in 5 ml of a solution containing 10% sucrose, 50 mM Tris-HCl (pH 8.0), 50 mM EDTA, add 1 ml of lysozyme solution (10 mg / ml) and incubate at 37 ° C. for 15 minutes, then 1 ml of 10% SDS The solution was added. An equal amount of chloroform / phenol solution (1: 1) was added to this solution, mixed with stirring, separated into an aqueous layer and a solvent layer by centrifugation at 10,000 rpm for 3 minutes, and the aqueous layer was separated. This aqueous layer was gently overlaid with twice the amount of ethanol, and while slowly stirring with a glass rod, the DNA was wrapped around the glass rod and separated.

This DNA was dissolved in 10 mM Tris-HCl, pH 8.0 solution (hereinafter abbreviated as TE) containing 1 mM EDTA. After treating this with an equal volume of chloroform / phenol solution, the aqueous layer was separated by centrifugation, and twice the amount of ethanol was added to separate the DNA again by the above method and dissolved in 2 ml of TE.
Reference example 2
Preparation of DNA fragment containing gene encoding creatinine amidinohydrolase and recombinant vector having the DNA fragment 20 μg of DNA obtained in Reference Example 1 was partially decomposed with restriction enzyme Sau3AI (manufactured by Toyobo) and sucrose density gradient centrifugation To recover fragments of 2-10 kbp. On the other hand, pBluescript KS (+) cleaved with the restriction enzyme BamHI (Toyobo) was dephosphorylated with bacterial alkaline phosphatase (Toyobo), and then both DNAs were treated with 1 unit of T4 DNA ligase (Toyobo) at 16 ° C. for 12 hours. The DNA was ligated by reacting. Ligated DNA was transformed using Escherichia coli JM109 competent cell (Toyobo Co., Ltd.) and agar medium for detecting creatine amidinohydrolase activity [0.5% yeast extract, 0.2% meat extract, 0.5% Polypeptone, 0.1% NaCl, 0.1% KH ₂ PO ₄ , 0.05% MgSO ₄ / 7H ₂ O, 1.15% creatine, 10 U / ml sarcosine oxidase (Toyobo), 0.5 U / ml Peroxidase (Toyobo Co., Ltd., 0.01% o-dianisidine, 50 μg / ml ampicillin, 1.5% agar). The detection of creatine amidinohydrolase activity was performed using colonies that grew in the above medium and stained brown as an index. Approximately 1 × 10 ⁵ transformant colonies were obtained per μg of DNA used.

  As a result of screening about 12,000 colonies, 6 strains of colonies stained brown were found. These strains were cultured in LB liquid medium (1% polypeptone, 0.5% yeast extract, 0.5% NaCl, 50 μg / ml ampicillin), and creatine amidinohydrolase activity was measured. Hydrolase activity was detected. Among these strains, a plasmid possessed by the strain having the highest creatine amidinohydrolase activity has an inserted DNA fragment of about 5 kbp, and this plasmid was designated as pCRH17.

Next, the inserted DNA of pCRH17 was excised with restriction enzymes EcoRV (Toyobo) and PstI (Toyobo) and ligated to pBluescript KS (+) cleaved with the same restriction enzyme to prepare pCRH173.
Example 1 Preparation of Recombinant Plasmid pCRH273 Using the recombinant plasmid pCRH173 of Reference Example 2, the upstream part of the creatine amidinohydrolase structural gene in the inserted DNA from the structural gene of β-galactosidase derived from the vector was a synthetic DNA described in SEQ ID NO: 3. And a commercially available mutagenesis kit (Transformer ™; manufactured by Clonetech) to prepare a recombinant plasmid pCRH173M. The detailed method for introducing the mutation was in accordance with the protocol attached to the kit.

Further, pCRH173M was cleaved with a restriction enzyme EcoRI (manufactured by Toyobo) and then self-ligated to prepare pCRH273 (FIG. 1).
Example 2 Introduction of Mutation into Creatine Amidinohydrolase Gene Commercially available E. coli competent cells (E. coli) with pCRH273 prepared in Example 1
After transformation, XLI-Red (manufactured by Clonetech) 3 ml of LB liquid medium (1% polypeptone, 0.5% yeast extract, 1.0% NaCl) containing ampicillin in total (50 μg / ml; manufactured by Nacalai Tesque) And incubating overnight at 37 ° C. The plasmid was recovered from the whole culture broth by a conventional method. This plasmid was again transformed into a commercially available E. coli competent cell (E. coli JM109; manufactured by Toyobo Co., Ltd.), applied to an agar medium for detecting creatine amidinohydrolase activity, and cultured overnight at 37 ° C. From the mutant creatine amidinohydrolase library thus obtained, a strain that strongly expressed creatine amidinohydrolase activity (a highly colored strain) was selected.
Example 3 Screening for Creatine Amidinohydrolase with Reduced Km Value The candidate strain selected in Example 2 was ampicillin-containing (200 μg / ml) 3 ml TB medium (1.2% polypeptone, 2.4% yeast extract, 0 4% glycerol, 0.0231% KH ₂ PO ₄ , 0.1254% K ₂ HPO ₄ ), and cultured with shaking at 37 ° C. overnight. The cells were collected from 1 ml of the culture solution by centrifugation, and a crude enzyme solution was prepared by crushing glass beads. Using the obtained crude enzyme solution, creatine amidinohydrolase was measured by the activity measuring method described above. In addition, creatine amidinohydrolase activity was measured in the same manner using an activity measurement reagent in which only the substrate concentration was reduced to 1/10. Strains whose ratio of these two types of activity values (activity of 1/10 substrate concentration ÷ ordinary activity) was increased compared to wild type creatine amidinohydrolase were selected as mutants with decreased Km values.

As a result of screening about 20,000 strains by the above-mentioned method, mutants with reduced Km values for 3 strains of creatine can be obtained. Recombinant plasmids possessed by each of them are pCRH273M1 (FERM BP-5374), pCRH273M2 (FERM BP-5375) and pCRH273M3 (FERM BP-5376).
Example 4 Production of creatine amidinohydrolase from Escherichia coli JM109 (pCRH273M1) 6 L of TB medium was dispensed into a 10 L-jar fermenter, autoclaved at 121 ° C. for 15 minutes, allowed to cool, and then separately sterile filtered 50 mg 6 ml each of / ml ampicillin (manufactured by Nacalai Tesque) and 200 mM IPTG (manufactured by Nippon Seika) were added. This culture medium was inoculated with 60 ml of a culture solution of Escherichia coli JM109 (pCRH273M1) (FERM BP-5374) that had been shaken and cultured in an LB medium at 30 ° C. for 24 hours in advance and cultured at 37 ° C. for 24 hours with aeration and agitation. The creatine amidinohydrolase activity at the end of the culture was 8.7 U / ml.

The cells were collected by centrifugation and suspended in 50 mM phosphate buffer, pH 7.0.
The cell suspension was crushed with a French press and centrifuged to obtain a supernatant. The obtained crude enzyme solution was fractionated with ammonium sulfate, desalted by Sephadex G-25 (Pharmacia Biotech) gel filtration, and purified by octyl Sepharose CL-6B (Pharmacia Biotech) column chromatography to obtain a purified enzyme preparation. . The creatine amidinohydrolase preparation obtained by this method showed an almost single band by SDS-PAGE, and the specific activity at this time was 18.4 U / mg-protein.
Table 1 shows the summary of purification so far. Table 2 shows the physicochemical properties of creatine amidinohydrolase obtained by the above method.

Example 5 Production of creatine amidinohydrolase from Escherichia coli JM109 (pCRH273M2) 6 L of TB medium was dispensed into 10 L-jar fermenter, autoclaved at 121 ° C. for 15 minutes, allowed to cool, and then separately sterile filtered 50 mg 6 ml each of / ml ampicillin (manufactured by Nacalai Tesque) and 200 mM IPTG (manufactured by Nippon Seika) were added. This culture medium was inoculated with 60 ml of a culture solution of Escherichia coli JM109 (pCRH273M2) (FERM BP-5375) previously shake-cultured in LB medium at 30 ° C. for 24 hours, and cultured at 37 ° C. for 24 hours with aeration and agitation. The creatine amidinohydrolase activity at the end of the culture was 5.6 U / ml.

The cells were collected by centrifugation and suspended in 50 mM phosphate buffer, pH 7.0.
The cell suspension was crushed with a French press and centrifuged to obtain a supernatant. The obtained crude enzyme solution was fractionated with ammonium sulfate, desalted by Sephadex G-25 (Pharmacia Biotech) gel filtration, and purified by octyl Sepharose CL-6B (Pharmacia Biotech) column chromatography to obtain a purified enzyme preparation. . The creatine amidinohydrolase preparation obtained by this method showed an almost single band in SDS-PAGE, and the specific activity at this time was 14.3 U / mg-protein.
Table 3 shows the summary of purification so far. Table 4 shows the physicochemical properties of creatine amidinohydrolase obtained by the above method.

Example 6 Cres from Escherichia coli JM109 (pCRH273M3)
Manufacture of atine amidinohydrolase 6 L of TB medium was dispensed into a 10 L-jar fermenter, autoclaved at 121 ° C. for 15 minutes, allowed to cool, and then separately filtered aseptically, 50 mg / ml ampicillin (manufactured by Nacalai Tesque), and 200 mM IPTG ( 6 ml each of Nippon Seika Co., Ltd. was added. This medium was inoculated with 60 ml of a culture solution of Escherichia coli JM109 (pCRH273M3) (FERM BP-5376) that had been shaken and cultured in advance at 30 ° C. for 24 hours in LB medium, and cultured at 37 ° C. for 24 hours with aeration and agitation. The creatine amidinohydrolase activity at the end of the culture was 8.3 U / ml.

The cells were collected by centrifugation and suspended in 50 mM phosphate buffer, pH 7.0.
The cell suspension was crushed with a French press and centrifuged to obtain a supernatant. The obtained crude enzyme solution was fractionated with ammonium sulfate, desalted by Sephadex G-25 (Pharmacia Biotech) gel filtration, and purified by octyl Sepharose CL-6B (Pharmacia Biotech) column chromatography to obtain a purified enzyme preparation. . The creatine amidinohydrolase preparation obtained by this method showed an almost single band in terms of SDS-PAGE, and the specific activity at this time was 14.8 U / mg-protein.
Table 5 shows a summary of the purification so far. Table 6 shows the physicochemical properties of creatine amidinohydrolase obtained by the above method.

Table 7 below summarizes the Km values for creatine of the novel creatine amidinohydrolase and wild type creatine amidinohydrolase of the present invention. As is apparent from Table 7, it can be seen that the Km value of the novel creatine amidinohydrolase of the present invention was lower than that of the wild type creatine amidinohydrolase.

Example 7
Using the purified creatine amidinohydrolase and wild type creatine amidinohydrolase prepared in Example 5, preparation solutions for creatinine having the following composition were prepared, and the required amounts of creatine amidinohydrolase in the creatinine measurement test solution were compared.

Creatine amidinohydrolase of Example 5, 20, 40, 60 U / ml
Or wild type creatine amidinohydrolase creatinine amide hydrolase 150 U / ml
Sarcosine oxidase 7U / ml
Peroxidase 3PU / ml
MOPS buffer 0.1M, pH 8.0
Triton X-100 0.1%
4-Aminoantipyrine 0.15 mM
TOOS (aniline derivative) 0.2 mM
3 ml of the above test solution was added to 60 μl of a sample containing 100 mg / dl of creatinine, and the change in absorbance was measured at 37 ° C. and a wavelength of 546 nm. The measurement result (time course) is shown in FIG. In the figure, Wild represents wild type creatine amidinohydrolase, and pCRH273M2 represents the creatine amidinohydrolase of the present invention.

  As is apparent from FIG. 2, when the measurement reaction is completed in 5 minutes, the creatine amidinohydrolase of the present invention can be measured with a smaller amount of enzyme (about 1/3 amount) than the wild type creatine amidinohydrolase. there were. In addition, it was confirmed that the reactivity in the latter half of the measurement, that is, the reactivity when the amount of creatinine in the sample decreased was good.

  (Sequence Listing)

1 is a view showing a recombinant plasmid pCRH273. It is drawing which shows the measurement result (time course) at the time of measuring the creatinine in a sample using the creatine amidinohydrolase and wild type creatine amidinohydrolase of this invention.

Claims (7)

A novel creatine amidinohydrolase having the following physicochemical properties:
Action: Creatine + H ₂ O → Sarcosine + Urea Optimal temperature: about 40 ° C to 50 ° C
Optimum pH: about 8.0 to 9.0
Thermal stability: about 50 ° C. or less (pH 7.5, 30 minutes)
Km value for creatine when sarcosine oxidase and peroxidase are used as conjugate enzymes and the activity is measured: about 3.5 to 10.0 mM
Molecular weight: about 43,000 (SDS-PAGE)
Isoelectric point: about 4.5 A novel creatine amidinohydrolase having the following physicochemical properties:
Action: Creatine + H ₂ O → Sarcosine + Urea Optimal temperature: about 40 ° C to 50 ° C
Optimum pH: about 8.0 to 9.0
Thermal stability: about 50 ° C. or less (pH 7.5, 30 minutes)
Km value for creatine when measuring activity using sarcosine oxidase and peroxidase as conjugate enzymes: about 4.5 ± 1.0
Molecular weight: about 43,000 (SDS-PAGE)
Isoelectric point: about 4.5 A novel creatine amidinohydrolase having the following physicochemical properties:
Action: Creatine + H ₂ O → Sarcosine + Urea Optimal temperature: about 40 ° C to 50 ° C
Optimum pH: about 8.0 to 9.0
Thermal stability: about 50 ° C. or less (pH 7.5, 30 minutes)
Km value for creatine when sarcosine oxidase and peroxidase are used as conjugate enzymes and the activity is measured: about 6.5 ± 1.0
Molecular weight: about 43,000 (SDS-PAGE)
Isoelectric point: about 4.5 A novel creatine amidinohydrolase having the following physicochemical properties:
Action: Creatine + H ₂ O → Sarcosine + Urea Optimal temperature: about 40 ° C to 50 ° C
Optimum pH: about 8.0 to 9.0
Thermal stability: about 50 ° C. or less (pH 7.5, 30 minutes)
Km value for creatine when sarcosine oxidase and peroxidase are used as conjugate enzymes and the activity is measured: about 9.0 ± 1.0
Molecular weight: about 43,000 (SDS-PAGE)
Isoelectric point: about 4.5 A method for producing a novel creatine amidinohydrolase, comprising culturing a microorganism producing a novel creatine amidinohydrolase having the following physicochemical properties in a nutrient medium and collecting the novel creatine amidinohydrolase.
Action: Creatine + H ₂ O → Sarcosine + Urea Optimal temperature: about 40 ° C to 50 ° C
Optimum pH: about 8.0 to 9.0
Thermal stability: about 50 ° C. or less (pH 7.5, 30 minutes)
Km value for creatine when sarcosine oxidase and peroxidase are used as conjugate enzymes and the activity is measured: about 3.5 to 10.0 mM
Molecular weight: about 43,000 (SDS-PAGE)
Isoelectric point: about 4.5 A reagent for quantifying creatine in a sample, comprising the novel creatine amidinohydrolase, sarcosine oxidase and hydrogen peroxide detection composition according to claim 1. A reagent for quantifying creatinine in a sample, comprising creatinine amide hydrolase, the novel creatine amidinohydrolase according to claim 1, sarcosine oxidase and a composition for detecting hydrogen peroxide.

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