JP3422197B2 - Stable creatine amidinohydrolase - Google Patents

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a stable creatine amidinohydrolase as compared with wild-type creatine amidinohydrolase and a method for producing the creatine amidinohydrolase.

Creatinine and creatine are found in blood or urine, and the rapid and accurate detection and measurement of the amount of creatinine and creatine is associated with diseases such as uremia, chronic nephritis, acute nephritis, giant dystrophy and ankylosing dystrophy. It is very important to diagnose such as. In order to make such a diagnosis, it is common to quantify creatinine and creatine in blood or urine.

As a conventional method for quantifying creatine, creatine in a sample is quantified by reacting creatine in the sample with creatine amidinohydrolase and sarcosine oxidase, and measuring the produced hydrogen peroxide by a hydrogen peroxide measuring means. To do. Further, there is a method of quantifying creatinine in a sample by causing creatinine amide hydrolase, creatine amidinohydrolase and sarcosine oxidase to act on creatinine and measuring the hydrogen peroxide produced by a hydrogen peroxide measuring means.

On the other hand, creatinine amide hydrolase,
Creatine amidinohydrolase or sarcosine oxidase is widely found in the microbial world, and various products have already been industrially produced and used as clinical test agents.

[0005]

However, creatine amidinohydrolase produced from various known bacterial cells has low thermostability, and particularly long-term stability in liquid form was not sufficient. For example, an enzyme derived from Bacillus (Japanese Patent Publication No. 61-17465) has a low thermostability of 40 ° C. or lower.
In addition, enzymes derived from the genus Corynebacterium, Micrococcus, Actinobacillus or Bacillus (see
-76915) has a thermal stability of 50 ° C. or lower, but its long-term stability in a liquid state was unknown.

[0006] Attempts have also been made to introduce a mutant creatine amidinohydrolase having improved stability by introducing a mutation into the wild-type creatine amidinohydrolase gene (Japanese Patent Publication No. 3-36514, (Kaihei 7-255485 gazette), there is no description as a high-purity enzyme purified as an enzyme for clinical laboratory drugs, and the stability of liquid in the liquid for a long time as an important index as an enzyme for clinical laboratory drugs is not described. There was no description, and the effect was not clear.

[0007]

As a result of intensive studies to solve the above problems, the present inventors have found that the bacteria belonging to the genus Alcaligenes have excellent thermal stability and Km against creatine. It has been found that creatine amidinohydrolase having a relatively low value is produced (Japanese Patent Application No. 6-63363). However, it is hard to say that the creatine amidinohydrolase is still sufficiently stable as an enzyme corresponding to a liquid reagent which is a mainstream of clinical test drugs at present, and further, a creatine amidinohydrolase which is stable in a liquid form for a long time is demanded. It had been.

[0008]

Means for Solving the Problems As a result of intensive studies to solve the above problems, the present inventors have used a creatine amidinohydrolase gene derived from a bacterium of the genus Alcaligenes,
We succeeded in creating a novel creatine amidinohydrolase with excellent long-term liquid stability by a protein engineering method.

That is, it is a stable creatine amidinohydrolase characterized by being a mutant creatine amidinohydrolase having improved long-term stability in a neutral buffer as compared with the wild type creatine amidinohydrolase.

The present invention also has a residual activity of 60% or more, preferably 70% or more, more preferably 80% or more after storage in a PIPES buffer or a potassium phosphate buffer at 45 ° C. for 1 week. Creatine amidinohydrolase.

The present invention is also a creatine amidinohydrolase gene encoding these stable creatine amidinohydrolases.

Furthermore, the present invention is a recombinant plasmid containing a gene encoding these creatine amidinohydrolases.

The present invention is also a transformed cell transformed with these recombinant plasmids.

Furthermore, the present invention is a method for producing a stable creatine amidinohydrolase, which comprises culturing these transformed cells in a nutrient medium and collecting creatine amidinohydrolar.

[0015]

BEST MODE FOR CARRYING OUT THE INVENTION The novel creatine amidinohydrolase of the present invention is an enzyme that acts on creatine in the presence of water to have sarcosine and urea, which is higher than wild-type creatine amidinohydrolase. It is a stable creatine amidinohydrolase with improved long-term stability, especially in liquid form. The wild type creatine amidinohydrolase in the present invention,
Alcaligenes faecaris T
It is an enzyme produced by E3581 (FERM P-14237), and its properties are as follows. Action: creatine + H ₂ O → sarcosine +
Urea optimal temperature: 40 ° C to 45 ° C Optimal pH: 8.0 to 9.0 Thermal stability: Approximately 50 ° C or less (50 mM potassium phosphate buffer, pH 7.5, 30 minutes) pH stability: 4 to 10 (25 ° C, 18 hours) Liquid stability: 45 to 50% (0.1M PIPES, p
Km for creatine when activity is measured with sarcosine oxidase and peroxidase as a coupling enzyme in H7.0 at 45 ° C for 1 week)
Value: 16 to 30 mM Molecular weight: about 43,000 (SDS-PAGE) The amino acid sequence of the enzyme is shown in Sequence Listing, SEQ ID NO: 1.

The long-term stability in liquid state in the present invention means
It means the residual activity after storage for 1 week at 45 ° C. in neutral buffer, eg PIPES buffer or potassium phosphate buffer. One embodiment of the present invention is a stable creatine amidinohydrolase having a residual activity of 60% or more after storage at 45 ° C. for 1 week in PIPES buffer or potassium phosphate buffer. Another embodiment is PI
45 ° C. in PES buffer or potassium phosphate buffer,
It is a stable creatine amidinohydrolase having a residual activity of 70% or more after storage for 1 week. Furthermore, another embodiment is a stable creatine amidinohydrolase having a residual activity of 80% or more after storage at 45 ° C. for 1 week in PIPES buffer or potassium phosphate buffer.

One embodiment of the present invention is that at least one amino acid in the amino acid sequence of SEQ ID NO: 1 in the sequence listing, for example, arginine at position 135, glutamic acid at position 15 and / or arginine at position 104 is another amino acid. Is a stable creatine amidinohydrolase substituted with. Further, as a specific example, SEQ ID NO: 1
The 135th arginine in the amino acid sequence described is alanine, the 15th glutamic acid is glycine, or 1
There is an enzyme in which the 04th arginine is replaced with histidine, or an enzyme in which two or more substitution sites selected from the group consisting of these 15th glutamic acid, 104th arginine and 135th arginine are substituted. .

An example of the present invention is creatine amidinohydrolase having the following physicochemical properties, which is a novel enzyme having excellent liquid stability as compared with the wild type enzyme. Action: creatine + H ₂ O → sarcosine +
Urea optimal temperature: 40 ° C to 50 ° C Optimal pH: 6.5 to 9.0 Thermal stability: Approximately 50 ° C or less (50 mM potassium phosphate buffer, pH 7.5, 30 minutes) pH stability: 4 to 10 (25 ° C., 18 hours) Liquid stability: 60-80% (0.1M PIPES, p
Km for creatine when activity is measured with sarcosine oxidase and peroxidase as a coupling enzyme in H7.0 at 45 ° C for 1 week)
Value: 16-30 mM Molecular weight: about 43,000 (SDS-PAGE)

One of the novel creatine amidinohydrolases obtained by the present invention has the following physicochemical properties. Action: creatine + H ₂ O → sarcosine +
Urea optimal temperature: 45 ° C to 50 ° C Optimal pH: 6.5 to 7.5 Thermal stability: Approximately 50 ° C or less (50 mM potassium phosphate buffer, pH 7.5, 30 minutes) pH stability: 4 to 10 (25 ° C., 18 hours) Liquid stability: 68% (0.1M PIPES, pH 7.
Km for creatine when sarcosine oxidase and peroxidase are used as conjugate enzymes for activity measurement
Value: 16 mM Molecular weight: about 43,000 (SDS-PAGE)

Another novel creatine amidinohydrolase has the following physicochemical properties. Action: creatine + H ₂ O → sarcosine +
Urea optimal temperature: 40 ° C Optimal pH: 8.0-9.0 Thermal stability: Approximately 50 ° C or less (50 mM potassium phosphate buffer, pH 7.5, 30 minutes) pH stability: 4 to 10 (25 Liquid stability: 72% (0.1M PIPES, pH 7.
Km for creatine when sarcosine oxidase and peroxidase are used as conjugate enzymes for activity measurement
Value: 30 mM Molecular weight: about 43,000 (SDS-PAGE)

Furthermore, another novel creatine amidinohydrolase has the following physicochemical properties. Action Creatine + H ₂ O → Sarcosine +
Urea optimal temperature: 40 ° C Optimal pH: 8.0-9.0 Thermal stability: Approximately 50 ° C or less (50 mM potassium phosphate buffer, pH 7.5, 30 minutes) pH stability: 4 to 10 (25 Liquid stability: 79% (0.1M PIPES, pH 7.
Km for creatine when sarcosine oxidase and peroxidase are used as conjugate enzymes for activity measurement
Value: 30 mM Molecular weight: about 43,000 (SDS-PAGE)

Further, the present invention introduces a mutation into a gene encoding the amino acid sequence shown in SEQ ID NO: 1 of the Sequence Listing to compare with wild-type creatine amidinohydrolase,
It is a gene encoding creatine amidinohydrolase, which has improved stability, particularly long-term stability in liquid form.

The gene encodes an amino acid sequence in which at least one of the 15th, 104th and 135th amino acids in the amino acid sequence shown in SEQ ID NO: 1 is replaced with another amino acid. An example is a mutant gene that is a gene.

Further, as a specific example, an amino acid sequence in which the 15th amino acid, the 104th amino acid or the 135th amino acid of the amino acid sequence shown in SEQ ID NO: 1 is substituted with another amino acid is coded. Examples of such genes include

As an embodiment of the present invention, a gene encoding an amino acid sequence in which glutamic acid at position 15 of the amino acid sequence shown in SEQ ID NO: 1 is replaced with glycine, and arginine at position 104 is replaced with histidine. A gene encoding a different amino acid sequence, and 135
There is a gene encoding an amino acid sequence in which the second arginine is replaced with alanine.

A gene encoding an amino acid sequence in which at least two of the 15th, 104th and 135th amino acid sequences of the amino acid sequence shown in SEQ ID NO: 1 is replaced with another amino acid, for example, the 135th amino acid. There is a gene encoding an amino acid sequence in which the arginine of is replaced with alanine and the 15th glutamic acid is replaced with glycine, or an amino acid sequence in which the 104th arginine is replaced with histidine.

As the gene of the present invention, at least one gene among the genes shown in the sequence listing / SEQ ID NO: 2 is replaced with another gene, and compared with the wild-type creatine amidinohydrolase, the PIPES buffer solution or phosphorus is used. There is a creatine amidinohydrolase gene that encodes a mutant creatine amidinohydrolase with improved long term stability in potassium phosphate buffer.

Further, the present invention provides a recombinant plasmid containing a gene encoding the creatine amidinohydrolase, a transformed cell transformed with the recombinant plasmid, or the cell cultured in a nutrient medium to obtain the creatine amidinohydrolase. Is a method for producing the creatine amidinohydrolase. As the vector and host cell, those generally used in the art are exemplified.

One of the methods for producing the creatine amidinohydrolase of the present invention is to introduce a mutation into a gene encoding a wild-type creatine amidinohydrolase, which is more stable than the wild-type creatine amidinohydrolase by a protein engineering method. In particular, it is a method for producing a novel creatine amidinohydrolase having improved long-term liquid stability.

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

Another embodiment of the present invention is to introduce a mutation into the gene encoding the amino acid sequence set forth in SEQ ID NO: 1 in the sequence listing to improve stability, particularly long-term stability, as compared to wild-type creatine amidinohydrolase. A method for producing a novel creatine amidinohydrolase with improved liquid stability.

As a method for introducing a mutation into the wild-type creatine amidinohydrolase gene, any known method can be used. That is, a protein engineering method such as a PCR method or a site-directed mutagenesis method can be used from a method of contacting the gene DNA with a drug serving as a mutation source, a method of ultraviolet irradiation, and the like. In addition, it is also possible to introduce mutations in vivo using Escherichia coli in which gene mutations occur frequently due to defective gene repair mechanism.

The mutant creatine amidinohydrolase gene obtained as described above was transformed into Escherichia coli, and then agar medium for detecting creatine amidinohydrolase activity (J. Ferment. Bioeng., Vol. 76 No. 2 77-81 ( 1993)), and colonies colored brown by expressing creatine amidinohydrolase activity were selected. The selected colonies are fed to a nutrient medium such as LB medium or 2 × YT.
After inoculating the medium and culturing at 37 ° C. overnight, the cells are crushed to extract a crude enzyme solution. Any known method may be used as a method for disrupting the cells, for example, a physical disruption method such as ultrasonic treatment or glass bead disruption, or a method using a lytic enzyme such as lysozyme can be used. Stabilized creatine amidinohydrolase can be selected by measuring the residual activity after treatment at 55 ° C. for 30 minutes using this crude enzyme solution. Then, the creatine amidinohydrolase gene having the mutation introduced therein can be recovered by recovering the recombinant plasmid from the strain producing the stabilized creatine amidinohydrolase.

As a method for identifying a mutation site from a gene into which a mutation has been introduced, a conventionally known DNA nucleotide sequence determination method may be used, and it is convenient to use a commercially available kit.

The method for obtaining purified creatine amidinohydrolase from the strain selected by the above method may be any known method and is not limited to the following method. After recovering the bacterial cells obtained by culturing in a nutrient medium, the crude enzyme solution can be obtained by crushing and extracting by an enzymatic or physical crushing method. A creatine amidinohydrolase fraction is recovered from the obtained crude enzyme extract by ammonium sulfate precipitation. This crude enzyme solution was used as Sephadex G-25.
(Pharmacia Biotech) Desalination can be performed by a method such as gel filtration. After this operation, octyl sepharose CL-6B (Pharmacia Biotech) column chromatography can be used for separation and purification to obtain a purified enzyme preparation. This purified enzyme preparation is SDS-
It has been purified to the extent that it shows an almost single band by PAGE.

The microorganisms producing the novel creatine amidinohydrolase used in the present invention include Escherichia coli JM109 (pCRH273S1),
Escherichia Collie JM109 (pCRH273S
2), Escherichia coli JM109 (pCRH27
3S3) is exemplified. The method for culturing these microorganisms and collecting the creatine amidinohydrolase of the present invention from the culture is not particularly limited and may be a conventional method.

[0037]

EXAMPLES The present invention will be specifically described below with reference to examples. In the examples, creatine amidinohydrolase activity was measured as follows. In the definition of the enzyme activity of the present invention, the amount of enzyme that produces 1 μmol of sarcosine per minute under the following conditions is defined as 1 unit (U). 3 ml of the following reaction mixture is placed in a cuvette (d = 1 cm) and preheated at 37 ° C. for about 3 minutes. Next, add 0.1 ml of enzyme solution,
After gently mixing, the change in absorbance at 500 nm was recorded for 5 minutes with a spectrophotometer controlled at 37 ° C. with water as a control, and the change in absorbance per minute was calculated from the linear portion of 2 to 5 minutes (ΔOD test). In the blind test, instead of the enzyme solution, an enzyme diluent (50 mM potassium phosphate buffer, pH 7.
5) Add 0.1 ml and perform the same operation as above to
The change in absorbance per minute is determined (ΔOD blank). Measuring principle

[0038]

[Chemical 1]

Composition of reaction mixture 0.3M HEPES, pH 7.6 0.005% 4-aminoantipyrine 0.015% phenol 1.8% creatine 6U / ml sarcosine oxidase 6U / ml peroxidase

[0040]

[Equation 1]

13.3: mmol extinction coefficient (cm ² / μM) 1/2 of quinoneimine dye under the above-mentioned measurement conditions 1/2: 1/2 of quinoneimine dye formed from one molecule of H ₂ O ₂ produced by enzymatic reaction Coefficient of being a molecule 1.0: optical path length (cm) 0.1: amount of added enzyme solution (ml)

Reference Example 1 Separation of chromosomal DNA Alcaligenes faecalis TE3581 (FERM
The chromosomal DNA of P-14237) was isolated by the following method. The cells were cultured in 150 ml of normal broth medium with shaking at 30 ° C. overnight, followed by centrifugation (8,000 rpm, 1
The cells were collected for 0 minutes. 10% sucrose, 50
Suspended in 5 ml of a solution containing mM Tris-hydrochloric acid (pH 8.0) and 50 mM EDTA, added 1 ml of a lysozyme solution (10 mg / ml) dissolved in the above-mentioned buffer solution, and added at 37 ° C 1
Incubate for 5 minutes, then add 1 ml of 10% SDS solution. To this solution, an equal amount of chloroform / phenol solution (1: 1) was added, mixed by stirring, and 10,000 rpm,
The water layer and the solvent layer were separated by centrifugation for 3 minutes, and the water layer was separated. Gently layer twice the amount of ethanol on this water layer,
The DNA was separated by wrapping it around the glass rod while gently stirring with the glass rod.

This DNA was added to 1 mM EDTA
It was dissolved in 0 mM Tris-HCl, pH 8.0 solution (hereinafter abbreviated as TE). After treating this with an equal amount of chloroform / phenol solution, the aqueous layer was separated by centrifugation, twice the amount of ethanol was added, the DNA was separated again by the above method, and dissolved with 2 ml of TE.

Reference Example 2 A gene encoding creatine amidinohydrolase
Recombinant vector containing the DNA and the DNA fragment
Preparation of 20 μg of the DNA obtained in Reference Example 1 with the restriction enzyme Sau3AI
(Toyobo Co., Ltd.) partially decomposed and a 2-10 kbp fragment was recovered by a sucrose density gradient centrifugation method. On the other hand, pBI cut with the restriction enzyme BamHI (manufactured by Toyobo)
After dephosphorylating uescript KS (+) with bacterial alkaline phosphatase (Toyobo), both DNAs were reacted with 1 unit of T4 DNA ligase (Toyobo) at 16 ° C. for 12 hours to ligate the DNAs.
The ligated DNA was transformed with a competent cell of Escherichia coli JM109 (manufactured by Toyobo), 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 (manufactured by Toyobo),
0.5 U / ml peroxidase (manufactured by Toyobo), 0.0
1% o-dianisidine, 50 μg / ml ampicillin,
1.5% agar]. The creatine amidinohydrolase activity was detected using colonies that grew in the above medium and were stained brown as an index. Used DNA 1μ
About 1 × 10 ⁵ transformant colonies were obtained per g.

As a result of screening about 12,000 colonies, 6 strains were found to be stained brown. LB liquid medium (1% polypeptone, 0.5% yeast extract, 0.5% NaCl, 50 μg)
/ Ml ampicillin) and the creatine amidinohydrolase activity was measured, and the creatine amidinohydrolase activity was detected in all the strains. Of these strains, the plasmid possessed by the strain with the highest creatine amidinohydrolase activity contained an inserted DNA fragment of about 5 kbp.
It was set to H17. Then, the inserted DNA of pCRH17 was cut out with restriction enzymes EcoRI (Toyobo) and PstI (Toyobo) and cleaved with the same restriction enzymes pBluescr.
It was ligated to iptKS (+) to prepare pCRH173.

Reference Example 3 Recombinant plasmid pCRH27
Preparation of 3 Using the recombinant plasmid pCRH173 of Reference Example 2, the creatine amidinohydrolase structural gene upstream portion of the inserted DNA from the vector-derived β-galactosidase structural gene was used as a synthetic DNA described in Sequence Listing / SEQ ID NO: 3.
And Mutagenesis Kit (TransformerTM; Cl
Onetech) and then the recombinant plasmid p
CRH173M was made. The detailed method for producing the recombinant plasmid was performed according to the protocol attached to the kit. Furthermore, pCRH173M is a restriction enzyme E
After cutting with coRI (manufactured by Toyobo), self-ligation was performed to prepare pCRH273.

Example 1 Creatine amidinohydrolar
Introduction of a mutation into the ze gene Commercially available E. coli XLI-Red; Clo with pCRH273 produced in Reference Example 1
3 m containing ampicillin (50 μg / ml; Nacalai Tesque) after transformation
1 LB liquid medium (1% polypeptone, 0.5% yeast extract, 1.0% NaCl) was inoculated and shake-cultured overnight at 37 ° C. The plasmid was recovered from the total amount of this culture solution by a conventional method. Commercially available E. coli JM109 (manufactured by Toyobo) was transformed with this plasmid, applied to an agar medium for detecting creatine amidinohydrolase activity, and cultured at 37 ° C. overnight. From the mutant creatine amidinohydrolase library thus obtained, a strain strongly expressing creatine amidinohydrolase activity (a strain with strong coloring) was selected.

Example 2 Creatine with improved thermal stability
Screening for amidinohydrolase The candidate strains selected in Example 1 contained ampicillin (2
00 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 HP
O ₄ ), and cultured overnight at 37 ° C. with shaking. The bacterial cells were recovered from 1 ml of the culture by centrifugation, and glass beads were crushed to prepare a crude enzyme solution. The creatine amidinohydrolase activity was measured by the above-mentioned activity measuring method using the obtained crude enzyme solution. In addition, the same crude enzyme solution at 55 ° C,
After heat treatment for 30 minutes, creatine amidinohydrolase activity was measured. By comparing these two types of activity values, a strain having a higher residual ratio of creatine amidinohydrolase activity after heat treatment than that of wild-type creatine amidinohydrolase as compared with wild-type creatine amidinohydrolase was selected as a thermostability-improving mutant. As a result of screening about 10,000 strains by the above-mentioned method, three strains with improved thermostability can be obtained, and recombinant plasmids each of which has pCRH273S.
1, pCRH273S2 and pCRH273S3 were named.

PCRH273S1, pCRH273S
2. To identify the mutation site of pCRH273S3, Ra
The nucleotide sequence of all creatine amidinohydrolase genes was confirmed using a dioactive Sequencing Kit (Toyobo). As a result, in pCRH273S1, the 135th arginine (codon; CGC) became alanine (codon; GCC) (R135A), and in pCRH273S2, the 15th glutamic acid (codon; GAG) and 135.
The second arginine (codon; CGC) is glycine (codon; GGG) and alanine (codon; GCC) (E15G-R135A), and pCRH273S3, respectively.
Then the 104th arginine (codon; CGC) and 13
The fifth arginine (codon; CGC) is histidine (codon; CAC) alanine (codon: GCC) (R1
04H-R135A) was confirmed to be substituted.

Example 3 Escherichia coli JM10
9 (pCRH273S1) creatine amidinohi
Preparation of Drorase 6 L of TB medium was dispensed to 10 L-jar fermenter, autoclaved at 121 ° C. for 15 minutes, and then left to cool, and then aseptically filtered at 50 mg / ml ampicillin (manufactured by Nacalai Tesque), and 200 mM IPTG (Nippon Seisei). Chemicals) was added in an amount of 6 ml. Escherichia, which had been preliminarily shake-cultured in LB medium at 30 ° C. for 24 hours in this medium,
Collie JM109 (pCRH273S1) culture medium 60
ml was inoculated and the cells were cultured at 37 ° C. for 24 hours with aeration and stirring. The creatine amidinohydrolase activity at the end of culture was 3.
It was 6 U / ml. Collect the cells by centrifugation,
The cells were suspended in 50 mM phosphate buffer, pH 7.0.

The above-mentioned cell suspension was crushed with a French press and centrifuged to obtain a supernatant. The crude enzyme solution thus obtained was subjected to ammonium sulfate fractionation, 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 table product. . The specific activity of the creatine amidinohydrolase preparation obtained by this method was 13.3 U / mg-protein. Table 1 shows a summary of the purification so far. Table 2 shows the physicochemical properties of creatine amidinohydrolase obtained by the above method.

[0052]

[Table 1]

[0053]

[Table 2]

Example 4 Escherichia coli JM10
9 (pCRH273S2) creatine amidinohi
Production of Drorase 6 L of TB medium was dispensed into 10 L-jar fermenter, autoclaved at 121 ° C. for 15 minutes, and allowed to cool, then separately sterile filtered 50 mg / ml ampicillin (manufactured by Nacalai Tesque), and 200 mM IPTG (Japan). 6 ml of each product was added. Culture medium 6 of Escherichia coli JM109 (pCRH273S2) which had been shake-cultured in LB medium at 30 ° C. for 24 hours in advance in this medium.
0 ml was inoculated and cultured at 37 ° C. for 24 hours with aeration and stirring.
The creatine amidinohydrolase activity at the end of the culture was 6.5 U / ml. The cells were collected by centrifugation and suspended in 50 mM phosphate buffer, pH 7.0.

The bacterial cell suspension was crushed with a French press and centrifuged to obtain a supernatant. The crude enzyme solution thus obtained was subjected to ammonium sulfate fractionation, 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 table product. . The specific activity of the creatine amidinohydrolase preparation obtained by this method was 14.8 U / mg-protein. Table 3 shows a summary of the purification so far. Table 4 shows the physicochemical properties of creatine amidinohydrolase obtained by the above method.

[0056]

[Table 3]

[0057]

[Table 4]

Example 5 Escherichia coli JM10
9 (pCRH273S3) creatine amidinohi
Preparation of Drorase 6 L of TB medium was dispensed to 10 L-jar fermenter, autoclaved at 121 ° C. for 15 minutes, and then left to cool, and then aseptically filtered at 50 mg / ml ampicillin (manufactured by Nacalai Tesque), and 200 mM IPTG (Nippon Seisei). Chemicals) was added in an amount of 6 ml. Escherichia, which had been preliminarily shake-cultured in LB medium at 30 ° C. for 24 hours in this medium,
Collie JM109 (pCRH273M3) culture medium 60
ml was inoculated and the cells were cultured at 37 ° C. for 24 hours with aeration and stirring. Creatine amidinohydrolase activity at the end of culture is 1
It was 2.3 U / ml. The cells were collected by centrifugation and suspended in 50 mM phosphate buffer, pH 7.0.

The bacterial cell suspension was crushed with a French press and centrifuged to obtain a supernatant. The crude enzyme solution thus obtained was subjected to ammonium sulfate fractionation, 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 table product. . The creatine amidinohydrolase preparation obtained by this method showed an almost single band by SDS-PAGE, and the specific activity at this time was 16.2 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.

[0060]

[Table 5]

[0061]

[Table 6]

Example 6 Various mutants and wild-type creatine
Comparison of Thermostability of Amidinohydrolase The results of comparing the thermostability of the mutant creatine amidinohydrolase obtained in Examples 3, 4 and 5 with the wild type creatine amidinohydrolase are shown in FIG. In Figure 1,
■ indicates wild type, ◆ indicates pCRH273S2, ▲ indicates pCRH2
73S1, ● indicates the thermostability of the enzyme obtained from pCRH273S3. As can be seen from this figure, the inflection point for heat denaturation of all creatine amidinohydrolases is 50 ° C.
However, it was confirmed that the mutant creatine amidinohydrolase of the present invention has a significantly improved residual activity at 55 ° C.

Example 7 Various mutants and wild type creatine
Comparison of liquid stability of amidinohydrolase Furthermore, the results of comparing the long-term stability of the mutant creatine amidinohydrolase obtained in Examples 3, 4 and 5 with the wild type creatine amidinohydrolase in liquid form are shown in FIG. 2 (PIPES buffer). Liquid) and FIG. 3 (potassium phosphate buffer). 2 and 3, ■ indicates wild type, ◆ indicates pCRH273S2, ▲ indicates pCRH273S1, and ● indicates p.
3 shows the liquid stability of the enzyme obtained from CRH273S3. As a result, surprisingly, each mutant creatine amidinohydrolase greatly increased the stability of the wild-type creatine amidinohydrolase in the long-term liquid stability, which could not be estimated only by comparing the thermal stability in a short time. It was confirmed that it exceeded.

[0064]

INDUSTRIAL APPLICABILITY According to the present invention, a novel creatine amidinohydrolase which is useful as an enzyme for a diagnostic agent and is excellent in liquid stability can be obtained, and it has become possible to industrially produce the creatine amidinohydrolase in large quantities.

[0065]

[Sequence list]

SEQ ID NO: 1 Sequence length: 401 Sequence type: Amino acid topology: Linear sequence type: Protein origin Organism name: Alcaligenes faecalis Strain name: TE3581 (MICRO deposit number FERM P14)
237) Sequence information: protein sequence having creatine amidinohydrolase activity 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 1 80 185 190 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 3 90 395 400 Ile Ile Arg Asn 404

SEQ ID NO: 2 Sequence length: 1212 Sequence type: Nucleic acid (DNA) Number of strands: Double-stranded topology: Linear Sequence type: genomic DNA Origin organism name: Alcaligenes faecalis Strain name: TE3581 (Ministry of Engineering deposit number FERM P14
237) Sequence 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 1 00 105 110 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 GAC 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 Thr T rp 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 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

SEQ ID NO: 3 Array length: 39 Sequence type: nucleic acid (DNA) Number of chains: Single chain Topology: linear Sequence type: Synthetic DNA Array CAACA TGTCG TCAGT CATAT GTGTT TCCTG TGTGA AATT 39

C

[Brief description of drawings]

FIG. 1 shows the thermostability of the stable creatine amidinohydrolase of the present invention.

FIG. 2 shows the liquid stability of the stable creatine amidinohydrolase of the present invention in PIPES buffer.

FIG. 3 is a graph showing the liquid stability of the stable creatine amidinohydrolase of the present invention in a potassium phosphate buffer.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI (C12N 1/21 C12N 15/00 ZNAA C12R 1:19) (C12N 9/78 C12R 1:19) (56) Reference Flat 8-308579 (JP, A) JP-A 8-89255 (JP, A) (58) Fields investigated (Int. Cl. ⁷ , DB name) C12N 9/48-9/86 C12N 15/00-15 / 90 SwissProt / PIR / GeneS eq GenBank / EMBL / DDBJ / GeneSeq BIOSIS / WPI (DIALOG) PubMed EUROPAT (QUESTEL) JISST file (JOIS)

Claims (5)

(57) [Claims] 1. An amino according to any one of the following (a) to (c):
Creatine amidinohydrolase consisting of an acid sequence: (A) 13 in the amino acid sequence of SEQ ID NO: 1
Amino acid in which arginine at position 5 is replaced with alanine
Array; (B) 13 in the amino acid sequence of SEQ ID NO: 1
Arginine at the 5th position is alanine, and glutamic acid at the 15th position is
An amino acid sequence in which is substituted with glycine; (C) 13 in the amino acid sequence of SEQ ID NO: 1
Arginine at the 5th position is alanine, and arginine at the 104th position
The amino acid sequence in which is replaced with histidine. 2. A creatine amidinohydrolase gene encoding click rare Chin amidinohydrolase of claim 1, wherein. 3. A recombinant plasmid containing the creatine amidinohydrolase gene according to claim 2 . 4. A transformed cell obtained by transforming a host cell with the recombinant plasmid according to claim 3 . 5. The transformed cell of claim 4, wherein cultured in a nutrient medium, preparation features and to torque rare Chin amidinohydrolase and collecting the creatine amidinohydrolase.