JP2001252088A - Gene encoding creatine amidinohydrase - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for producing a new creatine amidinohydrase showing excellent thermostability and a smaller Km value toward creatine by the gene recombinant method. SOLUTION: This method is the method for producing the creatine amidinohydrase from a creatine amidinohydrase gene containing the base sequence encoding the amino acid sequence represented by the sequence table (see the specification)-sequence number 1 or the amino acid sequence which is obtained by adding, deleting or substituting one or a plurality of amino acids to the above amino acid sequence and exhibits creatine amidinohydrase enzymatic activity, a recombinant vector containing the gene, a transformant obtained by means of transforming by the recombinant vector and the transformant.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a gene encoding a novel creatine amidinohydrolase which is excellent in thermostability and has a smaller Km value for creatine, a recombinant vector containing the gene, and a gene encoding the recombinant vector. The present invention relates to a transformed transformant and a method for producing a novel creatine amidinohydrolase using the transformed transformant.

[0002] Creatinine and creatine are found in blood or urine, and the rapid and accurate detection and measurement of the amount is associated with diseases such as uremia, chronic nephritis, acute nephritis, giantosis, ankylosing dystrophy and the like. Very important for diagnosis. In order to make such a diagnosis, quantification of creatinine and creatine in blood or urine is generally performed.

[0003] As a conventional method for quantifying creatine, creatine in a sample is allowed to act with creatine amidinohydrolase and sarcosine oxidase. The generated hydrogen peroxide is measured by a hydrogen peroxide measuring means to quantify creatine in the sample. In addition, there is a method in which creatinine is reacted with creatinine amide hydrolase, creatine amidinohydrolase, and sarcosine oxidase, and the generated hydrogen peroxide is measured by a hydrogen peroxide measuring means to quantify creatinine in the sample.

On the other hand, creatinine amidohydrolase,
Creatine amidinohydrolase and sarcosine oxidase have been widely found in the microbial community, and have already been industrially manufactured and used as clinical test agents.

[0005]

However, creatine amidinohydrolase produced from various known cells has low thermostability and a high Km value for creatine. For example, enzymes derived from the genus Bacillus (Japanese Patent Publication No.
No. 7465) has low thermal stability of 40 ° C. or less. Furthermore, the enzyme derived from Pseudomonas putida has a small Km value for creatine of 1.33 mM. Enzymes derived from Corynebacterium, Micrococcus, Actinobacillus and Bacillus (Japanese Patent Publication No. 3-76915) have a heat stability of 50 ° C. or less and a Km value of 20 for creatine.
Relatively small as mM. However, there has been a demand for a novel creatine amidinohydrolase which is more excellent in thermostability and has a small Km value for creatine.

As a result of intensive studies to solve the above problems, it has been found that bacteria belonging to the genus Alkaligenes have excellent thermostability and produce creatine amidinohydrolase having a smaller Km value for creatine. I found it (Japanese Patent Application No. 6-63363).

However, in order to obtain creatine amidinohydrolase from the above-mentioned Alcaligenes bacterium, it is necessary to add an expensive inducer such as creatinine or creatine to the medium, and the production amount is low. As a result, the production cost is increased, and there is a problem as a source of the enzyme for clinical laboratory use.

[0008]

Means for Solving the Problems In order to solve the above-mentioned problems, the present inventors have proposed as a creatine amidinohydrolase producing bacterium Alcaligenes faecalis TE3581.
(Alcaligenes faecalis TE3581) was selected, the creatine amidinohydrolase gene was successfully isolated from chromosomal DNA extracted from the cells, and the entire nucleotide sequence of the DNA was determined. Furthermore, the creatine amidinohydrolase was successfully produced in a transformant at a high level by a genetic engineering technique, and it became possible to supply a large amount of highly pure creatine amidinohydrolase at low cost.

That is, the present invention relates to an amino acid sequence described in SEQ ID NO: 1 or an amino acid sequence wherein one or more amino acids are added, deleted or substituted in the amino acid sequence and brings about the enzymatic activity of creatine amidinohydrolase. A gene encoding creatine amidinohydrolase, which comprises a nucleotide sequence encoding the sequence.

The gene encoding the creatine amidinohydrolase of the present invention is a gene containing the nucleotide sequence described in SEQ ID NO: 2 in the Sequence Listing.

Further, the gene encoding creatine amidinohydrolase of the present invention has one or more bases added, deleted or substituted in the nucleotide sequence described in SEQ ID NO: 2 in the sequence listing, and creatine amidinohydrolase. Is a nucleotide sequence encoding an amino acid sequence that provides the enzyme activity of

[0012] The present invention is a recombinant vector containing the above gene.

Further, the present invention relates to a transformant transformed with the above-mentioned recombinant vector.

According to the present invention, the above transformant is cultured in a medium,
A method for producing creatine amidinohydrolase, which comprises producing creatine amidinohydrolase and collecting the creatine amidinohydrolase.

The gene encoding the creatine amidinohydrolase of the present invention is, for example, Alcaligenes faecalis TE3581 (FERM).
P-14237). As a medium for culturing this strain, a medium containing a carbon source, a nitrogen source, inorganic ions, and if necessary, nitrate, phosphate and the like is used. As the carbon source, sugars such as glucose and lactose, and sugar alcohols such as glycerol and sorbitol can be used. As the nitrogen source, polypeptone, tryptone, meat extract, yeast extract and the like can be used. Furthermore, for the production of creatine amidinohydrolase, it is necessary to add creatinine or creatine as an enzyme inducer. In culturing the cells, the cells are cultured under aerobic conditions at a temperature and pH at which the cells can grow best, and cultured until the production of creatine amidinohydrolase is maximized.

To purify creatine amidinohydrolase from the cultured cells, the following method is used. The cells are collected from the culture solution by centrifugation, and then the cells are crushed to extract creatine amidinohydrolase. As a method for disrupting cells, for example, treatment with a cell wall lysing enzyme such as lysozyme, ultrasonic disruption,
Physical treatment such as glass bead crushing and French press crushing or a combination of these processes can be performed. The creatine amidinohydrolase fraction is recovered from the crude enzyme extract thus obtained by ammonium sulfate precipitation. The creatine amidinohydrolase fraction thus obtained is desalted by, for example, Sephadex G-25 (Pharmacia Biotech) gel filtration or the like, and then subjected to various combinations of column chromatography, for example, D
EAE Sepharose CL-6B (Pharmacia Biotech), Phenyl Sepharose CL-6B (Pharmacia Biotech), Sephadex G-200 (Pharmacia Biotech) can be purified to high purity by gel filtration.

The protein purified by the above method is SDS
-PAGE showed a uniform band. At this time, the specific activity of creatine amidinohydrolase was about 19 U / mg protein. Note that the combination of column chromatography need not be limited to the above steps, but several steps of column chromatography are required to obtain an electrophoretically uniform band.

The physicochemical properties of the above creatine amidinohydrolase are as follows. Action: creatine + H2O → sarcosine +
Urea Optimum temperature: 40 ° C to 45 ° C Optimum pH: 8.0 to 9.0 Thermal stability: about 50 ° C or less (pH 7.5, 30 minutes) pH stability: pH about 4 to 10 Km value for creatine : About 15.2 mM Molecular weight: about 43,000 (SDS-PAGE) Isoelectric point: about 3.5

The gene encoding the novel creatine amidinohydrolase of the present invention may be extracted from the cells of Alcaligenes faecalis TE3581, or may be chemically synthesized. As the sequence of the above gene,
For example, insertion into the DNA encoding the amino acid sequence described in Sequence Listing / SEQ ID NO: 1, the DNA having the nucleotide sequence described in Sequence Listing / SEQ ID NO: 2, or the nucleotide sequence described in Sequence Listing / SEQ ID NO: 2 , Deletion, substitution, and the like.

The gene of the present invention is obtained, for example, by separating and purifying the chromosomal DNA of Alcaligenes faecalis TE3581 and then cutting the DNA with sonication, restriction enzymes and the like, and a linear expression vector. A blunt end or cohesive end is ligated and closed with DNA ligase or the like to construct a recombinant vector. After the thus obtained recombinant vector is transferred to a replicable host microorganism, screening is performed using the marker of the vector and the expression of creatine amidinohydrolase activity as an index to obtain a microorganism holding the recombinant vector. Next, the microorganism is cultured, the recombinant vector is separated and purified from the cultured cells, and the creatine amidinohydrolase gene may be collected from the recombinant vector.

The DNA derived from the gene donor, Alcaligenes faecalis TE3581, is specifically collected as follows. That is, the donor microorganism is, for example, 1
A culture obtained by stirring and culturing for 日間 3 days is collected by centrifugation and then lysed to prepare a lysate containing the creatine amidinohydrolase gene. Lysis methods include, for example, lysozyme and β
-Treatment with a lytic enzyme such as glucanase, protease and other enzymes and, if necessary, a surfactant such as sodium lauryl sulfate (SDS), and physical disruption such as freeze-thaw or French press treatment It may be combined with the method.

From the lysate obtained in this manner, DNA
Can be separated and purified by appropriately combining conventional methods such as protein removal treatment with phenol treatment or protease treatment, ribonuclease treatment, and alcohol precipitation treatment. The method of cutting DNA isolated and purified from microorganisms can be performed by, for example, ultrasonic treatment, restriction enzyme treatment, or the like. Preferably, a type II restriction enzyme that acts on a specific nucleotide sequence is suitable.

As the vector, a vector constructed for gene recombination from a phage or a plasmid capable of autonomous propagation in a host microorganism is suitable. As the phage, for example, Escherichia coli
Is used as a host microorganism, λgt · 10, λgt ·
11 or the like can be used. As a plasmid, for example, when Escherichia coli is used as a host microorganism, pBR322, pUC19, pBl
and the like can be used. Such a vector can be cleaved with the restriction enzyme used for cutting the microorganism DNA as the creatine amidinohydrolase gene donor described above to obtain a vector fragment, but the restriction enzyme used for cutting the microorganism DNA is not necessarily used. It is not necessary to use the same restriction enzymes. The method for binding the microbial DNA fragment to the vector DNA fragment may be a method using a known DNA ligase. For example, after annealing the cohesive end of the microbial DNA fragment and the cohesive end of the vector fragment, an appropriate DNA ligase is used. Use of microbial DNA
A recombinant vector of the fragment and the vector DNA fragment is prepared. If necessary, after annealing, it can be transferred to a host microorganism and a recombinant vector can be prepared using in vivo DNA ligase.

The host microorganism may be any one as long as the recombinant vector can be stably and autonomously propagated and can express a foreign gene. Generally, Escherichia coli W3
110, Escherichia coli C600, Escherichia coli HB101, Escherichia coli JM109
Etc. can be used. When a vector of pBR322 or a derivative thereof is used, Serratia (Serrati) is used.
a) bacteria of the genus, such as Serratia rickefaciens (Serrati
a liquefaciens) can be used.

As a method for transferring the recombinant vector to the host microorganism, for example, when the host microorganism is Escherichia coli, a competent cell method or an electroporation method by calcium treatment can be used. When the host microorganism is a bacterium of the genus Serratia, an electroporation method or the like can be used. By culturing the thus obtained transformant microorganism in a nutrient medium, it was found that a large amount of creatine amidinohydrolase could be stably produced. Selection of the presence or absence of the transfer of the target recombinant vector to the host microorganism may be performed by searching for a microorganism that simultaneously expresses a drug resistance marker and a microorganism that simultaneously express creatine amidinohydrolase activity of the vector that holds the target DNA, For example, a microorganism that grows on a selective medium based on a drug resistance marker and that produces creatine amidinohydrolase may be selected.

The nucleotide sequence of the creatine amidinohydrolase gene obtained by the method described above is
ence, 214, 1205-1210, 1981), and the amino acid sequence of creatine amidinohydrolase was deduced from the determined nucleotide sequence. The thus-selected recombinant vector having the creatine amidinohydrolase gene can be easily removed from a transformed microorganism and transferred to another microorganism. In addition, a DNA that is a creatine amidinohydrolase gene is recovered from a recombinant vector holding the creatine amidinohydrolase gene by a restriction enzyme or a PCR method, and ligated to another vector fragment,
Transfer to the host microorganism can also be easily performed.

The culture form of the host microorganism, which is a transformant, may be selected by taking into consideration the nutritional and physiological properties of the host. Usually, liquid culture is used in many cases. Advantageously, a culture is performed. As the carbon source of the medium, those commonly used for culturing microorganisms can be widely used. As long as the host microorganism can assimilate, for example, glucose, sucrose, lactose, maltose, fructose, molasses, pyruvic acid and the like can be used. The nitrogen source may be any nitrogen compound that can be used by the host microorganism, such as peptone, meat extract, casein hydrolyzate, organic nitrogen compounds such as soybean meal alkaline extract, and inorganic salts such as ammonium sulfate and salt ammonium. Nitrogen compounds can be used. In addition, salts such as phosphate, carbonate, sulfate, magnesium, calcium, potassium, iron, manganese, and zinc, specific amino acids, specific vitamins, and the like can be used as necessary.

The cultivation temperature can be appropriately changed within a range in which the host microorganism grows and produces creatine amidinohydrolase. In the case of Escherichia coli, the cultivation temperature is preferably 20 to 40%.
It is about 42 ° C., and in the case of Serratia rickefaciens, it is preferably about 20 to 35 ° C. The culturing time varies somewhat depending on the culturing conditions, but it may be terminated at an appropriate time in consideration of the timing when creatine amidinohydrolase reaches the maximum yield, and is usually about 20 to 48 hours. The pH of the medium can be appropriately changed within a range in which the host microorganism grows and produces creatine amidinohydrolase, and is usually preferably about pH 6.0 to 9.0. The method of recovering the cells from the culture solution may be a commonly used method, and for example, can be recovered by centrifugation or filtration. When creatine amidinohydrolase in the culture solution is secreted outside the cells, this cell separation solution may be used, and creatine amidinohydrolase can be separated and purified according to the following method after cell disruption. When creatine amidinohydrolase is present in the cells, it can be crushed and extracted by the enzymatic or physical crushing method as described above. The creatine amidinohydrolase fraction is recovered from the crude enzyme extract thus obtained by ammonium sulfate precipitation. The crude enzyme solution can be desalted by a method usually used, for example, dialysis using a semi-permeable membrane or Sephadex G-25 (Pharmacia Biotech) gel filtration. After this operation, DEAE Sepharose CL-6
B (Pharmacia Biotech) and octyl sepharose CL6-B (Pharmacia Biotech) can be separated and purified by column chromatography to obtain a purified enzyme preparation. This purified enzyme preparation was prepared by SDS-PAGE.
It is purified to the extent that it shows almost a single band.

The protein having creatine amidinohydrolase activity obtained by the production method of the present invention has the following properties. Action: creatine + H2O → sarcosine +
Urea Optimum temperature: 40 ° C to 45 ° C Optimum pH: 8.0 to 9.0 Thermal stability: about 50 ° C or less (pH 7.5, 30 minutes) pH stability: pH about 4 to 10 Km value for creatine : About 15.2 mM Molecular weight: about 43,000 (SDS-PAGE) Isoelectric point: about 3.5

[0030]

The present invention will be described below in more detail with reference to examples. In the examples, the activity of creatine amidinohydrolase was measured as follows. First, 0.9 ml of a substrate solution (creatine dissolved in 50 mM phosphate buffer, pH 7.5 to a concentration of 0.1 M) was placed in a test tube.
Preheat at 37 ° C for about 5 minutes. Next, 0.1 ml of enzyme solution
Was added, and the reaction was started. After reacting at 37 ° C. for exactly 10 minutes, 2.0 ml of a DAB solution (dissolve 2.0 g of dimethylaminobenzaldehyde in 100 ml of dimethyl sulfoxide and add 15 ml of concentrated hydrochloric acid) To stop the reaction. After standing at 25 ° C. for 20 minutes, the yellow dye (Ehrich reaction product) formed by the condensation of urea formed with dimethylaminobenzaldehyde is measured by absorbance at 425 nm. Blind: 0.9 ml of substrate solution 3
After leaving at 7 ° C. for 10 minutes, 2.0 ml of DAB solution is added and mixed, and then 0.1 ml of enzyme solution is added to prepare the mixture. After standing at 25 ° C. for 20 minutes, absorbance is measured.
The amount of the enzyme that produces 1 micromol of the yellow pigment per minute under the above conditions is defined as 1 unit (U).

Reference Example 1 Alkaligenes faecalis T
Purification of creatine amidinohydrolase from E3581 Alcaligenes faecalis TE3581 (FERM P-1423
7) was cultured in a 10 L jar fermenter. The medium composition was 1% creatine, 1% polypeptone, and 0.1% yeast extract.
This was a medium (pH 7.0) containing 5% and 0.5% NaCl, and cultured at 30 ° C. for 24 hours with aeration and stirring. The productivity at this time was about 2 U / ml-b. The cells were collected by centrifugation and suspended in a 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, DEAE Sepharose CL-6B (Pharmacia Biotech) column chromatography, phenyl Sepharose CL-6B (Pharmacia Biotech). Biotech) column chromatography,
It was purified by Sephadex G-200 (Pharmacia Biotech) gel filtration to obtain a purified enzyme preparation. The creatine amidinohydrolase preparation obtained by the method showed a uniform band on SDS-PAGE, and the specific activity at this time was 19.5 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.

[0033]

[Table 1]

[0034]

[Table 2]

Example 1 Isolation of Chromosomal DNA Chromosome DN of Alcaligenes faecalis TE3581
A was separated by the following method. The strain was cultured with shaking at 30 ° C. overnight in 150 ml of ordinary broth medium, and then centrifuged (8
000 rpm for 10 minutes). 10% sucrose, 50 mM Tris-HCl (pH 8.0), 50
The suspension was suspended in 5 ml of a solution containing mM EDTA, 1 ml of a lysozyme solution (10 mg / ml) was added, the mixture was kept at 37 ° C. for 15 minutes, and then 1 ml of a 10% SDS solution was added.
An equal volume of a chloroform / phenol solution (1:
1) was added, and the mixture was stirred and mixed, and separated into an aqueous layer and a solvent layer by centrifugation at 10,000 rpm for 3 minutes. The aqueous layer was separated. The aqueous layer was gently overlaid with twice the volume of ethanol, and the DNA was wound around a glass rod while being slowly stirred with a glass rod to separate the DNA. This DNA was added to 10 mM containing 1 mM EDTA.
It was dissolved in a solution of mM Tris-HCl, pH 8.0 (hereinafter abbreviated as TE). This was treated with an equal volume of a chloroform / phenol solution, and the aqueous layer was separated by centrifugation, and twice the amount of ethanol was added.
Dissolved in ml of TE.

Example 2 Preparation of a DNA Fragment Containing a Gene Encoding Creatine Amidinohydrolase and a Recombinant Vector Having the DNA Fragment 20 μg of the DNA obtained in Example 2 was digested with the restriction enzyme Sau3AI.
(Toyobo), and fragments of 2 to 10 kbp were recovered by sucrose density gradient centrifugation. On the other hand, pBlue digested with the restriction enzyme BamHI (manufactured by Toyobo)
After the script KS (+) was dephosphorylated with bacterial alkaline phosphatase (manufactured by Toyobo), both DNAs were reacted with one unit of T4 DNA ligase (manufactured by Toyobo) at 16 ° C. for 12 hours to ligate the DNA. The ligated DNA was transformed using competent cells 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% KH2 PO4, 0.05%
MgSO4.7H2O, 1.15% creatine, 10U
/ Ml sarcosine oxidase (manufactured by Toyobo), 0.5 U
/ Ml peroxidase (Toyobo), 0.01% ο-
Dianisidine, 50 μg / ml ampicillin, 1.5%
Agar]. The detection of creatine amidinohydrolase activity was carried out using colonies that grew in the above medium and stained brown. About 1 × 10 5 transformant colonies were obtained per μg of DNA used. As a result of screening about 12,000 colonies,
Six colonies stained brown were found. These strains were cultured in an LB liquid medium (1% polypeptone, 0.5% yeast extract, 0.5% NaCl, 50 μg / ml ampicillin), and the creatine amidinohydrolase activity was measured. Hydrolase activity was detected. Among these strains, the plasmid possessed by the strain having the highest creatine amidinohydrolase activity had an inserted DNA fragment of about 5 kbp, and this plasmid was designated as pCRH17. FIG. 1 shows a restriction enzyme map of the inserted DNA of pCRH17. Next, the inserted DNA of pCRH17 was cut out with restriction enzymes EcoRV (manufactured by Toyobo) and PstI (manufactured by Toyobo), and pBluescript cleaved with the same restriction enzymes was used.
Ligation to tKS (+) created pCRH173.

Example 3 Determination of Nucleotide Sequence A subclone was prepared using various restriction enzymes for an about 3.3 kbp inserted DNA fragment of pCRH173. Various subclones can be obtained by Radioactive Sequencing
The nucleotide sequence was determined using Kit (manufactured by Toyobo). The determined base sequence and amino acid sequence are shown in the sequence listing. The molecular weight of the protein determined from the amino acid sequence is about 46,000
And the molecular weight of the creatine amidinohydrolase of Alcaligenes faecalis TE3581 (about 43,00
0).

Example 4 Preparation of Transformant A transformant of E. coli was prepared by Escherichia coli JM.
109 competent cells (manufactured by Toyobo) in pCRH17
3 and transformant Escherichia coli JM
109 (pCRH173) was obtained. The preparation of a transformant of Serratia sp. Is performed by Serratia liquefaciens IF.
O12979 was transformed by electroporation using Gene Pulser (manufactured by Bio-Rad) to obtain Serratia rickefaciens (pCRH173).

Example 5 Escherichia coli JM10
Production of creatine amidinohydrolase from 9 (pCRH173) TB medium (1.2% polypeptone, 2.4% yeast extract, 0.4% glycerol, 0.0231% KH2 PO
4, 0.1254% KH2 PO4) was dispensed into a 10 L jar fermenter, autoclaved at 121 ° C. for 15 minutes, allowed to cool, and then sterile-filtered separately at 50 mg / m 2.
1 Ampicillin (manufactured by Nacalai Tesque) and 200 mM
6 ml each of IPTG (manufactured by Nippon Seika) was added.
Escherichia coli JM109 (pCRH17
60 ml of the culture solution of 3) was inoculated, and cultured with aeration and stirring at 37 ° C. for 24 hours. The creatine amidinohydrolase activity at the end of the culture was 8.9 U / ml. The above cells were collected by centrifugation, and 50 mM phosphate buffer, pH 7.0.
Suspended in water.

The cell suspension was crushed by a French press and centrifuged to obtain a supernatant. The resulting crude enzyme solution is fractionated with ammonium sulfate, desalted by Sephadex G-25 (Pharmacia Biotech) gel filtration, DEAE Sepharose CL-6B (Pharmacia Biotech) column chromatography, octyl Sepharose CL-6B (Pharmacia Biotech) column. Purification by chromatography gave a purified enzyme preparation. The creatine amidinohydrolase preparation obtained by this method was used for SDS-PA
It shows almost a single band in terms of GE, and the specific activity at this time is 1
It was 8.9 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.

[0041]

[Table 3]

[0042]

[Table 4]

Example 6 Production of Creatine Amidinohydrolase from Serratia rickefaciens (pCRH173) In the same manner as in Example 5, production of creatine amidinohydrolase from Serratia rickefaciens (pCRH173) was performed. The cultivation was performed at 30 ° C. without adding IPTG. At this time, the creatine amidinohydrolase activity at the end of the culture was 11.0 U
/ Ml. The specific activity of the obtained purified enzyme preparation was 19.1 U / mg-protein. Table 5 summarizes the purification. Table 6 shows the physicochemical properties of creatine amidinohydrolase obtained by the above method.

[0044]

[Table 5]

[0045]

[Table 6]

By comparing Table 1, Table 3 and Table 5, it can be seen that creatine amidinohydrolase can be prepared in a large amount by a simpler method according to the production method of the present invention. In particular, when Serratia liquefaciens (pCRH173) is used, a large amount of an inducer required to express creatine amidinohydrolase in large amounts, such as creatine required for Alcaligenes faecalis TE3581 or IPTG required for Escherichia coli, is not used at all. It is excellent in that creatine amidinohydrolase can be produced.

[0047]

Industrial Applicability According to the present invention, the nucleotide sequence and amino acid sequence of the novel creatine amidinohydrolase gene become clear, and creatine amidinohydrolase can be industrially produced in large quantities using this sequence.

[0048]

[Sequence List] <110> Toyo Boseki Kabushiki Kaisya <120> <130> 01-0161 <141> 2001-02-26 <160> 2 <170> Patentin version 2.0 <210> 1 <211> 404 <212> PRT <213> Alkaligenes faecalis TE3581 (FERM P14237) <400> 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 385 390 395 400 400 Ile Ile Arg Asn 404

<210> 2 <211> 1212 <212> DNA <213> Alkaligenes faecalis TE3581 (FERM P14237) <220> <400> actactattc cggctggctg 180 tactgctatt tcggacgcaa gtacggcatg gtcatcgacc acaacaacgc cacgacgatt 240 tcggccggca tcgacggcgg ccagccctgg cgccgcagct tcggcgacaa catcacctac 300 accgactggc gccgcgacaa tttctatcgc gccgtgcgcc agctgaccac gggcgccaag 360 cgcatcggca tcgagttcga ccacgtcaat ctcgacttcc gccgccagct cgaggaagcc 420 ctaccgggcg tcgacttcgt cgacatcagc cagccctcga tgtggatgcg caccatcaag 480 tcgctcgaag agcagaagct gatccgcgaa ggcgcccgcg tgtgtgacgt cggcggcgcg 540 gcctgcgcgg ctgccatcaa ggccggcgtg cccgagcatg aagtggcgat cgccaccacc 600 aatgcgatga tccgcgagat cgccaaatcg ttccccttcg tggagctgat ggacacctgg 660 acctggttcc agtcgggcat caacaccgac ggcgcgcaca atccggtcc caaccgcatc 720 gtgcaatccg gccgcctac gcctccct gcc cgg ctactacacc 780 gcgctggagc gcacgctgtt ctgcgaccat gtcgatgacg ccagcctcga catctgggag 840 aagaacgtgg ccgtgcatcg ccgcgggctc gagctgatca agccgggcgc gcgctgcaag 900 gacatcgcca tcgagctcaa cgagatgtac cgcgagtggg acctgctgaa gtaccgctcc 960 ttcggctatg gccactcctt cggcgtgctg tgccactact acggtcgcga ggccggcgtg 1020 gagctgcgcg aggacatcga caccgagctg aagcccggca tggtggtctc catggagccg 1080 atggtgatgc tgccggaggg catgcccggt gccggcggct atcgcgagca cgacatcctg 1140 atcgtcgggg aggacggtgc cgagaacatc accggcttcc cgttcggtcc ggaacacaac 1200 atcatccgca ac 1212

[Brief description of the drawings]

FIG. 1 shows a restriction map of plasmid pCRH17.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification FI FI Theme Court ゛ (Reference) (C12N 1/21 (C12N 9/78 C12R 1: 425) C12R 1:05) (C12N 9/78 C12N 15 / 00 ZNAA C12R 1:05) C12R 1: 425)

Claims (11)

[Claims] 1. Encoding an amino acid sequence described in SEQ ID NO: 1 or an amino acid sequence in which one or more amino acids are added, deleted or substituted in the amino acid sequence and which brings about the enzymatic activity of creatine amidinohydrolase. A gene encoding creatine amidinohydrolase, characterized in that the gene comprises a base sequence of the present invention. 2. The gene encoding creatine amidinohydrolase according to claim 1, wherein the creatine amidinohydrolase has the following physicochemical properties. Action: creatine + H2O → sarcosine +
Urea Optimum temperature: 40 ° C to 45 ° C Optimum pH: 8.0 to 9.0 Thermal stability: about 50 ° C or less (pH 7.5, 30 minutes) pH stability: pH about 4 to 10 Km value for creatine : About 15.2 mM Molecular weight: about 43,000 (SDS-PAGE) Isoelectric point: about 3.5 3. The method of claim 1 wherein the creatine amidinohydrolase is Alcaligenes faecalis TE358.
The gene encoding creatine amidinohydrolase according to claim 1, which is an enzyme produced by 1 (FERM P-14237). 4. A gene encoding creatine amidinohydrolase, comprising the nucleotide sequence of SEQ ID NO: 2. 5. A nucleotide sequence described in SEQ ID NO: 2 in which one or more bases are added, deleted or substituted, and encodes an amino acid sequence which brings about the creatine amidinohydrolase enzymatic activity. A gene encoding creatine amidinohydrolase having a base sequence of 6. A recombinant vector comprising the gene encoding creatine amidinohydrolase according to claim 1, 4 or 5. 7. A transformant in which a host cell has been transformed with a recombinant vector containing the gene encoding creatine amidinohydrolase according to claim 1, 4 or 5. 8. The transformant according to claim 7, wherein the host cell is a gram-negative bacterium. 9. The transformant according to claim 7, wherein the host cell is a bacterium of the genus Serratia. 10. The transformant according to claim 9, wherein the Serratia bacterium is Serratialiquefaciens. 11. A transformant in which a host cell is transformed by a recombinant vector containing the gene according to claim 1, 4 or 5, is cultured in a medium to produce creatine amidinohydrolase, and the creatine amidino is produced. A method for producing creatine amidinohydrolase, comprising collecting a hydrolase.