JPH10257890A - New creatine amidinohydrolase - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject new enzyme, comprising a creatine amidinohydrolase capable of decomposing creatine into sarcosine and urea in the presence of water, available in a large amount in high purity according to a genetic engineering technique and useful for determination, etc., of creatine and creatinine. SOLUTION: This creatine amidinohydrolase has actions on creatine in the presence of water and production of sarcosine and urea, about 7.0-8.5 optimum pH, is stable at about <=40 deg.C (by treatment at pH7.5 for 30min) and has the pH stability of about 5.0-9.0 (by treatment at 25 deg.C for 16hr), about 46mM value of Km for the creatine and a molecular weight of about 50,000 [measured by a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)], 47,146 (a calculated value from the amino acid composition) and about 78,000 (measured by a gel filtration) and further about 4.3 isoelectric point and is represented by the formula. The creatine amidinohydrolase is used for the determination, etc., of the creatine and creatinine. The enzyme is obtained by expressing a gene cloned from a chromosomal DNA of Arthrobacter sp. TE1826.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a novel enzyme having creatine amidinohydrolase activity which can be used for quantifying creatine and creatinine, a gene encoding the enzyme, and a method for producing the enzyme by genetic engineering techniques.

[0002]

2. Description of the Related Art Conventionally, creatine amidinohydrolase (EC 3.5.3.3) has been used as an enzyme for measuring creatine and creatinine in body fluids, which has been clinically used as an indicator for diagnosis of muscular and renal diseases. For example, creatinine amidohydrolase, sarcosine oxidase and peroxidase. Creatine amidinohydrolase is an enzyme that acts on creatine in the presence of water to catalyze a reaction that produces sarcosine and urea.

[0003] Such a creatine amidinohydrolase is known from the genus Pseudomonas (Journal of Biochemistry, Vo.
l.79, 1381-1383 (1976)) or bacteria of the genus Bacillus (Japanese Patent Publication No. 61-17465).
Further, other bacteria include genus Flavobacterium, genus Corynebacterium, genus Micrococcus (JP-A-51-11884), genus Alcaligenes, and genus Penicillium (JP-A-47-43281). Bacteria are only known.

[0004]

SUMMARY OF THE INVENTION It is an object of the present invention to isolate a novel creatine amidinohydrolase from a new bacterium which has not been produced so far and to clone a gene encoding the enzyme to produce a novel creatine amidinohydrolase. Another object of the present invention is to provide a method for producing the enzyme in large quantities. Another object of the present invention is to derive a method for producing a novel creatine amidinohydrolase having improved physicochemical properties by modifying the gene.

[0005]

Means for Solving the Problems The present inventors have conducted various studies to achieve the above object, and as a result, as a creatine amidinohydrolase producing bacterium, Arthrobacter sp. Arthrobacter sp. TE1826 (FERM P-
10637), a novel creatine amidinohydrolase was isolated from the strain, and a gene encoding creatine amidinohydrolase was successfully isolated from chromosomal DNA extracted from the cell, and expressed by the gene. Recombinant creatine amidinohydrolase was isolated. Further, determining the entire nucleotide sequence of the gene,
The enzyme was successfully produced by a genetic engineering technique without the need to add an expensive inducer such as creatinine to the medium, and the enzyme was successfully produced at high cost, and it was possible to supply a large amount of the enzyme with high purity at low cost.

That is, the present invention is a novel creatine amidinohydrolase having the following physicochemical properties. Action: Acts on creatine in the presence of water to produce sarcosine and urea. Optimum temperature: about 40 ° C Optimum pH: about 7.0 to 8.5 Thermal stability: about 40 ° C or less (pH 7.5, treated for 30 minutes) pH stability: about 5.0 to 9.0 (25 Km value for creatine: about 46 mM Molecular weight: about 50,000 (SDS-PAGE) 47,146 (calculated from amino acid composition) about 78,000 (gel filtration) Isoelectric point: about 4 .3

The present invention also relates to creatine amidinohydrolase which is a protein of the following (a) or (b): (A) a protein consisting of the amino acid sequence of SEQ ID NO: 1 in the sequence listing; (B) a protein comprising an amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence (a), and which has creatine amidinohydrolase activity

The present invention relates to a gene encoding creatine amidinohydrolase, which is a recombinant protein of the following (a) or (b): (A) a protein consisting of the amino acid sequence of SEQ ID NO: 1 in the sequence listing; (B) a protein comprising an amino acid sequence in which one or more amino acids have been deleted, substituted or added in the amino acid sequence (a), and having creatine amidinohydrolase activity;

Further, the present invention is a gene encoding creatine amidinohydrolase comprising the following DNA (c), (d) or (e). (C) DNA consisting of the nucleotide sequence of SEQ ID NO: 2 in the sequence listing (d) One or more bases are added, deleted or substituted in the above-mentioned nucleotide sequence (c), and creatine amidino DNA encoding an amino acid sequence having hydrolase activity (e) Bacteria derived from a bacterium that hybridizes under stringent conditions to DNA comprising the nucleotide sequence of (c) above and encodes a protein having creatine amidinohydrolase activity DNA of

[0010] The present invention is a recombinant vector containing the gene coding for creatine amidinohydrolase.

[0011] The present invention also relates to a transformant obtained by transforming a host cell with the above-described recombinant vector.

Further, the present invention provides a method for producing creatine amidinohydrolase, comprising culturing the above transformant, producing creatine amidinohydrolase, and collecting the creatine amidinohydrolase.

[0013] The present invention also relates to Arthrobacter sp. TE1826 (FERM P-1).
0637) is cultured to produce creatine amidinohydrolase, and the creatine amidinohydrolase is collected.

[0014]

DETAILED DESCRIPTION OF THE INVENTION The creatine amidinohydrolase of the present invention is a creatine amidinohydrolase-producing microorganism, for example, Arthrobacter sp.
sp.) TE1826 (FERM P-10637). However, by isolating the gene encoding creatine amidinohydrolase and isolating the creatine amidinohydrolase expressed from the gene, the creatine amidinohydrolase of the present invention can be conveniently prepared.
And it can be obtained efficiently. In one embodiment of the present invention, (a) a protein consisting of the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing or (b) one or more amino acids in the amino acid sequence (a) are deleted,
Consisting of a substituted or added amino acid sequence, and
There are proteins that have creatine amidinohydrolase activity. The degree of amino acid deletion, substitution, or addition includes those in which the basic characteristics are not changed or the characteristics are improved. The method for producing these mutants follows a conventionally known method.

The gene encoding the creatine amidinohydrolase of the present invention is, for example, Arthrobacter sp. TE1826 (FERM P-
10637) or can be chemically synthesized. By using the polymerase chain reaction (PCR), it is also possible to obtain a DNA fragment containing the creatine amidinohydrolase gene.

Examples of the above genes include (a) DNA encoding a protein consisting of the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing, and (b) one or several amino acids in the amino acid sequence (a). Has a deletion, substitution or addition of an amino acid sequence, and encodes a protein having creatine amidinohydrolase activity. The degree of DNA deletion, substitution, and addition includes those in which the basic properties are not changed or the properties are improved. The method for producing these mutants follows a conventionally known method.

Alternatively, (c) one or several bases are added, deleted or substituted in the base sequence of SEQ ID NO: 2 in the base sequence of DNA, (d) or (c), And a protein that hybridizes under stringent conditions to DNA encoding an amino acid sequence having creatine amidinohydrolase activity or DNA comprising the nucleotide sequence of (e) and (c), and encodes a protein having creatine amidinohydrolase activity There is DNA derived from bacteria. Here, the stringent condition is x2 SSC (300 mM NaCl, 30 mM
M citric acid) at 65 ° C. for 16 hours.

As a method for obtaining the gene of the present invention, for example, Arthrobacter sp.
Chromosome DN of E1826 (FERM P-10637)
A, after separating and purifying A, the DNA cleaved by sonication, restriction enzyme treatment, etc., and a linear expression vector were ligated with DN at the blunt end or cohesive end of both DNAs.
A recombinant vector is constructed by binding and closing with A ligase or the like. After the thus obtained recombinant vector is transferred to a replicable host microorganism, screening is performed using the vector marker and the expression of the enzyme activity as indices, and the microorganism carrying the recombinant vector containing the gene encoding creatine amidinohydrolase is screened. Get. Next, the microorganism is cultured, and the recombinant vector is separated from the cultured cells.
It may be purified and the creatine amidinohydrolase gene may be collected from the recombinant vector.

The gene donor Arthrobacter sp. TE1826 (FERM P
Specifically, the chromosomal DNA of (-10637) is 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. As a lysis method, for example, treatment with a lytic enzyme such as lysozyme or β-glucanase is performed, and if necessary, a protease, another enzyme, or a surfactant such as sodium lauryl sulfate (SDS) is used in combination, and further frozen. It may be combined with a physical crushing method such as melting or French pressing.

From the lysate thus obtained, DNA
Can be separated and purified by a conventional method, for example, by appropriately combining 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 acting 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 growth in a host microorganism is suitable. Examples of the phage include Escherichia coli (Escherichia coli).
If li) is used as the host microorganism, Lambda-gt10, Lambda
da-gt11 etc. can be used. As a plasmid,
For example, when Escherichia coli is used as a host microorganism, pBR322, pUC19,
pBluescript, pLED-M1 (Journal of Fermentati
on and Bioenzineering, Vol. 76, 265-269 (1993)). Such a vector is used for the above-mentioned creatine amidinohydrolase gene donor, microorganism D.
A vector fragment can be obtained by digestion with the restriction enzyme used for cleavage of NA, but it is not always necessary to use the same restriction enzyme as that used for cleavage of the microorganism DNA. 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. Microbial DNA fragment and vector DNA
Create a recombinant vector with the fragment. If necessary, after annealing, it can be transferred to a host microorganism and a recombinant vector can be prepared using in vivo DNA ligase.

As the host microorganism, any microorganism can be used as long as the recombinant vector is stable, capable of autonomous propagation and capable of expressing a foreign gene, and is generally Escherichia coli W3.
110, Escherichia coli C600, Escherichia coli HB101, Escherichia coli JM109, etc. 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.

The microorganism, which is a transformant thus obtained, can stably produce a large amount of creatine amidinohydrolase by being cultured in a nutrient medium. Selection of the presence or absence of transfer of the target recombinant vector to the host microorganism may be performed by searching for a drug resistance marker of the vector holding the target DNA and a microorganism that simultaneously expresses creatine amidinohydrolase activity. Microorganisms that grow on a selective medium based on creatine amidinohydrolase may be selected.

The nucleotide sequence of the creatine amidinohydrolase gene obtained by the above-described method is Scientific (Scie).
nce, Vol. 214, 1205-1210 (1981)), and the amino acid sequence of creatine amidinohydrolase was estimated 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, DNA, which is a creatine amidinohydrolase gene, can be recovered from a recombinant vector having the creatine amidinohydrolase gene by restriction enzyme or PCR, coupled to another vector fragment, and transferred to a host microorganism.

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 stirred culture is performed. As the nutrient source of the medium, those commonly used for culturing microorganisms can be widely used. The carbon source may be any carbon compound that can be assimilated, such as glucose, sucrose, lactose, maltose, fructose, molasses, and pyruvic acid. As the nitrogen source, any available nitrogen compound may be used. For example, peptone, meat extract, yeast extract, casein hydrolyzate, alkali extract of soybean meal and the like are used. In addition, phosphate, carbonate, sulfate,
Magnesium, calcium, potassium, iron, manganese,
Salts such as zinc, specific amino acids, specific vitamins and the like are used as needed. The culture temperature can be appropriately changed within a range in which the bacteria grow and produce creatine amidinohydrolase. In the case of Escherichia coli, it is preferably about 20 to 42 ° C. The culturing time varies somewhat depending on the conditions, but the culturing may be terminated at an appropriate time in consideration of the time when creatine amidinohydrolase reaches the maximum yield, and is usually about 6 to 48 hours. The pH of the medium can be changed as appropriate within a range in which the bacteria grow and produce creatine amidinohydrolase, but the pH is particularly preferably 6.0 to 6.0.
It is about 9.0.

[0027] A culture solution containing cells producing creatine amidinohydrolase in the culture can be directly collected and used. However, in general, when creatine amidinohydrolase is present in the culture solution according to a conventional method,
It is used after separating a creatine amidinohydrolase-containing solution from microbial cells by filtration, centrifugation, or the like. If creatine amidinohydrolase is present in the cells, collect the cells from the resulting culture by filtration or centrifugation, and then destroy the cells by a mechanical method or an enzymatic method such as lysozyme. If necessary, a chelating agent such as EDTA and / or a surfactant are added to solubilize creatine amidinohydrolase, and separated and collected as an aqueous solution.

The creatine amidinohydrolase-containing solution thus obtained is subjected to, for example, concentration under reduced pressure, membrane concentration,
Further, precipitation may be performed by a salting-out treatment with ammonium sulfate, sodium sulfate or the like, or by a fractional precipitation method using a hydrophilic organic solvent such as methanol, ethanol, acetone or the like. Heat treatment and isoelectric point treatment are also effective purification means. After that, purified creatine amidinohydrolase can be obtained by performing gel filtration using an adsorbent or a gel filtration agent, adsorption chromatography, ion exchange chromatography, and affinity chromatography.

For example, Sephadex G-25
(Pharmacia Biotech), etc., gel filtration, DE
AE Sepharose CL-6B (Pharmacia Biotech),
Octyl Sepharose CL6-B (Pharmacia Biotech) can be separated and purified by column chromatography to obtain a purified enzyme preparation. This purified enzyme preparation has been purified to the extent that it shows almost a single band by electrophoresis (SDS-PAGE).

The creatine amidinohydrolase of the present invention has the following physicochemical properties. Action: Acts on creatine in the presence of water to produce sarcosine and urea. Optimum temperature: about 40 ° C Optimum pH: about 7.0 to 8.5 Thermal stability: about 40 ° C or less (pH 7.5: treated for 30 minutes) pH stability: about 5.0 to 9.0 (25 Km value for creatine: about 46 mM Molecular weight: about 50,000 (SDS-PAGE) 47,146 (calculated from amino acid composition) about 78,000 (gel filtration) Isoelectric point: about 4 .3

Table 1 shows a comparison of properties between the creatine amidinohydrolase of the present invention and a known creatine amidinohydrolase. The homology between the amino acid sequence of the creatine amidinohydrolase of the present invention and the amino acid sequence of the known creatine amidinohydrolase produced by Pseudomonas putida is 6
3.4%, and a comparison between the two is shown in FIG. The creatine amidinohydrolase of the present invention is a novel enzyme having different properties from known enzymes that catalyze the same reaction.

[0032]

[Table 1]

[0033]

The present invention will be described below in more detail with reference to examples. In the examples, the measurement of creatine amidinohydrolase activity was performed using the following reagents and measurement conditions. <Reagent> Reagent mixture composition 0.3M HEPES pH 7.6 1.8% Creatine 0.015% Phenol 0.005% 4-aminoantipyrine 6U / ml Sarcosine oxidase 6U / ml Peroxidase

<Measurement conditions> 3 ml of the above reagent mixture was mixed at 37 ° C.
After preheating for about 3 minutes with 0.1 ml of enzyme solution,
The reaction was started at 7 ° C., reacted for 4 minutes, and then 500 nm
Is measured with a spectrophotometer. In the blind test, distilled water is added to the reagent mixture instead of the enzyme solution, and the absorbance change is measured in the same manner as described below. The amount of the enzyme that produces 1 micromol of sarcosine per minute under the above conditions is defined as 1 unit (U).

Example 1 Isolation of Chromosomal DNA Arthrobacter sp. TE1826 (FERM)
P-10637) was isolated by the following method. The strain was diluted with 100 ml of 2 × YT medium (1.6% polypeptone, 1% yeast extract, 0.5% sodium chloride (pH 7.2)).
After culturing with shaking at 7 ° C overnight, centrifugation (8,000 rpm, 10
Min). 15 mM sodium citrate,
After washing the cells with a solution containing 0.15 M sodium chloride, 20% sucrose and 50 mM Tris-HCl (p
H7.6) Suspend in 5 ml of a solution containing 1 mM EDTA, add 1 ml of lysozyme solution (100 mg / ml),
The mixture was kept at 37 ° C. for 30 minutes, and then 11 ml of a solution containing 1% lauroyl sarcosine acid, 0.1 M EDTA (pH 9.6) was added. To this suspension was added ethidium bromide solution at 0.5% and cesium chloride at about 100%, followed by stirring and mixing.
DNA was collected by ultracentrifugation at 55,000 rpm for 20 hours. The fractionated DNA was 10m containing 1mM EDTA.
It was dialyzed against M Tris-HCl, pH 8.0 solution (hereinafter abbreviated as TE) to obtain a purified DNA sample. This was treated with an equal volume of a chloroform / phenol solution, and the aqueous layer was separated by centrifugation. Two times the amount of ethanol was added, the DNA was separated again by the above method, and dissolved with 2 ml of TE.

Example 2 DNA fragment containing a gene encoding creatine amidinohydrolase and D
Preparation of Recombinant Vector Having NA Fragment 5 μg of the DNA obtained in Example 1 was partially digested with a restriction enzyme Sau3AI (manufactured by Toyobo), digested into fragments of 2 kbp or more, and then cut with a restriction enzyme BamHI (manufactured by Toyobo)
1 μg of ptKS (+) and T4-DNA ligase (Toyobo)
One unit was reacted at 16 ° C. for 12 hours to ligate DNA. The ligated DNA was transformed using competent cells of Escherichia coli JM109 (manufactured by Toyobo), and agar medium for detecting creatine amidinohydrolase activity [1.0%
Polypeptone, 0.5% yeast extract, 0.5% NaCl, 1% creatine, 10U / ml sarcosine oxidase (Toyobo), 5U
/ ml peroxidase (manufactured by Toyobo), 0.01% o-dianisidine, 50 µg / ml ampicillin, 1.5% agar]. The creatine amidinohydrolase activity was detected by using, as an indicator, a colony that grew in the above medium and stained brown.

Approximately 100,000 transformant colonies were obtained per μg of DNA used, and as a result of the above screening, one colony stained brown was found. This strain was 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. The activity was detected. The plasmid contained in this strain had an inserted DNA fragment of about 6.6 kbp, and this plasmid was designated as pCNAB1.

Next, the inserted DNA of pCNAB1 was excised with the restriction enzyme ApaI (manufactured by Toyobo), ligated to pBluescriptKS (+) cut with the same restriction enzyme, and ligated into pCNHAA-1.
It was created. FIG. 2 shows a restriction map of this pCNHAA-1.

Example 3 Determination of Nucleotide Sequence About 4.6 kbp of the inserted DNA fragment of pCNHAA-1, subclones were prepared using various restriction enzymes. Various subclones can be obtained by Radioactive Sequencin
The nucleotide sequence was determined using g 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 was 47,146, which was almost the same as the molecular weight of creatine amidinohydrolase of Arthrobacter sp. TE1826.

Example 4 Recombinant vector pCRHA-
Preparation of No. 1 In order to remove the part other than the creatine amidinohydrolase gene from the inserted DNA fragment of pCNHAA-1, PCR
Was performed to reduce the size of the inserted DNA fragment. PCR was performed under the following reaction solution composition and amplification conditions. <Reaction solution composition> Pfu DNA polymerase (manufactured by Stratagene) 5U / 100 μl 10-fold concentration buffer for Pfu DNA polymerase 10 μl / 100 μl pCNHAA-1 (template DNA) 0.1 μg / 100 μl dATP, dTTP, dGTP, dCTP 0.2 each mM 2 types of primers 1 μM each (described in SEQ ID Nos. 3 and 4 in the sequence listing)

<Amplification Conditions> Denaturation 95 ° C., temperature change for 30 seconds 1 minute 30 seconds Annealing 45 ° C., 1 second temperature change 1 second Reaction 75 ° C., 2 minutes 30 seconds Temperature change 1 second (30 cycles in total)

About 1.2 kbp of the amplified DNA fragment was ligated to pBluescript SK (-) digested with the
RHAR-1 was made. FIG. 3 shows a restriction map of pCRHAR-1. The inserted DNA fragment of pCRHARA-1 does not contain any portion other than the creatine amidinohydrolase gene.

Example 5 Preparation of Transformant Competent cells of Escherichia coli JM109 (manufactured by Toyobo) were transformed with pCRHAR-1 to obtain a transformant Escherichia coli JM109 (pCRHAR-1).

Example 6 Escherichia coli JM109 (pC
Production of creatine amidinohydrolase from RHAR-1) 500 ml of LB medium was dispensed into a 2 L flask, and
The mixture was autoclaved at 15 ° C. for 15 minutes, allowed to cool, and then 0.5 ml of sterile-filtered 50 mg / ml ampicillin (manufactured by Nacalai Tesque) was separately added. Escherichia cultivated beforehand in this medium with shaking at 30 ° C. for 7 hours in LB medium.
Inoculate 5 ml of a culture of Collie JM109 (pCRHAR-1)
The culture was carried out with aeration and stirring at 18 ° C. for 18 hours. The creatine amidinohydrolase activity at the end of the culture was about 5 U / ml.

The above cells were collected by centrifugation, and 20 mM
Suspended in phosphate buffer, pH 7.5. The cell suspension was crushed by a French press and centrifuged to obtain a supernatant. After performing the nucleic acid removal treatment and the ammonium sulfate fractionation treatment on the resulting crude enzyme solution with polyethyleneimine, Sephadex
(Sephadex) Demineralized by gel filtration with G-25 (Pharmacia Biotech), separated and purified by DEAE Sepharose CL-6B (Pharmacia Biotech) column chromatography, Octyl Sepharose CL6-B (Pharmacia Biotech) column chromatography, and purified An enzyme preparation was obtained. The creatine amidinohydrolase preparation obtained by this method shows a substantially single band by electrophoresis (SDS-PAGE), and the specific activity at this time is about 21 U /
mg-protein.

The properties of creatine amidinohydrolase obtained by the above method are shown below. Action: Acts on creatine in the presence of water to produce sarcosine and urea. Optimum temperature: about 40 ° C Optimum pH: about 7.0 to 8.5 Thermal stability: about 40 ° C or less (pH 7.5: treated for 30 minutes) pH stability: about 5.0 to 9.0 (25 Km value for creatine: about 46 mM Molecular weight: about 50,000 (SDS-PAGE) 47,146 (calculated from amino acid composition) about 78,000 (gel filtration) Isoelectric point: about 4 .3

Example 7 Arthrobacter sp. TE1
826 (FERM P-10637) was cultured in 2XYT medium (1.6% polypeptone, 1% yeast extract, 0.5% sodium chloride, pH 7.2) at 37 ° C. for about 1 to 3 days, and the culture was centrifuged. And then lysing this to produce creatine amidinohydrolase,
The creatine amidinohydrolase was collected.

[0048]

Industrial Applicability According to the present invention, a novel creatine amidinohydrolase gene is isolated from a bacterium belonging to the genus Arthrobacter, a method for producing the enzyme by genetic engineering techniques has been established, and a large-scale supply of the enzyme with high purity and creatinine. Can be used for quantitative determination of Further, a method for producing a novel creatine amidinohydrolase having improved physicochemical properties can be derived by modifying the gene.

[0049]

[Sequence list] SEQ ID NO: 1 Sequence length: 411 Sequence type: Amino acid Topology: Linear Sequence type: Protein Origin Organism name: Arthrobacter sp.
p.) Strain name: TE1826 (FERM P-10637) Sequence Met Gln Lys Ile Thr Asp Leu Glu Arg Thr Lys Val Leu His Asn Gly 1 5 10 15 Glu Lys Phe Lys Gly Thr Phe Ser Lys Ala Glu Met Asp Arg Arg Asn 20 25 30 Thr Asn Leu Arg Asn Tyr Met Ala Glu Lys Asp Ile Asp Ala Val Leu 35 40 45 Phe Thr Ser Tyr His Asn Ile Asn Tyr Tyr Ser Asp Phe Leu Tyr Thr 50 55 60 Ser Phe Asn Arg Asn Tyr Gly Leu Val Val Thr Gln Asn Lys His Val 65 70 75 80 Thr Val Ser Ala Asn Ile Asp Gly Gly Met Pro Trp Arg Arg Ser Tyr 85 90 95 Asp Glu Asn Ile Val Tyr Thr Asp Trp Arg Arg Asp Asn Tyr Phe Tyr 100 105 110 Ala Ile Gln Lys Val Leu Glu Glu Ala Gly Val Lys Lys Ala Arg Leu 115 120 125 Gly Ile Glu Glu Asp His Val Ser Ile Asp Leu Leu Arg Lys Phe Ser 130 135 140 Asp Thr Phe Pro Asn Phe Glu Leu Val His Val Ser Gln Asp Val Met 145 150 155 160 Lys Gln Arg Met Ile Lys Ser Ala Glu Glu Ile Arg His Ile Lys Asn 165 170 175 Gly Ala Arg Ile Ala Asp Ile Gly Gly Tyr Ala Val Val Glu Ala Ile 180 185 190 Gln Glu Gly Val Pro Glu Tyr Glu Val Ala Leu Ala Gly S er Lys Ala 195 200 205 Met Thr Arg Glu Ile Ala Lys Leu Tyr Pro Gln Ser Glu Leu Arg Asp 210 215 220 Thr Trp Val Trp Phe Gln Ala Gly Ile Asn Thr Asp Gly Ala His Ser 225 230 235 240 Trp Ala Thr Ser Lys Lys Val Gln Lys Gly Glu Ile Leu Ser Leu Asn 245 250 255 Thr Phe Pro Met Ile Ala Gly Tyr Tyr Thr Ala Leu Glu Arg Thr Leu 260 265 270 Phe Leu Glu Glu Val Ser Asp Ala His Leu Lys Tyr Trp Glu Ile Asn 275 280 285 Val Glu Val His Lys Arg Gly Leu Glu Leu Ile Lys Pro Gly Ala Val 290 295 300 Cys Lys Asp Ile Cys Ala Glu Leu Asn Glu Met Phe Arg Glu His Asp 305 310 315 320 Leu Val Lys Asn Arg Thr Phe Gly Tyr Gly His Ser Phe Gly Val Leu 325 330 335 Ser His Tyr Tyr Gly Arg Glu Ala Gly Leu Glu Leu Arg Glu Asp Ile 340 345 350 Asp Thr Ile Leu Glu Pro Gly Met Val Ile Ser Met Glu Pro Met Ile 355 360 365 Leu Ile Pro Glu Gly Gln Pro Gly Ala Gly Gly Tyr Arg Glu His Asp 370 375 380 Ile Leu Val Ile Gln Glu Asn Gly Val Val Glu Asp Ile Thr Gly Phe 385 390 395 400 400 Pro Phe Gly Pro Glu Tyr Asn Ile Ile Lys Lys 405 410

SEQ ID NO: 2 Sequence length: 1236 Sequence type: nucleic acid Topology: linear Sequence type: genomic DNA Origin Organism name: Arthrobacter sp.
p.) Strain name: TE1826 (FERM P-10637) Sequence ATG CAA AAA ATC ACT GAT CTT GAA AGA ACA AAA GTT CTG CAC AAT GGC 48 Met Gln Lys Ile Thr Asp Leu Glu Arg Thr Lys Val Leu His Asn Gly 1 5 10 15 GAA AAA TTT AAA GGT ACT TTC TCA AAA GCG GAA ATG GAC CGC AGG AAT 96 Glu Lys Phe Lys Gly Thr Phe Ser Lys Ala Glu Met Asp Arg Arg Asn 20 25 30 ACG AAC CTG CGT AAT TAC ATG GCT GAA AAA GAT ATT GAC GCT GTC CTA 144 Thr Asn Leu Arg Asn Tyr Met Ala Glu Lys Asp Ile Asp Ala Val Leu 35 40 45 TTT ACT TCT TAC CAC AAT ATT AAC TAT TAC AGC GAT TTC TTA TAT ACA 192 Phe Thr Ser Tyr His Asn Ile Asn Tyr Tyr Ser Asp Phe Leu Tyr Thr 50 55 60 TCT TTT AAC AGG AAT TAT GGA TTG GTT GTT ACC CAG AAC AAA CAC GTA 240 Ser Phe Asn Arg Asn Tyr Gly Leu Val Val Thr Gln Asn Lys His Val 65 70 75 80 ACA GTT AGT GCA AAC ATA GAT GGC GGG ATG CCT TGG AGA AGA AGC TAC 288 Thr Val Ser Ala Asn Ile Asp Gly Gly Met Pro Trp Arg Arg Ser Tyr 85 90 95 GAT GAA AAT ATT GTA TAC ACC GAC TGG AGA AGA GAC AAC TAT TTC TAT 336 Asp Glu Asn Ile Val Tyr Thr Asp Trp Ar g Arg Asp Asn Tyr Phe Tyr 100 105 110 GCA ATT CAA AAA GTA CTA GAA GAA GCA GGA GTT AAG AAA GCC CGC TTA 384 Ala Ile Gln Lys Val Leu Glu Glu Ala Gly Val Lys Lys Ala Arg Leu 115 120 125 GGC ATT GAA GAG GAC CAT GTG TCC ATC GAT CTT CTG AGA AAA TTC 432 Gly Ile Glu Glu Asp His Val Ser Ile Asp Leu Leu Arg Lys Phe Ser 130 135 140 GAC ACA TTT CCT AAC TTT GAA TTG GTT CAT GTT TCT CAA GAT GTT ATG 480 Asp Thr Phe Pro Asn Phe Glu Leu Val His Val Ser Gln Asp Val Met 145 150 155 160 AAA CAG CGG ATG ATC AAA TCT GCT GAG GAA ATT AGG CAT ATA AAA AAT 528 Lys Gln Arg Met Ile Lys Ser Ala Glu Glu Ile Arg His Ile Lys Asn 165 170 175 GGG GCA AGG ATT GCT GAC ATT GGC GGC TAC GCA GTT GTT GAA GCT ATT 576 Gly Ala Arg Ile Ala Asp Ile Gly Gly Tyr Ala Val Val Glu Ala Ile 180 185 190 CAA GAA GGT GTT CCC GAA TAT GAA GTA GCA CCT GCC GGC TCC AAG GCA 624 Gln Glu Gly Val Pro Glu Tyr Glu Val Ala Leu Ala Gly Ser Lys Ala 195 200 205 ATG ACT CGT GAG ATT GCC AAG CTA TAT CCG CAA TCA GAG TTA AGA GAC 672 Met Thr Arg Glu Ile Ala Ly s Leu Tyr Pro Gln Ser Glu Leu Arg Asp 210 215 220 ACT TGG GTC TGG TTC CAG GCT GGT ATT AAT ACT GAT GGA GCT CAC AGC 720 Thr Trp Val Trp Phe Gln Ala Gly Ile Asn Thr Asp Gly Ala His Ser 225 230 235 240 TGG GCA ACC TCC AAA AAA GTA CAA AAA GGT GAA ATT CTA AGC CTC AAC 768 Trp Ala Thr Ser Lys Lys Val Gln Lys Gly Glu Ile Leu Ser Leu Asn 245 250 255 ACA TTC CCG ATG ATT GCG GGT TAC TAC ACA GCG CTG GAA CGA ACT TTG 816 Thr Phe Pro Met Ile Ala Gly Tyr Tyr Thr Ala Leu Glu Arg Thr Leu 260 265 270 TTC TTA GAA GAA GTT TCT GAT GCC CAT CTA AAA TAT TGG GAA ATA AAC 864 Phe Leu Glu Glu Val Ser Asp Ala His Leu Lys Tyr Trp Glu Ile Asn 275 280 285 GTG GAA GTT CAC AAA CGC GGT CTT GAA TTA ATT AAG CCC GGT GCA GTA 912 Val Glu Val His Lys Arg Gly Leu Glu Leu Ile Lys Pro Gly Ala Val 290 295 300 TGT AAG GAT ATC TGT GCT GAG TTA AAT GAA ATG TTC CGT GAG CAT GAC 960 Cys Lys Asp Ile Cys Ala Glu Leu Asn Glu Met Phe Arg Glu His Asp 305 310 315 320 CTG GTT AAA AAC CGG ACG TTT GGT TAT GGC CAT TCA TTC GGA GTT CTT 1008 00u Val L ys Asn Arg Thr Phe Gly Tyr Gly His Ser Phe Gly Val Leu 325 330 335 TCC CAC TAC TAT GGC CGT GAA GCC GGG CTT GAG CTT CGT GAA GAT ATC 1056 Ser His Tyr Tyr Gly Arg Glu Ala Gly Leu Glu Leu Arg Glu Asp Ile 340 345 350 GAC ACC ATT CTC GAG CCA GGT ATG GTC ATT TCA ATG GAA CCG ATG ATC 1104 Asp Thr Ile Leu Glu Pro Gly Met Val Ile Ser Met Glu Pro Met Ile 355 360 365 TTG ATT CCT GAA GGA CAA CCG GGA GCC GGC GGA TAC CGC GAG CAT GAT 1152 Leu Ile Pro Glu Gly Gln Pro Gly Ala Gly Gly Tyr Arg Glu His Asp 370 375 380 ATC TTA GTG ATA CAA GAA AAT GGT GTA GTT GAA GAT ATT ACT GGC TTC 1200 Ile Leu Val Ile Gln Glu Asn Gly Val Val Glu Asp Ile Thr Gly Phe 385 390 395 400 CCA TTT GGC CCT GAA TAT AAT ATT ATC AAA AAG TAA 1236 Pro Phe Gly Pro Glu Tyr Asn Ile Ile Lys Lys 405 410

SEQ ID NO: 3 Sequence length: 49 Sequence type: number of nucleic acid chains: single strand Topology: linear Sequence type: synthetic DNA sequence AGGAA GGCAG GAGAT TAAGG ATGCA AAAAA TCACT GATC 49

SEQ ID NO: 4 Sequence length: 39 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Synthetic DNA sequence AATGG TGAAT TACTT TTTGA TAATA TTATA TTCAG GGCC 39

[Brief description of the drawings]

FIG. 1 shows the amino acid sequence of the creatine amidinohydrolase of the present invention and Pseudomonas puida.
FIG. 4 shows a comparison with the amino acid sequence of creatine amidinohydrolase produced by Tida).

FIG. 2 shows a restriction map of pCNAA-1.

FIG. 3 shows a restriction map of pCRHRA-1.

Continued on the front page (51) Int.Cl. ⁶ Identification code FI C12R 1:06) (C12N 1/21 C12R 1:19) (C12N 9/80 C12R 1:06) (C12N 9/80 C12R 1:19)

Claims (10)

[Claims] 1. A novel creatine amidinohydrolase having the following physicochemical properties: Action: Acts on creatine in the presence of water to produce sarcosine and urea. Optimum temperature: about 40 ° C Optimum pH: about 7.0 to 8.5 Thermal stability: about 40 ° C or less (pH 7.5, treated for 30 minutes) pH stability: about 5.0 to 9.0 (25 Km value for creatine: about 46 mM Molecular weight: about 50,000 (SDS-PAGE) 47,146 (calculated from amino acid composition) about 78,000 (gel filtration) Isoelectric point: about 4 .3 2. Creatine amidinohydrolase, which is a protein of the following (a) or (b): (A) a protein consisting of the amino acid sequence of SEQ ID NO: 1 in the sequence listing; (B) a protein comprising an amino acid sequence in which one or more amino acids have been deleted, substituted or added in the amino acid sequence (a), and having creatine amidinohydrolase activity; 3. The creatine amidinohydrolase according to claim 1, which has the amino acid sequence of SEQ ID NO: 1 in the sequence listing. 4. A gene encoding creatine amidinohydrolase which is the following protein (a) or (b): (A) a protein consisting of the amino acid sequence of SEQ ID NO: 1 in the sequence listing; (B) a protein comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence (a), and having creatine amidinohydrolase activity 5. A gene encoding creatine amidinohydrolase, which is a protein consisting of the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing. 6. The following D of (c), (d) or (e):
A gene encoding creatine amidinohydrolase consisting of NA. (C) From the base sequence described in SEQ ID NO: 2 in the sequence listing, D
NA (d) DNA encoding the amino acid sequence having creatine amidinohydrolase activity, wherein one or more bases are added, deleted or substituted in the base sequence of (c), and (e) Bacterial DNA which hybridizes with the DNA consisting of the nucleotide sequence of c) under stringent conditions and encodes a protein having creatine amidinohydrolase activity 7. A recombinant vector containing a gene encoding creatine amidinohydrolase according to claim 4, 5 or 6. 8. A transformant obtained by transforming a host cell with the recombinant vector described in claim 7. 9. A method for producing creatine amidinohydrolase, which comprises culturing the transformant according to claim 8, generating creatine amidinohydrolase, and collecting the creatine amidinohydrolase. 10. An arthrob sp.
acter sp.) TE1826 (FERM P-10637)
A creatine amidinohydrolase, and producing the creatine amidinohydrolase, and collecting the creatine amidinohydrolase.