JPH09154574A - New creatinine amidohydrolase - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a higher-purity creatinine amidohydrolase as compared with a conventional enzyme. SOLUTION: This creatinine amidohydrolase is obtained from Alcaligenes faecalis TE3581 (FERM P-14237) and has the following physicochemical properties: A gene is capable of coding the enzyme and a recombinant vector contains the gene. Furthermore, a transformant is transformed with the recombinant vector. The creatinine amidohydrolase is obtained by culturing the transformant in a culture medium, producing the creatinine amidohydrolase and collecting the produced creatinine amidohydrolase. optimum temperature: 60 deg.C; optimum pH: 7.5-8.5; thermal stability: about <=50 deg.C (at pH7.5 for 30min); pH stability: about 8.0-10.0 (at 4 deg.C for 16hr); molecular weight: about 31,500 (SDS-PAGE) and about 120,000 (gel filtration) and isoelectric point: about 4.8.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel enzyme having creatinine amide hydrolase activity which can be used for quantifying creatinine, a gene encoding the enzyme and a method for producing the enzyme by a genetic engineering technique.

[0002]

2. Description of the Related Art Conventionally, creatinine amide hydrolase (EC 3.5.2.10) has been used as an enzyme for measuring creatinine in body fluids, which is an index for diagnosing muscle diseases and renal diseases.
For example, it is used in combination with creatine amidinohydrolase, sarcosine oxidase, and peroxidase. That is, creatinine amide hydrolase is allowed to act on creatinine in a sample, creatine amidinohydrolase is allowed to act on creatine to be produced, and sarcosine oxidase is allowed to act on sarcosine to be produced, and the produced hydrogen peroxide is converted to peroxidase and a coloring agent ( For example, a color is developed with 4-aminoantipyrine and an aniline derivative, and the intensity of the developed color is measured by absorbance to measure the amount of creatinine in the sample.

Creatinine amidohydrolase is an enzyme that catalyzes a reversible reaction that acts on creatinine to produce creatine. Such creatinine amidohydrolase is a Pseudomonas genus (Journal of Biochem).
istry, Vol.86, 1109-1117 (1979)) or Alcaligenes (Chemical and Pharmaceutical Bulletin, Vol.
34, No. 1, 269-274 (1986)) and other bacteria are known to be produced. Of these, Pseudomonas putida (Ps
The gene encoding creatinine amide hydrolase produced by eudomonas putida) PS-7 has already been isolated and its amino acid sequence has been published (Japanese Patent Laid-Open No. 3-19690).
However, Pseudomonas putid
a) The production level of PS-7 creatinine amide hydrolase by genetic engineering technology is 15.7 U / ml according to the description in JP-A-3-19690, and higher productivity is desired. On the other hand, Pseudomonas Petida (Ps
Genetic information of creatinine amidohydrolase produced by microorganisms other than eudomonas putida) PS-7 and the primary structure of the enzyme have not yet been obtained.

[0004]

DISCLOSURE OF THE INVENTION The object of the present invention is to provide a creatinine amide hydrolase having a higher purity than conventional enzymes. The method for producing the enzyme will be established, and it will be possible to supply a large amount of the enzyme with high purity and use it for quantification of creatinine.

[0005]

Means for Solving the Problems As a result of various studies to achieve the above object, the present inventors have found that as a creatinine amide hydrolase-producing bacterium, Alcaligenes faecalis TE3581 (FERM P-1).
4237) was selected, and a gene encoding creatinine amide hydrolase was successfully isolated from the chromosomal DNA extracted from the cells, and a novel creatinine amide hydrolase expressed by the gene was isolated. Furthermore, the whole nucleotide sequence of the gene was determined, and the enzyme was successfully produced by a genetic engineering technique without the need to add an expensive inducer such as creatinine to the medium. It is possible to supply a large amount to.

That is, the present invention is a creatinine aminohydrolase having the following physicochemical properties.

Embedded image Optimal temperature: 60 ° C Optimal pH: 7.5-8.5 Thermal stability: Approximately 50 ° C or less (pH 7.5, 30 minutes) pH stability: Approximately 8.0-10.0 (4 ° C, 16 Time) Molecular weight: about 31,500 (SDS-PAGE) about 120,000 (gel filtration) Isoelectric point: about 4.8

The present invention also relates to the amino acid sequence set forth in SEQ ID NO: 1 in the sequence listing or the amino acid sequence
Alternatively, a gene encoding creatinine amide hydrolase characterized in that it has a plurality of amino acids added, deleted or substituted and contains a nucleotide sequence encoding a polypeptide which brings about the enzyme activity of creatinine amide hydrolase. . Preferred is a gene encoding creatinine amidohydrolase containing a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 1 in the sequence listing.

The present invention also provides the nucleotide sequence shown in SEQ ID NO: 2 of the Sequence Listing or one or more nucleotides added, deleted or substituted in the nucleotide sequence and brings about the enzyme activity of creatinine amide hydrolase. It is a gene encoding creatinine amidohydrolase characterized by a base sequence encoding a polypeptide. The gene encoding creatinine aminohydrolase of the present invention is preferably a gene containing the nucleotide sequence shown in SEQ ID NO: 2 in the sequence listing.

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

Further, the present invention is a transformant transformed with the above recombinant vector.

The present invention comprises culturing the above transformant in a medium,
A method for producing creatinine aminohydrolase, which comprises producing creatinine aminohydrolase and collecting the creatinine amide hydrolase.

Alcaligenes faecalis (Alca
ligenes faecalis) TE3581 (FERM P-14
It is also possible to culture 237) in a medium to produce creatinine amidohydrolase and collect the creatinine amidohydrolase.

[0013]

BEST MODE FOR CARRYING OUT THE INVENTION The creatinine amide hydrolase of the present invention is a creatinine amide hydrolase-producing microorganism such as Alcaligenes faecalis.
faecalis) TE3581 (FERM P-14237), but preferably, the gene encoding creatinine amide hydrolase is isolated from these microorganisms, thereby transforming other host cells to express creatinine amide hydrolase. Then, the creatinine amide hydrolase of the present invention is obtained simply and efficiently by isolation.

The gene encoding the creatinine amide hydrolase of the present invention is, for example, Alcaligenes faecalis TE3581 (FERM P-1.
4237) It may be extracted from bacterial cells or may be chemically synthesized. Further, it is also possible to obtain a DNA fragment containing the creatinine amide hydrolase gene by using the polymerase chain reaction method (PCR).

As the gene encoding creatinine amidohydrolase, for example, the amino acid sequence set forth in SEQ ID NO: 1 in the Sequence Listing or one or more amino acids in the amino acid sequence added, deleted or substituted, and The base sequence encoding a polypeptide that provides the enzyme activity of creatinine aminohydrolase can be mentioned. In addition, it encodes a base sequence shown in SEQ ID NO: 2 of the Sequence Listing or a polypeptide having one or more bases added, deleted or substituted in the base sequence and which brings about the enzyme activity of creatinine amide hydrolase. A gene encoding creatinine amide hydrolase, which has a nucleotide sequence of

As a method for obtaining the gene of the present invention, for example, Alcaligenes faecali
s) Isolation and purification of the chromosomal DNA of TE3581 (FERM P-14237), followed by sonication, restriction enzyme treatment, etc., and a linear expression vector to form a blunt end or adhesive of both DNAs. A recombinant vector is constructed by ligating and closing the ends with DNA ligase or the like. The recombinant vector thus obtained is transferred to a replicable host microorganism and then screened using the expression of the marker and enzyme activity of the vector as an index, and a recombinant vector containing a gene encoding creatinine amide hydrolase. To obtain a microorganism that retains.
Next, the microorganism is cultured, the recombinant vector is separated and purified from the cultured cells, and the creatinine amidohydrolase gene may be collected from the recombinant vector.

The gene donor Alcaligenes faecalis TE3581 (FERM P
Specifically, the chromosomal DNA of (14237)) is collected as follows. That is, the donor microorganism is, for example, 1 to 3
The culture obtained by culturing with stirring for one day is collected by centrifugation and then lysed to prepare a lysate containing the creatinine amide hydrolase gene. As a lysing method, for example, a treatment with a lysing enzyme such as lysozyme or β-glucanase is performed, and if necessary, a protease or other enzyme or a surfactant such as sodium lauryl sulfate (SDS) is used in combination, and further freeze-thawing or It may be combined with a physical crushing method such as French press treatment.

DNA from the lysate thus obtained
To separate and purify the product, follow the usual method. For example, deproteinization treatment by phenol treatment or protease treatment, ribonuclease treatment, alcohol precipitation treatment and the like can be appropriately combined. The method of cleaving the DNA separated and purified from the microorganism can be performed by, for example, ultrasonic treatment or restriction enzyme treatment. Type II restriction enzymes that act on specific nucleotide sequences are preferred.

Suitable vectors are those constructed for gene recombination from phages or plasmids capable of autonomous growth in host microorganisms. As the phage, for example, Escherichia coli
When i) is used as the host microorganism, Lambda-gt10, Lambd
a-gt11 etc. can be used. As a plasmid,
For example, when Escherichia coli is used as the host microorganism, pBR322, pUC19, pBluescrip
t, pLED-M1 (Journal of Fermentation and Bioenzin
eering, Vol.76, 265-269 (1993)) can be used.
A vector fragment can be obtained by cutting such a vector with the above-mentioned creatinine amide hydrolase gene donor, the restriction enzyme used for cutting the microbial DNA, but not necessarily the same as the restriction enzyme used for cutting the microbial DNA. It is not necessary to use the restriction enzyme of. Microbial DNA
The method for ligating the fragment with the vector DNA fragment may be a method using a known DNA ligase, for example, after annealing the cohesive ends of the microbial DNA fragment and the cohesive end of the vector fragment, use a suitable DNA ligase. A recombinant vector of a microbial DNA fragment and a vector DNA fragment is prepared. If necessary, after annealing, it can be transferred into a host microorganism and utilized in vivo DNA ligase to prepare a recombinant vector.

The host microorganism may be any one so long as the recombinant vector can stably and autonomously propagate and can express the trait of the foreign gene. Generally, Escherichia coli W3110, Escherichia coli C600, Escherichia. Collie HB101, Escherichia coli JM109, etc. can be used.

As a method for transferring the recombinant vector into the host microorganism, for example, when the host microorganism is Escherichia coli, the competent cell method by calcium treatment or the electroporation method can be used. The obtained transformant microorganism can stably produce a large amount of creatinine amide hydrolase by culturing in a nutrient medium. Selection as to whether or not the target recombinant vector is transferred to the host microorganism may be carried out by searching for a microorganism that simultaneously expresses a drug resistance marker of the vector carrying the target DNA and a microorganism that simultaneously expresses creatinine amidohydrolase activity, For example, a microorganism that grows in a selective medium based on a drug resistance marker and that produces creatinine amidohydrolase may be selected.

The nucleotide sequence of the creatinine amidohydrolase gene obtained by the above method is
ience, Vol. 214, 1205-1210 (1981)), and the amino acid sequence of creatinine amidohydrolase was deduced from the determined nucleotide sequence. The recombinant vector carrying the creatinine amide hydrolase gene once selected in this way can be easily taken out from the transformed microorganism and transferred to other microorganisms. In addition, it is also possible to easily recover the creatinine amidohydrolase gene DNA from a recombinant vector carrying the creatinine amidohydrolase gene by restriction enzyme or PCR, ligate it with other vector fragments, and transfer it to a host microorganism.

Regarding the culture form of the host microorganism which is a transformant, the culture conditions may be selected in consideration of the nutritional physiological properties of the host. Usually, in most cases liquid culture is carried out, but industrially aeration is carried out. It is advantageous to carry out agitation culture. 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, salts such as phosphates, carbonates, sulfates, magnesium, calcium, potassium, iron, manganese and zinc, specific amino acids, specific vitamins and the like are used as necessary.

The culturing temperature can be appropriately changed within the range where the bacterium grows and produces creatinine amide hydrolase, but in the case of Escherichia coli, it is preferably about 20 to 42 ° C. Although the culturing time varies slightly depending on the conditions, it is sufficient to finish the culturing at an appropriate time in consideration of the time when the maximum yield of creatinine amidohydrolase is reached, and it is usually about 6 to 48 hours. The pH of the medium can be appropriately changed within the range in which the bacterium grows and produces creatinine amide hydrolase, but the pH is preferably about 6.0 to 9.0.

The culture solution containing the creatinine amide hydrolase-producing cells in the culture can be directly collected and used, but generally, when creatinine amide hydrolase is present in the culture solution according to a conventional method,
The creatinine amide hydrolase-containing solution is separated from the microbial cells by filtration, centrifugation, etc. before use. If creatinine amidohydrolase is present in the cells, collect the cells from the resulting culture by means such as filtration or centrifugation, and then destroy the cells by a mechanical method or an enzymatic method such as lysozyme. The creatinine amidohydrolase is solubilized by adding a chelating agent such as EDTA and / or a surfactant, if necessary, and separated and collected as an aqueous solution.

The creatinine amide hydrolase-containing solution thus obtained is subjected to, for example, vacuum concentration, 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. Further, heat treatment and isoelectric point treatment are also effective refining means. Then, a purified creatinine aminohydrolase 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 (Se
phadex) G-25 (Pharmacia Biotech), gel filtration, DEAE Sepharose CL-6B (Pharmacia Biotech), octyl Sepharose CL6
A purified enzyme preparation can be obtained by separating and purifying by -B (Pharmacia Biotech) column chromatography. This purified enzyme preparation has been purified to the extent that it shows an almost single band by SDS-PAGE.

An example of the creatinine amide hydrolase of the present invention has the following physicochemical properties.

Embedded image Optimal temperature: 60 ° C Optimal pH: 7.5-8.5 Thermal stability: Approximately 50 ° C or less (pH 7.5, 30 minutes) pH stability: Approximately 8.0-10.0 (4 ° C, 16 Time) Km value for creatinine: about 42 mM Molecular weight: about 31,500 (SDS-PAGE) about 120,000 (gel filtration) Isoelectric point: about 4.8

A comparison of the properties of the creatinine amide hydrolase of the present invention and known creatinine amide hydrolase is shown in Table 1 below. From this Table 1, the enzyme of the present invention differs from the conventional enzyme in the molecular weight, the number of subunits, and the Km value for creatinine.

The homology between the amino acid sequence of the creatinine amide hydrolase of the present invention and the known amino acid sequence of creatinine aminohydrolase produced by Pseudomonas putida PS-7 is 6
It is 5.5%, and a comparison between the two is shown in FIG. Therefore, the creatinine amide hydrolase of the present invention is a novel enzyme having different properties from the known enzyme that catalyzes the same reaction. The information on the amino acid sequence of creatinine amide hydrolase of the present invention facilitates the improvement of the enzymatic properties of the enzyme by substitution, insertion or deletion at the amino acid level.

[0030]

[Table 1]

[0031]

The present invention will be described below in more detail with reference to examples. In the examples, the creatinine amide hydrolase activity was measured using the following reagents under the following measurement conditions. Reagent Reagent A: Diacolor CRE Creatinine assay reagent (manufactured by Toyobo) A solution (enzyme reagent A vial containing 90 ml of solution A dissolved) Reagent B: 400 mM creatinine solution Reagent C: 1.5% phenol Aqueous solution Reagent D: 0.5% 4-aminoantipyrine aqueous solution measurement conditions First, reagent A, reagent B, reagent C, and reagent D were 3: 1: 0.0.
Mix at a ratio of 4: 0.04 (volume ratio) to prepare a reagent mixture. After preliminarily heating 3 ml of this reagent mixture at 37 ° C for about 5 minutes, 0.1 ml of enzyme solution was added, the reaction was started at 37 ° C, and after reacting for 4 minutes, the absorbance change per minute at 500 nm was measured. Measure 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 similarly measured. One unit (U) is defined as the amount of enzyme that decomposes 1 micromol of creatinine per minute under the above conditions.

Example 1 Isolation of chromosomal DNA Alcaligenes faecalis TE3581 (FERM P-1423
The chromosomal DNA of 7) was isolated by the following method. 15 strains
After culturing with shaking in 0 ml of a normal broth medium at 30 ° C. overnight, the cells were collected by centrifugation (8,000 rpm, 10 minutes).
10% sucrose, 50 mM Tris-HCl (pH 8.0)
Suspended in 5 ml of a solution containing 50 mM EDTA,
Add 1 ml of lysozyme solution (10 mg / ml),
Incubate for 15 minutes, then add 1 ml of 10% SDS solution. To this solution, an equal amount of chloroform / phenol solution (1: 1) was added, mixed with stirring, and centrifuged at 10,000 rpm for 3 minutes to separate into an aqueous layer and a solvent layer, and the aqueous layer was separated. The aqueous layer was gently overlaid with 2 volumes of ethanol, and the DNA was wound around the glass rod while gently stirring with a glass rod to separate the DNA. 1 with 1 mM EDTA
It was dissolved in a 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, and twice the amount of ethanol was added to separate the DNA again by the above method, and dissolved with 2 ml of TE.

Example 2 DNA fragment containing a gene encoding creatinine aminohydrolase and D
Preparation of Recombinant Vector Having NA Fragment 20 μg of the DNA obtained in Example 1 was partially digested with a restriction enzyme Sau3AI (manufactured by Toyobo), and a fragment of 2 to 10 kbp was recovered by a sucrose density gradient centrifugation method. On the other hand, pBluescriptKS digested with restriction enzyme BamHI (Toyobo)
After dephosphorylating (+) with bacterial alkaline phosphatase (Toyobo), both DNAs were treated with T4DN.
One unit of A ligase (manufactured by Toyobo) was reacted at 16 ° C for 12 hours to ligate the DNA. The ligated DNA was transformed using Escherichia coli JM109 competent cells (manufactured by Toyobo) and agar medium for detecting creatinine amide hydrolase activity [1.0% polypeptone, 0.5% yeast extract, 0.5% NaCl, 1% creatinine, 200U]. / ml Creatine amidinohydrolase (Toyobo), 10U / ml sarcosine oxidase (Toyobo), 5U / ml peroxidase (Toyobo), 0.01% o-dianisidine, 50μg / ml
Ampicillin, 1.5% agar]. The creatinine amide hydrolase activity was detected by growing in the above medium,
The colony stained brown was used as an index.

Approximately 100,000 transformant colonies were obtained per 1 μg of the DNA used, and as a result of the above-mentioned screening, one colony that was stained brown was found.
This strain was cultured in LB liquid medium (1% polypeptone, 0.5% yeast extract, 0.5% NaCl, 50 μg / ml ampicillin), and the creatinine amide hydrolase activity was measured. The activity was detected. The plasmid possessed by this strain has an inserted DNA fragment of approximately 5 kbp,
This plasmid was designated as pCNHA17. Then pCN
The inserted DNA of HA17 was cut out with restriction enzymes SacII and PstI (Toyobo) and cleaved with the same restriction enzymes pBluescript
Ligation into KS (+) created pCNHA-1. A restriction enzyme map of these pCNHA17 and pCNHA-1 is shown in FIG.

Example 3 Determination of Nucleotide Sequence Subclones of the approximately 1.2 kbp inserted DNA fragment of pCNHA-1 were prepared with various restriction enzymes. Various subclones can be obtained by Radioactive Sequencin
The nucleotide sequence was determined using g Kit (manufactured by Toyobo). The determined nucleotide sequence and amino acid sequence are shown in the sequence listing. The molecular weight of the protein determined from the amino acid sequence is 28,744.
And almost coincided with the molecular weight of creatinine aminohydrolase of Alcaligenes faecalis TE3581.

Example 4 Recombinant vector pCNHA-1
Preparation of 1 In order to remove the portion other than the creatinine aminohydrolase gene from the inserted DNA fragment of pCNHA-1, the inserted DNA fragment was miniaturized by PCR. PCR was performed under the following reaction solution composition and amplification conditions. Reaction liquid
Composition Pfu DNA polymerase (manufactured by Stratagene) 5U / 100 µl 10-fold concentration buffer for Pfu DNA polymerase 10 µl / 100 µl pCNHA-1 (template DNA) 0.1 µg / 100 µl dATP, dTTP, dGTP, dCTP 0.2 mM each 1 µM each Amplification conditions Denaturation 94 ° C, 30 seconds Temperature change 1 minute, 30 seconds Annealing 45 ° C, 1 second Temperature change 1 second Reaction 75 ° C, 30 seconds Temperature change 1 second (described above) Repeat 30 times)

The amplified DNA fragment, about 0.8 kbp, was treated with restriction enzymes NdeI and BamHI (Toyobo Co., Ltd.) and ligated to pLED-M1 cut with the same restriction enzymes to obtain pCNHA-1.
1 was created. Figure 2 shows a restriction map of pCNHA-11.
Shown in The inserted DNA fragment of pCNHA-11 does not contain a portion other than the creatinine amide hydrolase gene. That is, NdeI and BamH were added to both ends of the inserted DNA fragment.
Each I restriction enzyme cleavage site exists, NdeI overlaps with the start codon (ATG) of the creatinine amide hydrolase gene, and BamHI is located immediately after the stop codon (TGA) of the creatinine amide hydrolase gene.

Example 5 Preparation of Transformant Escherichia coli JM109 competent cells (manufactured by Toyobo) were transformed with pCNHA-11 to obtain a transformant Escherichia coli JM109 (pCNHA-11).

Example 6 Escherichia coli JM109 (pC
Production of creatinine aminohydrolase from NHA-11) 500 ml of LB medium was dispensed into a 2 L flask, and 121
The mixture was autoclaved at 15 ° C for 15 minutes, allowed to cool, and then 0.5 ml of 50 mg / ml ampicillin (manufactured by Nacalai Tesque), which had been separately sterilized and filtered, was added. This medium was inoculated beforehand with 5 ml of a culture solution of Escherichia coli JM109 (pCNHA-11), which had been shake-cultured in LB medium at 30 ° C. for 7 hours, and cultured with aeration and stirring at 37 ° C. for 18 hours. The creatinine amide hydrolase activity at the end of the culture was about 50 U / ml. The above-mentioned bacterial cells were collected by centrifugation to obtain 50 mM phosphate buffer,
Suspended in pH 7.8. The cell suspension was crushed by a French press and centrifuged to obtain a supernatant. The resulting crude enzyme solution was subjected to nucleic acid removal treatment with polyethyleneimine and ammonium sulfate fractionation treatment, desalted by gel filtration with Sephadex G-25 (Pharmacia Biotech), and then DEAE Sepharose CL-6B (Pharmacia Biotech). Separation and purification were performed by column chromatography and octyl sepharose CL6-B (Pharmacia Biotech) column chromatography to obtain a purified enzyme table product. The creatinine aminohydrolase preparation obtained by the method is SD
S-PAGE electrophoresis showed almost a single band, and the specific activity at this time was 83 U / mg-protein.

The properties of creatinine amide hydrolase obtained by the above method are shown below.

Embedded image Optimal temperature: 60 ° C Optimal pH: 7.5-8.5 Thermal stability: Approximately 50 ° C or less (pH 7.5, 30 minutes) pH stability: Approximately 8.0-10.0 (4 ° C, 16 Time) Km value for creatinine: about 42 mM Molecular weight: about 31,500 (SDS-PAGE) about 120,000 (gel filtration) Isoelectric point: about 4.8

[0041]

INDUSTRIAL APPLICABILITY The enzyme of the present invention is a novel enzyme because it has a difference in molecular weight, the number of subunits, and the Km value for creatinine as compared with the conventionally known creatinamide hydrolase. In addition, the productivity of creatinine amidohydrolase produced by the gene recombination technology using the conventional enzyme Pseudomonas putida PS-7 gene is 15.7 U / ml, whereas The productivity is 50 U / ml, and high-purity enzyme can be produced. The information on the amino acid sequence of the creatinine amide hydrolase of the present invention has the effect of facilitating the improvement of the enzymatic properties of the enzyme by substitution, insertion or deletion at the amino acid level.

[0042]

[Sequence list]

SEQ ID NO: 1 Sequence length: 258 Sequence type: Amino acid Topology: Linear Sequence type: Protein Origin organism name: Alcaligenes faculis
ecalis) Strain name: TE3581 (FERM P-14237) Sequence Met Arg Asp Thr Val Phe Ala Ala Glu Leu Thr Trp Pro Asp Tyr His 1 5 10 15 Ser Arg Val Gln Asp Gly Ser Val Pro Ile Leu Leu Pro Val Gly Ser 20 25 30 Met Glu Gln His Gly His His Met Pro Met His Val Asp Val Leu Leu 35 40 45 Pro Val Glu Phe Ala Arg Arg Val Ala Gly Glu Val Gly Ala Leu Val 50 55 60 Ala Pro Phe Thr Tyr Gly Tyr Lys Ser His Gln Lys Ser Gly Gly 65 70 75 80 Gly Asn His Leu Pro Gly Thr Thr Ser Leu Asp Gly Val Thr Leu Val 85 90 95 Ala Ala Leu Arg Asp Val Ile Lys Glu Phe Ala Arg His Gly Val Arg 100 105 110 Arg Met Cys Leu Val Asn Gly His Phe Glu Asn Ser Trp Phe Ile Ile 115 120 125 Glu Gly Ile Asp Leu Ala Leu Arg Glu Leu Arg Trp Asp Gly Ile Asp 130 135 140 Asn Met Lys Ile Val Val Leu Ser Tyr Trp Asp Phe Val Asp Lys Glu 145 150 155 160 Thr Ile Ala Arg Leu Tyr Pro Asn Gly Phe Thr Gly Trp Asp Leu Glu 165 170 175 His Gly Gly Val Leu Glu Thr Ser Leu Met Leu Ala Leu Tyr Pro His 180 185 190 Leu Val Ser Leu Asp Arg Ala Val His His Glu Pro Ala Ser Phe Pro 195 200 205 Pro Tyr Asp Val Tyr Pro Pro Lys Pro Glu Trp Thr Pro Ala Ser Gly 210 215 220 Thr Leu Ser Ser Pro Lys Glu Ala Ser Glu Glu Lys Gly Glu Ile Leu 225 230 235 240 Leu Glu Val Cys Val Asn Gly Ile Val Asn Ala Leu Lys Thr Glu Phe 245 250 255 Pro Arg

SEQ ID NO: 2 Sequence length: 777 Sequence type: Nucleic acid Topology: Linear Sequence type: Genomic DNA Origin organism name: Alcaligenes faculis
ecalis) Strain name: TE3581 (FERM P-14237) Sequence ATG CGC GAT ACA GTT TTT GCT GCC GAA TTG ACC TGG CCG GAC TAT CAC 48 Met Arg Asp Thr Val Phe Ala Ala Glu Leu Thr Trp Pro Asp Tyr His 1 5 10 15 AGC CGT GTC CAG GAC GGT TCG GTT CCG ATC CTG CTG CCG GTC GGA TCG 96 Ser Arg Val Gln Asp Gly Ser Val Pro Ile Leu Leu Pro Val Gly Ser 20 25 30 ATG GAG CAG CAC GGC CAT CAC ATG CCG ATG CAT GTC GAC GTC CTG CTG 144 Met Glu Gln His Gly His His Met Pro Met His Val Asp Val Leu Leu 35 40 45 CCG GTG GAA TTC GCG CGC CGT GTT GCC GGC GAG GTC GGC GCT CTC GTC 192 Pro Val Glu Phe Ala Arg Arg Val Ala Gly Glu Val Gly Ala Leu Val 50 55 60 GCG CCG CCC TTC ACC TAT GGC TAC AAA TCG CAC CAG AAG TCC GGC GGC 240 Ala Pro Pro Phe Thr Tyr Gly Tyr Lys Ser His Gln Lys Ser Gly Gly 65 70 75 80 GGC AAC CAC CTG CCC GGC ACC ACC AGC CTG GAT GGC GTC ACG CTG GTG 288 Gly Asn His Leu Pro Gly Thr Thr Ser Leu Asp Gly Val Thr Leu Val 85 90 95 GCG GCA CTG CGC GAC GTG ATC AAG GAA TTC GCC CGC CAC GGC GTG CGG 336 Ala Ala Leu Arg Asp Val Ile Lys G lu Phe Ala Arg His Gly Val Arg 100 105 110 CGC ATG TGC CTG GTC AAT GGC CAT TTC GAG AAC TCC TGG TTC ATC ATC 384 Arg Met Cys Leu Val Asn Gly His Phe Glu Asn Ser Trp Phe Ile Ile 115 120 125 GAG GGC ATC GAC CTG GCG CTG CGC GAG CTG CGC TGG GAC GGC ATC GAC 432 Glu Gly Ile Asp Leu Ala Leu Arg Glu Leu Arg Trp Asp Gly Ile Asp 130 135 140 AAC ATG AAG ATC GTG GTG CTG TCC TAT TGG GAC TTC GTG GAC AAG GAG 480 Asn Met Lys Ile Val Val Leu Ser Tyr Trp Asp Phe Val Asp Lys Glu 145 150 155 160 ACG ATC GCG CGC CTT TAT CCC AAT GGC TTC ACC GGC TGG GAC CTC GAA 528 Thr Ile Ala Arg Leu Tyr Pro Asn Gly Phe Thr Gly Trp Asp Leu Glu 165 170 175 CAT GGC GGC GTG CTG GAA ACC TCG CTG ATG CTG GCG CTC TAT CCG CAT 576 His Gly Gly Val Leu Glu Thr Ser Leu Met Leu Ala Leu Tyr Pro His 180 185 190 CTG GTA AGC CTC GAC CGC GCC GTC CAC CAC GAG CCC GCT TCC TTC CCG 624 Leu Val Ser Leu Asp Arg Ala Val His His Glu Pro Ala Ser Phe Pro 195 200 205 CCT TAT GAC GTC TAT CCG CCC AAG CCC GAA TGG ACG CCG GCC AGC GGC 672 Pro Tyr Asp Val Tyr P ro Pro Lys Pro Glu Trp Thr Pro Ala Ser Gly 210 215 220 ACG CTC TCC TCG CCC AAG GAA GCC TCC GAG GAG AAG GGC GAG ATC CTG 720 Thr Leu Ser Ser Pro Lys Glu Ala Ser Glu Glu Lys Gly Glu Ile Leu 225 230 235 240 CTG GAA GTC TGC GTC AAC GGC ATC GTC AAC GCG CTG AAG ACC GAA TTC 768 Leu Glu Val Cys Val Asn Gly Ile Val Asn Ala Leu Lys Thr Glu Phe 245 250 255 CCG CGC TGA 777 Pro Arg

SEQ ID NO: 3 Sequence Length: 49 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Synthetic DNA Sequence AACATCAAAA TGGGAGATGA TGAACATATG CGCGAGATACAG TTTTTCCTG 49

SEQ ID NO: 4 Sequence length: 49 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Synthetic DNA sequence TTTTTTGGAT CCTCAGCGCG GGAATTCGGT CTTCAGCGCG TTGACGATG 49

[Brief explanation of chart]

FIG. 1 shows the amino acid sequence of the creatinine amide hydrolase of the present invention and Pseudomonas puida.
tida) shows a comparison with the amino acid sequence of creatinine amide hydrolase produced by PS-7.

FIG. 2 pCNHA17, pCNHA-1, pCNHA
The restriction enzyme map of -11 is shown.

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Claims (10)

[Claims] 1. A novel creatinine amidohydrolase having the following physicochemical properties: Embedded image Optimum temperature: 60 ° C Optimum pH: 7.5-8.5 Thermal stability:
About 50 ° C or lower (pH 7.5, 30 minutes) pH stability: about 8.0 to 10.0 (4 ° C, 16 hours) Molecular weight: about 31,
500 (SDS-PAGE) about 120,000 (gel filtration) Isoelectric point: about 4.8 2. The creatinine amide hydrolase according to claim 1, which contains the amino acid sequence set forth in SEQ ID NO: 1 in the Sequence Listing. 3. Alcalige faecalis
nes faecalis) TE3581 (FERM P-1423)
The creatinine amide hydrolase according to claim 1, which is an enzyme produced by 7). 4. An amino acid sequence set forth in SEQ ID NO: 1 of the Sequence Listing, or a polypeptide having one or more amino acids added, deleted or substituted in the amino acid sequence and which brings about the enzyme activity of creatinine aminohydrolase. A gene encoding creatinine amidohydrolase, which contains a base sequence encoding the gene. 5. A gene encoding creatinine amide hydrolase, which comprises a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 1 in the Sequence Listing. 6. A base sequence set forth in SEQ ID NO: 2 of the Sequence Listing or a polypeptide which has one or more bases added, deleted or substituted in the base sequence and brings about the enzyme activity of creatinine amidohydrolase. A gene encoding creatinine amidohydrolase, which is characterized in that it has the encoding base sequence. 7. A gene encoding creatinine amidohydrolase, which comprises the nucleotide sequence set forth in SEQ ID NO: 2 in the Sequence Listing. 8. A recombinant vector containing the gene encoding the creatinine amide hydrolase according to claim 4, 5, 6 or 7. 9. A transformant obtained by transforming a host cell with a recombinant vector containing the gene encoding the creatinine amide hydrolase according to claim 4, 5, 6, or 7. 10. The host cell according to claim 4, 5, 6 or 7.
The creatinine amide hydrolase is produced by culturing a transformant transformed with a recombinant vector containing a gene encoding creatinine amide hydrolase described above in a medium, and collecting the creatinine amide hydrolase. Method for producing hydrolase.