JP3508871B2 - DNA having genetic information of protein having creatinine deiminase activity and method for producing creatinine deiminase - Google Patents

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a DNA fragment having the genetic information of a protein having creatinine deiminase activity, a recombinant vector having the DNA fragment, a transformant transformed with the recombinant vector, and the trait. It relates to a method for producing creatinine deiminase using a transformant.

[0002]

2. Description of the Related Art Creatinine deiminase (EC 3.5.4.2
1) is an enzyme that catalyzes the reaction of hydrolyzing creatinine into N-methylhydantoin and ammonia, and is used as a reagent for measuring creatinine in body fluids.
Sources of creatinine deiminase include Bacillus bacteria (JP-A-61-219383), Corynebacterium bacteria (JP-A-52-34976),
Flavobacterium (The Journal of Biologica
Chemistry Vol.260 No.7 p3915-3922, 1985) is known. However, the amount of creatinine deiminase produced by these bacteria was not sufficient. Further, in order to produce creatinine deiminase in these bacteria, it is necessary to add creatinine as an inducer to the medium. However, since creatinine is expensive, there has been a demand for an inexpensive production method that does not require creatinine when industrially producing creatinine deiminase.

By introducing a gene for an enzyme from a cell that requires an inducer into another cell to produce the enzyme by genetic engineering, it is possible to inexpensively produce the enzyme without the need for the inducer. , N-acetylneuraminate aldolase and other enzymes have been practiced. However, creatinine deiminase has an unknown amino acid sequence and gene, and has not been produced by genetic engineering until now.

[0004]

The object of the present invention is to isolate a DNA fragment having the genetic information of a protein having creatinine deiminase, to elucidate its molecular structure, and to identify a protein having a creatinine deiminase activity. The purpose is to provide a means that can be mass-produced in pure form at low cost by an engineering method.

[0005]

[Means for Solving the Problems] In order to achieve the above-mentioned object, the present inventors have found that a DNA having a genetic information of a protein having a creatinine deiminase activity is extracted from a chromosomal DNA extracted from cells of a creatinine deiminase-producing bacterium. The fragment was successfully isolated and the overall structure of its DNA was determined. Furthermore, the present creatinine deiminase was successfully produced by a transformant using a genetic engineering technique under high conditions without inducers, and it was possible to inexpensively supply a large amount of highly pure creatinine deiminase.

That is, the present invention is a DNA fragment having the genetic information of a protein having creatinine deiminase activity, and an example thereof is a DNA fragment encoding the amino acid shown in SEQ ID NO: 2 in the Sequence Listing, for example, described in SEQ ID NO: 1. One containing a base sequence to be described.

The present invention is also a recombinant vector having the above DNA fragment.

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

Further, the present invention is a method for producing creatinine deiminase, which comprises culturing the above transformant in a medium to produce creatinine deiminase and collecting the creatinine deiminase.

The creatinine deiminase-producing bacterium of the present invention is not particularly limited as long as it is a strain that produces creatinine deiminase, but in the examples of the present invention, Bacillus sp. CNI-1365 (Microtechnology Research Institute No. 8138) was used.

As a medium for culturing these bacteria, there is a medium containing a carbon source, a nitrogen source, an inorganic ion, and if necessary, a nitrate, a phosphate and the like. As the carbon source, starch or starch hydrolyzate, sugar condensate, peptones and the like are used. As the nitrogen source, polypeptone, tryptone, meat extract, yeast extract and the like can be used. It is desirable to appropriately adjust the pH and temperature of the culture medium under aerobic conditions.
The culture time is until the creatinine deiminase activity of the culture is maximized.

The following method is used to purify creatinine deiminase from the culture. The lysate containing creatinine deiminase is prepared by centrifuging the culture to collect the cells and then lysing the cells. As a lysis method, for example, a treatment with a cell wall lysing enzyme such as lysozyme or β-glucanase, or a physical disruption method such as ultrasonic disruption or dynomill disruption is suitable. The lysate thus obtained is subjected to ammonium sulfate precipitation fractionation, desalted, and then subjected to column chromatography fractionation on a carrier such as a cation exchange column, anion exchange column or hydroxylapatite column.

The DNA having the genetic information of the protein having creatinine deiminase activity of the present invention (hereinafter, also referred to as creatinine deiminase gene) may be extracted from creatinine deiminase-producing bacteria or may be synthesized. Examples of the base sequence include the base sequence encoding the amino acid sequence described in SEQ ID NO: 2 in the sequence listing or the base sequence described in SEQ ID NO: 1 in the sequence listing. The DNA fragment of the present invention may be modified by gene recombination technology without changing the basic characteristics of creatinine deiminase encoded by the DNA at a specific site of the basic DNA or improving the characteristics thereof. Artificially mutate to, for example, replace, delete,
It also includes those that have been inserted.

The DNA fragment of the present invention is obtained by, for example, isolating and purifying DNA of a bacterium of the genus Bacillus, and then cleaving the DNA using ultrasonic waves, restriction enzymes, etc. and a linear expression vector to smooth both DNAs. Alternatively, the cohesive ends are ligated and closed with a DNA ligase to give a recombinant vector. The recombinant vector thus obtained is transferred to a replicable host microorganism, and then screened using the vector marker and creatinine deiminase activity or hybridization with a probe specific for the creatinine deiminase gene as an index to perform the recombination. Obtain a microorganism carrying the vector. Culturing the microorganism,
The recombinant vector may be separated and purified from the cultured cells, and then the creatinine deiminase gene may be collected from the recombinant vector.

Next, the method for collecting DNA will be described in more detail. DNA derived from a microorganism that is a gene donor is collected as follows. That is, for example, the above-described bacterium as a donor microorganism is aerobically stirred and cultured in a liquid medium for about 1 to 3 days, the obtained culture is centrifuged to collect the cells, and then the cells are lysed to contain the creatinine deiminase gene. Lysates can be prepared. As a lysing method, for example, a treatment with a cell wall lysing enzyme such as lysozyme or β-glucanase is performed, and if necessary, another enzyme such as a protease or a surfactant such as sodium lauryl sulfate is used in combination, and a physical crushing method of the cell wall is further used. The freeze-thawing or French press treatment may be performed in combination with the above-mentioned lysis method.

In order to separate and purify DNA from the lysate thus obtained, a method such as deproteinization treatment by phenol extraction, protease treatment, ribonuclease treatment, alcohol precipitation centrifugation etc. is appropriately combined according to a conventional method. Can be done. As a method for cleaving the DNA separated and purified from the microorganism, for example, ultrasonic treatment or restriction enzyme treatment can be carried out. However, in order to facilitate the binding between the obtained microbial DNA fragment and the vector, the restriction enzyme is particularly specified. Type II restriction enzymes that act on the nucleotide sequence, such as EcoRI, HindIII, BamHI, are suitable.

Suitable vectors are those constructed for gene recombination from phages or plasmids capable of autonomously growing in host microorganisms. As the phage, for example, when Escherichia coli is used as the host microorganism, λgt · 10, λgt · 11 and the like can be used. As a plasmid, for example, when Escherichia coli is used as a host microorganism, pBluescr
You can use ipt, pUC18, etc.

It is desirable to obtain a vector fragment by cleaving such a vector with the same restriction enzyme as that used for cleaving the creatinine deiminase gene donor microbial DNA described above. The method of ligating the microbial DNA fragment with the vector fragment may be a method using a known DNA ligase. For example, after annealing the cohesive ends of the microbial DNA fragment and the vector fragment, an appropriate DNA ligase is used. A recombinant vector of a microbial DNA fragment and a vector fragment is prepared. If necessary, it is also possible to transfer to a host microorganism after annealing and utilize a DNA ligase in vivo to prepare a recombinant vector.

The host microorganism may be any one so long as the recombinant vector can be stably and autonomously propagated and can express the trait of foreign DNA. For example, the host microorganism can be Escherichia coli W3110, Escherichia coli C60.
0, Escherichia collie JM109, Escherichia collie HB101, Escherichia collie DH5 α, etc. can be used.

As a method of transferring the recombinant vector into the host microorganism, for example, when the host microorganism is a microorganism belonging to the genus Escherichia, a method of transferring the recombinant DNA in the presence of calcium ions can be adopted. You can
Further, an electroporation method may be used. The thus obtained transformant microorganism can stably produce a large amount of creatinine deiminase by culturing in a nutrient medium. The selection as to whether or not the target recombinant vector has been transferred into the host microorganism may be carried out by searching for a microorganism capable of simultaneously expressing the drug resistance marker of the vector carrying the target DNA and creatinine deiminase. A microorganism that grows in a selection medium based on the above and produces creatinine deiminase may be selected.

The base sequence of the creatinine deiminase gene obtained by the above method is Proc. Natl. Acad. Sc
i. USA, 74, 5463-5467 (1977), the amino acid sequence of creatinine deiminase was deduced by the dideoxy method and deduced from the nucleotide sequence. In this way, the recombinant vector carrying the creatinine deiminase gene once selected can be easily taken out from the transformed microorganism and transferred to another host microorganism. In addition, a recombinant vector containing the creatinine deiminase gene is cleaved with a restriction enzyme or the like to cut out a DNA containing the creatinine deiminase gene, and a vector fragment obtained by cleaving this by the same method is ligated, Transfer to host microorganisms is also easy.

The culture form of the transformant host microorganism may be selected by considering the culture conditions in consideration of the nutritional physiological properties of the host. In most cases, liquid culture is carried out, but industrially aeration stirring culture is carried out. Is advantageous. As the nutrient source of the medium, those usually used for culturing microorganisms can be widely used. The carbon source may be any assimilable carbon compound, for example, glucose, sucrose, lactose,
Maltose, fructose, molasses, pyruvic acid, etc. are used. Any available nitrogen compound may be used as the nitrogen source, and for example, peptone, meat extract, yeast extract, casein hydrolyzate, soybean meal alkali extract, etc. 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 culture temperature can be appropriately changed within the range in which the bacterium grows and produces creatinine deiminase, but in the case of Escherichia coli, it is preferably about 20 to 42 ° C.
Although the culturing time varies 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 deiminase is reached, and it is usually about 6 to 48 hours. The medium pH can be appropriately changed within a range in which the bacterium grows and produces creatinine deiminase, but particularly preferably pH 6.0.
It is about 9.0.

The culture solution containing the creatinine deiminase-producing cells in the culture can be collected and used as it is. Generally, when creatinine deiminase is present in the culture solution, filtration and centrifugation are carried out. It is used after separating the creatinine deiminase-containing solution and the microbial cells by separation or the like. When creatinine deiminase is present in the microbial cells, the obtained culture is harvested by means such as filtration or centrifugation, and then the microbial cells are destroyed by a mechanical method or an enzymatic method such as lysozyme. The creatinine deiminase 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 deiminase-containing solution thus obtained is concentrated under reduced pressure, membrane concentrated, salted out with ammonium sulfate, sodium sulfate or the like, or fractionally precipitated with a hydrophilic organic solvent such as methanol, ethanol or acetone. It may be precipitated by the method. Further, heat treatment and isoelectric point treatment are also effective refining means. Purified creatinine deiminase can be obtained by gel filtration using an adsorbent or a gel filtration agent, adsorption chromatography, ion exchange chromatography, and affinity chromatography.

[0026]

EXAMPLES The present invention will be specifically described below with reference to examples. In the examples, creatinine deiminase activity was measured as follows. That is, 49 mM phosphate buffer (p
H7.5), 38 mM creatinine, 0.29 mM NADPH, 0.95 mM
Incubate the enzyme in α-ketoglutarate, 16 U / ml glutamate dehydrogenase at 37 ℃ for 3-4 minutes. One molecule of NADPH is consumed when one molecule of ammonia generated by hydrolysis of creatinine and α-ketoglutarate are bound by the action of glutamate dehydrogenase to form glutamate. This N
The decrease in ADPH is measured by the decrease in absorbance at 340 nm per minute. One unit of enzyme activity was the amount of enzyme that produced 1 micromol of ammonia per minute under this condition.

Example 1 Determination of amino acid sequence of creatinine deiminase Creatinine deiminase powder (manufactured by Toyobo) was used as Superd.
Gel filtration chromatography on an ex200 column and Mono
Purified by ion exchange column chromatography using a -Q column. When the final purified sample was checked by SDS-PAGE, a nearly uniform band was obtained, and the molecular weight of the subunit was calculated to be about 43,000 daltons. This preparation was further purified by reverse phase HPLC using a Vydac C4 column also for desalting, and the amino acid sequence from the N terminus was determined by the manual Edman degradation method (SEQ ID NO: 3 in the sequence listing). In addition, the peptide mixture obtained by degrading the same sample with Asp-N, a protease specific for aspartic acid residues, was separated and purified by reverse-phase HPLC, and the amino acid sequence of the peptide fragment was determined by the manual Edman degradation method. (SEQ ID NO: 4 in the sequence listing).

Example 2 Isolation of chromosomal DNA The chromosomal DNA of Bacillus sp. CNI-1365 was isolated by the following method. The strain was added to 100 ml of LB medium (1.0%
Polypeptone, 0.5% yeast extract, 0.5% sodium chloride (p
After shaking culture at 37 ° C overnight in H7.2), the cells were collected by centrifugation (8000 rpm, 10 minutes). After washing the cells with a solution containing 15 mM sodium citrate and 0.15 M sodium chloride, the cells were suspended in 5 ml of a solution containing 20% sucrose, 1 mM EDTA and 50 mM Tris-HCl (pH 7.6), and 0.5 ml of Lysozyme solution (100 mg / m
l) was added and the mixture was kept warm at 37 ° C for 30 minutes. Then 11 ml of a solution containing 1% lauroyl sarcosinic acid, 0.1 M EDTA (pH 9.6) was added. To this suspension was added ethidium bromide solution 0.5% cesium chloride (about 100%), and the mixture was stirred and mixed at 55.000 rpm, 2
The DNA was collected by ultracentrifugation for 0 hours. Fractionated DNA
Is a solution containing 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (T
It was dialyzed with E) to obtain a purified DNA sample. Competent cells of Escherichia coli DH5α were prepared by the method of Hanahan and used as a host for library preparation.

Example 3 Preparation of DNA Fragment Containing Creatinine Deiminase Gene and Recombinant Vector Having the DNA Fragment Two types of probes (SEQ ID NO: 5 in the sequence listing, based on the amino acid sequence determined in Example 1) 6) was prepared, and the creatinine deiminase gene fragment was amplified by the PCR method using the chromosomal DNA obtained in Example 2 as a template to give about 1 kbp.
The amplified fragment of Next, Southern hybridization of the chromosomal DNA obtained in Example 2 was performed using the obtained fragment as a probe, and as a result, a specific band of 6 kbpKpnI was detected. Therefore, the DNA of this part was extracted and cleaved with restriction enzyme KpnI (manufactured by Toyobo) to produce pBluescript and T4-DN.
The DNA was ligated by reacting with A ligase (manufactured by Toyobo). The ligated DNA was transformed with Escherichia coli DH5α competent cells. The obtained colonies were screened by colony hybridization using a fragment amplified by the PCR method as a probe to obtain 6 kbp KpnI DNA containing the creatinine deiminase gene.
A recombinant vector containing the fragment was obtained and named pCD1.

Then, the inserted DNA fragment of pCD1 was digested with various restriction enzymes and subcloned into pBluescript to obtain a derivative plasmid having various inserted DNA fragments, and Escherichia coli DH5α was transformed. Insertion DNA restriction maps of pCD1 and its derivative plasmids are shown in FIG.

Example 4 Determination of nucleotide sequence Insertion DNA of pCD2 which is a derivative plasmid of pCD1
The fragment was cleaved with various restriction enzymes to prepare subclones. The various subclones are
The nucleotide sequence was determined using ASE VERSION2.0 7-deaza-dGTP kit (manufactured by Toyobo). The determined nucleotide sequence and amino acid sequence are shown in SEQ ID NO: 1 and SEQ ID NO: 2 in the sequence listing.

Example 5 Cultivation of transformants and production of creatinine deiminase (1) Creatinine deiminase production medium (0.5% yeast extract,
500 ml of 50 ml of 1.0% polypeptone, 0.5% NaCl (pH 7.5))
The mixture was dispensed into a flask, autoclaved at 121 ° C. for 15 minutes, allowed to cool, and then an aseptically filtered antibiotic (50 mg / ml ampicillin (manufactured by Nacalai Tesque)) was added. This medium was inoculated with 5 ml of a culture solution of the pCD2 recombinant which had been shake-cultured at 30 ° C. for 18 hours in a medium having the same composition as the above, and cultured at 30 ° C. with aeration and stirring. Twenty hours after the start of culture, the pCD2 recombinant showed a creatinine deiminase activity of about 0.15 U / ml without adding the inducer creatinine to the medium. Under the same conditions, Bacillus sp. CNI-
The creatinine deiminase activity of 1365 was below the detection limit. That is, it was revealed that the creatinine deiminase gene of the present invention was efficiently expressed in Escherichia coli without an inducer.

Example 6 Cultivation of transformant and production of creatinine deiminase (2) The pCD2 recombinant was cultured under the same conditions as in Example 5, except that the culture temperature was changed to 37 ° C. 2 from the start of culture
After 0 hours, the pCD2 recombinants showed a creatinine deiminase activity of about 1.7 U / ml without the addition of the inducer creatinine to the medium. That is, it was revealed that the productivity can be significantly improved by optimizing the culture temperature.

[0034]

According to the present invention, the nucleotide sequence and amino acid sequence of the creatinine deiminase gene have been clarified,
Creatinine deiminase can be easily mass-produced in high purity by genetic engineering techniques. In addition, creatinine, which is an inducer required for the production of creatinine deiminase, became unnecessary by using the production method of the present invention. Furthermore, by using the creatinine deiminase gene of the present invention and various protein engineering techniques, it becomes possible to produce a novel creatinine deiminase having higher activity or higher stability.

[0035]

[Sequence list]

SEQ ID NO: 1 Sequence length: 1591 Sequence type: nucleic acid (DNA) Number of chains: double-stranded Topology: linear Sequence type: genomicDNA origin Organism name: Bacillus sp. Strain name: CNI-1365 Array CTGCAGCTGT CCTCGGACCA GTTACCTCGA CCACCTCAAC CCCCGATGAA AACTATCGAG 60 GTTCTCGGAC AAGGCTAAGC GGAGGTCCCC GATGCAGAGG CAATGCTGAG AAACTATCTA 120 CGACAGCTCA CCATTTTTAT TTAACTCCAC ACTAATACCT GTCAAGCAAT AGAATAGATA 180 GCATCTGGAT CCTCCAGAGT TTGAAAATAT GCCTTGACAT GTAGAAATGG AGTTCTT 237 GTG CGC ATT ACA AAC GCC CAG GTT AAG AAC TAC GCA GAG TTA GTT GAT 285 Met Arg Ile Thr Asn Ala Gln Val Lys Asn Tyr Ala Glu Leu Val Asp   1 5 10 15 ATC ACC ATA GAG GGT GAA AGA ATC TCA ACT ATT ACC CCC TCC GCG CTT 333 Ile Thr Ile Glu Gly Glu Arg Ile Ser Thr Ile Thr Pro Ser Ala Leu              20 25 30 CGA CCA GAA GAA GAT CGC CGC GCG GAC GAT TAC GAT GCC GCA GGA AGA 381   Arg Pro Glu Glu Asp Arg Arg Ala Asp Asp Tyr Asp Ala Ala Gly Arg          35 40 45 CTG GTC TCA CCC CAG TTC GCC GAA GCA CAC ATC CAC CTT GAC TAC GCA 429   Leu Val Ser Pro Gln Phe Ala Glu Ala His Ile His Leu Asp Tyr Ala      50 55 60 AAC ACC GCG GGA GTA CCT CGC GAA AAT TCT TCT GGC ACA CTT TTT GAA 477 Asn Thr Ala Gly Val Pro Arg Glu Asn Ser Ser Gly Thr Leu Phe Glu  65 70 75 80 GCC ATC GAA ATC TGG GCC GAC CGC AAA ACC CAA GGC TTC CAC ATC AAA 525 Ala Ile Glu Ile Trp Ala Asp Arg Lys Thr Gln Gly Phe His Ile Lys                  85 90 95 GAG GAT ATT AAA GCG AAA GCT CTC CAG GCA GCC CGT CGG GCA GCA GAA 573 Glu Asp Ile Lys Ala Lys Ala Leu Gln Ala Ala Arg Arg Ala Ala Glu             100 105 110 CAC GGC GTT GGC TTC ATC CGC ACC CAT GTA GAC GTT ACA GAT CCC ACC 621 His Gly Val Gly Phe Ile Arg Thr His Val Asp Val Thr Asp Pro Thr         115 120 125 TTC GCC GGG TTT GAA GCA ATT GCG GAG CTG CGC GAT GAA GTC CGC GAG 669 Phe Ala Gly Phe Glu Ala Ile Ala Glu Leu Arg Asp Glu Val Arg Glu     130 135 140 TGG TGC GAT ATC CAG ATT GTC GCC TTC CCG CAA AAT GGC ATT TAC GCC 717 Trp Cys Asp Ile Gln Ile Val Ala Phe Pro Gln Asn Gly Ile Tyr Ala 145 150 155 160 TAC GAA GGT GGC CAG AAG CTA ATC TCA GAT GCA ATG TCT GCA GGT GCA 765 Tyr Glu Gly Gly Gln Lys Leu Ile Ser Asp Ala Met Ser Ala Gly Ala                 165 170 175 GAT GTC GTT GGT GGC ATC CCA CAC CTT GAA CCC ACC CGA GAT GAT GGT 813 Asp Val Val Gly Gly Ile Pro His Leu Glu Pro Thr Arg Asp Asp Gly             180 185 190 GTC GAG TCG GTG AAA TGG CTT TTC GAC CTT GCA GAG AAG CAC TCA GCC 861 Val Glu Ser Val Lys Trp Leu Phe Asp Leu Ala Glu Lys His Ser Ala         195 200 205 CCC ATC GAT ATC CAC ACC GAT GAA ATT GAC GAT CCA CAT TCC CGA TTT 909 Pro Ile Asp Ile His Thr Asp Glu Ile Asp Asp Pro His Ser Arg Phe     210 215 220 GTC GAA GTC CTC GCC GCA GAA GCC GCA AAA CGT GAC ATG GGC GCA CAA 957 Val Glu Val Leu Ala Ala Glu Ala Ala Lys Arg Asp Met Gly Ala Gln 225 230 235 240 ACC GTG GTG TCC CAT TCT GTT GCG ATG GCA TAT TAT TCA CCT GGC TAC 1005 Thr Val Val Ser His Ser Val Ala Met Ala Tyr Tyr Ser Pro Gly Tyr                 245 250 255 ATG GCG CGA CTT TTA CCC AAG CTC GCA GCA TCA AAG GTT CGT TTT GCA 1053 Met Ala Arg Leu Leu Pro Lys Leu Ala Ala Ser Lys Val Arg Phe Ala             260 265 270 GTA TGC CCC AAT GAA AAC CTC CAT CTG CAA GGA CTT GGT TTC CAA GGA 1101 Val Cys Pro Asn Glu Asn Leu His Leu Gln Gly Leu Gly Phe Gln Gly         275 280 285 CCC GTC CCC CGA GGT GTT GCA CCG GTA AAG CAA CTT ACC GAA TGG GGA 1149 Pro Val Pro Arg Gly Val Ala Pro Val Lys Gln Leu Thr Glu Trp Gly     290 295 300 ATT CCA GTA AGT TTT TGC CAG GAC TCA CTC AAT GAC CCC TTC TAC CCC 1197 Ile Pro Val Ser Phe Cys Gln Asp Ser Leu Asn Asp Pro Phe Tyr Pro 305 310 315 320 ATG GGC GAT GGA GAT CTA CTC CGC ATT CTC GAT TCT GGA TTA CAC GTG 1245 Met Gly Asp Gly Asp Leu Leu Arg Ile Leu Asp Ser Gly Leu His Val                 325 330 335 TCC CAC ATG CTC ACA GCC AGC CAC TTG AAG AAT GCA CTA TCG TTC ATC 1293 Ser His Met Leu Thr Ala Ser His Leu Lys Asn Ala Leu Ser Phe Ile             340 345 350 ACC ACC AAT CCA GCC GGA AAC CTA GGC CTG GAC AAT TAC GAC ATT GCA 1341 Thr Thr Asn Pro Ala Gly Asn Leu Gly Leu Asp Asn Tyr Asp Ile Ala         355 360 365 GAA AAC TCC CCG GCG AAC CTG CTG GTT CTT GAT GCG AGC AGC GAG AAG 1389 Glu Asn Ser Pro Ala Asn Leu Leu Val Leu Asp Ala Ser Ser Glu Lys     370 375 380 GAA GCT GTA CAA AGA AAA GCT TCC GTA CTT TGAGCATCCA CCGCGGCAAA 1439 Glu Ala Val Gln Arg Lys Ala Ser Val Leu 385 390 AAGGTGCTCT CCAGGGAGCC CGAACAGGTG GACTGGAACA TCTAACAGCC CAGTTGGGCC 1499 TCCTTAAATT TTGTGGCACT CCCCACATTT CTATCAATCT ATAGAAAGTA TGACTTAAGT 1559 CGATTTTGCA AGTTTCTATA GATTGATAGA AA 1591

SEQ ID NO: 2 Sequence length: 394 Sequence type: Amino acid Topology: linear Sequence Type: Protein origin Organism name: Bacillus sp. Strain name: CNI-1365 Array Val Arg Ile Thr Asn Ala Gln Val Lys Asn Tyr Ala Glu Leu Val Asp   1 5 10 15 Ile Thr Ile Glu Gly Glu Arg Ile Ser Thr Ile Thr Pro Ser Ala Leu              20 25 30 Arg Pro Glu Glu Asp Arg Arg Ala Asp Asp Tyr Asp Ala Ala Gly Arg          35 40 45 Leu Val Ser Pro Gln Phe Ala Glu Ala His Ile His Leu Asp Tyr Ala      50 55 60 Asn Thr Ala Gly Val Pro Arg Glu Asn Ser Ser Gly Thr Leu Phe Glu  65 70 75 80 Ala Ile Glu Ile Trp Ala Asp Arg Lys Thr Gln Gly Phe His Ile Lys                  85 90 95 Glu Asp Ile Lys Ala Lys Ala Leu Gln Ala Ala Arg Arg Ala Ala Glu             100 105 110 His Gly Val Gly Phe Ile Arg Thr His Val Asp Val Thr Asp Pro Thr         115 120 125 Phe Ala Gly Phe Glu Ala Ile Ala Glu Leu Arg Asp Glu Val Arg Glu     130 135 140 Trp Cys Asp Ile Gln Ile Val Ala Phe Pro Gln Asn Gly Ile Tyr Ala 145 150 155 160 Tyr Glu Gly Gly Gln Lys Leu Ile Ser Asp Ala Met Ser Ala Gly Ala                 165 170 175 Asp Val Val Gly Gly Ile Pro His Leu Glu Pro Thr Arg Asp Asp Gly             180 185 190 Val Glu Ser Val Lys Trp Leu Phe Asp Leu Ala Glu Lys His Ser Ala         195 200 205 Pro Ile Asp Ile His Thr Asp Glu Ile Asp Asp Pro His Ser Arg Phe     210 215 220 Val Glu Val Leu Ala Ala Glu Ala Ala Lys Arg Asp Met Gly Ala Gln 225 230 235 240 Thr Val Val Ser His Ser Val Ala Met Ala Tyr Tyr Ser Pro Gly Tyr                 245 250 255 Met Ala Arg Leu Leu Pro Lys Leu Ala Ala Ser Lys Val Arg Phe Ala             260 265 270 Val Cys Pro Asn Glu Asn Leu His Leu Gln Gly Leu Gly Phe Gln Gly         275 280 285 Pro Val Pro Arg Gly Val Ala Pro Val Lys Gln Leu Thr Glu Trp Gly     290 295 300 Ile Pro Val Ser Phe Cys Gln Asp Ser Leu Asn Asp Pro Phe Tyr Pro 305 310 315 320 Met Gly Asp Gly Asp Leu Leu Arg Ile Leu Asp Ser Gly Leu His Val                 325 330 335 Ser His Met Leu Thr Ala Ser His Leu Lys Asn Ala Leu Ser Phe Ile             340 345 350 Thr Thr Asn Pro Ala Gly Asn Leu Gly Leu Asp Asn Tyr Asp Ile Ala         355 360 365 Glu Asn Ser Pro Ala Asn Leu Leu Val Leu Asp Ala Ser Ser Glu Lys     370 375 380 Glu Ala Val Gln Arg Lys Ala Ser Val Leu 385 390

SEQ ID NO: 3 Sequence length: 9 Sequence type: Amino acid topology: Linear sequence type: Peptide origin Organism name: Bacillus sp. Strain name: CNI-1365

SEQ ID NO: 4 Sequence length: 6 Sequence type: Amino acid topology: Linear sequence type: Peptide origin Organism name: Bacillus sp. Strain name: CNI-1365 Sequence Sequence characteristics Other information Xaa is Leu or Met.

SEQ ID NO: 5 Sequence length: 20 Sequence type: nucleic acid (DNA) Number of chains: Single chain Homology: linear Sequence type: Synthetic DNA Array ATGCGIATIA CIAATGCICA 20

SEQ ID NO: 6 Sequence length: 17 Sequence type: nucleic acid (DNA) Number of chains: Single chain Topology: linear Sequence type: Synthetic DNA Array CTRGGIAAGA TGGGIGA 17

[Brief description of drawings]

FIG. 1 Inserted DNA of pCD1 and its derivative plasmids
The restriction enzyme map of is shown.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI (C12N 9/78 C12R 1:19) (C12N 15/09 ZNA C12R 1:07) (58) Fields investigated (Int.Cl. ⁷ , DB name) C12N 15/09 ZNA C12N 1/21 C12N 9/78 C12N 1/21 SwissProt / PIR / GeneS eq GenBank / EMBL / DDBJ / GeneSeq BIOSIS / MEDLINE / WPID S (STN)

Claims (3)

(57) [Claims] 1. A base sequence represented by SEQ ID NO : 1 in the sequence listing.
Amino acids listed in a row or in SEQ ID NO: 2 in the sequence listing
It has a nucleotide sequence encoding the sequence and
A DNA fragment containing the genetic information of a protein having an enzyme activity
Recombinant vector with fragments, belonging to the genus Escherichia
It is necessary to add creatinine during culturing after transforming a microorganism.
Traits that do not require creatinine deiminase
Convertible. 2. A method for producing creatinine deiminase, which comprises culturing the transformant according to claim 1 in a medium to produce creatinine deiminase and collecting the creatinine deiminase. 3. The method for producing creatinine deiminase according to claim 2 , which comprises culturing at a temperature of about 37 ° C.