JP3091202B2 - Method for producing acetylpolyamine amide hydrolase - Google Patents

Description

The present invention relates to a DNA belonging to the genus Escherichia and carrying genetic information involved in the production of acetylpolyamine amide hydrolase (hereinafter abbreviated as APH) (hereinafter referred to as APH gene DNA). This is abbreviated.) The present invention relates to a method for producing APH using a microorganism having a fragment.

APH is used, for example, as a clinical diagnostic enzyme for quantifying urinary polyamine, which is a tumor marker.

2. Description of the Related Art Methods for producing APH include Streptomyces genus (JP-A-56-144088) and Mycoplana genus (JP-A-6464).
A method using a microorganism belonging to, for example, -85080) is known.

Even if these microorganisms are cultured in a medium usually used for enzyme production, the production amount is extremely small, which is economically disadvantageous.

SUMMARY OF THE INVENTION An object of the present invention is to provide a method for producing APH inexpensively and in large quantities by using a microorganism.

Means for Solving the Problems According to the present invention, an APH gene DNA derived from a microorganism belonging to the genus Escherichia and belonging to the genus Mycoplana is used as a vector DN.
By culturing a microorganism having a recombinant DNA obtained by incorporating it into A in a medium, a method for producing APH at low cost and in large quantities can be provided.

 Hereinafter, the present invention will be described in detail.

APH gene DNA belongs to the genus Mycoplana and can be obtained from chromosomal DNA of a microorganism capable of producing APH. Specifically, Mycoplana bullata (FER)
Chromosomal DNA obtained from the MBP-1845 strain can be mentioned. Isolation of APH gene DNA from chromosomal DNA of a microorganism belonging to the genus Mycoplana and capable of producing APH can be performed by a conventional method, for example, Current Topics in Microbiology and Immunology.
and Immunology) Vol. 96 (1982).

The obtained APH gene DNA is integrated into a vector DNA to prepare a recombinant DNA. Incorporation of APH gene DNA into vector DNA is performed by a conventional method, for example, after cutting chromosomal DNA and vector DNA with restriction enzymes to prepare an APH gene DNA fragment and a vector DNA fragment, and treating a mixture of both with DNA ligase. Can be performed.

The vector DNA used here may be any vector as long as it can use Escherichia coli as a host, and pUC118, pBR322 and the like are particularly preferably used. As restriction enzymes, for example, BamH I, Sau3A
I, Bgl II, EcoR I, Pst I and the like. Since the cleavage site of Sau3A I has a protruding end having the same structure as the cleavage site of BglII or BamHI, ligation for recombination is possible. A DNA fragment obtained by subjecting chromosomal DNA to limited or complete digestion with Sau3A I or Bgl II is preferably ligated to a vector DNA fragment digested with BamHI.

As DNA ligase, T4D from T. phage-infected Pseudomonas aeruginosa
NA ligase is preferably used.

The recombinant DNA obtained by the above method can be used in a conventional manner, for example, in the Journal of Molecular Biology (J. of
Molecular Biology) Vol.166P.557 (1983) can be used to introduce Escherichia coli. Methods for selecting strains containing recombinant DNA (ie, vector DNA incorporating DNA carrying the genetic information involved in APH production) are routinely selected, for example, by molecular cloning (Molecular Cloning: T. Maniatis, EF Flitch, J. Am.
By Sambrook, Cold Spring Harbor Publishers,
1982: Hereinafter, molecular cloning refers to this document. ) Can be performed as follows.

The strain containing the recombinant DNA was transformed into an LM medium (1% Bactotryptone, 0.5% yeast extract, 10 mM NaC).
_{l, 10mM MgSO 4, 50mg /} l ampicillin, and cultured in 1.5% Bacto) on the resulting colonies transferred to a filter,
After lysis, the DNA was immobilized on a filter.
A colony to which a DNA probe prepared based on the N-terminal amino acid sequence of an APH protein produced by a microorganism of the genus Mycoplana described in JP-B-85080 and which binds under the following hybridization conditions is selected.

Hybridization conditions The DNA-immobilized filter and the DNA probe were combined with a prehybridization solution (50% formamide, 5%
nhardt's solution, a solution containing 5 × SSPE, 0.1% SDS, 100 μg / ml salmon sperm) at 42 ° C. After the hybridization is completed, the hybridization solution is drained. The obtained filter was washed in 2 × SSC and 0.1% SDS for 5 to 10 minutes, and the washing operation was performed for 3 to 4 minutes.
Repeat several times. Filter the filter with 1 × SSC and 0.1% SDS
Washing at 68 ° C for 1 to 1.5 hours.

When stringent conditions are required, soak the filter in 0.2 × SSC and 0.1% SDS at 68 ° C. for 60 minutes.

The SSC solution (20 ×) is a solution containing 17.53% NaCl and 8.82% sodium citrate and adjusted to pH 7.0, Denha
The rdt's solution (50 ×) is a solution containing 1% ficoll, 1% polyvinylpyrrolidone, and 1% BSA. SSPE (20 ×) is 17.
Contains 4% NaCL, 2.76% NaH ₂ PO ₄ .H ₂ O, 0.74% EDTA, pH
This is a solution adjusted to 7.4.

From the selected colonies, those having further APH activity are selected to obtain a microorganism containing the vector DNA into which the APH gene DNA has been completely incorporated.

From the obtained microorganism, a recombinant DNA can be obtained by a conventional method described in Molecular Cloning.

In order to express APH in a large amount by using Escherichia coli as a host microorganism using the recombinant DNA contained in the obtained microorganism, the following procedure is performed.

The obtained recombinant DNA is first prepared with a restriction enzyme map using a conventional method, for example, the method described in Molecular Cloning, and a DNA probe prepared based on the N-terminal amino acid sequence of the APH protein is bound. D
NA fragments are found by Southern hybridization.

 This DNA fragment containing the 5 'untranslated region of the APH gene was
3Incorporated into phage DNA and its base sequence united
・ State Biochemical Corporation (Unit
ed State Biochemical Corporation)
-Se Is determined by the method using.

From the obtained nucleotide sequence information, the APH gene promoter, the APH structural gene region not containing the SD sequence
The DNA is excised from NA, and the start codon of the APH gene is ligated downstream of the high expression promoter of Escherichia coli and the SD sequence so that the distance between the promoter SD sequence is appropriate.

As the promoter used herein, any one that works in Escherichia coli is effective, and particularly, a tric (trc) promoter, a trip (trp) promoter, a lambda PL promoter and the like can be mentioned.

The APH-producing ability of the microorganism containing the recombinant DNA thus obtained is examined, and a microorganism having a higher APH-producing ability is selected. Specific examples of the microorganism include Escherichia coli TRC- containing a recombinant DNA ptrc NMAPH obtained by incorporating a DNA fragment of APH gene derived from Mycoplana brata into PUC118 (manufactured by Takara Shuzo).
APH strain.

The transformant obtained by the present invention produces and accumulates APH in the culture by culturing by a usual bacterial culture method.

As a medium used for culturing the microorganism of the present invention, any of a synthetic medium and a natural medium containing a carbon source, a nitrogen source, an inorganic substance, and other nutrients can be used.

As the carbon source, for example, various carbohydrates such as glucose, starch, starch hydrolyzate, molasses and the like are used, and the use amount thereof is preferably about 5 to 70 g /.

Examples of the nitrogen source include various inorganic and organic ammonium salts such as ammonium sulfate, ammonium phosphate and ammonium carbonate, or nitrogen-containing organic substances such as peptone, yeast extract, corn steep liquor, and casein hydrolyzate. The amount used is 5
About 70 g / is preferable.

As the inorganic substance, for example, potassium hydrogen phosphate,
Potassium phosphate, magnesium sulfate, manganese sulfate, zinc sulfate, etc. are used, and the amount used is 0.05 to 5 g /
The degree is preferred.

The cultivation is performed under aerobic conditions such as shaking culturing or aeration stirring culturing. The culture temperature is preferably from 25 to 37 ° C, and the culture is usually completed in about 16 to 48 hours.

When purifying APH from cells that have been cultured, the cells are collected from the culture by centrifugation or the like,
The obtained cells are disrupted by ultrasonic treatment, trituration using glass beads, French press, etc., and the enzyme is extracted. The extract is treated by a conventional method such as ammonium sulfate salting out method, chromatography using an ion exchange resin, or gel permeation method to obtain purified APH.

APH produced by the recombinant obtained in the present invention is disclosed in
It has the same enzymatic activity as APH described in -85080.

 Hereinafter, examples of the present invention will be described.

Example 1. Cloning of APH gene (1) Preparation of chromosomal DNA containing APH gene Mycoplana brata strain FERM BP-1845 was cultured in KM102 medium (1
0 g / peptone, 7 g / meat extract, 5 g / yeast extract and 3 g NaCl).
The cells obtained by shaking culture for one day are cooled by Hitachi cooling centrifuge (RP
The cells were collected by centrifugation at 10,000 rpm for 10 minutes at 4 ° C using an R20-2 rotor). Bacteria in current topics in
Microbiology and immunology Vol.96
(1982), and chromosomal DNA was extracted to obtain about 1 mg of chromosomal DNA.

(2) Introduction of chromosomal DNA fragment into vector DNA Take 10 µg of the chromosomal DNA obtained in the preceding section (1), and add M buffer [1
0 mM Tris-HCl buffer (pH 7.5), 10 mM MgCl ₂ , 50 mM
NaCl, 1 mM dithiothreitol (DTT)], and 50 units of the restriction enzyme BglII was added. The mixture was kept at 37 ° C for 4 hours to completely decompose. This was then purified according to the method described in Molecular Cloning.
Fractionation was performed by 4040% sucrose density gradient centrifugation. Centrifugation at 20 ° C using a Hitachi ultracentrifuge (SRP28 rotor)
This was performed at 6,000 rpm for 16 hours. After centrifugation, fractionation was performed, a part of each fraction was subjected to agarose electrophoresis according to the method described in Molecular Cloning, and the size of the DNA fragment was measured. 5-8Kb of DNA
After collecting only fractions containing the fragments and performing ethanol precipitation, ligation buffer [66 mm Tris-HCl buffer (pH 7.6), 5 mM MgCl ₂ , 5 mM DTT, 1 mM ATP] 60 μl
To obtain a DNA fragment containing about 4 μg of the APH gene.
5 μg of vector pUC118 (Takara Shuzo) was added to M buffer.
After dissolving in 150 µm, 20 units of restriction enzyme BamH I was added and reacted at 37 ° C for 3 hours to completely decompose. Thereafter, 20 μl of 1M Tris-HCl buffer (pH 8.6) and 2 units of alkaline phosphatase derived from calf small intestine (manufactured by Boehringer Mannheim) were added, and the mixture was reacted at 37 ° C. for 1 hour to perform dephosphorylation. Heat at 65 ° C for 10 minutes to inactivate the enzyme, perform ethanol precipitation,
μl of ligation buffer. Then Bgl
16 μl of a solution containing 4 μg of APH gene DNA obtained by complete digestion with II and 5 μg of pUC118 obtained by digestion with BamHI
20 μl of a solution containing the mixture, and the total volume was adjusted to 75 μl with a ligation buffer.
A ligation reaction was performed at 6 ° C. for 16 hours to obtain a solution containing the recombinant DNA.

(3) Transformation of Escherichia coli MM294 The obtained solution containing the recombinant DNA was subjected to transformation of Escherichia coli MM294.

A competent cell of Escherichia coli MM294 strain was prepared according to the method described in J. of Molecular Biology, Vol. 166, p. 557 (1983). Mix 10 μm of the solution containing the recombinant DNA obtained in the above paragraph (2) with 210 μm of the MM294 strain competent cell, allow to stand on ice for 30 minutes, and heat at 42 ° C for 90 minutes.
Heat treated for 800 seconds, and add 800μC SOC medium (2% Bacto Tryptone, 0.5% Yeast Extract, 10mM NaC
l, 2.5 mM KCl, 10 mM MgCl ₂ , 10 mM MgSO ₄ , 20 mM glucose) and shake-cultured at 37 ° C. for 1 hour.
100 μl was applied to an LM plate (Bacto agar added to LM medium to a final concentration of 1.5%). This, 3
After culturing at 7 ° C. for 16 hours, a total of 3143 transformants were obtained on 101 plates.

(4) Selection of transformed strain Escherichia coli obtained as described in (3) above
The transformed strain of MM294 was transformed into nylon
It was transferred to a filter (Hybond-N, manufactured by Amersham). The 101 filters thus obtained were subjected to lysis, denaturation and fixation of DNA by the method described in Molecular Cloning, and then colony hybridization was performed using a probe labeled with ³² P.

The probe used for colony hybridization was a 50mer having a DNA base sequence corresponding to the amino acid sequence of alanine at position 29 from aspartic acid at position 13 from the N-terminus of APH described in JP-A-64-85080. Whose sequence is AACCGCCAAGA from the 5 'side
CCGAGCTCTACGGCGGCGAGCTGGTCCCCCCGTTCGAGGC.

The obtained filter was subjected to autoradiography to discriminate the colonies to which the probe was bound, and seven colonies were selected. The obtained seven colonies (that is, the transformants) were transformed into 60 ml of LB medium (10 g / tryptone, 5 g).
/ Yeast extract, 5 g / NaCl) for 16 hours. Thereafter, cells were collected according to a conventional method, and a TE buffer [10 mM Tris-HCl buffer (pH 8.
0), 1 mM EDTA] and suspended in 6 ml, and lysed by sonication. Using the resulting extract, JP-A-64-85080
The enzymatic activity of APH was measured using acetylputrescine as a substrate according to the method described in Japanese Patent Application Laid-Open Publication No. H07-157,078. As a result, a clone obtained from a certain transformant exhibited an activity of about 1 [milliunit / ml culture solution]. The transformant was named Escherichia coli APHW. The plasmid contained in the APHW strain was isolated and analyzed for its structure. As a result, it was found that the plasmid had the structure shown in FIG. This plasmid is called pAPHW
It was named.

Example 2. Preparation of APH high expression plasmid (1) Structural analysis of APH gene promoter, SD sequence and initiation codon APH itself has a promoter that works in Escherichia coli because it does not work effectively in Escherichia coli. It is necessary to insert the APH gene into the expression vector DNA. At that time, it is necessary to construct a suitable distance between the translation initiation codon of the inserted APH gene and the SD sequence of the vector. for that purpose,
Information on the DNA base sequence of the 5 'upstream region including the translation initiation site of the APH gene is required. The method for determining the DNA base sequence is described below.

 First, 10 mM Tris-HCl buffer (pH 8.0), 5 mM MgCl
_TwoFrom 100 mM NaCl and 1 mM 2-mercaptoethanol
Solution (hereinafter referred to as A solution) 1 μg in 50 μl
Plasmid DNA pAPHW with 10 units of EcoRI and 10 units of
The mixture was reacted with BamHI at 37 ° C. for 3 hours to completely digest.
This solution was subjected to electrophoresis using a 0.7% agarose gel.
No, in a 0.5 μg / ml ethidium bromide solution by a conventional method.
After staining, use Nylon according to the capillary transfer method.
Filter (Gene Screen Plus, manufactured by Dupont)
Transcribed. The obtained filter is referred to as γ³²P-ATP (3000Ci
/ m mol, Amersham) and T4 kinase (Takara Shuzo)
1.0 × 10⁷cpm / pmol end-labeled probe
[Example 1 (4)], 6 × SSC solution, 5 × Denhardt's
Solution, 0.5% SDS solution and 100 μg calf thymus DNA (Signal
In a 50 ml solution consisting of
Distillation was performed. Then 0.3 × SSC solution and
30 minutes at 55 ° C in 50 ml of a solution consisting of
Afterwards, autoradiation with X-ray film (Fujicolor)
Ophographic signal obtained with ethidium bromide staining
The EcoR I- shown in FIG.
The probe was found to bind to the BamHI fragment. this
Therefore, the structure containing the start codon of the APH gene in this region
The presence of the 5 'region and the upstream regulatory region of the gene
Was expected. Next, the DNA base sequence of this fragment
Ited State Biochemical Corporation
(United State Biochemical Corporation)
Sequinase Was determined by the method using. this
Sequence and translation initiation code of the fragment thus determined
Table 2 shows the corresponding amino acid sequences downstream of
did. N-terminal of APH protein determined from base sequence
The amino acid sequence is the APH disclosed in JP-A-64-85080.
48 N-terminal amino acid sequences.

(2) Construction of APH expression vector using trc promoter Based on the information on the nucleotide sequence obtained in Example 2. (1), Nco I-Hind III of high expression plasmid pKK233-2 (Pharmacia) having trc promoter was used. First in restriction enzyme site
The Nde I-Hind III fragment of the plasmid pAPHW shown in the figure was inserted.

First, 1 μg of pKK233-2 was added to 50 mM Tris-HCl buffer (pH 7.5), 10 mM MgCl ₂ , 100 mM CaC
l and a solution consisting of 1 mM dithioerythritol (hereinafter referred to as
It is called B solution. ) 37 with 3 units of Nco I in 50μ
After the reaction at 37 ° C. for 37 hours, the plasmid digested with NcoI was treated with 30 mM sodium acetate (pH 5.0), 100 mM NaCl, 1 mM
The protruding ends were scraped off with 50 units of mung bean nuclease in a 50 μ solution of zinc acetate and 10% glycerol to make the ends blunt. Plasmids with blunt Nco I cut surfaces were added to 10 units of Hind I
Reaction was carried out at 37 ° C. for 3 hours using II to obtain an Nco I-Hind III fragment.

Then, 1 μg of pAPHW was added to 5 units of N in 50 μ of the B solution.
The reaction was carried out at 37 ° C. for 3 hours using deI, and then the plasmid digested with NdeI was treated with a 7 mM Tris-HCl buffer (pH 7.
5), 0.1mM EDTA, 20mM NaCl , 7mM MgCl 2, 0.1mM d
Using 50 units of a solution consisting of ATP, 0.1 mM dGTP, 0.1 mM dCTP and 0.1 mM dTTP (hereinafter referred to as C solution) using 2 units of the Klenow fragment of DNA polymerase in 50 μl.
The reaction was carried out at 15 ° C. for 15 minutes to make the ends blunt. Then Nde I
Plasmid with a smooth cut surface was placed in 50 μA solution,
Reaction was carried out at 37 ° C. for 3 hours using 10 units of Hind III, and Nde
An I-Hind III fragment was obtained.

The thus obtained plasmid pKK233-2 Nco
I-Hind III fragment and plasmid pAPHW Nde I-Hind III
The fragments were ligated by a conventional method using DNA ligase to obtain plasmid ptrcNHAPH. In this way, tie A
The PH start codon can be located at an optimal position downstream of the SD sequence of the vector DNA.

Next, 1 μg of the recombinant plasmid ptrcNHAPH obtained to remove an extra portion on the 3 ′ side of the insert fragment was reacted with 5 units of Hind III in 50 μl of A solution at 37 ° C. for 3 hours.
The plasmid digested with II was combined with 5 units of
The reaction was performed at 37 ° C. for 3 hours with Mlu I. The plasmid digested with MluI was further reacted at 37 ° C. for 15 minutes using 2 units of Klenow fragment in 50 μl of the C solution to make both ends blunt. Both ends that had become blunt were joined by a conventional method using DNA ligase.

FIG. 2 shows the structure of the plasmid ptrcNMAPH constructed by the above method.

The thus-obtained plasmid ptrcNMAPH was transformed into Escherichia coli MM294 according to a conventional method to obtain an APH-producing strain, Escherichia coli TRC-APH.

Escherichia coli TRC-APH has been deposited under the Budapest Treaty on June 25, 1990 with the Institute of Microbial Industry and Technology, National Institute of Advanced Industrial Science and Technology under the name of Microtechnical Research Institute No. 2986 (FERM BP-2986).

Example 3. Comparison of production titers of various strains The three strains shown in Table 3 were replaced with 60 ml of the medium shown in Table 3
Was inoculated into a 300 ml Erlenmeyer flask containing, and cultured with shaking at 30 ° C for 18 hours.

Composition of medium I: 1% broth, 1% yeast extract, (pH 7.0) Composition of medium II: 0.5% yeast extract, 0.5%
Tryptone, 0.05% NaCl, 0.1% KH ₂ PO ₄ , 0.01% MnSO ₄ ,
Composition of 1.0% acetyl putrescine 2XYT medium: 16 g / tryptone, 10 g / yeast extract, 5 g / NaCl After cultivation, cells were collected by centrifugation, suspended in TE buffer, disrupted by sonication, A cell extract was prepared, and the APH activity in the extract was measured according to the method described in JP-A-64-85080. The results are shown in Table 3.

Example 4. Production of APH by recombinant strain TRC-APH (1) Culture of TRC-APH strain in 5 culture tanks TRC-APH strain was cultured at 1.5% Na ₂ HPO ₄ , 0.3% KH ₂ PO ₄ , 0.5% NaC
l, 0.1% NH ₄ Cl, 0.5% peptone, 0.1% trace element solution [25g
/ (NH ₄ ) ₂ HPO ₄ , 5 g / K ₂ SO ₄ , 0.75 g / NaCl, 5 g / Mg
_{SO 4 · 7H 2 O, 0.75g} / FeSO 4 · 7H 2 O, 0.17g / ZnSO 4 · 7H 2
_{O, 0.075g / CuSO 4 · 5H} 2 O, 0.038g / MnSO 4 · 4~5H
₂ O, 0.15 g / CaCl ₂・ 2H ₂ O, 0.017 g / Na ₂ B ₄ O ₇ , 10 H ₂ O,
_{0.0075g / (NH 4) 6 Mo} 7 O 24 · 10H 2 O ], 0.5% _{glucose, 0.0246% MgSO 4 · 7H 2} O, 4mg / Vitamin B1,50mg /
Containing a medium consisting of ampicillin (pH 6.6) 2.0
The cells were inoculated in a volume culture tank and cultured with aeration and stirring at 28 ° C. for 30 hours.
Eight hours after the start of the culture, a total of 70 ml of a feed medium having a composition of 16.7% peptone, 16.7% glucose and 0.4% trace element solution was continuously injected.

This bacterium showed an APH productivity of 38 [unit / ml culture solution] under these conditions.

(2) Purification of APH Cells were collected from the culture solution obtained in Example 4. (1) by centrifugation. The obtained cells were suspended in 200 ml of a 0.01 M Tris-HCl buffer (pH 8.0) and subjected to ultrasonic treatment to disrupt the cells, thereby extracting enzymes in the cells. The obtained crude extract is
The soluble fraction was obtained by centrifugation at 12,000 rpm for 20 minutes.

Next, solid ammonium sulfate is added to the obtained soluble fraction.
After addition to 40% saturation, the precipitated portion was collected.

The precipitate is washed with 50 ml of 0.01 M Tris-HCl buffer (pH 8.
0) and 0.005 M mercaptoethanol. This solution was dialyzed against the same buffer 10 for 24 hours.
The dialysate was passed through DEAE-Sepharose (Pharmacia) (1000 ml, diameter 10 cm) equilibrated with the same buffer to adsorb APH. Further, impure proteins were washed away with the same buffer.
Next, elution was carried out with a concentration gradient from 0 to 0.5 M of sodium chloride in the same buffer. The eluted active fractions are combined, ammonium sulfate is added to the active fraction, and the portion that precipitates at 90% saturation is collected by centrifugation (12,000 xg, 20 minutes).
It was dissolved in 50 ml of Tris-HCl buffer (pH 8.0). This solution was dialyzed against the same buffer solution 2 for 24 hours. The dialysate was freeze-dried to obtain 2 g (specific activity 29 units / mg) of an APH powder purified enzyme preparation.

According to the present invention, a microorganism that belongs to the genus Escherichia and has a recombinant DNA obtained by incorporating a DNA fragment carrying genetic information related to APH production from a microorganism belonging to the genus Mycoplana into a vector DNA is used. Thus, a method for producing APH inexpensively and in large quantities can be provided.

[Brief description of the drawings]

FIG. 1 is a restriction map of a recombinant plasmid pAPHW contained in the Escherichia coli APHW strain. FIG. 2 shows the structure of the plasmid ptrcNMAPH.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI (C12N 9/80 C12R 1:19) (C12N 15/09 C12R 1:01) (58) Investigation field (Int.Cl. ⁷ , DB name) WPI (DIALOG) BIOSIS (DIALOG) DDBJ / EMBL / GenBank

Claims (5)

(57) [Claims] The present invention is characterized in that AACGCCAAGACCGAGCTCTACGGCGGCGAGCTGGTCCC is responsible for genetic information relating to acetylpolyamine amide hydrolase production derived from a microorganism belonging to the genus Mycoplana.
DNA that hybridizes with a DNA probe having the nucleotide sequence represented by CCCGTTCGAGGC under stringent conditions
A microorganism belonging to the genus Escherichia having a recombinant DNA obtained by incorporating the fragment into a vector is cultured in a medium, acetylpolyamine amide hydrolase is produced and accumulated in the culture, and acetyl polyamine amide hydrolase is collected from the culture. A method for producing acetylpolyamine amide hydrolase. (2) AACGCCAAGACCGAGCTCTACGGCGGCGAGCTGGTCCC, which carries genetic information related to acetylpolyamine amide hydrolase production derived from a microorganism belonging to the genus Mycoplana.
DNA that hybridizes with a DNA probe having the nucleotide sequence represented by CCCGTTCGAGGC under stringent conditions
A microorganism belonging to the genus Escherichia, having a recombinant DNA obtained by incorporating a fragment into a vector. (3) AACGCCAAGACCGAGCTCTACGGCGGCGAGCTGGTCCCCC, which carries genetic information related to the production of acetylpolyamine amide hydrolase derived from a microorganism belonging to the genus Mycoplana.
A recombinant DNA obtained by incorporating a DNA fragment that hybridizes under stringent conditions with a DNA probe having a base sequence represented by CGTTCGAGGC into a vector. 4. A gene encoding an acetylpolyamine amide hydrolase polypeptide derived from a microorganism belonging to the genus Mycoplana represented by the amino acid sequence represented by the following formula (1). 5. A gene represented by the base sequence represented by the following formula (2), which encodes an acetylpolyamine amide hydrolase polypeptide derived from a microorganism belonging to the genus Mycoplana.