JP2999954B2 - Enzymatic production of 4-carbamoyl-1-β-D-ribofuranosyl-imidazolium-5-oleate - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to 4-carbamoyl-1-β-D-ribofuranosyl-imidazolium-5-
The present invention relates to an enzymatic production method of oleate (mizoribine).

[0002]

2. Description of the Related Art Mizoribine is eupenicillium (Eupenicilium).
cillium), a nucleic acid-related substance found in a culture solution of a microorganism belonging to the genus Cillium (J. Antibiotics., 27 (10), 775 (197
4)) It has excellent immunosuppressive activity and is used clinically for the purpose of suppressing rejection in kidney transplantation.
As an enzymatic production method of mizoribine, a method using nucleoside phosphorylase has already been reported (Japanese Patent Publication No. 3-65757, J. Ferment. Technol., 53 (8), 609).
(1975)).

[0003]

However, the above conventional methods have not always been satisfactory in terms of synthesis yield. For example, when 1 mol of 4-carbamoyl-imidazolium-5-oleate is used, the synthesis amount of mizoribine is at most about 0.5 mol, and the synthesis yield is 50%.
It was extremely low as follows. For this reason, establishment of an efficient method for producing mizoribine has been desired. Therefore, an object of the present invention is to provide a method for efficiently producing mizoribine.

[0004]

Means for Solving the Problems The inventors of the present invention have conducted intensive studies to achieve the above object, and as a result, have found that the use of purine nucleoside phosphorylase having specific properties makes it possible to synthesize mizoribine efficiently. Have found and completed the present invention. That is, the present invention relates to a method for producing mizoribine from 4-carbamoyl-imidazolium-5-oleate and ribose-1-phosphate using a nucleoside phosphorylase, wherein the purine nucleoside phosphorylase has high substrate specificity for adenosine as a nucleoside phosphorylase. The present invention relates to a method for the enzymatic production of mizoribine, characterized by using

[0005]

BEST MODE FOR CARRYING OUT THE INVENTION (1) Enzyme Used in the Present Invention Purine nucleoside phosphorylase used in the method of the present invention is characterized by having a high substrate specificity to adenosine especially among purine nucleosides. That is, a purine nucleoside phosphorylase having a substrate specificity for adenosine higher than that for inosine or guanosine is selected and used in the method of the present invention.

Several enzymes having such characteristics have already been reported, for example, purine nucleoside phosphorylase reported by Yamauchi (JP-A-4-4883).
Publication), Purine nucleoside phosphorylase reported by Hori et al. (Agric. Biol. Chem., 53 (12), 3219 (1989))
And the like. All of these enzymes are thermophilic bacteria belonging to the genus Bacillus, specifically, Bacillus stearothermophilus.
The purine nucleoside phosphorylase reported by Yamauchi having the following amino acid sequence is particularly suitable for the enzymatic production of mizoribine.

[0007]

(Equation 2)

(2) Preparation of Enzyme The enzyme used in the present invention can be easily prepared by a conventional recombinant DNA technique. The DNA molecule containing the purine nucleoside phosphorylase structural gene used in the preparation of the enzyme is not particularly limited as long as it contains the structural gene of purine nucleoside phosphorylase having high substrate specificity for adenosine. Specifically, a DNA molecule containing a region encoding the enzyme reported in Yamauchi having the above amino acid sequence and defined by the restriction map shown in FIG. 1 is exemplified. In addition, "punB" in FIG. 1 means a gene encoding a purine nucleoside phosphorylase having high substrate specificity for adenosine (702 bp, encoding a polypeptide having a molecular weight of 25775 and consisting of 274 amino acid residues).

[0009] Such a DNA molecule has a structural gene for purine nucleoside phosphorylase and an upstream SD gene.
It is desirable to contain the sequence. By containing the SD sequence, the production amount of the target enzyme can be significantly increased as compared with the case where a DNA molecule containing only the structural gene is used. Explaining by giving a specific example, in the base sequence shown in FIGS. 2 and 3 including the gene encoding the reported enzyme in Yamauchi having the amino acid sequence, at least the base sequence from the SD sequence to the stop codon The productivity of the target purine nucleoside phosphorylase can be improved by using a DNA molecule containing

Such a DNA molecule can be cloned from a thermophilic bacterium belonging to the genus Bacillus using the nucleoside degrading activity at a high temperature as an index as in the method of Yamauchi (JP-A-4-4883). it can. Alternatively, as is usually done, a partial amino acid sequence such as the amino terminus of a purified purine nucleoside phosphorylase derived from a thermophile belonging to the genus Bacillus is determined by a known method, or a partial sequence of the amino acid sequence is referred to. Alternatively, a method of synthesizing an oligonucleotide corresponding thereto and selecting a DNA fragment containing a gene encoding purine nucleoside phosphorylase from a gene bank of a thermophile belonging to the genus Bacillus using the oligonucleotide as a probe can also be adopted. The host used for cloning is not particularly limited, but Escherichia coli is suitable as the host because of operability and simplicity. Further, referring to FIG. 2 and FIG.
Chemical synthesis may be performed using an NA synthesizer.

Usually, even when these fragments are cloned into a plasmid vector or the like, the DNA fragment often does not have a promoter, or even if it has a promoter, it cannot function efficiently in a heterologous microorganism. It is said that high expression of the encoded gene usually does not occur. In addition, when extra DNA other than the coding region is present, even if the gene is read through from the promoter of another gene present on the plasmid vector,
hrough) transcription can also result in its high expression, which is not preferred. Therefore, in order to realize high expression of the target gene, the nucleotide sequence of the cloned DNA fragment is analyzed, the coding region of the gene is identified, and unnecessary sequences are removed. Thus, it is necessary to construct a recombinant expression vector in which an expression control signal (transcription initiation and translation initiation signal) is ligated 5 ′ upstream thereof so that the gene is highly expressed in the microbial cells. The determination of the DNA base sequence can be performed by a conventional method, for example, the method of Maxamugilbert (Methods in Enzymology, 65, 499 (1980)) or the dideoxy chain terminator method (Methods in E
nzymology, 101, 20 (1983)).

The expression control signal used to express the target purine nucleoside phosphorylase gene in a large amount in a heterologous microorganism can be artificially controlled.
It is desirable to use a strong transcription initiation and translation initiation signal that dramatically increases the expression level of the purine nucleoside phosphorylase gene. As such a transcription initiation signal, when E. coli is used as a host,
lac promoter, trp promoter, tac pro
Motor (Proc. Natl. Acsd. Sci. U
SA. , 80, 21 (1983), Gene, 20, 2
31 (1982)), trc promoter (J. Bio
l. Chem. , 260, 3539 (1985)) and glyceraldehyde-3 when yeast is used as a host.
-Phosphate dehydrogenase (J. Bio
l. Chem. 254, 2078 (1980)) and inhibitory acid phosphatase (Nucl. Acids. R).
es. , 11, 1657 (1983)) and the like.

As the vector, various plasmid vectors, phage vectors, and the like can be used. The vector can be replicated in the microbial cell, has an appropriate drug resistance marker and a specific restriction enzyme cleavage site, and is It is desirable to use a plasmid vector with a high copy number. When Escherichia coli is used as a host, pBR322 (Gen
e, 2, 95 (1975)), pUC18, pUC19 (Gene, 33, 10
3 (1985)). When yeast is used as a host, YEp13 (ATCC 37115), YEp13
p24 (ATCC 37051). Methods for preparing a purine nucleoside phosphorylase structural gene, linking a cloned gene to an expression preparation signal, and the like are well-known techniques for ordinary engineers, particularly those who belong to the fields of molecular biology and genetic engineering, and Specifically, for example, “Molecular Cloning” (Maniatis
Ed., Cold Spring Harbor, New York (1982)).

A host microorganism is transformed with the prepared recombinant vector. The host microorganism is not particularly limited as long as it has high safety and is easy to handle. For example, microorganisms commonly used in DNA recombination operations such as Escherichia coli and yeast can be used. Among them, Escherichia coli is advantageous in handling and in synthesizing nucleoside analogs, and the K-12 strain used in recombination experiments, for example, C
600 bacteria, JM105 bacteria, JM109 bacteria, etc. can be used
It is. Many methods for transforming microorganisms have already been reported, and they may be appropriately selected depending on the microorganism used as a host. For example, when Escherichia coli is used as a host, a method of introducing a plasmid into the cells by treatment with calcium chloride at a low temperature (J. Mol Biol., 5
3,159 (1970)). When yeast is used as a host, the protoplast method (Proc. Natl. Acsd. Sc.
i. USA. , 75, 1929 (1978)) or an alkali metal treatment method (J. Bacteriol., 1).
53, 163 (1983)).

The obtained transformant is grown in a culture in which the microorganism can grow, and furthermore, the expression of the cloned purine nucleoside phosphorylase gene is induced, and the culture is continued until the enzyme is accumulated in a large amount in the cells. . The culture of the transformant may be performed according to a conventional method using a medium containing nutrients necessary for the growth of the microorganism, such as a carbon source and a nitrogen source. For example, when using E. coli as a host,
As a medium, 2xYT medium (Methods in En
zymology, 100, 20 (1983)), LB
Medium, M9CA medium (Molecular Clonin
g, described above) and the like, and culturing at a culturing temperature of 20 to 40 ° C. with aeration and stirring as necessary. When a plasmid is used as a vector, an appropriate amount of an antibiotic (ampicillin, tetracycline, etc., depending on the drug resistance marker of the plasmid) is added to the culture medium to prevent the omission of the plasmid during culture. I do.

When it is necessary to induce the expression of the purine nucleoside phosphorylase gene during the culture, the expression of the gene is induced by a method commonly used for the promoter used. For example, when the lac promoter or the trc promoter is used, the expression inducer isopropyl-β-thiogalactopyranoside (IP
TG) is added in an appropriate amount. After inducing the expression of the purine nucleoside phosphorylase gene, the culture is continued for several hours to obtain a culture as an enzyme source in order to accumulate a large amount of the gene product in the cells.

Examples of the enzyme source used for the production of mizoribine include the above-mentioned culture, cultured cells, processed cells obtained by treating the cells, or crude or purified enzymes prepared from the cells. be able to. For example, when cells are used as an enzyme source, cells obtained by suspending cells recovered from a culture by membrane separation or centrifugation in an appropriate buffer may be used as the enzyme source. Also, the recovered cells are suspended in an appropriate buffer, lysed by ultrasonic treatment, French press treatment, or the like, and a cell-free extract obtained by removing cell residue by centrifugation is used as a crude enzyme solution. be able to. Even when further purification is required, heat treatment, ammonium sulfate salting out treatment, dialysis treatment, solvent treatment such as ethanol, various kinds of chromatographic treatments, etc., which are usually used for enzyme purification alone or at most two kinds of treatments A highly purified enzyme preparation can be prepared by simple means that are only combined. When Escherichia coli is used as the host microorganism, the cell-free extract is subjected to a heat treatment (1 hour at 60 to 80 ° C.).
After about 10 minutes, most of the Escherichia coli-derived proteins are denatured, can be easily removed by centrifugation, and very efficient enzyme purification is possible.

(3) Enzymatic production method of mizoribine The method of the present invention comprises the steps of preparing a specific enzyme obtained as described above from 4-carbamoyl-imidazolium-5-oleate and ribose-1-phosphate. Mizoribine is to be prepared. The ribose-1-phosphate used in the reaction may be prepared from nucleosides or nucleotides by the action of nucleoside phosphorylase, and the reaction for producing ribose-1-phosphate and the reaction for synthesizing mizoribine may be the same. It can also be performed in a system.

The reaction conditions may be determined by appropriately determining the optimum conditions of the enzyme used.
The reaction pH can be appropriately selected from the range of 3 to 10 as the reaction pH. The reaction is performed by adding 1 to 100 m of phosphoric acid if necessary.
M in a buffer containing 1-1000 mM 4-carbamoyl-imidazolium-5-oleate, 1-1000 mM ribose-1-phosphate and 0.1-100 (U / m
1) using purine nucleoside phosphorylase,
The reaction can be performed for about 72 hours. After the reaction, the obtained mizoribine can be isolated and purified by a conventional method.

[0020]

According to the method of the present invention, it is possible to synthesize mizoribine in a yield of 80% or more based on the 4-carbamoyl-imidazolium-5-oleate used, as shown in the following Examples. Yes, a very practical method.

[0021]

EXAMPLES Hereinafter, the present invention will be described specifically with reference to examples, but the present invention is not limited thereto. The preparation of DNA, cleavage with restriction enzymes, ligation of DNA with T4 DNA ligase, and transformation of Escherichia coli in this example are all referred to as "Molcular Clonin".
g "(supra). In addition, various restriction enzymes, T
4DNA ligase, plasmid vectors pUC118 and pUC119 and a deletion kit for kilosequencing were all obtained from Takara Shuzo.

Example (1) Determination of DNA base sequence [0022] The thermophilic bacterium Bacillus stellarothermophilus ( Bacillus s.
2.4 kbp DN containing a gene encoding a purine nucleoside phosphorylase with high substrate specificity for adenosine from TH6-2 strain of tearothermophilus )
The Eco RV- Sph I DNA fragment was subcloned into pUC119 from the plasmid pPUR1.ΔS into which the A fragment had been inserted (see No. 11196 from Seikagaku Kenkyusho, JP-A-4-4883). The recombinant plasmid was converted into a reference (Gene, 28, 28) using a deletion kit for kilosequencing.
351 (1981)), a part of the inserted portion was removed, and various clones having different chain lengths were produced. The inserts of the various clones obtained were subjected to the dideoxy chain terminator method (Science, 214, 1295 (198
The DNA base sequence was determined according to 1)). As a result, FIG.
And the DNA sequence of FIG. 3 was obtained. This base sequence is 70
It is 2 bp and encodes a polypeptide having a molecular weight of 25775 consisting of 234 amino acid residues starting with methionine (Met).

(2) Preparation of recombinant vector for expression (see FIGS. 4 and 5) A recombinant vector pTrc-B56 for purine nucleoside phosphorylase production was prepared by the following method. That is, pPUR1 · ΔS was replaced with restriction enzymes Eco RV and Sph I.
2.4 kbp DN containing purine nucleoside phosphorylase structural gene and SD sequence obtained by cleavage with
A fragment and HincI of plasmid vector pUC119
I and SphI digested fragments were ligated using T4 DNA ligase, and Escherichia coli JM109 was transformed using the ligation reaction solution. From the obtained ampicillin-resistant transformant,
A plasmid pUC9PUNB was obtained in which a DNA fragment containing the purine nucleoside phosphorylase structural gene and the SD sequence was inserted into pUC119. Next, pUC9PU
After cutting NB with restriction enzymes Bam HI and Kpn I, a plasmid in which the 5 ′ upstream region of the SD sequence was deleted was prepared using a deletion kit.
Was transformed.

From the obtained ampicillin-resistant transformants, a plasmid pUC9B56 was obtained. EcoRI-HindIIIDN containing purine nucleoside phosphorylase structural gene and SD sequence from the plasmid
A fragment was cut out with the same restriction enzyme, and this fragment and restriction enzyme E
co RI, the plasmid vector pTrc99A cut with HindIII (Gene, 69,301 (198
8), obtained from Pharmacia) and T4DNA
Ligation was carried out using ligase, and Escherichia coli JM109 strain was transformed using the ligation reaction solution. From the obtained transformant, a recombinant vector pTrc-B5 into which an SD sequence and a purine nucleoside phosphorylase structural gene were inserted immediately after the trc promoter for expression of pTrc99A.
6 was obtained.

(3) Culture of Transformant and Enzyme Extraction A transformant of Escherichia coli carrying the pTrc-B56 plasmid vector was inoculated into 100 ml of 2 × TY medium containing 100 μ / ml ampicillin, and cultured with shaking at 37 ° C. . When the concentration reached 4 × 10 ⁸ cells / ml, IPTG was added to the culture solution to a final concentration of 1 mM, and further 3 mM was added.
Shaking culture was continued at 7 ° C. for 5 hours. After completion of the culture, the cultured cells are collected by centrifugation (9,000 xg, 10 minutes)
Then , 20 ml of a buffer (50 mM, Tris-HCl buffer (pH 7.8), 5 mM EDTA, 0.1% tri
ton X100). Lysozyme was added to the cell suspension to a final concentration of 1 mg / ml,
For 1 hour to lyse the transformant, and then centrifuged (2,000 × g, 10 minutes) to remove cell body residues.

The supernatant fraction thus obtained was used as a cell extract, and purine nucleoside phosphorylase activity in the cell extract was measured. The results were compared with the control bacteria [pTr
E. coli JM109 carrying c99A and pUC1
18 PURS EV-S (JP-A-4-4883)
Are shown in Table 1 below.
As a result, as shown in Table 1, in the transformant holding the produced recombinant vector, the control bacterium (pTrc9
Adenosine phosphorolytic activity at high temperature, which was hardly observed in E. coli JM109 carrying 9A, was confirmed. Moreover, the transformant (pTr
The transformant (pUC118PURS.EV-) produced by a conventional method (Japanese Unexamined Patent Publication No. 4-4883) was used.
It was also confirmed to produce purine nucleoside phosphorylase about 40 times that of S). Purine nucleoside phosphorylase activity was determined by the method of Yamauchi (JP-A-4-48).
No. 83) and the phosphorylation activity of adenosine at 70 ° C. was measured and calculated.

[0027]

[Table 1]

(4) Characteristics of Purified Enzyme The above-mentioned bacterial cell extract is subjected to a conventional method (eg, WO90 / 10080).
Purine nucleoside phosphorylase. As a result, the properties of the purine nucleoside phosphorylase are as follows. Action International Patent Classification: C. It belongs to 2.4.2.1 and catalyzes the following phosphorolysis reaction.

[0029]

(Equation 3)

Substrate Specificity Table 1 shows the results of phosphorolysis when various purine nucleosides were used as substrates. As specified in Table 1, adenosine, 2'-deoxyadenosine, inosine, 2'-deoxyinosine, guanosine, 2'-
It is specific for deoxyguanosine and exhibits high substrate specificity, especially for adenosine and 2'-deoxyadenosine.

[0031]

[Table 2]

Optimum pH and pH stability The optimum pH is 7.0-7.5, and the stable pH range is 5.0-5.0.
9.4 (see FIGS. 6 and 7). Optimum temperature and temperature stability The optimum temperature is 70 ° C, and the stable temperature range is up to 60 ° C (see FIGS. 8 and 9). Molecular weight The molecular weight measured by gel filtration method (using Sephacryl S-300 gel) is about 109,000,
The molecular weight measured by acrylamide gel electrophoresis is about 27,000 (the molecular weight calculated from the amino acid sequence is 2
5,775. ), It is presumed that the present enzyme in a native state forms a tetramer composed of the same subunit. Isoelectric point Isoelectric focusing (PastSystem IEF3
-9 gel was used), and the isoelectric point was 5.2.

(5) Synthesis of Mizoribine Using Cultured Cells 10 ml of a culture solution of Escherichia coli JM109 strain carrying pTrcB56 obtained by the same method as in (3) above and pTrc-plasmid which is a plasmid for expressing pyrimidine nucleoside phosphorylase. Cultured cells were collected by centrifugation from 10 ml of a culture solution of Escherichia coli JM109 strain (JP-A-6-253854) holding pyn, suspended in 1 ml of physiological saline, and mixed so as to be uniform. 9 ml of a 40 mM phosphate buffer (pH 7.0) containing 40 mM 4-carbamoyl-imidazolium-5-oleate and 60 mM uridine was added to 1 ml of the cell suspension mixture, and the mixture was reacted at 45 ° C. for 8 hours. Mizoribine was synthesized. High performance liquid chromatography (column:
The yield of the substance was measured using YMC-PACK ODS-AQ), and as a result, mizoribine was synthesized at a ratio of 4-carbamoyl-imidazolium-5-oleate of 85%.

(6) Synthesis of Mizoribine Using Cell Extract The enzyme extract derived from the transformant JM109 (pTrcB56) prepared in the above (3) was added to 10 ml of a buffer having the same composition as the above (5). 50 μl of the transformant JM109 (p
Trc-pyn) -derived enzyme extract (JP-A-6-253)
No. 854) (20 μl) (purine nucleoside phosphorylase 10 unit / ml, pyrimidine nucleoside phosphorylase 1 unit / ml as final concentrations)
The mixture was added and reacted at 50 ° C. for 8 hours. As a result of measuring the production rate of mizoribine using high performance liquid chromatography, it was confirmed that mizoribine was synthesized at a ratio of 4-carbamoyl-imidazolium-5-oleate of 92%.

[0035]

[Brief description of the drawings]

FIG. 1 shows a 2.4 kbp Sau3AI containing a purine nucleoside phosphorylase structural gene from a thermophilic bacterium Bacillus stearothermophilus TH6-2.
1 shows a restriction map of an SphI DNA fragment .

FIG. 2 shows the first half of the base sequence of a DNA fragment containing a purine nucleoside phosphorylase structural gene derived from a thermophilic bacterium Bacillus stearothermophilus TH6-2. In the figure, → 56 is pUC9B5
6. The 5 ′ end of the DNA fragment containing the enzyme structural gene in the pTrcB56 plasmid was D. Is SD
In the sequence, Met indicates the translation initiation codon of the purine nucleoside phosphorylase structural gene, respectively.

FIG. 3 also shows the latter half of the base sequence of a DNA fragment containing a purine nucleoside phosphorylase structural gene derived from a thermophilic bacterium Bacillus stearothermophilus TH6-2. In the figure, STOP indicates a stop codon.

FIG. 4 shows the recombinant plasmid vector pTrcB.
56 shows the first half of the 56 construction method.

FIG. 5 shows the recombinant plasmid vector pTrcB.
56 shows the latter half of the 56 construction method.

FIG. 6 shows the optimal pH range of purine nucleoside phosphorylase.

FIG. 7 shows the stable pH range of purine nucleoside phosphorylase.

FIG. 8 shows the optimal temperature range of purine nucleoside phosphorylase.

FIG. 9 shows the stable temperature range of purine nucleoside phosphorylase.

Continuation of the front page (56) References JP-A-4-4883 (JP, A) JP-A-2-222677 (JP, A) JP-A-2-303485 (JP, A) JP-A-3-127986 (JP) JP-A-51-1693 (JP, A) JP-B 3-65957 (JP, B2) (58) Fields investigated (Int. Cl. ⁷ , DB name) BIOSIS (DIALOG) WPI (DIALOG)

Claims (4)

(57) [Claims] 1. Use of a nucleoside phosphorylase to produce 4
-Carbamoyl-imidazolium-5-oleate and ribose-1-phosphate from 4-carbamoyl-1-β-D
-A method for producing ribofuranosyl-imidazolium-5-oleate, wherein a purine nucleoside phosphorylase having high substrate specificity for adenosine is used as a nucleoside phosphorylase. 2. The method according to claim 1, wherein the purine nucleoside phosphorylase is derived from a thermophile belonging to the genus Bacillus. 3. The method according to claim 1, wherein the purine nucleoside phosphorylase is derived from a thermophile belonging to the genus Bacillus and has the following amino acid sequence. (Equation 1) 4. The method according to claim 1, wherein the ribose-1-phosphate is obtained by phosphorolysis of a nucleoside or nucleotide.