JPH10286097A - Enzymatic production of purine arabinoside - Google Patents

Abstract

(57) [Problem] To provide an efficient method for producing purine arabinoside. SOLUTION: In the method for producing purine arabinoside from purine and uracil arabinoside using nucleoside phosphorylase, purine nucleoside phosphorylase and uridine phosphorylase are used in combination as nucleoside phosphorylase, and the use ratio of the two enzymes is an enzyme unit ( The present invention relates to an enzymatic production method of purine arabinoside, characterized in that purine nucleoside phosphorylase is used at least 1.5 times as much as uridine phosphorylase as a unit).

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for producing purine arabinoside enzymatically.

[0002]

2. Description of the Related Art Purine arabinoside represented by vidarabine (adenine arabinoside) and fludarabine (fluoroadenine arabinoside) or its 5'-phosphate has an antiviral activity or an anticancer activity. Some compounds are actually used clinically as pharmaceuticals. As an enzymatic production method of purine arabinoside, a method using nucleoside phosphorylase has already been reported (JP-B-60-41598, JP-B-61-348).
0, Japanese Patent Publication No. 62-10157, Japanese Patent Publication No. 62-1427
7, Japanese Patent Publication No. 62-14278, Japanese Patent Publication No. 62-1427
9. JP-B-62-15199, JP-B-62-1520
0, JP-A-56-8695, JP-A-56-8696, JP-A-57-118797, etc.).

[0003]

Among the enzymatic production methods described above, a method in which purine nucleoside phosphorylase and uridine phosphorylase are used in combination as nucleoside phosphorylases has already been reported (Japanese Unexamined Patent Publication No. 56-8 / 1981).
695), but this method could not always be a method that was very satisfactory in terms of the synthesis yield. Therefore, the present invention
An object of the present invention is to provide an efficient method for producing purine arabinoside.

[0004]

Means for Solving the Problems The present inventors have made intensive studies to achieve the above object, and as a result, in the above-mentioned conventional method (JP-A-56-8695), purine nucleoside phosphorylase was replaced with uridine phosphorylase. Focusing on the fact that these two enzymes were used in an amount or slightly higher, the use ratio of these two enzymes was examined. As a result, the purine nucleoside phosphorylase was used at least 1.5 times that of uridine phosphorylase, resulting in the synthesis yield of purine arabinoside. Can be significantly improved, and the present invention has been completed. That is, the present invention provides a method for producing purine arabinoside from purine and uracil arabinoside using nucleoside phosphorylase, wherein purine nucleoside phosphorylase and uridine phosphorylase are used in combination as nucleoside phosphorylase, and the use ratio of the two enzymes is Purine nucleoside phosphorylase is a unit of uridine phosphorylase.
The present invention relates to a method for producing purine arabinoside enzymatically, which is used at least five times.

[0005]

BEST MODE FOR CARRYING OUT THE INVENTION

(1) Preparation of enzyme Purine nucleoside phosphorylase (EC 2.4.2.1) and uridine phosphorylase (EC 2.4.2.3) used in the present invention are known enzymes;
It is not limited to a special one as long as it can be used for the synthesis of purine arabinoside. Specifically, any purine nucleoside phosphorylase may be used as long as it catalyzes a reaction for synthesizing purine arabinoside from a purine base and arabinose-1-phosphate.Examples of uridine phosphorylase include uracil arabinoside as uracil and arabinose-. Any catalyst can be used as long as it catalyzes a reaction decomposing into 1-phosphoric acid. Many such enzymes have already been reported, and some of them have been reported up to the nucleotide sequence of a gene encoding the enzyme (Japanese Patent Application Laid-Open No. 56-8955, Proc.
Natil.Acad.Sci. USA, 88, 7185-7189 (1991), Nucl.
eic Acids Res., 17, 6741 (1989), Biosci. Biotech. B
iochem., 59, 1987-1990 (1995)).

[0006] Therefore, the preparation of the enzyme used in the present invention can be easily carried out by ordinary recombinant DNA techniques. That is, an oligonucleotide corresponding to a part of the structural gene of a purine nucleoside phosphorylase or a uridine phosphorylase that has been reported and can be used in the method of the present invention is synthesized, and the oligonucleotide is used as a probe to obtain a purine from a gene bank of a microorganism such as Escherichia coli. A DNA fragment containing a gene encoding nucleoside phosphorylase or uridine phosphorylase may be selected. The host used for cloning is not particularly limited, but Escherichia coli is suitable as the host because of operability and simplicity.

[0007] Usually, even if the selected DNA fragment is cloned into a plasmid vector or the like, the DNA fragment does not have a promoter, or even if it has a promoter, it cannot function efficiently in a heterologous microorganism. In many cases, high expression of the encoded gene is not usually expected. In addition, extra DNA other than the coding region
Is not preferable, because even when read-through transcription from a promoter of another gene present on a plasmid vector is performed, its high expression may occur. 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 carried out by a conventional method, for example, the method of Maxamugilbert (Methods in Enzymology, 65, 499 (198
0)) or the dideoxy chain terminator method (Methods in Enzymology, 101, 20 (1983)).

As an expression control signal used to express a target purine nucleoside phosphorylase gene or uridine phosphorylase gene in a large amount in a heterologous microorganism, artificial control is possible, and the expression level of the enzyme gene is dramatically increased. It is desirable to use such strong transcription initiation and translation initiation signals. When Escherichia coli is used as a host, such a transcription initiation signal may be lac promoter, trp promoter, ta
c promoter (Proc. Natl. Acsd. Sci. USA., 80, 2
1 (1983), Gene, 20, 231 (1982)), trc promoter (J. Biol. Chem., 260, 3539 (1985)), and glyceraldehyde-3-phosphate when yeast is used as a host. Dehydrogenase (J. Biol. Chem., 254, 2
078 (1980)) and inhibitory acid phosphatase (Nucl. Aci
ds. Res., 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 such as preparation of each structural gene of purine nucleoside phosphorylase or uridine phosphorylase, ligation of a cloned gene with an expression preparation signal, are well known to general engineers, particularly those who belong to the field of molecular biology and genetic engineering. Technology, specifically, for example, “Molecular Cloning” (edited by Maniatis et al., Cold Spring H
arbor, New York (1982)).

[0010] A host microorganism is transformed with the produced 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 purine arabinoside synthesis. For example, the K-12 strain and C
600 bacteria, JM105 bacteria, JM109 bacteria and the like can be used. 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 cells by treatment with potassium chloride at a low temperature (J. Mol. Biol., 53, 159 (197
0)) can transform E. coli. Also,
When yeast is used as a host, the protoplast method (Proc.
Natl. Acsd. Sci. USA., 75, 1929 (1978)) or an alkali metal treatment method (J. Bacteriol., 153, 163 (1983)).

The obtained transformant is grown in a culture in which the microorganism can grow, and the expression of the cloned purine nucleoside phosphorylase gene or uridine phosphorylase gene is induced to accumulate a large amount of the enzyme in the cells. Cultivate until up to. 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 E. coli is used as a host, 2 ×
TY medium (Methods in Enzymology, 100, 20 (1983)),
Using a medium commonly used for culturing E. coli, such as an LB medium and an M9CA medium (Molcular Cloning, supra),
Cultivate at a culture temperature of 40 ° C. with aeration and agitation as necessary. When a plasmid is used as a vector, an appropriate amount of an antibiotic (ampicillin, tetracycline, or the like, depending on the drug resistance marker of the plasmid) is added to the culture solution to prevent the plasmid from dropping out during the culture. I do.

When it is necessary to induce the expression of a purine nucleoside phosphorylase gene or a uridine phosphorylase gene during culture, the expression of the gene is induced by a method commonly used for the promoter used. For example, when the lac promoter or trc promoter is used, an appropriate amount of isopropyl-β-thiogalactopyranoside (IPTG), which is an expression inducer, is added at the middle stage of the culture. After inducing the expression of the nucleoside phosphorylase gene, culturing 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.

The enzyme source used in the method of the present invention includes:
The culture, the cultured cells, the treated cells obtained by treating the cells, and the crude or purified enzyme prepared from the cells can be exemplified. 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. If further purification is required,
Advanced treatments commonly used in enzyme purification such as heat treatment, ammonium sulfate salting out treatment, dialysis treatment, solvent treatment such as ethanol, various chromatographic treatments, etc., alone or in combination of at most two types, are highly advanced. A purified enzyme preparation can be prepared.

(2) Enzymatic method for producing purine arabinoside In the method of the present invention, the purine nucleoside phosphorylase and uridine phosphorylase obtained as described above are used in combination, and the use ratio of the two enzymes is determined in enzyme units (units). ), Purine nucleoside phosphorylase is used at least 1.5 times that of uridine phosphorylase to produce purine arabinoside from purine and uracil arabinoside. Purine and uracil arabinoside used in the reaction are both known compounds, and may be appropriately selected according to the target purine arabinoside.

The reaction conditions may be determined by appropriately determining the optimum conditions for 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
M in a buffer containing 1 to 1000 mM purine, 1 to 10 mM
00 mM uracil arabinoside and 0.1-100
(U / ml) purine nucleoside phosphorylase and uridine phosphorylase to carry out the reaction for about 1 to 100 hours. Purine nucleoside phosphorylase and uridine phosphorylase may be used in an amount of at least 1.5 times, more specifically, 1.5 to 100 times, preferably 1.5 to 10 times, as urine phosphorylase as an enzyme unit. More preferably, it is used about 1.5 to 5 times. After the reaction, the obtained purine arabinoside can be isolated and purified by a conventional method.

[0016]

According to the method of the present invention, the desired purine arabinoside is synthesized in a yield of 60% or more per used purine and, depending on the compound, 90% or more, as shown in the following Examples. It is possible and a very practical method.

[0017]

EXAMPLES The present invention will be described below in detail with reference to examples, but it is apparent that the present invention is not limited to these examples. In the examples, DNA preparation, restriction enzyme digestion, DNA ligation using T4 DNA ligase, and transformation of Escherichia coli are all referred to as “Molecular cloning” (Maniati
s et al., Cold spring Harbor Laboratory, Cold Spring
Harbor, New York (1982)). In addition, restriction enzymes, AmpliTaq DNA polymerase, alkaline phosphatase, T4 polynucleotide kinase,
T4 DNA ligase was obtained from Takara Shuzo.

Example 1: Synthesis of adenine arabinoside (ara A) (1) Cloning of large intestine purine nucleoside phosphorylase gene Escherichia coli K12 strain JM109 (obtained from Takara Shuzo Co., Ltd.)
Of chromosomal DNA from Saito and Miura (Biochim. Biophy
s. Acta., 72, 619 (1963)). This DNA
Was used as a template, the following two types of primer DNAs were synthesized according to a conventional method, and the Escherichia coli purine nucleoside phosphorylase (deoD) gene was amplified by the PCR method.

Primer (A): 5'-ATGTTCTGATGGATTTGGGCGGAG-3 'Primer (B): 5'-GATACTAAAAAGCCGGAGCAGTCT-3'

The amplification of the deoD gene by PCR is performed in 100 ml of a reaction solution (50 mM potassium chloride, 10 mM
Tris-HCl (pH 8.3), 1.5 mM magnesium chloride, 0.001% gelatin, temperate DNA
0.1 mg, primer DNA (A) and (B) 0.2 mM each, AmpliTaq DNA polymerase (2.5 units) were added to Perkin-Elmer Ce
tus Instrument DNA Ther
Using a Mal Cycler, heat denaturation (94 ° C, 1
Min), annealing (57 ° C., 1.5 minutes), and polymerization (72 ° C., 1.5 minutes) were repeated 25 times.

After gene amplification, the reaction solution was treated with a phenol / chloroform (1: 1) mixed solution, and twice the volume of ethanol was added to the water-soluble fraction to precipitate DNA. The precipitated and recovered DNA was separated by agarose gel electrophoresis according to the method described in the literature (Molecular cloning, described above), and 850 was collected.
The DNA fragment corresponding to b was purified. Blunt the DNA
After blunting with ing kit (obtained from Takara Shuzo), the plasmid pUC18 (obtained from Takara Shuzo) and phosphorylated at the 5 'end with T4 polynucleotide kinase, and then treated with restriction enzymes SmaI and alkaline phosphatase, and T4 DNA Ligation was performed using ligase. Escherichia coli JM109 was transformed using the ligation reaction solution, and plasmid pUC was isolated from the resulting ampicillin-resistant transformant.
-EPuF was isolated. pUC-EPuF is pUC18
The DNA fragment containing the Escherichia coli deoD gene was inserted in the forward direction into the SmaI cleavage site downstream of the lac promoter.

Next, the plasmid pUCEPuF was digested with restriction enzymes EcoRI and SalI, and the resulting DNA fragment corresponding to 860b was separated by agarose gel electrophoresis.
Collected. Similarly to the DNA fragment, plasmid pTrc99A (PhRc99A) digested with restriction enzymes EcoRI and SalI
armasia Biotech. And T4
Ligation was performed using DNA ligase. Escherichia coli JM109 was transformed using this ligation reaction solution, and plasmid pTrc-EPu was transformed from the obtained ampicillin-resistant transformant.
Was isolated. pTrc-EPu is the t of pTrc99A.
EcoRI-Sal containing the E. coli deoD gene in the EcoRI-SalI digestion fragment downstream of the rc promoter.
It is the one into which the IDNA fragment has been inserted.

(2) Preparation of Escherichia coli purine nucleoside phosphorylase Escherichia coli JM10 carrying plasmid pTrc-EPu
9 bacteria were added to 2 containing 100 mg / ml ampicillin.
The cells were inoculated into 300 ml of xYT medium and cultured with shaking at 37 ° C. When the culture reached 4 × 10 ⁸ bacteria / ml, IPTG was added to the culture solution to a final concentration of 1 mM.
The shaking culture was continued for 3 hours. After completion of the culture, the cells were collected by centrifugation (9,000 × g, 10 minutes),
ml of buffer (50 mM Tris-HCl (pH 7.5),
5 mM EDTA, 0.1% Triton X-100,
0.2 mg / ml lysozyme). 1 at 37 ° C
After keeping the temperature for a period of time, ultrasonic treatment was carried out to disrupt the cells, and the cell residues were removed by centrifugation (20,000 × g, 10 minutes). The supernatant fraction thus obtained was used as an enzyme preparation. Purine nucleoside phosphorylase activity in the enzyme preparation is shown in the following table together with a control bacterium (Escherichia coli JM109 strain carrying pTrc99A). The purine nucleoside phosphorylase activity in the present invention was calculated by measuring the phosphorylation activity of inosine at 50 ° C. according to the method of Yamauchi (JP-A-4-4882).

[0024]

[Table 1]

(3) Cloning of colorectal uridine phosphorylase gene Escherichia coli K12 strain JM109 (obtained from Takara Shuzo Co., Ltd.)
Of chromosomal DNA from Saito and Miura (Biochim. Biophy
s. Acta., 72, 619 (1963)). This DNA
Was used as a template, the following two types of primer DNAs were synthesized according to a conventional method, and the E. coli purine nucleoside phosphorylase (udp) gene was amplified by the PCR method.

Primer (A): 5′-AGTGTCTTTTTGCTTCTTCTGACT-3 ′ Primer (B): 5′-TACGCAAAAATACAAAAGGCCGAA-3 ′

The amplification of the udp gene by PCR is performed in 100 ml of a reaction solution (50 mM potassium chloride, 10 mM
Tris-HCl (pH 8.3), 1.5 mM magnesium chloride, 0.001% gelatin, temperate DNA 0.1
mg, primer DNA (A) and (B) 0.2 each
mM, AmpliTaq DNA polymerase (2.5
Unit) by Perkin-Elmer Cetus
Instrument DNA Thermal
Using a cycler, the steps of heat denaturation (94 ° C., 1 minute), annealing (55 ° C., 1.5 minutes), and polymerization (72 ° C., 1.5 minutes) were repeated 25 times.

After gene amplification, the reaction solution was treated with a phenol / chloroform (1: 1) mixed solution, and twice the volume of ethanol was added to the water-soluble fraction to precipitate DNA. The precipitated and recovered DNA was separated by agarose gel electrophoresis according to the method described in the literature (Molecular cloning, described above), and 850 was collected.
The DNA fragment corresponding to b was purified. Blunt the DNA
After blunting with ing kit (obtained from Takara Shuzo), the plasmid pUC18 (obtained from Takara Shuzo) and phosphorylated at the 5 'end with T4 polynucleotide kinase, and then treated with restriction enzymes SmaI and alkaline phosphatase, and T4 DNA Ligation was performed using ligase. Escherichia coli JM109 was transformed using the ligation reaction solution, and plasmid pU was transformed from the resulting ampicillin-resistant transformant.
C-UrdF was isolated. pUC-UrdF is pUC1
The DNA fragment containing the Escherichia coli udp gene was inserted in the SmaI cleavage site downstream of the lac promoter of No. 8 in the forward direction.

Next, the plasmid pUCEPuF was digested with restriction enzymes KpnI and SalI, and the obtained DNA fragment corresponding to 870b was separated and recovered by agarose gel electrophoresis. As with the DNA fragment, restriction enzymes KpnI and Spn
The plasmid pTrc99A (Phar
macia Biotech. From the company) and T4DN
Ligation was performed using A ligase. Escherichia coli JM109 was transformed using this ligation reaction solution, and plasmid pTrc-Urd was isolated from the resulting ampicillin-resistant transformant. pTrc-Urd is the trc of pTrc99A
KpnI-SalI DNA containing Escherichia coli udp gene in EcoRI-SalI digestion fragment downstream of promoter
The fragment was inserted.

(4) Preparation of E. coli uridine phosphorylase E. coli JM10 carrying plasmid pTrc-Urd
9 bacteria were added to 2 containing 100 mg / ml ampicillin.
The cells were inoculated into 300 ml of xYT medium and cultured with shaking at 37 ° C. When the culture reached 4 × 10 ⁸ bacteria / ml, IPTG was added to the culture solution to a final concentration of 1 mM.
The shaking culture was continued for 3 hours. After completion of the culture, the cells were collected by centrifugation (9,000 × g, 10 minutes),
ml of buffer (50 mM Tris-HCl (pH 7.5),
5 mM EDTA, 0.1% Triton X-100,
0.2 mg / ml lysozyme). After incubating at 37 ° C for 1 hour, sonication was performed to crush the cells,
Further, cell residue was removed by centrifugation (20,000 × g, 10 minutes). The supernatant fraction thus obtained was used as an enzyme preparation. The activity of uridine phosphorylase in the enzyme preparation was determined using the control bacteria (E. coli JM1 carrying pTrc99A).
09 bacteria) in the following table. The uridine phosphorylase activity in the present invention is determined by the method of Yamauchi (Japanese Unexamined Patent Publication No.
4882), the phosphorylation activity of uridine at 50 ° C. was measured and calculated.

[0031]

[Table 2]

(5) Synthesis of adenine arabinoside (ara A) 60 mM uracil arabinoside (ara U), 40 mM adenine, 20 mM potassium phosphate buffer (pH 7.0)
50 units (U) of Escherichia coli purine nucleoside phosphorylase and 25 U of Escherichia coli uridine phosphorylase prepared as described above were added to 5 ml, and reacted at 50 ° C. for 48 hours. As a result of qualitative and quantitative analysis by high performance liquid chromatography, 3
7.42 mM of ara A was produced (reaction yield: 93.6).
% Per adenine).

Example 2 Synthesis of 2,6-diaminopurine arabinoside (ara Dap) and guanine arabinoside 60 mM araU, 50 mM 2,6-diaminopurine, 2 mM
The same enzyme as in Example 1 was added to 5 ml of 0 mM potassium phosphate buffer (pH 7.0) in the same unit, and reacted at 50 ° C. for 65 hours. As a result of qualitative and quantitative analysis by high performance liquid chromatography, 48.18 mM ara Dap was produced (reaction yield: 96.4% vs. 2,6-diaminopurine). Further, it was also confirmed by high performance liquid chromatography that 50 U of Escherichia coli adenosine deaminase was added to 5 ml of the reaction solution, and all ara Dap produced by reacting at 40 ° C. for 5 hours was converted to guanine arabinoside.

Example 3 2-Fluoroadenine arabinoside (fludarabine) 2-Fluoroadenine was prepared with reference to US Pat. No. 5,180,824. That is, 42 g of 3 g of diaminopurine
Sodium nitrite was added to the HBF4 solution under ice cooling, followed by stirring. The reaction solution is diluted with water and adsorbed resin (elution solvent 5%
The obtained crystals were collected by filtration, and 1 g of 2-fluoroadenine (33% yield) was obtained. Next, 60 mM Ara U, 50 mM 2-fluoroadenine, 20 mM potassium phosphate buffer (pH 7.0),
The same enzyme as in Example 1 was added to 5 ml of 30% (v / v) dimethylsulfoxide in the same unit, and reacted at 50 ° C. for 65 hours. As a result of qualitative and quantitative analysis by high performance liquid chromatography, 32 mM fludarabine was produced (reaction yield: 64% vs. 2-fluoroadenine). The reaction solution was incubated in a boiling water bath to stop the enzymatic reaction, and then concentrated. The precipitated crystals were collected by filtration to obtain fludarabine.

Claims (4)

[Claims] 1. A method for producing purine arabinoside from purine and uracil arabinoside using nucleoside phosphorylase, wherein purine nucleoside phosphorylase and uridine phosphorylase are used in combination as nucleoside phosphorylase, and the use ratio of the two enzymes is an enzyme unit. A method for producing purine arabinoside enzymatically, wherein purine nucleoside phosphorylase is used as (unit) 1.5 times or more of uridine phosphorylase. 2. The method according to claim 1, wherein purine nucleoside phosphorylase is used in an amount of 1.5 to 100 times that of uridine phosphorylase as an enzyme unit (unit). 3. The method according to claim 1, wherein the ratio of the two enzymes used is 1.5 to 10 times the purine nucleoside phosphorylase as the uridine phosphorylase as the enzyme unit (unit). 4. The method according to claim 1, wherein the ratio of the use of the two enzymes is such that purine nucleoside phosphorylase is used 1.5 to 5 times as much as uridine phosphorylase as an enzyme unit.
The described method.