JP2002051781A - Method for enzymatically producing deoxynucleoside - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for producing a deoxynucleoside in a short time by efficiently producing a nucleoside deoxyribosyltransferase II. SOLUTION: This method for producing a deoxynucleoside comprises using nucleoside deoxyribosyltransferase II expressed by using a gene encoding an enzyme protein having a nucleoside deoxyribosyltransferase II activity.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a gene encoding nucleoside deoxyribosyltransferase-II, and a recombinant nucleoside deoxyribosyltransferase-II (Nucleoside) prepared using the gene.
deoxyribosyltransferase-II) and a method for producing deoxynucleosides using the enzyme.

[0002]

2. Description of the Related Art Deoxynucleosides are compounds useful as raw materials for various drugs including antisense drugs (oligomers of deoxynucleotides). Conventionally, these deoxynucleosides have been prepared by chemically synthesizing or enzymatically decomposing DNA such as milt. Furthermore, recently, methods for synthesizing deoxynucleosides using enzymes have been reported, and most of them are methods using nucleoside phosphorylase.

[0003] In addition to nucleoside phosphorylase, nucleoside deoxyribosyltransferase is one of the enzymes that can be used for the enzymatic synthesis of deoxynucleosides. Nucleoside deoxyribosyltransferase (EC2.4.2.6., Also known as trans-N-deoxyribosylase) is an enzyme that catalyzes a reaction to transfer a deoxyribosyl group of a deoxynucleoside to another base. Lactobacillus helveticus (Lactobacillus helveticus) and Lactobacillus leichmannii (Lactobacillus leichmannii), which belong to two types of lactic acid bacteria, belong to two types, nucleoside deoxyribosyltransferases I and II, and their enzymatic properties have already been reported. (Methods in Enzymology, Vol. 51.446 (1978),
Journal of Biotechnology, 23, 193-210 (1992), Biol.ch
em. Hoppe-Seyler, Vol. 377, 357-362 (1996)). In addition, the synthesis of 2 ', 3'-dideoxynucleosides and the like using nucleoside deoxyribosyltransferase has also been reported (WO90 / 06312, WO91 /).
04322, Biochemical And Biophysical Research Co
mmunications, Vol.155, No.2, 829-834 (1988)).

As described above, nucleoside deoxyribosyltransferase is an enzyme useful for enzymatic synthesis of deoxynucleosides. A crushed cell derived from a lactic acid bacterium belonging to the genus Lactobacillus or a cell lysate such as a cell wall derived from the crushed cell lactic acid The following problems have been pointed out in the conventional method in which a cell-free extract from which residues have been removed is used as an enzyme source as it is. (1) Lactic acid bacteria generally grow poorly, and it is difficult to prepare a large amount of cells. (2) Since nucleoside deoxyribosyltransferase is an intracellular enzyme, it is necessary to disrupt lactic acid bacteria. However, the cell wall of the lactic acid bacterium is hard, and the amount of nucleoside deoxyribosyltransferase produced in the cells is low. As a result, the extraction efficiency of nucleoside deoxyribosyltransferase is extremely low.

(3) In addition to nucleoside deoxyribosyltransferase, various enzymes such as deaminase and nucleoside phosphorylase coexist in the enzyme solution prepared from the cells. There is a high risk of lowering the yield of the deoxynucleoside of the synthetic target product (for example, when the target product is deoxyadenosine or deoxycytidine, the target product is deaminated when coexisting with adenosine deaminase or cytidine deaminase, and deoxyinosine or It is converted to deoxyuridine.
In the presence of phosphorylase, the synthesized deoxynucleoside is phosphorolyzed, resulting in a decrease in the synthesis yield of deoxynucleoside).

[0006]

Means for Solving the Problems In order to solve the above-mentioned problems, the present inventors have produced a large amount of nucleoside deoxyribosyltransferase by a genetic engineering technique and used it for the synthesis of deoxynucleoside. We thought that the problem could be solved and replaced nucleoside deoxyribosyltransferase with recombinant DN
A production was attempted by the A technique. However, conventionally,
Lactobacillus leichm
annii), the amino acid sequence corresponding to nucleoside deoxyribosyltransferase-II has been deduced (The Journal of Bioligical Chemistry,
Vol. 270, No. 26, 15551-15556, 15557-15562 (1995)), no report has been made on the nucleoside deoxyribosyltransferase gene, and nucleoside deoxyribosyltransferase obtained by genetic engineering techniques has not been reported. There is no report that it was used for the synthesis of deoxynucleosides. As a result of repeated trial and error, the present inventors succeeded in cloning a gene encoding nucleoside deoxyribosyltransferase-II derived from Lactobacillus helveticus, and produced a specific nucleoside deoxyribosyltransferase produced using this gene. By using -II, all the disadvantages of the above-mentioned conventional method were overcome, and it was found that deoxynucleosides could be efficiently synthesized, and the present invention was completed.

That is, the present invention relates to a recombinant nucleoside deoxyribosyltransferase-II having the amino acid sequence shown in SEQ ID NO: 1; and one or several amino acids are deleted or substituted in the amino acid sequence shown in SEQ ID NO: 1. , A modified nucleoside deoxyribosyltransferase-II having a modified or added amino acid sequence.

[0008] The present invention also relates to a gene encoding nucleoside deoxyribosyltransferase-II having the amino acid sequence shown in SEQ ID NO: 1; comprising the nucleotide sequence shown in SEQ ID NO: 2 and encoding nucleoside deoxyribosyltransferase-II. A gene that
In the base sequence shown in SEQ ID NO: 2, a gene consisting of a base sequence in which one or more bases have been deleted, substituted, inserted or added, and encoding nucleoside deoxyribosyltransferase-II, and such a gene And a gene encoding nucleoside deoxyribosyltransferase-II.

Further, the present invention provides a method for producing deoxynucleosides using nucleoside deoxyribosyltransferase-II.
The present invention relates to a method for producing deoxynucleoside, characterized in that any one of the following (a) and (b) is used as deoxyribosyltransferase-II. (A) Recombinant nucleoside deoxyribosyltransferase-I having the amino acid sequence shown in SEQ ID NO: 1
I, (b) a recombinant nucleoside deoxyribosyltransferase-II having an amino acid sequence in which one or several amino acids have been deleted, substituted, modified or added in the amino acid sequence shown in SEQ ID NO: 1.

[0010] Furthermore, the present invention relates to a method for producing deoxynucleoside using nucleoside deoxyribosyltransferase-II, wherein the nucleoside deoxyribosyltransferase-II is a gene described in the following (c) to (g): The present invention relates to a method for producing deoxynucleoside using a recombinant nucleoside deoxyribosyltransferase-II prepared using any one of the above. (C) a gene encoding nucleoside deoxyribosyltransferase-II having the amino acid sequence shown in SEQ ID NO: 1; (d) a gene consisting of the nucleotide sequence shown in SEQ ID NO: 2; A gene in which one or a plurality of bases have been deleted, substituted, inserted or added; (f) a gene which hybridizes under stringent conditions to the gene described in the above (d) or (e); A gene further comprising an SD sequence upstream of the gene described in (e) or (f).

[0011]

BEST MODE FOR CARRYING OUT THE INVENTION The nucleoside deoxyribosyltransferase-II of the present invention is an enzyme having at least the amino acid sequence represented by SEQ ID NO: 1, is substantially pure in terms of enzymatic activity, and is disadvantageous for deoxynucleoside synthesis. Does not possess nucleoside phosphorylase or deaminase activity. Here, nucleoside deoxyribosyltransferase-II refers to the following:
Refers to an enzyme that catalyzes one or several reactions of

<Reaction Catalyzed by Nucleoside Deoxyribosyltransferase-II> dRib-Pur (1) + Pur (2) = dRib-
Pur (2) + Pur (1) dRib-Pur + Pyr = dRib-Pyr + Pu
r dRib−Pyr (1) + Pyr (2) = dRib−
Pyr (2) + Pyr (1) (in the formula, dRib: deoxyribosyl group, Pur: purine base, Pyr: pyrimidine base)

The recombinant nucleoside deoxyribosyltransferase-II of the present invention having the amino acid sequence shown in SEQ ID NO: 1 has one or several amino acids deleted as long as it maintains the activity of catalyzing the above reaction.
It may be substituted, modified or added. The deletion, substitution, modification or addition of the amino acid sequence described above can be performed by site-directed mutagenesis, which is a well-known technique before filing the application, for example, Proc. Natl. Acad. Sci. USA, 81, 4662. −56
66 (1984), Nucleic Acid Res. 10,6487-6500 (1982),
WO85 / 00817; Nature 316, 601-605 (1985) and the like. The deletion, substitution, modification or addition of one or several amino acids means deletion, substitution, or substitution by a well-known method such as site-directed mutagenesis.
It means that a sufficient number of amino acids that can be modified or added are deleted, substituted, modified or added.

The recombinant nucleoside deoxyribosyltransferase-II having the amino acid sequence shown in SEQ ID NO: 1 is an enzyme derived from Lactobacillus helveticus. The enzyme is nucleoside deoxyribosyltransferase-II among enzymes derived from Lactobacillus. High activity and particularly preferred. Such a nucleoside deoxyribosyltransferase-II is
It can be prepared using a gene encoding an enzyme having the amino acid sequence shown in SEQ ID NO: 1, specifically, a gene consisting of the base sequence shown in SEQ ID NO: 2. For example,
Lactobacillus helvetica
icus) A gene derived from ATCC8018 will be described as a specific example. FIG. 2 shows HincI in the restriction enzyme map shown in FIG.
2 shows the result of analyzing a part of the base sequence of the DNA fragment cleaved by I, and shows base numbers 74 to 547 in FIG.
The second sequence corresponds to the structural gene of nucleoside 2'-deoxyribosyltransferase-II, and is the same as the nucleotide sequence shown in SEQ ID NO: 2 above.

In the present invention, as long as nucleoside deoxyribosyltransferase-II can be produced, one or more bases in the base sequence represented by SEQ ID NO: 2 are deleted, substituted, inserted or added. Genes which have been used, or genes which hybridize with those genes under stringent conditions, can also be used. In addition, a gene in which one or more bases have been deleted, substituted, inserted or added, as in the case of the amino acid sequence described above, deletion, substitution, by a known method such as site-directed mutagenesis. It means that the number of bases that can be modified or added is deleted, substituted, modified or added. In addition, the stringent condition is 5 ×
SSC (1 × SSC is 8.76 g of sodium chloride and 4.41 g of sodium citrate dissolved in 1 liter of water), 0.1% w / v N-lauroyl sarcosine.
Sodium salt, 0.02% w / v SDS, 0.5%
Using a solution containing w / v blocking reagent,
This means that the hybridization reaction is performed under a reaction temperature condition for about 0 hours.

Furthermore, in the present invention, an SD sequence (Shine-Dalgarno Sequencnc) is added upstream of the gene encoding nucleoside deoxyribosyltransferase-II.
Genes comprising e) can also be used, and the use of such genes is advantageous in that the production of enzymes can be significantly increased. If such a gene is specifically described with reference to FIG. 2, a gene having at least the sequence represented by base numbers 62 to 547 in FIG. 2 can be exemplified.

The cloning of such a gene, the preparation of an expression vector using the cloned DNA fragment, and the preparation of nucleoside deoxyribosyltransferase-II using the expression vector are performed by those skilled in the field of molecular biology. Is a well-known technique. Specifically, for example, “Molecular Cloning” (ed. By Maniatis et al., Col.
d Spring Harbor Laboratories, Cold Spring Harbor,
New York (1982)). For example, as shown in FIG. 3, a part of the amino acid sequence such as the N-terminal and C-terminal of nucleoside deoxyribosyltransferase-II purified from a microorganism belonging to the genus Lactobacillus is determined by a known method and corresponds to the determined amino acid sequence. Synthesize the oligonucleotide. A DNA fragment containing a gene encoding nucleoside deoxyribosyltransferase-II may be cloned from chromosomal DNA of a lactic acid bacterium belonging to the genus Lactobacillus using the synthesized oligonucleotide as a probe. The host used for cloning is not particularly limited, but Escherichia coli is suitably used as the host because of operability and simplicity.

In order to construct a high expression system for the cloned gene, for example, the method of Maxam-Gilbert (Me
thods in Enzymology, 65, 499 (1980)) or the dideoxy chain terminator method (Methods in Enzym).
, 101, 20 (1983)) to identify the coding region of the gene by analyzing the nucleotide sequence of the cloned DNA fragment, and self-express the gene in the microbial cells according to the host microorganism. A recombinant expression vector in which expression control signals (transcription initiation and translation initiation signals) are ligated upstream thereof is prepared so as to be possible.

As an expression control signal used for producing nucleoside deoxyribosyltransferase-II in a large amount in a heterologous microorganism, artificial control is possible, and the production amount of nucleoside deoxyribosyltransferase-II can be dramatically reduced. It is desirable to use strong transcription initiation and translation initiation signals that increase. Such a transcription initiation signal includes a lac promoter, tr
p promoter, tac promoter (Proc. Natl. Ac
ad. Sci. USA., 80, 21 (1983), Gene, 20, 231 (198
2)), trc promoter (J. Biol. Chem., 260, 35)
39 (1985)), and glyceraldehyde-3-phosphate dehydrogenase (J.
Biol. Chem. , 254, 2078 (1980)) and inhibitory acid phosphatase (Nucl. Acids Res., 11, 1657 (1983)).

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 having a high copy number of Specifically, when E. coli is used as a host, pBR322 (Gene,
2, 95 (1975)), pUC18, pUC19 (Gene, 3
3, 103 (1985)). Also,
When yeast is used as a host, YEp13 (ATCC3711
5), YEp24 (ATCC37051) and the like.

A microorganism is transformed using the recombinant vector thus prepared. The host microorganism is not particularly limited as long as it is highly safe and easy to handle. For example,
Microorganisms commonly used for DNA recombination operations, such as Escherichia coli and yeast, can be used. Among them, Escherichia coli is advantageous in handling and in deoxynucleoside synthesis,
For example, strain K12, C60 used for recombinant DNA experiments
0 bacteria, JM105 bacteria, JM109 bacteria (Gene, 33, 103-119
(1985)) 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 the cells by treating with calcium chloride at a low temperature (J. Mo.
l. Biol. , 53, 159 (1970)). When using yeast as a host,
Protoplast method (Proc. Natl. Acad. Sci. USA, 75,
1929 (1978)) or the alkali metal treatment method (J. Bact
eriol. 153, 163 (1983)).

The obtained transformant is grown in a medium in which the microorganism can grow, and further induces the production of cloned nucleoside deoxyribosyltransferase-II to accumulate a large amount of the enzyme in the cells. Cultivate until 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 Escherichia coli is used as a host, a 2xYT medium (Methods
in Enzymology, 100, 20 (1983)), LB medium, M9C
Using a medium commonly used for culturing Escherichia coli, such as an A medium (Molecular Cloning, described above), culturing can be carried out at a culturing temperature of 20 to 40 ° C. with aeration and stirring as needed. When a plasmid is used as a vector, an appropriate amount of an antibiotic (ampicillin, kanamycin, etc., depending on the drug resistance marker of the plasmid) is added to the culture solution to prevent the omission of the plasmid during the culture. I do.

When it is necessary to induce the expression of the nucleoside deoxyribosyltransferase-II gene during the culture, the expression of the gene is induced by a method commonly used for the promoter used. For example, when a lac promoter or a tac promoter is used, an appropriate amount of isopropyl-β-D-thiogalactopyranoside (hereinafter abbreviated as IPTG), which is an expression inducer, is added at the middle stage of the culture. When the promoter used has constitutive transcription activity, it is not necessary to add an expression inducing agent.

The bacterial cells are recovered from the thus prepared culture by membrane separation or centrifugation. The recovered cells can be used as nucleoside deoxyribosyltransferase-II, but the recovered cells are suspended in an appropriate buffer.
The cells are physically crushed by ultrasonic treatment, French press treatment, or lysed enzymatically by lysozyme treatment, etc., and the cell residue is removed by centrifugation to prepare a cell-free extract. It is preferable to use the extract as nucleoside deoxyribosyltransferase-II. Since nucleoside deoxyribosyltransferase-II is excessively present in the cell-free extract, it can be used as an enzyme source without any particular purification treatment. Furthermore, a crudely purified product or a purified product obtained by a single treatment or a combination of several treatments commonly used for enzyme purification such as heat treatment, ammonium sulfate salting-out treatment, dialysis treatment, treatment with a solvent such as ethanol, and various chromatographic treatments. May be used as nucleoside deoxyribosyltransferase-II.

The production of the deoxynucleoside of the present invention comprises
It is characterized by using the above specific nucleoside deoxyribosyltransferase-II which does not have nucleoside phosphorylase or deaminase activity which is substantially pure in enzymatic activity and which is not favorable for deoxynucleoside synthesis. The reaction conditions and the like can be performed according to a conventionally known method (for example, see International Patent Publication WO90 / 10080). That is, the acceptor of the deoxy sugar residue used in the reaction may be variously selected from known bases depending on the base of the deoxynucleoside to be synthesized, and specifically, a heterocyclic base or a substituent (for example, amino group). Derivatives having a group, substituted amino group, hydroxyl group, oxo group, mercapto group, acyl group, alkyl group, substituted alkyl group, alkoxyl group, halogen atom, etc.). Specific examples of the heterocyclic base include, for example, purine and its derivatives, pyrimidine and its derivatives, triazole and its derivatives, imidazole and its derivatives, deazapurine and its derivatives, azapurine and its derivatives, azapyrimidine and its derivatives or pyridine and its derivatives Derivatives.

Specifically, as the purine base and its derivatives, adenine, guanine, hypoxanthine, xanthine, 6-mercaptopurine, 6-thioguanine, N ⁶
Pyrimidines and derivatives thereof such as cytosine, uracil, thymine, and 5-halogenouracil (such as 5-fluorouracil and 5-iodouracil), such as -alkyl or acyladenine, 2-alkoxyadenine, 2-thioadenine, and 2,6-diaminopurine; , 5-halogeno-cytosine (such as 5-fluorocytosine), 5-trihalogenomethyl uracil (5-trifluoromethyl uracil, etc.), 2-thiocytosine, 4-thiouracil, N ⁴ - acyl, 5- halogenoalkyl vinyluracil like, Triazole and its derivatives include 1,2,4-triazole-3-carboxamide, 1,2,4-triazole-3-carboxylic acid, 1,2,4-triazole-3
-Imidazole and its derivatives such as alkyl carboxylate include 5-amino-4-imidazolecarboxamide, 4-carbamoyl-imidazolium-5
Oleates, benzimidazoles and the like, and deazapurines and derivatives thereof such as 1-deazaadenine, 3-deazaadenine, 7-deazaadenine and 7-deazaguanine; and azapurines and derivatives thereof such as 8-azaadenine and 7-deaza-8-azahypoxanthine ( Azapyrimidine and its derivatives such as 5-azathymine, 5-azacytosine and 6-azauracil; or pyridine and its derivatives as 3
-Deazauracil, nicotinic acid, nicotinamide and the like.

The donor of the deoxy sugar residue may be selected according to the desired deoxynucleoside, and specifically, 2′-deoxyadenosine, 2′-deoxyinosine, 2′-deoxyguanosine, 2'-deoxynucleosides such as' -deoxyuridine, thymidine, 2'-deoxyxanthosine, 3'-deoxynucleosides such as 3'-deoxyinosine and cordycepin, 2 ', 3'-dideoxyinosine, 2' And 3'-dideoxyadenosine, 2 ', 3'-dideoxyguanosine, 2', 3'-dideoxyuridine, and 2 ', 3'-dideoxynucleosides such as 3'-deoxythymidine.

The synthesis reaction of deoxynucleosides is carried out in 0.1.
Using nucleoside deoxyribosyltransferase-II of 001 unit / ml or more, preferably 0.01 unit / ml or more, and accepting and donating a deoxy sugar residue to a concentration of 1 mM to 1 M, preferably 50 to 500 mM. The body is dissolved or suspended in water or a buffer solution (pH 3 to 10), and 10 to 60 ° C., preferably 20 to 5 ° C.
The reaction can be carried out under a temperature condition of 0 ° C. for about 10 minutes to 50 hours with stirring as necessary. When the reaction temperature is lower than 10 ° C., the reaction rate is low and the reaction efficiency is poor. On the other hand, when the reaction temperature exceeds 60 ° C., the enzyme protein may be denatured or deactivated, which is not preferable. When the pH fluctuates during the reaction, the pH may be corrected to the above range using an acid or an alkali.

After completion of the reaction, the resulting deoxynucleoside can be separated and purified by a method generally used as a method for purifying nucleic acid-related substances or a method utilizing the same. For example, ion exchange chromatography,
Various types of chromatography such as adsorption chromatography, partition chromatography, and gel filtration, counter-current distribution, counter-current extraction, and other methods that use partitioning between two liquid phases, as well as concentration, cooling, and organic solvent addition, etc. A general separation and purification method used in separation and purification of deoxynucleosides, such as a method utilizing the difference, may be performed alone or in an appropriate combination.

[0030]

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. Preparation of DNA, cleavage with restriction enzymes, ligation of DNA with T4 DNA ligase, and transformation of Escherichia coli in the Examples were all performed according to "Molecular Cloning" (described above). In addition, various restriction enzymes, T4D
NA ligase, plasmid vectors pHSG398 and pHSG399 were all obtained from Takara Shuzo. A DNA labeling kit and a DNA detection kit for Southern hybridization were obtained from Boehringer Mannheim.

[0031]

Example 1 (1) Cloning of nucleoside deoxyribosyltransferase-II structural gene and determination of nucleotide sequence Lactobacillus helvetica (Lactobacillus helvetica)
icus) A purified enzyme preparation of nucleoside deoxyribosyltransferase-II (200 pmol: 4 μg) prepared from ATCC8018 by a conventional method was denatured at 37 ° C. for 1 hour in the presence of urea having a final concentration of 8 M, and trypsin (TaKaRa / TaKaRa). After treatment with Residue-specific Proteases Kit), the DNA was transferred to a PVDF membrane according to a standard method. The transcribed bands (two) were cut out and subjected to N-terminal amino acid sequence analysis. Based on the nucleotide sequence deduced from the obtained amino acid sequence, primers for PCR were designed as follows.

Primer (A): 5-CCTCTGCAGCCATGAAYA
ARAARAARACNYTNTAYTTYGG-3 Primer (B): 5-ACCAAGCTTRTTRTGIARRTAYTCIGGRTG
YTCRTC-3 (in the above primer sequence, Y is C or T
R represents A or G, N represents A, C, G or T;
Represents 2′-deoxyinosine, respectively. )

Amplification of a gene by PCR is performed in the reaction solution 10
In 0 μl [50 mM potassium chloride, 10 mM Tris-HCl (pH 8.3), 1.5 mM magnesium chloride, 0.0
01% gelatin, 0.2 mM dATP, 0.2 mM dG
TP, 0.2 mM dCTP, 0.2 mM dTTP, 0.1 μg of lactic acid bacteria chromosomal DNA, primer DNA (A)
(B) 5.0 μM each, 2.5 units of AmpliTaq DNA polymerase] and Perkin-Elmer
DNA Th manufactured by Cetus Instrument
Thermal denaturation (94 ° C.,
30 seconds), annealing (40 ° C., 30 seconds), and extension reaction (72 ° C., 30 seconds) were repeated 28 times. A probe for Southern hybridization was prepared using a mixed solution containing dNTP and DIG-labeled UTP under the above PCR conditions. Lactic acid bacterium chromosomal DNA was digested with various restriction enzymes, electrophoresed, and then subjected to Southern hybridization according to a conventional method. From these results, a restriction map as shown in FIG. 1 was obtained.

As shown in FIG. 1, the nucleoside deoxyribosyltransferase-II gene (ndtB g
ene) is SalI-EcoRI of chromosomal DNA of lactic acid bacteria.
Was found to be present in the 3.2 kb fragment. Therefore, 2.3- of SalI and EcoRI digests of the lactic acid bacteria chromosome.
The 4.0 kb fragment was recovered and this was
PCR was performed using the DNA ligated with the I and EcoRI digests as the template DNA. The primers used were primer (B) and sequencing primer M4 (manufactured by Takara Shuzo) under the above conditions at 94 ° C. for 30 seconds, 40 ° C. for 30 seconds,
The cycle of 72 ° C. for 120 seconds was repeated 30 times. The obtained DNA fragment was digested with SalI and HindIII and then incorporated into a cloning vector pHSG398. This plasmid was designated as p398-5SH.

From the nucleotide sequence analysis of the upstream region of the obtained ndtB gene, a primer located upstream of the initiation codon of the nucleoside deoxyribosyltransferase-II gene was designed as follows. Primer (C): 5-TAAGTCGACAGCAATTTTTATGGGGAG-3 DN obtained by ligating chromosomal DNA of lactic acid bacteria after EcoRI digestion
A DNA fragment containing the nucleoside deoxyribosyltransferase-II gene derived from lactic acid bacteria was amplified by PCR using A as a template and primer (C).

The PCR reaction

Using the same reaction composition and reactor as described above, heat denaturation (94 ° C., 30 seconds), annealing (50 ° C.
Sec) and the extension reaction (72 ° C, 2 minutes) were repeated 30 times. After gene amplification, SalI and E
After digestion with coRI, a 1.0 kb DNA fragment was purified. The recovered DNA was subjected to restriction enzymes SalI and EcoRI.
PHSG398 or pHSG3 digested with
Ligation was performed using 99 and T4 DNA ligase. Escherichia coli JM109 was transformed using the ligation reaction solution, and plasmids p398-T2F5 and p399-T2F5 were isolated from the obtained chloramphenicol-resistant transformants.
Each of the obtained transformants was JM109 [p398-
T2F5] and JM109 [p399-T2F5].

The DNA base sequences of the inserts of the clones of these recombinant plasmids were determined by the dideoxy chain terminator method (Science, 214, 1295 (1981)). As a result, the D of the nucleoside deoxyribosyltransferase-II structural gene shown in FIG.
The NA base sequence was obtained. This nucleotide sequence is 474 bp and encodes a polypeptide having a molecular weight of 18,317 consisting of 158 amino acids starting with Met. The amino acid sequence of the amino terminal 10 amino acids of this peptide completely coincided with that of the purified and isolated lactic acid bacterium nucleoside deoxyribosyltransferase-II.

(2) Preparation of recombinant vector for nucleoside deoxyribosyltransferase-II production Nucleoside deoxyribosyltransferase-
A recombinant vector for production II, pTrc-T2F4, was prepared by the following method (see FIG. 3). That is, p399-T
Using 2F5 as a template and the following primer (D) and sequencing primer M4 (manufactured by Takara Shuzo), a DNA fragment containing the nucleoside deoxyribosyltransferase-II gene derived from lactic acid bacteria was amplified by PCR. In addition, a BspHI recognition sequence was introduced into the primer (D) so as to link to the NcoI site of the vector. Primer (D): 5-AAATCATGAACAAGAAAAAGACTTTATAT-
Three

The PCR reaction uses the same reaction composition and reactor as in (1), and includes the steps of heat denaturation (94 ° C., 30 seconds), annealing (50 ° C., 30 seconds), and extension reaction (72 ° C., 2 minutes). Performed by repeating 30 times. After gene amplification, digestion with BspHI and EcoRI resulted in a 1.0 kb DN
The A fragment was purified. The recovered DNA is used as a restriction enzyme Nco
Plasmid pTrc99A digested with I and EcoRI
(Gene, 69, 301 (1988), obtained from Pharmacia) and ligated using T4 DNA ligase. Escherichia coli JM109 strain was transformed using the ligation reaction solution, and a recombinant vector in which a nucleoside deoxyribosyltransferase-II structural gene was inserted immediately after the trc promoter for expressing pTc99A from the obtained ampicillin-resistant transformant, pTrc-T2F4 was isolated.
The resulting transformant was transformed into JM109 [pTrc-T2F
4].

(3) Culture of Transformant and Preparation of Enzyme Escherichia coli transformant carrying the above recombinant vector was
20 μg / ml chloramphenicol or 100 μg
2xYT medium 100 containing g / ml ampicillin
The mixture was inoculated into a ml, and cultured with shaking at 37 ° C. 4 × 10 ⁸ /
At the time when the volume reached 100 ml, IPTG was added to the culture solution to a final concentration of 0.05 mM, and shaking culture was further continued at 37 ° C for 4 hours. After completion of the culture, centrifugation (9,000 ×
g, 10 minutes), and collect 10 ml of buffer (10 mM Tris-HCl (pH 7.8), 1 mM E
DTA). The cell suspension was treated with an ultrasonic crusher, and the cell residue was removed by centrifugation (12,000 × g, 10 minutes). The supernatant fraction thus obtained was used as a cell-free extract. Nucleoside deoxyribosyltransferase-II activity in the cell-free extract is shown in the following table together with a control bacterium (Escherichia coli JM109 carrying pHSG398).

The nucleoside deoxyribosyltransferase-II activity was measured and calculated using 5 mM deoxyadenosine and cytosine or thymidine and cytosine as substrates. That is, a cell-free extract is added to the reaction solution to start the reaction, and when the reaction is stopped, 1
Inactivate the enzyme by boiling for a minute. The amount of deoxycytidine produced at 37 ° C. was quantified by high performance liquid chromatography, and the activity of producing 1 μmole of deoxycytidine per minute at 37 ° C. was 1 unit (unit).
And

[0043]

[Table 1] * 1 unit = 1μmole 2'-deoxycytidine-production fro
m 2'-deoxyadenosine / min at 37 ℃ ** 1 unit = 1μmole 2'-deoxycytidine-production fr
om thymidine / min at 37 ℃

As shown in Table 1, the activity of nucleoside deoxyribosyltransferase-II was confirmed in the transformant containing the prepared recombinant vector, but the control bacterium (Escherichia coli JM109 carrying pHSG398) was used.
Was not detected. In addition, as shown in Table 2 below, the transformant (pTrc-T2
F4-bearing bacterium) shows the productivity of the original strain of lactic acid bacterium Lactobacillus helveticus ATCC.
This corresponds to 3,000 to 4,000 times the 8018 strain. In addition,
Nucleoside deoxyribosyltransferase-II prepared from an Escherichia coli transformant carrying this recombinant vector had properties equivalent to those of enzymes derived from lactic acid bacteria.

[0045]

[Table 2] * 1 unit = 1μmole 2'-deoxycytidine-production fr
om 2'-deoxyuridine / minat 37 ℃

(4) Synthesis of 2′-deoxycytidine The above transformant JM109 was added to 1 ml of an aqueous solution containing 20 mM of 2′-deoxyuridine and 20 mM of cytosine.
A cell-free extract equivalent to 1 ml of a culture solution of [pTrc-T2F4] was added and reacted at 37 ° C. for 1 hour. As a result of measuring the amount of 2′-deoxycytidine produced by the same method as in the above (3), 13.5 mM of 2′-deoxycytidine was synthesized. Further, as a result of reaction at 37 ° C. for 18 hours using a cell-free extract equivalent to 2.5 μl of the culture solution, 13
mM of 2'-deoxycytidine was synthesized. In addition, 20 mM 2'-deoxyuridine which is a conventional method and 20 mM
As a comparative example, a cell-free extract equivalent to 1 ml of a culture solution of the lactic acid bacterium Lactobacillus helveticus ATCC8018 was added to 1 ml of an aqueous solution containing M cytosine, and the reaction was carried out at 37 ° C. for 8 days. 2'-Deoxycytidine was synthesized. FIG. 4 shows the time course of the 2′-deoxycytidine synthesis reaction in the method of the present invention and the conventional method. Thus, in the present invention, it was confirmed that a small amount of cell-free extract was used to synthesize deoxynucleoside in a short time. In addition, in the case of the conventional method, the reaction time was long, so that it was observed that the synthesized 2′-deoxycytidine was decomposed, but in the present invention, no decomposition of 2′-deoxycytidine was observed.

[0047]

Example 2 Synthesis of 2′-deoxycytidine (2) Transformant JM109 [pTrc-T2F4] prepared in Example 1 was added to 1 ml of an aqueous solution containing 1 mM of 2′-deoxyadenosine and 1 mM of cytosine. 6 μl of a 200-fold diluted cell extract was added (9 unit / L)
Reaction solution) and reacted at 37 ° C. for 12 minutes. As a result of measuring the production rate of 2′-deoxycytidine, 0.2 mM of 2′-deoxycytidine (20% ratio of 2′-deoxycytidine to 2′-deoxycytidine) was synthesized.

[0048]

Example 3 Synthesis of 2′-deoxycytidine (3) The transformant JM109 [pTrc-T2F4] prepared in Example 1 was added to 1.5 L of an aqueous solution containing 133 mM of 2′-deoxyuridine and 133 mM of cytosine. 0.45 ml of the derived cell-free extract (9 unit / L reaction solution) was added and reacted at 37 ° C. for 18 hours. As a result of measuring the production rate of 2′-deoxycytidine, 72 mM 2′-deoxycytidine was obtained.
Deoxycytidine (to 2'-deoxyuridine ratio 54
%) Was confirmed to be synthesized.

[0049]

Example 4 Synthesis of Thymidine A cell-free extract derived from the transformant JM109 [pTrc-T2F4] prepared in Example 1 was added to 5 ml of an aqueous solution containing 100 mM 2'-deoxyuridine and 100 mM thymine. Add 8 μl (0.9 unit / L reaction solution)
Then, the reaction was carried out at 37 ° C. for 18 hours. As a result of measuring the thymidine generation rate, 48 mM thymidine (48% of 2'-deoxyuridine ratio) was synthesized.

[0050]

Example 5 Synthesis of 2'-deoxyadenosine Cell-free extraction from the transformant JM109 [pTrc-T2F4] prepared in Example 1 in 5 mL of an aqueous solution containing 100 mM 2'-deoxyuridine and 100 mM adenine 0.05 ml of the solution was added (15 unit / L reaction solution) and reacted at 37 ° C. for 20 hours. As a result of measuring the production rate of 2'-deoxyadenosine, 2'-deoxyadenosine was synthesized at a ratio of 2% to 2'-deoxyuridine of 77%.

[0051]

Example 6 Synthesis of 2′-deoxyguanosine Cell-free extraction from the transformant JM109 [pTrc-T2F4] prepared in Example 1 in 5 mL of an aqueous solution containing 100 mM 2′-deoxyuridine and 100 mM guanine 0.05 ml of the solution was added (15 unit / L reaction solution) and reacted at 37 ° C. for 20 hours. As a result of measuring the production rate of 2′-deoxyguanosine, 2′-deoxyguanosine was synthesized at a ratio of 2% to 2′-deoxyuridine of 30%.

[0052]

Example 7 Synthesis of 2′-deoxyguanosine (2) Transformant JM109 [pTrc-T2F4] prepared in Example 1 was added to 5 mL of an aqueous solution containing 10 mM of 2′-deoxyadenosine and 10 mM of guanine. Add 0.005 ml of bacterial cell extract (15 unit / L reaction solution)
Then, the reaction was carried out at 37 ° C. for 20 hours. As a result of measuring the production ratio of 2'-deoxyguanosine, 2'-deoxyguanosine was synthesized at a ratio of 2'-deoxyadenosine to 30%. In addition, 2'-deoxyinosine was not detected from the reaction solution.

[0053]

According to the conventional method for synthesizing deoxynucleosides using lactic acid bacteria prepared from lactic acid bacteria as an enzyme source, in addition to the low abundance of nucleoside deoxyribosyltransferase from the cells, the extraction efficiency is poor, and therefore the object of the present invention is to achieve the object. The enzyme activity is extremely low, a large amount of enzyme is required, and the substrate concentration cannot be increased. As a result, the synthesis efficiency of deoxynucleoside is extremely low, and it cannot be a practical method at all. Further, there is an inherent problem that the yield of deoxynucleoside is reduced due to a side reaction caused by other mixed enzymes. However,
According to the present invention, nucleoside deoxyribosyltransferase-II can be efficiently produced and a large amount of an enzyme having a high activity can be obtained. This is a very practical method that allows efficient synthesis of deoxynucleosides, particularly 2'-deoxynucleosides. Furthermore, since the recombinant nucleoside deoxyribosyltransferase-II of the present invention is enzymatically pure, side reactions are scarcely observed even when a crudely purified enzyme such as a cell-free extract is used, and the problem of the conventional method is problematic. It is a way to overcome the point.

[0054]

[Sequence List] SEQUENCE LISTING <110> YAMASA CORPORATION <120> Process for the enzymatically preparation of deoxynucleosides <130> YP2000-017 <140> <141> <160> 6 <170> PatentIn Ver. 2.1 <210> 1 <211 > 158 <212> PRT <213> Lactobacillus helveticus <400> 1 Met Asn Lys Lys Lys Thr Leu Tyr Phe Gly Ala Gly Trp Phe Asn Glu 1 5 10 15 Lys Gln Asn Lys Ala Tyr Lys Glu Ala Met Ala Ala Leu Lys Glu Asn 20 25 30 Pro Thr Val Asp Leu Glu Asn Ser Tyr Val Pro Leu Glu Asn Gln Tyr 35 40 45 Lys Gly Ile Arg Ile Asp Glu His Pro Glu Tyr Leu His Asn Ile Glu 50 55 60 Trp Ala Ser Ala Thr Tyr His Asn Asp Leu Val Gly Ile Lys Thr Ser 65 70 75 80 Asp Val Leu Leu Gly Val Tyr Leu Pro Gln Glu Glu His Val Gly Leu 85 90 95 Gly Met Glu Leu Gly Tyr Pro Leu Ser Gln Gly Lys Leu Phe Phe Trp 100 105 110 Phe Ser His Met Lys Asp Tyr Gly Lys Pro Ile Ile Leu Met Ser Trp 115 120 125 Gly Val Cys Asp Asn Ala Ser Gln Ile Ser Glu Leu Lys Asp Phe Asp 130 135 140 Phe Asn Lys Pro Arg Tyr Asn Phe Tyr Asp Gly Ala Val Tyr 145 150 155 <210> 2 <211> 474 <212> DNA <213> Lactobacillus helveticus <400> 2 atgaataaaa agaagacgct gtactttggt gccggttggt ttaatgaaaa gcaaaacaaa 60 gcttacaaag aagcaatggc agctttaaaa gaaaatccaa cagttgattt agaaaatagt 120 tatgtgcccc ttgaaaacca atacaagggt attcgcattg atgagcatcc agagtacttg 180 cacaacattg aatgggcttc tgcaacctac cacaatgatt tagtaggaat taagacttct 240 gatgtcctgc ttggcgtata tctgccacaa gaagaacacg tcggcttagg catggaactg 300 ggctacccat tatctcaagg aaaattattt ttttggtttt cccatatgaa agattacggc 360 aagccaatca tcttaatgag ctggggcgtt tgtgacaatg ccagtcagat cagtgaattr <filer> <a> <a> ndtB gene <400> 3 cctctgcagc catgaayaar aaraaracny tntayttygg 40 <210> 4 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> primer for amplification of ndtB gene <400> 4 accaagcttr ttrtgnarrt aytcnggrtg ytcrtc 36 < 210> 5 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer for amplification of ndtB gene <400> 5 taagtcgaca gcaattttta tggggag 27 <210> 6 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> primer for amplification of ndtB gene <400> 6 aaatcatgaa caagaaaaag actttatat 29

[0055]

[Brief description of the drawings]

FIG. 1 shows a 4.3 kb EcoRI-EcoRI containing the nucleoside deoxyribosyltransferase-II structural gene from the lactic acid bacterium Lactobacillus helveticus ATCC8018.
1 shows a restriction map of a DNA fragment. Figure 1
"NdtB" is a gene encoding nucleoside deoxyribosyltransferase-II (47
4bp, molecular weight 18,31 consisting of 158 amino acids
7 encoding 7 polypeptides).

FIG. 2 shows the nucleotide sequence of a DNA fragment containing a nucleoside deoxyribosyltransferase-II structural gene derived from Lactobacillus helveticus ATCC8018. In FIG. D. Indicates the SD sequence, Met indicates the translation initiation codon of the nucleoside deoxyribosyltransferase-II structural gene, and stop indicates the stop codon.

FIG. 3 shows a recombinant plasmid vector, pTrc.
Fig. 3 shows a method for constructing T2F4.

FIG. 4 shows a time course of a 2′-deoxycytidine synthesis reaction using 2′-deoxyuridine and cytosine.

Continued on the front page (51) Int.Cl. ⁷ Identification FI FI Theme Court II (Reference) (C12N 9/12 (C12P 19/38 C12R 1: 225) C12R 1:19) (C12P 19/38 C12N 15/00 ZNAA C12R 1:19) C12R 1:19)

Claims (10)

[Claims] 1. A recombinant nucleoside deoxyribosyltransferase-II having the amino acid sequence shown in SEQ ID NO: 1. 2. A recombinant nucleoside deoxyribosyltransferase-II having an amino acid sequence in which one or several amino acids are deleted, substituted, modified or added in the amino acid sequence shown in SEQ ID NO: 1. 3. A nucleoside deoxyribosyltransferase having the amino acid sequence shown in SEQ ID NO: 1.
Gene encoding II. 4. A gene consisting of the nucleotide sequence shown in SEQ ID NO: 2 and encoding nucleoside deoxyribosyltransferase-II. 5. In the base sequence shown in SEQ ID NO: 2,
A gene comprising a base sequence in which one or more bases have been deleted, substituted, inserted or added, and which encodes nucleoside deoxyribosyltransferase-II. 6. A gene that hybridizes with the gene according to claim 4 or 5 under stringent conditions and encodes nucleoside deoxyribosyltransferase-II. 7. A gene further comprising an SD sequence upstream of the gene according to claim 4, 5 or 6. 8. A method for producing deoxynucleoside using nucleoside deoxyribosyltransferase-II, wherein the recombinant nucleoside deoxyribosyltransferase-II according to claim 1 or 2 is used as nucleoside deoxyribosyltransferase-II. A method for producing deoxynucleosides. 9. A method for producing deoxynucleosides using nucleoside deoxyribosyltransferase-II, wherein the method is prepared by using any one of the genes according to claims 3 to 7 as nucleoside deoxyribosyltransferase-II. A method for producing deoxynucleoside, comprising using a recombinant nucleoside deoxyribosyltransferase-II. 10. The method according to claim 8, wherein the deoxynucleoside is a 2′-deoxynucleoside.