JP2001046097A - Enzymic production of deoxynucleoside - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject compound as a raw material, etc., for medicines by using a nucleoside-deoxyribosyltransferase II enzyme protein having a specific amino acid sequence and reacting a base receptor with a deoxysaccharide residue donor. SOLUTION: Either one of an enzyme protein composed of an amino acid sequence represented by the formula or an enzyme protein composed of an amino acid sequence in which one or several amino acids are deleted, substituted, modified or added in the amino acid represented by the formula and having a nucleoside-deoxyribosyltransferase II activity is used as the nucleoside-deoxyribosyltransferase II to obtain the objective deoxynucleoside useful as a raw material for various medicines including an antisense medicine in a method of producing the deoxynucleoside from a base receptor and a deoxysaccharide residue donor by using the nucleoside-deoxyribosyltransferase II.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a specific nucleoside
Deoxyribosyltransferase II (Nucleoside
The present invention relates to a method for producing deoxynucleosides using deoxyribosyltransferase II).

[0002]

2. Description of the Related Art Deoxynucleosides are compounds useful as raw materials for various drugs such as 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 the reaction of transferring the deoxyribosyl group of deoxynucleoside to another base, and belongs to, for example, the genus Lactobacillus. Lactobacillus helveticus (Lact.
obacillus helveticus) contains two types of nucleoside deoxyribosyltransferases I and II,
Their enzymatic properties have already been reported (Method
s in Enzymology, Vol.LI. 446 (1978). In addition, synthesis of 2 ′, 3′-dideoxynucleoside using such nucleoside deoxyribosyltransferase has been reported (WO90 / 06312, WO9).
1/04322, Journal of Biotechnology, 23 (1992) 1
93-210).

[0004]

As described above, although nucleoside deoxyribosyltransferase is an enzyme useful for enzymatic synthesis of deoxynucleoside, an enzyme extract or the like derived from lactic acid bacteria belonging to the genus Lactobacillus is directly used as an enzyme source. However, the following problems have been pointed out in the conventional method. (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 lactic acid bacteria is hard, and the expression level of nucleoside deoxyribosyltransferase in the cells is low. As a result, the extraction efficiency of nucleoside deoxyribosyltransferase is very low.

(3) In the enzyme solution prepared from the cells, various enzymes such as deaminase and nucleoside phosphorylase other than nucleoside deoxyribosyltransferase are present. The yield of the deoxynucleoside of the product may be reduced (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 deoxyuridine is produced. In addition, if nucleoside phosphorylase is present in the reaction system, the synthesized deoxynucleoside is phosphorolyzed to reduce the yield of deoxynucleoside.)

[0006]

Means for Solving the Problems In order to solve the above problems, the present inventors have expressed a large amount of nucleoside deoxyribosyltransferase by a genetic engineering technique and used it for the synthesis of deoxynucleosides. Considering that the problem could be solved, the recombinant DN of nucleoside deoxyribosyltransferase was
The expression by the A method was attempted. However, no report has been made on the nucleoside deoxyribosyltransferase gene, and as a result of repeated trial and error,
Nucleoside deoxyribosyltransferase I
Successful cloning of a gene encoding an enzyme protein having an I activity, and using the enzyme protein having a specific nucleoside deoxyribosyltransferase II activity expressed using this gene, overcomes the above-mentioned drawbacks of the conventional method. The present inventors have found that deoxynucleosides can be efficiently synthesized, and have completed the present invention.

That is, the present invention relates to a method for producing deoxynucleosides from a base acceptor and a deoxysugar residue donor using nucleoside deoxyribosyltransferase II, wherein the nucleoside deoxyribosyltransferase II has the sequence (a) Number 1
Or (b) an amino acid sequence represented by SEQ ID NO: 1;
One or several amino acids are deleted, substituted, modified or added. The amino acid sequence described in the amino acid sequence, and any one of enzyme proteins having nucleoside deoxyribosyltransferase II activity is used. And a method for producing deoxynucleosides.

Further, the present invention provides a method for producing deoxynucleosides from a base acceptor and a deoxysugar residue donor using nucleoside deoxyribosyltransferase II, wherein the nucleoside deoxyribosyltransferase II comprises the following (c) ) To (g)
A method for producing deoxynucleosides, characterized by using an enzyme protein having nucleoside deoxyribosyltransferase II activity prepared using any of the genes described in (1). (C) a gene encoding an enzyme protein consisting of the amino acid sequence represented by SEQ ID NO: 1; (d) a gene having the nucleotide sequence represented by SEQ ID NO: 2; A gene in which a plurality of bases have been deleted, substituted, inserted or added; (f) a gene that hybridizes under stringent conditions to the gene described in (d) or (e) above; d) A gene further comprising an SD sequence upstream of the gene described in (e) or (f)

Further, the present invention relates to an enzyme protein comprising the amino acid sequence represented by SEQ ID NO: 1 and having a nucleoside deoxyribosyltransferase II activity, or one or several amino acids in the amino acid sequence represented by SEQ ID NO: 1. Has an amino acid sequence deleted, substituted, modified or added, and has an activity of nucleoside deoxyribosyltransferase II.

Furthermore, the present invention relates to a gene comprising an amino acid sequence represented by SEQ ID NO: 1 and coding for an enzyme protein having nucleoside deoxyribosyltransferase II activity, and a base sequence represented by SEQ ID NO: 2; A gene encoding an enzyme protein having deoxyribosyltransferase II activity. In the base sequence represented by SEQ ID NO: 2,
One or a plurality of bases are deleted, substituted, inserted or added, comprising a base sequence, and encoding a gene encoding an enzyme protein having nucleoside deoxyribosyltransferase II activity, or under stringent conditions with such a gene. The present invention relates to a gene encoding an enzyme protein which hybridizes and has nucleoside deoxyribosyltransferase II activity.

[0011]

BEST MODE FOR CARRYING OUT THE INVENTION (1) Enzyme protein having nucleoside deoxyribosyltransferase II activity The enzyme protein having nucleoside deoxyribosyltransferase II activity of the present invention is SEQ ID NO: 1.
Is an enzyme protein consisting of the amino acid sequence represented by Here, the term "nucleoside deoxyribosyltransferase II activity" means an activity catalyzing one or several kinds of the following reactions, and as long as such activity is maintained, the amino acid sequence represented by SEQ ID NO: 1 Is not limited to an enzyme protein consisting of
An enzyme protein 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 is also used as the enzyme protein having nucleoside deoxyribosyltransferase II activity of the present invention. can do.

<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)

[0013] Such an enzyme protein having nucleoside deoxyribosyltransferase II activity has a gene encoding the enzyme protein having the amino acid sequence represented by SEQ ID NO: 1, specifically, the base sequence represented by SEQ ID NO: 2. It can be prepared using the gene. For example, a gene derived from Lactobacillus helveticus ATCC8018 will be described as a specific example. FIG. 2 shows D in the restriction enzyme map shown in FIG. 1 which is cut by HincII and EcoRI.
It shows the result of analyzing a part of the base sequence of the NA fragment, wherein the sequence represented by base numbers 74 to 564 in FIG. 2 corresponds to the structural gene of nucleoside 2′-deoxyribosyltransferase II, It is the same as the nucleotide sequence shown by No. 2.

In the present invention, one or more bases in the base sequence represented by SEQ ID NO: 2 may be deleted or substituted, as long as the enzyme protein having nucleoside deoxyribosyltransferase II activity can be expressed. , Inserted or added genes, or genes that hybridize with those genes under stringent conditions, can also be used. Note that the stringent conditions referred to herein are 5 × SSC (1 × SSC).
SSC is 8.76 g of sodium chloride and 4.41 g of sodium citrate dissolved in 1 liter of water).
Using a solution containing 1% w / v N-lauroyl sarcosine sodium salt, 0.02% w / v SDS, and 0.5% w / v blocking reagent at 60 ° C. for about 20 hours under a reaction temperature condition. Performing a hybridization reaction.

Furthermore, in the present invention, an SD (Shi) is further added upstream of a gene encoding an enzyme protein having nucleoside deoxyribosyltransferase II activity.
ne-Dalgarno Sequence) gene comprising a sequence can also be used, and by using such a gene,
This is preferable because the production amount of the enzyme protein can be significantly increased. Such a gene will be described in detail with reference to FIG.
A gene having the sequence shown at position 64 can be exemplified.

The cloning of such a gene, the preparation of an expression vector using the cloned DNA fragment, and the preparation of an enzyme protein having nucleoside deoxyribosyltransferase II activity using the expression vector are well known in the field of molecular biology. It is a well-known technique to the engineers to which it belongs, and specifically, for example, “Molecular Clon
ing ”(edited by Maniatis et al., Cold Spring Harbor Laboratori)
es, Cold Spring Harbor, New York (1982)). For example, as shown in FIG. 3, nucleoside deoxyribosyltransferase I purified from a microorganism belonging to the genus Lactobacillus
A part of the amino acid sequence such as the N-terminal and C-terminal of I is determined by a known method, and an oligonucleotide corresponding thereto is synthesized. A DNA fragment containing a gene encoding an enzyme protein having nucleoside deoxyribosyltransferase II activity 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 Makizamu Gilbert (Me
thods in Enzymology, 65, 499 (1980)) or the dideoxy chain terminator method (Methods in Enzym).
, 101, 20 (1983)) to analyze the nucleotide sequence of the cloned DNA fragment to identify the coding region of the gene, and the gene is automatically transformed 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 so as to enable expression is prepared.

The expression control signal used to express a protein having nucleoside deoxyribosyltransferase II activity in a large amount in a heterologous microorganism can be artificially controlled, and can be used to express a protein having nucleoside deoxyribosyltransferase II activity. It is desirable to use a strong transcription initiation and translation initiation signal that dramatically increases the amount. When Escherichia coli is used as a host, such a transcription initiation signal may be lac promoter, trp promoter, tac promoter (Proc. Natl. Acad. Sci. US
A., 80, 21 (1983), Gene, 20, 231 (1982)), trc promoter (J. Biol. Chem., 260, 3539 (1985)), and the like. Aldehyde-3-
Phosphate dehydrogenase (J. Biol. Chem., 2
54, 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 a microorganism, 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 cells by treating with calcium chloride at 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 the expression of the cloned protein having nucleoside deoxyribosyltransferase II activity is induced, whereby the enzyme protein is introduced into the cells. Culture until large accumulation. 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, 2xYT medium (Methods in Enzymology, 100,
20 (1983)), LB medium, M9CA medium (Molecular Cl
oning, described above), etc., and culturing can be carried out 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, 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 an enzyme protein having nucleoside deoxyribosyltransferase II activity during the culture, the expression of the genetic hand is induced by a method commonly used for the promoter used.
For example, when a lac promoter or a tac promoter is used, the expression inducer isopropyl-β-D-thiogalactopyranoside (hereinafter referred to as IPT)
G). When the promoter used has constitutive transcription activity, it is not necessary to add an expression inducing agent.

The cells are recovered from the thus prepared culture by membrane separation or centrifugation. The recovered cells can be used as an enzyme protein having nucleoside deoxyribosyltransferase II activity, but the recovered cells can be suspended in an appropriate buffer, subjected to ultrasonic treatment, and subjected to French treatment. The cells are physically crushed by pressing or the like, 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 it as an enzyme protein having deoxyribosyltransferase II activity. Since the cell extract contains an excess of an enzyme protein having nucleoside deoxyribosyltransferase II activity, it can be used as an enzyme source without any particular purification treatment. further,
Heat treatment, ammonium sulfate salting-out treatment, dialysis treatment, solvent treatment such as ethanol, various chromatographic treatments and other commonly used treatments for enzyme purification, alone or in combination -It may be used as an enzyme protein having deoxyribosyltransferase II activity.

(2) Enzymatic synthesis of deoxynucleoside The production of deoxynucleoside of the present invention is carried out by the above-mentioned specific nucleoside deoxyribosyltransferase I
It is characterized by using an enzyme protein having I activity, and other reaction conditions and the like can be performed according to conventionally known method conditions (for example, see International Patent Publication WO90 / 10080). That is, the base acceptor used in the reaction may be selected from various known bases depending on the base of the deoxynucleoside to be synthesized, and specifically, a heterocyclic base or a substituent (eg, an amino group, a substituted amino group) Base derivatives having a group, a hydroxyl group, an oxo group, a mercapto group, an acyl group, an alkyl group, a substituted alkyl group, an alkoxyl group, a 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, the purine bases and derivatives thereof include 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 deoxy sugar residue donor may be selected according to the desired deoxynucleoside, and specifically, 2′-deoxyinosine, 2′-deoxyguanosine, 2′-deoxyuridine, thymidine, 2'-deoxyadenosine, 2'-deoxyxanthosine and other 2'-deoxynucleosides, 3'-deoxyadenosine (cordesepine), 3'-deoxynucleosides such as 3'-deoxyinosine, 2 ', 3' -Dideoxyinosine, 2 ', 3'-dideoxyguanosine,
2 ′, such as 2 ′, 3′-dideoxyuridine, 3′-deoxythymidine, 2 ′, 3′-dideoxyadenosine,
3'-dideoxynucleosides are exemplified.

The synthesis reaction of deoxynucleoside is 1 m
The base acceptor and the deoxy sugar residue donor are dissolved or suspended in water or a buffer solution (pH 3 to 10) so as to have a concentration of M to 1 M, preferably 50 to 500 mM.
C., preferably 10 minutes to 5 minutes at a temperature of 20 to 50 ° C.
The reaction can be carried out for about 0 hours with stirring if 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. During the reaction, p
When H fluctuates, acid or alkali is used to make the above p
What is necessary is just to correct to H range.

After completion of the reaction, the produced 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, various types of chromatography such as ion exchange chromatography, adsorption chromatography, partition chromatography, and gel filtration, countercurrent distribution, countercurrent extraction, etc., methods utilizing partitioning between two liquid phases, concentration, cooling, organic solvents A general separation and purification method used in separation and purification of deoxynucleosides such as a method utilizing a difference in solubility such as addition may be used alone or in an appropriate combination.

[0029]

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, an enzyme protein having nucleoside deoxyribosyltransferase II activity can be efficiently expressed, and a large amount of highly active enzyme protein can be obtained, so that a small amount of enzyme protein is sufficient. This is an extremely practical method that can increase the substrate concentration and efficiently synthesize deoxynucleosides, particularly 2'-deoxynucleosides, in a short time. Further, as will be described in Examples below, side reactions are hardly observed even when a crudely purified enzyme protein such as a cell-free extract is used, and this method overcomes the problems of the conventional method.

[0030]

[Example] Lactobacillus helveticus (Lactobacil
The present invention will be specifically described with reference to Examples relating to the synthesis of deoxynucleoside using an enzyme protein having nucleoside deoxyribosyltransferase II activity derived from ATCC8018 (Lus helveticus). Further, in this example, preparation of DNA, cleavage with a restriction enzyme, T4
DNA ligation using DNA ligase and the method for transforming Escherichia coli were all performed according to “Molecular Cloning” (described above). Various restriction enzymes, T4 DNA 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 according to a conventional method was subjected to denaturation treatment at 37 ° C. for 1 hour in the presence of urea having a final concentration of 8 M, and a trypsin solution (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 analysis. Based on the nucleotide sequence deduced from the amino acid sequence, P
The primer for CR was 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 01 μl (50 mM potassium chloride, 10 mM Tris-HCl (pH 8.3), 1.5 mM magnesium chloride, 0.1 mM
001% gelatin, 0.2 mM dATP, 0.2 mMd
GTP, 0.2 mM dCTP, 0.2 mM dTTP, lactic acid bacteria chromosomal DNA 0.1 μg, primer DNA
(A) (B) 0.2 mM each, AmpliTaq DNA
Polymerase 2.5 units) with Perkin-El
Mer Cetus Instrument DNA
Using a Thermal Cycler, heat denaturation (94
C., 30 seconds), annealing (40.degree. C., 30 seconds), and extension reaction (72.degree. C., 30 seconds) were repeated 28 times. A probe for Southern hybridization was prepared by adding DIG-labeled UT to dNTP under the above PCR conditions.
It was prepared using a mixed solution containing P. Lactic acid bacteria chromosome DN
A 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 (NdRTge
ne) was found to be present in the 3.2 kb SalI-EcoRI fragment of the lactic acid bacterium chromosomal DNA. Therefore, 2.3 to 4. of SalI and EcoRI digests of the lactic acid bacteria chromosome.
The 0 kb fragment was recovered and used as the SalI of pUC18,
The ligation with EcoRI digest was used as template DN.
PCR was performed as A. The primers were primer (B) and primer M4 for sequencing (TaKaRa)
At 94 ° C. for 30 seconds and 40 ° C. at 30 ° C.
A 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 obtained upstream region, 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).

In the PCR reaction, the same reaction composition and the same reactor as in Example 1 were used, and the steps of thermal denaturation (94 ° C., 30 seconds), annealing (50 ° C., 30 seconds), and extension reaction (72 ° C., 2 minutes) were performed. Performed by repeating 30 times. After gene amplification, digestion with SalI and EcoRI resulted in 1.0 kb DN
The A fragment was purified. The recovered DNA was subjected to restriction enzyme Sal.
I and EcoRI digested plasmids pHSG398 or pHSG399 were ligated with T4 DNA ligase. Escherichia coli JM109 strain was transformed using the ligation reaction solution, and plasmids p398-T2F5 and p399-T2F were obtained from the obtained chloramphenicol-resistant transformants.
5 was isolated. Each of the obtained transformants was JM10
9 [p398-T2F5], JM109 [p399-T
2F5].

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 DN of the nucleoside deoxyribosyltransferase II structural gene shown in FIG.
The A base sequence was obtained. This nucleotide sequence is 495 bp and encodes a polypeptide consisting of 165 amino acids starting with Met and having a molecular weight of 19,128. The amino acid sequence of the amino terminal 10 of this peptide completely matched that of the purified and isolated lactic acid bacterium nucleoside deoxyribosyltransferase II.

(2) Preparation of recombinant vector for high expression of nucleoside deoxyribosyltransferase II nucleoside deoxyribosyltransferase I
The recombinant vector pTrc-T2F4 for I production was produced by the following method (see FIG. 3). That is, p399-T2
Using F5 as a template and the following primer (D) and sequencing primer RV (TaKaRa),
A DNA fragment containing the nucleoside deoxyribosyltransferase II gene derived from lactic acid bacteria was amplified by the method. In addition, a BspHI recognition sequence was introduced into the primer D so as to link to the NcoI site. Primer (D): 5-AAATCATGAACAAGAAAAAGACTTTATAT-
Three

The PCR reaction uses the same reaction composition and reactor as in Example 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. The ligation reaction solution was used to transform Escherichia coli JM109 strain, and from the resulting ampicillin-resistant transformant, a recombinant vector in which a nucleoside deoxyribosyltransferase II structural gene was inserted immediately after the trc promoter for expression of pTc99A, pTrc -T2F4 was isolated. The obtained transformant was transformed into JM109 [pTrc-T2F4].
It was named.

(3) Culture of Transformant and Preparation of Enzyme Escherichia coli transformant carrying the above recombinant vector was
Inoculate 100 ml of 2 × YT medium containing 100 μg / ml chloramphenicol or ampicillin,
Shaking culture was performed at 7 ° C. At the time of reaching 4 × 10 ⁸ cells / ml, IPTG was added to the culture solution to a final concentration of 0.05 mM.
Was further added and shaking culture was continued at 37 ° C. for 4 hours. After completion of the culture, the cultured cells were collected by centrifugation (9,000 × g, 10 minutes), and suspended in 10 ml of a buffer solution (10 mM Tris-HCl (pH 7.8), 1 mM EDTA).
The cell suspension was treated with an ultrasonic crusher, and the cell residue was further removed by centrifugation (12,000 × g, 10 minutes). The supernatant fraction thus obtained was used as a bacterial cell extract. The activity of nucleoside deoxyribosyltransferase II in the bacterial cell extract was determined using a control strain (pHSG
It is shown in the table below together with E. coli JM109 carrying 398). The nucleoside deoxyribosyltransferase II activity was measured using 5 mM deoxyadenosine and cytosine or thymidine and cytosine as substrates. The reaction was started by adding a cell extract to the reaction solution, and the enzyme was inactivated by boiling for 1 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 defined as one unit (unit).

[0041]

[Table 1] * 1 unit = 1μmole deoxycytidine-production from d
eoxyadenosine / min at 37 ℃ ** 1 unit = 1μmole deoxycytidine-production from
thymidine / min at 37 ℃

As shown in Table 1, control bacteria (pHSG398)
Is not detected by Escherichia coli JM109 carrying the recombinant vector, but in the transformant carrying the prepared recombinant vector,
Nucleoside deoxyribosyltransferase I
I activity was confirmed. Further, as shown in Table 2 below, the productivity of the transformant (pTrc-T2F4 retaining bacterium) constructed according to the present invention was 3,000 of the original strain, Lactobacillus helveticus ATCC8018 strain. It corresponds to ~ 4,000 times. Nucleoside deoxyribosyltransferase I prepared from a transformant of Escherichia coli carrying the recombinant vector was used.
I had properties equivalent to those of enzymes derived from lactic acid bacteria.

[0043]

[Table 2] * 1 unit = 1μmole deoxycytidine-production from
uridine / min at 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 production rate of 2′-deoxycytidine by the same method as in the above (3), 13.5 mM of 2′-deoxycytidine was synthesized. Further, as a result of reacting 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. As a comparative example, 20 mM 2'-deoxyuridine and 20 mM
To 1 ml of an aqueous solution containing M cytosine, a cell-free extract equivalent to 1 ml of a culture solution of a lactic acid bacterium Lactobacillus helveticus ATCC8018 was added, and reacted at 37 ° C. for 8 days.
As a result of measuring the production rate of deoxycytidine, 10.6 m
M 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.

[0045]

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.

[0046]

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 bacterial cell extract derived from the plant (9 unit / L reaction solution)
Then, the reaction was carried out at 37 ° C. for 18 hours. As a result of measuring the production rate of 2'-deoxycytidine, it was confirmed that 72 mM of 2'-deoxycytidine (ratio to 2'-deoxyuridine: 54%) was synthesized.

[0047]

Example 4 Synthesis of Thymidine 1. Cell extract 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. After adding 8 μl (0.9 unit / L reaction solution), the mixture was reacted 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.

[0048]

Example 5 Synthesis of 2'-deoxyadenosine Extraction of cells derived 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. Add 0.05 ml of solution (15 unit / L reaction solution)
Then, the reaction was carried out at 37 ° C. for 20 hours. As a result of measuring the production rate of 2'-deoxyadenosine, 2'-deoxyadenine was synthesized at a ratio of 2'-deoxyuridine to 77%.

[0049]

Example 6 Synthesis of 2'-deoxyguanosine (1) Derived from the transformant JM109 [pTrc-T2F4] prepared in Example 1 in 5 mL of an aqueous solution containing 100 mM of 2'-deoxyuridine and 100 mM of guanine. Add 0.05 ml of 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 rate of 2′-deoxyguanosine, 2′-deoxyguanosine was synthesized at a ratio of 2% to 2′-deoxyuridine of 30%.

[0050]

Example 7 Synthesis of 2'-deoxyguanosine (2) Derived from the transformant JM109 [pTrc-T2F4] prepared in Example 1 in 5 mL of an aqueous solution containing 10 mM 2'-deoxyadenosine and 10 mM 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.

[0051]

[Sequence List] SEQUENCE LISTING <110> YAMASA CORPORATION <120> Process for the enzymatically preparation of deoxynucleosides <130> YP99-00006 <140> <141> <160> 6 <210> 1 <211> 165 <212> PRT < 213> Lactobacillus helveticus <400> 1 Met Asn Lys Lys Lys Thr Leu Tyr Phe Gly Ala Gly Trp Phe Asn 1 5 10 15 Glu Lys Gln Asn Lys Ala Tyr Lys Glu Ala Met Ala Ala Leu Lys 20 25 30 Glu Asn Pro Thr Val Asp Leu Glu Asn Ser Tyr Val Pro Leu Glu 35 40 45 Asn Gln Tyr Lys Gly Ile Arg Ile Asp Glu His Pro Gln Tyr Leu 50 55 60 His Asn Ile Glu Trp Ala Ser Ala Thr Tyr His Asn Asp Leu Val 65 70 75 Gly Ile Lys Thr Ser Asp Val Leu Leu Gly Val Tyr Leu Pro Gln 80 85 90 Glu Glu His Val Gly Leu Gly Met Glu Leu Gly Tyr Pro Leu Ser 95 100 105 Gln Gly Lys Leu Phe Phe Trp Phe Ser His Met Lys Asp Tyr Gly 110 115 120 Lys Pro Ile Ile Leu Met Ser Trp Gly Val Cys Asp Asn Ala Ser 125 130 135 Gln Ile Ser Glu Leu Lys Asp Phe Asp Phe Asn Lys Pro Arg Tyr 140 145 150 Asn Phe Tyr Asp Gly Ser Cys Ile Leu Lys 155 160 Asn Lys Gln Thr Lys 165 <210> 2 <211> 491 <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 acagtacttg 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 cagtgaatta 420 aaagacttcg actttaacaa gcctcgctac aatttctacg acgggagctg tatattaaaa 480 aataagcaaa c 491 <210> 3 <211> 40 <212> DNA <213> Artificial Sequence <220 > <223> primer for amplification of NdRT gene <400> 3 cctctgcagc catgaayaar aaraaracny tntayttygg 40 <210> 4 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> primer for amplification of NdRT gene <400 > 4 accaagcttr ttrtgiarrt aytciggrtg ytcrtc 36 <210> 5 <211> 27 <212> DNA <213> Artific ial Sequence <220> <223> primer for amplification of NdRT gene <400> 5 taagtcgaca gcaattttta tggggag 27 <210> 6 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> primer for amplification of NdRT gene <400> 6 aaatcatgaa caagaaaaag actttatat 29

[0052]

[Brief description of the drawings]

FIG. 1 shows a 4.3 kb EcoRI-EcoRID containing a nucleoside deoxyribosyltransferase II structural gene from lactic acid bacteria Lactobacillus helveticus ATCC8018.
It shows a restriction enzyme map of the NA fragment. “Ndt2” in FIG. 1 is a gene encoding nucleoside deoxyribosyltransferase II (495b
p, which encodes a polypeptide of molecular weight 19,128 consisting of 165 amino acids).

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 when 2′-deoxyuridine is used as a deoxy sugar residue donor and cytosine is used as a base acceptor.

Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat II (reference) (C12N 15/09 ZNA C12R 1: 225)

Claims (9)

[Claims] 1. A method for producing deoxynucleosides from a base acceptor and a deoxy sugar residue donor using nucleoside deoxyribosyltransferase II, wherein (a) SEQ ID NO: 1 is used as nucleoside deoxyribosyltransferase II. Or (b) the amino acid sequence represented by the amino acid sequence shown in SEQ ID NO: 1 in which one or several amino acids are deleted, substituted, modified or added, A method for producing deoxynucleosides, characterized by using any one of enzyme proteins having nucleoside deoxyribosyltransferase II activity. 2. A method for producing deoxynucleosides from a base acceptor and a deoxysugar residue donor using nucleoside deoxyribosyltransferase II, wherein the nucleoside deoxyribosyltransferase II is represented by the following (c) to (g). A method for producing deoxynucleosides, characterized by using an enzyme protein having nucleoside deoxyribosyltransferase II activity prepared using any of the genes described in (1). (C) a gene encoding an enzyme protein consisting of the amino acid sequence represented by SEQ ID NO: 1; (d) a gene having the nucleotide sequence represented by SEQ ID NO: 2; A gene in which a plurality of bases have been deleted, substituted, inserted or added; (f) a gene that hybridizes under stringent conditions to the gene described in (d) or (e) above; d) A gene further comprising an SD sequence upstream of the gene described in (e) or (f) 3. The method according to claim 1, wherein the deoxynucleoside is 2′-deoxynucleoside. 4. An enzyme protein comprising the amino acid sequence represented by SEQ ID NO: 1 and having nucleoside deoxyribosyltransferase II activity. 5. An enzyme comprising an amino acid sequence shown in SEQ ID NO: 1 wherein one or several amino acids have been deleted, substituted, modified or added, and which has nucleoside deoxyribosyltransferase II activity. protein. 6. A gene comprising an amino acid sequence represented by SEQ ID NO: 1 and encoding an enzyme protein having nucleoside deoxyribosyltransferase II activity. 7. It comprises a base sequence represented by SEQ ID NO: 2,
Nucleoside deoxyribosyltransferase I
A gene encoding an enzyme protein having I activity. 8. In the base sequence represented by SEQ ID NO: 2,
A gene encoding an enzyme protein having a nucleoside deoxyribosyltransferase II activity, comprising a base sequence in which one or more bases have been deleted, substituted, inserted or added. (9) a nucleic acid which hybridizes with the gene according to (6) or (7) under stringent conditions;
A gene encoding an enzyme protein having deoxyribosyltransferase II activity.