KR102000927B1 - An N-deoxyribosyl transferase mutant, and a method for producing nucleoside using the same - Google Patents

Abstract

The present invention relates to a N-deoxyribosyltransferase mutant, a polynucleotide encoding the mutant, a vector containing the polynucleotide and a microorganism, and a method for producing a nucleoside using the polynucleotide.

Description

Deoxyribosyl transferase mutant, and a method for producing a nucleoside using the mutant N-deoxyribosyl transferase mutant, and a method for producing nucleoside using the same,

The present invention relates to a N-deoxyribosyltransferase mutant, a polynucleotide encoding the mutant, a vector containing the polynucleotide and a microorganism, and a method for producing a nucleoside using the polynucleotide.

The oligonucleotide therapeutic agent directly binds DNA or RNA having genetic information in vivo to block disease-related protein production (antisense drug), aptamer that binds protein, small-interferring RNA functioning as double strand such as siRNA, micro-RNA and antagomir, Decoy acting on transcription factors, and CpG oligonucleotides that cause immune activation.

In order for these oligonucleotide therapeutic agents to be introduced into desired cells and exhibit activity, it is necessary to increase the stability to resist in vivo conditions including rapid degradation by various nucleic acid degrading enzymes. Accordingly, various studies have been made to increase the stability of oligonucleosides by using an unnatural nucleoside (a nucleotide which is a component of nucleic acid or a nucleoside introducing a chemical modification into sugar).

In particular, the 2'-deoxy-2'-fluoronucleosides are excellent in stability against in vivo nucleic acid degrading enzymes, and are thermodynamically stable with an increase in the melting temperature of about 1.8 ° C. per the number of nucleotides in the formation of the RNA duplexes The results have been confirmed and are recently attracting attention as a raw material for an oligonucleotide therapeutic agent. 2'-deoxy-2'-fluoro nucleoside has been known as an industrially difficult substance using an enzyme as a raw material of oligonucleotides so far. Up to now, two kinds of enzymatic production methods using biotransformation of 2'-deoxy-2'-fluoro nucleoside have been reported. One was confirmed by using N-deoxyribosyltransferase derived from Lactobacillus reuteri to react with 2'-fluoro-2 'deoxynucleoside, and the other was purine phosphorylase and An enzymatic preparation method using biotransformation using pyrimidine phosphorylase is known. However, the conventional bioconversion process has a difficulty in the manufacturing process because it shows low purity and yield such as a large amount of enzyme use, low substrate concentration and long reaction time.

In addition, the enzyme synthesis method is a method which enables the industrial production of 2'-deoxy-2'-fluoro nucleoside in terms of cost and efficiency, but unlike a chemical synthesis method or a hydrolysis method, an enzyme which is a biological catalyst is used There is a problem in that the yield and stable production of the final product differ greatly depending on differences in the types of enzymes, differences in the origins and amino acid sequences, plasmid constructs, host strains, and culture conditions for gene expression .

Therefore, in order to produce an efficient and stable 2'-deoxy-2'-fluoro nucleoside, an enzyme gene having a strong activity and stable expression is searched, and a gene expression system capable of stably expressing these genes .

Under these technical backgrounds, the present inventors have made intensive efforts to develop novel N-deoxyribosyltransferases having high activity, and have found that they have high substrate reactivity to 2'-deoxy-2'-fluoronucleosides It was found that a novel N-deoxyribosyltransferase mutant can be produced and that an oligonucleoside can be produced with high yield and high purity using this N-deoxyribosyltransferase mutant Respectively.

One object of the present invention is to provide an N-deoxyribosyltransferase mutant having excellent transglycosylation activity and substrate reactivity.

Another object of the present invention is to provide a polynucleotide encoding the mutant, a vector containing the polynucleotide, a microorganism into which the vector is introduced, and a method for producing an N-deoxyribosyltransferase mutant using the microorganism will be.

It is still another object of the present invention to provide a method for producing a nucleoside using the N-deoxyribosyltransferase mutant or a microorganism expressing the mutant.

This will be described in detail as follows. On the other hand, each description and embodiment disclosed in the present invention can be applied to each other description and embodiment. That is, all combinations of various elements disclosed in the present invention fall within the scope of the present invention. Further, the scope of the present invention is not limited by the detailed description described below.

One aspect of the present invention for attaining the above object is to provide a method for producing an N-deoxyribosyltransferase comprising the L59Q mutation in which the 59th leucine in the amino acid sequence of SEQ ID NO: 2 is substituted with glutamine (nucleoside deoxyribosyl transferase (NDT) variant.

The N-deoxyribosyltransferase mutant of the present invention exhibits an activity of highly efficiently catalyzing the transfer or transglycosylation reaction of 2'-deoxy-2'-fluororibos, There is a characteristic of exhibiting high activity and substrate reactivity compared to the known N-deoxyribosyltransferase.

The N-deoxyribosyltransferase mutant may have an increased substrate reactivity to 2'-Deoxy-2'-fluoronucleoside, The 2'-deoxy-2'-fluoro nucleoside may be, but is not limited to, 2'-Deoxy-2'-fluorouridine.

The N-deoxyribosyltransferase having the amino acid sequence of SEQ ID NO: 2 was obtained from Lactobacillus strain < RTI ID = 0.0 > delbrueckii . < / RTI > The NDT mutant may have the amino acid sequence of SEQ ID NO: 10, in which leucine (L), which is the 59th amino acid in SEQ ID NO: 2, is substituted with glutamine (Q).

The term "N-deoxyribosyltransferase (NDT)" in the present invention refers to an enzyme also called trans-N-deoxyribosylase, which is a strain of Lactobacillus helveticus (Biochem. J. 50: 383-397, 1952), two isotopes of DRTase I and II are known. Among them, DRTase I catalyzes the transfer reaction of deoxyribose between two purines and DRTase II catalyzes the transfer of purine, pyrimidine or purine and pyrimidine deoxyribose Catalyzed. Lactobacillus leichmannii DRTase II has the activity of hydrolyzing the nucleoside to base and deoxyribose in the absence of the acceptor base, as well as this transfer reaction. The DRTase II enzyme derived from L. helveticus or L. leichmannii strain has low specificity for the receptor base and stereospecificity for producing only the β-anomer in the sugar transfer reaction, Crude extracts of these two strains are widely used for analog synthesis of antiviral or anticancer nucleosides.

In one embodiment of the present invention, the NDT variant comprising the L59Q mutation in the amino acid sequence of SEQ ID NO: 2 may be a mutant comprising the amino acid sequence of SEQ ID NO: 10.

In one specific embodiment of the present invention, NDT random mutation strain library was obtained by error prone PCR using the NDT gene containing the nucleotide sequence of SEQ ID NO: 1 as a template, The variants of the present invention screened through substrate specificity screening with 2'-Deoxy-2'-Fluorouridine (2FDU) are higher than NDT with the amino acid sequence of SEQ ID NO: 2 Activity and substrate reactivity (Examples 3 and 4).

In another aspect, the present invention provides a polynucleotide encoding the NDT variant, a recombinant vector comprising the polynucleotide, a microorganism into which the recombinant vector is introduced, and a method for producing the NDT mutant using the microorganism.

The NDT mutant is as described above.

As used herein, the term "polynucleotide" has the meaning inclusive of DNA (gDNA and cDNA) and RNA molecules, and in the polynucleotide, the nucleotide which is a basic constituent unit is not only a natural nucleotide, Also included are modified analogues (Scheit, Nucleotide Analogs, John Wiley, New York (1980); Uhlman and Peyman, Chemical Reviews, (1990) 90: 543-584). The polynucleotide may be an isolated polynucleotide.

The polynucleotide encoding the NDT variant may be derived from Lactobacillus delbrueckii . The sequence of a polynucleotide encoding an NDT variant of the present invention may be modified, and the modification includes addition, deletion, or non-conservative substitution or conservative substitution of nucleotides.

The polynucleotides of the present invention are also interpreted to include nucleotide sequences that exhibit substantial identity to the nucleotide sequences described above. The above substantial identity is determined by aligning the nucleotide sequence of the present invention with any other sequence as much as possible and analyzing the aligned sequence using algorithms commonly used in the art, , Specifically at least 90% homology, more specifically at least 95% homology.

In one embodiment of the present invention, the NDT variant comprising the amino acid sequence of SEQ ID NO: 10 in which the 59th leucine in the amino acid sequence of SEQ ID NO: 2 is replaced with glutamine may be encoded by the nucleotide sequence of SEQ ID NO:

As used herein, the term "vector" refers to a plasmid vector as a means for expressing a gene of interest in a host cell; Cosmeptide vector; And viral vectors such as a bacteriophage vector, an adenovirus vector, a retrovirus vector, and an adeno-associated viral vector, and specifically, a virus vector prepared by the present applicant and deposited in the Korea Culture Center of Microorganisms (KCCM) pFRPT (Accession No .: KCCM-10320).

A polynucleotide encoding an NDT variant in the vector of the present invention may be operatively linked to a promoter.

As used herein, the term "operably linked" means a functional linkage between a nucleic acid expression control sequence (e.g., an array of promoter, signal sequence, or transcription factor binding site) and another nucleic acid sequence, The regulatory sequence regulates transcription and / or translation of the different nucleic acid sequences.

The recombinant vector system of the present invention can be constructed through various methods known in the art, and specific methods for this are disclosed in Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press (2001) , Which is incorporated herein by reference.

The vector of the present invention can typically be constructed as a vector for cloning or as a vector for expression. In addition, the vector of the present invention can be constructed by using prokaryotic cells or eukaryotic cells as hosts.

For example, when the vector of the present invention is an expression vector and a prokaryotic cell is used as a host, a strong promoter capable of promoting transcription (such as a tac promoter, lac promoter, lacUV5 promoter, lpp promoter, pL 貫 promoter, , rac5 promoter, amp promoter, recA promoter, SP6 promoter, trp promoter, and T7 promoter), a ribosome binding site for initiation of decoding, and a transcription / translation termination sequence. The promoter and operator site of the E. coli tryptophan biosynthetic pathway (Yanofsky, C., J. Bacteriol., (1984) 158: 1018-8), when E. coli (e.g. HB101, BL21, 1024) and the left promoter of phage lambda (pL? Promoter, Herskowitz, I. and Hagen, D., Ann. Rev. Genet., (1980) 14: 399-445). When a Bacillus bacterium is used as a host cell, the promoter of the toxin protein gene of Bacillus subtilis (Appl. Environ. Microbiol. (1998) 64: 3932-3938; Mol. Gen. Genet. (1996) 250: 734-741 ) Or any promoter capable of expressing in Bacillus may be used as a regulatory region.

Meanwhile, the recombinant vector of the present invention can be prepared by using a plasmid (for example, pCL, pSC101, pGV1106, pACYC177, ColE1, pKT230, pME290, pBR322, pUC8 / 9, pUC6, pBD9, pHC79, pIJ61, pLAFR1, pGEX series, pET series, pUC19, etc.), phage (e.g., λgt4 · λB, λ-Charon, λΔz1 and M13) or viruses (eg SV40 and the like). For example, the recombinant vector of the present invention can be produced by manipulating an expression vector for E. coli, specifically, pFRPT (Korean Patent Registration No. 10-0449639), but is not limited thereto.

On the other hand, when the vector of the present invention is an expression vector and a eukaryotic cell is used as a host, a promoter derived from a genome of a mammalian cell such as a metallothionine promoter, a beta -actin promoter, a human hemoglobin promoter, Promoter), or a mammalian virus (e.g., adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus (CMV) promoter, HSV tk promoter, mouse breast tumor virus (MMTV) promoter, The LTR promoter of HIV, the promoter of Moloney virus promoter Epstein bar virus (EBV) and the promoter of rosacekoma virus (RSV)), and generally has a polyadenylation sequence as a transcription termination sequence.

On the other hand, the recombinant vector of the present invention includes an antibiotic resistance gene commonly used in the art as a selection marker, for example, ampicillin, gentamycin, carbenicillin, chloramphenicol, streptomycin, kanamycin, And resistance genes for tetracycline.

The microorganism capable of continuously cloning and expressing the vector of the present invention in a stable manner can be any microorganism known in the art. For example, Escherichia coli , Bacillus subtilis and Bacillus turginensis such as Bacillus sp, Streptomyces (Streptomyces), Pseudomonas (Pseudomonas) (e.g., Pseudomonas footage is (Pseudomonas putida)), Proteus Mira Billy's (Proteus mirabilis) or Staphylococcus (Staphylococcus) (e.g. , Staphylococcus ( Staphylocus carnosus ), and the like).

Suitable eukaryotic microbes of said vector include fungi such as Aspergillus species , fungi such as Pichia pastoris), Saccharomyces with my access Servigroup Asia (Saccharomyces cerevisiae), Iasi survey car Rome Seth (Schizosaccharomyces) and Neuro Castello La yeast, such as Chrysler Corporation (Neurospora crassa), or other lower eukaryotic cells, insect-high, such as cells derived from Eukaryotic cells, and cells derived from plants or mammals.

In one embodiment of the present invention, the microorganism into which the recombinant vector is introduced is Escherichia coli E. coli , e . coli DH5 [alpha], JM109, and may specifically be E. coli DH5 [alpha].

In the present invention, "transformation" and / or "transfection" to a microorganism includes any method of introducing the polynucleotide into an organism, cell, tissue or organ, Technology can be selected and performed. Such methods include electroporation, protoplast fusion, calcium phosphate (CaPO ₄ ) precipitation, calcium chloride (CaCl ₂ ) precipitation, agitation with silicon carbide fibers, Agrobacterium mediated transformation, PEG, dextran Sulfate, lipofectamine, and dry / inhibition-mediated transformation methods, and the like.

In one embodiment of the present invention, a microorganism comprising a polynucleotide encoding an NDT variant having the amino acid sequence of SEQ ID NO: 10 is activated or catalyzed by a transglycosylation reaction or a 2'-deoxy-2'-fluoro The substrate reactivity to the nucleoside may be increased and the activity to catalyze the N-glucoside bond between the base and the sugar moiety of the nucleoside may be increased. The polynucleotide may comprise the nucleotide sequence of SEQ ID NO: 9.

The present invention provides a method for producing an NDT variant using the microorganism. Specifically, the production method comprises the steps of: (a) culturing a microorganism transformed with the recombinant vector of the present invention; And (b) recovering the N-deoxyribosyltransferase mutant from the culture.

The cultivation of the transformed microorganism in the NDT variant production can be carried out according to a suitable culture medium and culture conditions known in the art. Such a culturing process can be easily adjusted according to the strain selected by those skilled in the art. Such various culturing methods are disclosed in various publications (e.g., James M. Lee, Biochemical Engineering, Prentice-Hall International Editions, 138-176). Cell culture can be classified into batch, infusion, and continuous culture depending on the suspension culture, adhesion culture, and culture method according to the cell growth method. The medium used for the culture should suitably meet the requirements of the particular strain.

In animal cell cultures, the medium comprises various carbon sources, nitrogen sources and trace element components. Examples of carbon sources that can be used include carbohydrates such as glucose, sucrose, lactose, fructose, maltose, starch and cellulose, fats such as soybean oil, sunflower oil, castor oil and coconut oil, fatty acids such as palmitic acid, stearic acid, and linoleic acid, Alcohols such as glycerol and ethanol, and organic acids such as acetic acid, and these carbon sources may be used singly or in combination.

Nitrogen sources that can be used in the present invention include, but are not limited to, organic nitrogen sources such as peptone, yeast extract, gravy, malt extract, corn steep liquor (CSL) and soybean wheat, and urea, ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate, And the like. These nitrogen sources may be used alone or in combination. The medium may include potassium dihydrogenphosphate, dipotassium hydrogenphosphate and the corresponding sodium-containing salts as a source. It may also include metal salts such as magnesium sulfate or iron sulfate. In addition, amino acids, vitamins, and suitable precursors and the like may be included.

During the culture, compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid and sulfuric acid can be added to the culture in an appropriate manner to adjust the pH of the culture. In addition, foaming can be suppressed by using a defoaming agent such as fatty acid polyglycol ester during the culture. In addition, oxygen or an oxygen-containing gas (e.g., air) is injected into the culture to maintain the aerobic state of the culture.

The NDT mutant obtained by culturing the transformed microorganism can be used in an unpurified state and can be further purified and used in high purity by various conventional methods such as dialysis, salt precipitation and chromatography.

In another aspect, the present invention provides a method for producing a nucleoside by forming an N-glucoside bond between a donor and a donor of a sugar residue using a NDT mutant of the present invention or a microorganism expressing the mutant .

The nucleoside preparation method comprises reacting a base donor and a sugar residue donor using a microorganism expressing an NDT variant to form an N-glucoside bond between the base portion of the base donor and the sugar portion of the sugar residue donor, Lt; RTI ID = 0.0 > a < / RTI >

The NDT mutant and the microorganism expressing the NDT mutant are as described above.

As used herein, the term "base donor" refers to a material that provides a base for the N-glucoside binding reaction of a nucleoside. The base donor used in the present invention may be appropriately selected according to the objective nucleoside, and may be a heterocyclic base capable of forming N-glucoside bond with a sugar moiety of the sugar residue donor by the action of NDT But is not limited thereto.

Wherein the heterocyclic base is selected from the group consisting of purine and derivatives thereof, pyrimidine and derivatives thereof, triazole and derivatives thereof, imidazole and derivatives thereof, deazapurine and derivatives thereof, azapurine and derivatives thereof, Derivatives or pyridine and derivatives thereof, and the heterocyclic base itself may be a nucleoside having a heterocyclic base as well.

Specifically, a substituent (for example, an amino group, a substituted amino group, a hydroxyl group, an oxo group, a mercapto group, an acyl group, an alkyl group, a substituted alkyl group, Alkoxyl groups such as adenine, guanine, hypoxanthine, xanthine, 6-mercaptopyrin, 6-thioguanine, N-alkyl or acyladenyl, 2-alkoxyadenine , 2-thioadenine, 2,6-diaminopurine and the like;

Pyrimidine derivatives having substituents similar to those described above at one or two or more positions of 2 ', 4' or 5 'of pyrimidine, such as cytosine, uracil, thymine, 5-halogenothrulose (5-fluorouracil , 5-iodoacyl, etc.), 5-halogenothiocin (such as 5-fluorocytosine), 5-trihalogenomethyluracil (such as 5-trifluoromethyluracil) Uracil, N-acyl cytosine, 5-halogenovinyl uracil, and the like;

1,2,4-triazole derivatives having a substituent at the 3 'position of 1,2,4-triazole such as 1,3,4-triazole-3-carboxamide 1,2,4-triazole 3-carboxylic acid, 1,2,4-triazole-3-carboxylic acid alkyl ester, and the like;

Imidazole derivatives having substituents at 4 ' and 5 ' of imidazole, such as 5-amino-4-imidazolecarboxamide, 4-carbamoyl-imidazol-5-olate, benzimidazole and the like;

Deazapurine derivatives such as 1-deaza adenine, 3-deaza adenine, 3-deaza guanine, 7-deaza adenine, 7-deazaguanine or 7-deaza- Compounds having substituents similar to those of the electrophilic derivatives;

Azaporidine derivatives such as 8-azaadenine, 7-deaza-8-azahepoxatin (azopurinol);

Azapyrimidine derivatives such as 5-azathimine, 5-azasitonin, 6-azauracil and the like; or

Pyridine derivatives such as 3-deazaiacyl, nicotinic acid, nicotinic acid amide and the like.

Specifically, it may be adenine, 2,6-diaminopurine, or cytosine.

As used herein, the term "sugar residue donor" refers to a substance that feeds a sugar residue to the N-glucoside binding reaction of a nucleoside. The sugar residue donor used in the present invention may be appropriately selected according to the objective nucleoside and may be a heterocyclic ribose capable of forming N-glucoside bond with the base portion of the base donor by the action of NDT Compound, or deoxyribose compound, but is not limited thereto.

The sugar residue donor may be a ribose compound such as a ribonucleoside such as inosine, guanosine, uridine, ribofuranosyl thymine, and ribose-1-phosphate; or

Examples of deoxyribose compounds include 2'-deoxyinosine, 2'-deoxyguanosine, 2'-deoxyuridine, thymidine, 2 ', 3'-dideoxyinosine, 2' Deoxyribose-1-phosphate, 2,3-dideoxyribose-1-phosphate and the like, such as deoxyuridine, deoxyuridine, deoxyuridine, deoxyuridine, deoxyuridine, Lt; / RTI >

Specifically, it may be thymidine, 2'-deoxyuridine, 2'-deoxy-2'-fluorouridine.

As an embodiment of the present invention, a method for producing a nucleoside comprises treating the N-deoxyribosyltransferase mutant of the present invention with 2'-deoxy-2'-fluorouridine and adenine, Deoxy-2'-fluoroadenosine, which comprises the step of preparing 2'-deoxy-2'-fluoroadenosine.

The method for producing the above 2'-deoxy-2'-fluoroadenosine can be carried out under the temperature condition of 45 ° C to 55 ° C, preferably 48 ° C to 52 ° C, more preferably about 50 ° C. The preparation of the above 2'-deoxy-2'-fluoroadenosine can be carried out at pH conditions of 5.8 to 6.8, preferably 6.1 to 6.5, more preferably about 6.3. The method for preparing deoxy-2'-fluoroadenosine has a substrate concentration of 1.6 to 2.4 M, preferably 1.8 to 2.2 M, more preferably about 2.0 M, as the substrate concentration conditions. In the method for preparing deoxy-2'-fluoroadenosine, the concentration of the dried cells to be fed under the enzyme input conditions is 50 to 150 g / L, preferably 80 to 120 g / L, more preferably about 100 g / L .

The process for preparing 2'-deoxy-2'-fluoroadenosine of the present invention may further comprise a purification process for removing impurities enzymes and uracil after the enzyme is converted. The above purification process comprises filtering under purified water and MeOH; And neutralizing the filtered filtrate to a pH of 7 to 8. Purified water and MeOH may be 1.5 to 3.5 times, preferably 2.5 times, and MeOH may be 9.5 to 11.5 times, preferably 10.5 times, of purified water in comparison to 2FDU doses. It may further include a crystallization step of 2FDA after the purification process.

In one specific embodiment of the present invention, 2'-deoxy-2'-fluoro-uridine and adenine are used as the starting material, using the NDT variant expression strain ( E. coli E206) 2'-fluoroadenosine was prepared, and it was confirmed that high purity 2'-deoxy-2'-fluoroadenosine can be produced with high efficiency (Example 5).

In another embodiment of the present invention, a method for producing a nucleoside comprises contacting an N-deoxyribosyltransferase mutant of the present invention with 2'-deoxy-2'-fluorouridine and 2,6-diaminopurine To produce 2'-deoxy-2'-fluoro-2,6-diaminopurine; And 2'-deoxy-2'-fluoro-2,6-diaminopurine was treated with adenosine deaminase derived from lactococcus lactis to prepare 2'-deoxy-2'-fluoroguanosine 2'-deoxy-2'-fluoro-guanosine.

The method for producing the above 2'-deoxy-2'-fluoroguanosine can be carried out under the temperature condition of 45 ° C to 55 ° C, preferably 48 ° C to 52 ° C, more preferably about 50 ° C. The method of preparing the above 2'-deoxy-2'-fluoroguanosine can be carried out under pH conditions of 5.5 to 6.5, preferably 5.8 to 6.3, more preferably about 6.0. The method for preparing deoxy-2'-fluoro-guanosine is a substrate concentration of 1.4 to 1.8 M, preferably 1.5 to 1.7 M, and more preferably about 1.6 M, of 2FDU as a substrate concentration condition, . In the method for producing deoxy-2'-fluoro-guanosine, the amount of the dried cells injected under the enzyme loading conditions is 1.5 to 2.5 times, preferably about 2.0 times the 2FDU, and the dried cells can be divided and administered.

The method for preparing 2'-deoxy-2'-fluoro-guanosine of the present invention may further include a purification step for removing an enzyme and URA which are impurities after the enzyme is converted. Wherein the purification process comprises: at least one filtration under purified water and MeOH; And neutralizing the filtered filtrate to a pH of 7 to 8. And may further include a crystallization step of 2FDG after the purification process.

In one specific embodiment of the present invention, 2'-deoxy-2'-fluorouridine and 2,6-diaminopurine are used as a substrate to express N-deoxyribosyltransferase mutant ( E. coli E206) was used for primary conversion to 2'-deoxy-2'-fluoro-2,6-diaminopurine, adenosine deaminase was used to convert 2'-deoxy-2'-fluoroguanosine , And it was confirmed that high purity 2'-deoxy-2'-fluoroguanosine can be produced with high efficiency (Example 6).

In another embodiment of the present invention, a method for producing a nucleoside comprises treating the N-deoxyribosyltransferase variant of the present invention with 2'-deoxy-2'-fluorouridine and adenine to produce 2 ' - < / RTI >deoxy-2'-fluoroadenosine; And 2'-deoxy-2'-fluoroadenosine to produce 2'-deoxy-2'-fluoroinosine by treating adenosine deaminase derived from lactococcus lactis. -Deoxy-2 ' -fluoroinosine. ≪ / RTI >

In one specific embodiment of the present invention, 2'-deoxy-2'-fluoroadenosine is used as a substrate to express N-deoxyribosyltransferase mutant ( E. coli E206) was used to prepare 2'-deoxy-2'-fluoroinosine, and it was confirmed that 2'-deoxy-2'-fluoroinosine could be produced with high purity and high efficiency (Example 7).

The NDT mutant of the present invention or the production method using the microorganism expressing the mutant can mass-produce various nucleosides with high purity and high yield.

The N-deoxyribosyltransferase mutant of the present invention exhibits high activity against a non-natural type 2'-fluoro substrate compared with the conventional N-deoxyribosyltransferase, and has high yield and high purity To produce various nucleosides.

1 shows a process for preparing 2'-Deoxy-2'-fluoroadenosine according to the present invention.
2 shows a process for preparing 2'-Deoxy-2'-fluoroguanosine according to the present invention.
3 shows the conversion of 2'-deoxy-2'-fluoroguanosine according to the present invention.
4 shows the results of HPLC analysis of the secondary crystallization of 2'-deoxy-2'-fluoroguanosine according to the present invention.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example  1. Mistake inducing ( Error prone ) PCR method  by NDT  Random mutation of the gene

The present inventors have found that the existing Lactobacillus The nucleoside deoxyribosyl transferase (NDT) gene derived from delbrueckii KCCM35470 is a gene having a total size of 474 bp (SEQ ID NO: 1) and encodes NDT having the amino acid sequence of SEQ ID NO: 2. Since the NDT has a very low activity or no activity of the unnatural nucleoside compared to the natural nucleoside, it has been difficult to use the NDT in the enzymatic production method of the nucleoside.

Thus, Applicants have conducted real-time PCR to increase the reactivity of existing NDTs, particularly 2'-Deoxy-2'-Fluorouridine (2FDU) A random mutagenesis of the NDT gene was performed. Real-time PCR is a method of randomly inducing a mutation in a gene between primers by inducing an error in the polymerase reaction that amplifies the gene. Common mutagenesis methods include random mutagenesis using a mutagenic agent, UV irradiation, and site direct mutagenesis to add, remove, and mutate specific nucleotide sequences. However, Induced mutation of the NDT gene, the real - time PCR method was chosen.

The PCR mutagenesis was performed by using the pFRPT-NDT gene (SEQ ID NO: 3) as a template, and the primer sequences for real-time PCR were as shown in Table 1 below.

[Table 1]

NdeI and XbaI were inserted at the start codon and stop codon sites of pFRPT-NDT, respectively. The composition of reaction solution and PCR conditions for error prone PCR are shown in Table 2 below.

[Table 2]

Realization of MnSO _{4 in} PCR reaction solution And the concentration of dNTP was controlled to control the frequency of errors. The condition used in this example was designed to cause the deformation of 5.8 genes per 1000 bp. In the case of the NDT gene, the modification was designed to occur at 2 to 3 nucleotides, and the Diversify PCR Random Mutagenesis Kit (Cat No. 630703, Real-time PCR was performed using Clontech, www.clontech.com).

Next, 484 bp PCR product amplified by agarose gel electrophoresis was confirmed after PCR reaction, and the extracted PCR product was digested with restriction enzymes XbaI and NdeI . The vector to be inserted with the restriction enzyme digested PCR product was constructed using pFRPT-PUNP (purine nucleoside phosphorylase) (SEQ ID NO: 6) prepared by the applicant and pFRPT-PUNP was digested with restriction enzymes XbaI and NdeI The vector with the PUNP gene removed was treated with CIAP (Calf Intestinal Alkaline Phosphatase) to prevent self ligation. The truncated gene and vector were ligated with PCR products and vectors using T4 ligase, and this product was named pFRPT-mNDT. Using Gene pulser, pFRPT-mNDT was transformed into E. coli DH5α, and 1 mL of fresh SOC medium was added and cultured at 37 ° C for 1 hour. The culture broth was plated on LB agar plate medium containing kanamycin and then further cultured at 37 ° C for 20 hours.

Example  2. Random Mutation Verification

In order to construct the NDT random mutagenesis library, it is first necessary to verify the transformed colonies. In order to isolate colonies in which pFRPT-mNDT was normally inserted in the colonies, PCR screening was performed as follows, and primer sequences for PCR screening were as shown in Table 3 below.

[Table 3]

The PCR primers were designed using the RBS (ribosome binding site) region of the pFRPT and the base sequence of MCS (multi cloning site). PCR screening was performed using SapphireAmp Fast PCR Master Mix (Cat No. RR350A, TAKARA, co.) was performed according to the method shown in Table 4 below.

[Table 4]

PCR was performed under the conditions shown in Table 4. As a result, 20 to 30 colonies of 50 colonies were found to be normal mutations, and most of the remaining colonies were satellite, and 2 to 3 colonies were PCR ptRPT-NDT bp).

A total of 214 mutants were obtained through repetitive transformation and PCR screening of pFRPT-mNDT. Among them, 10 colonies were randomly selected to extract the plasmid, and then the plasmid was extracted from Bionics (www.bionicsro.co.kr) .

As a result, it was confirmed that nine mutants were modified in one nucleotide, and one mutation was modified in three nucleotides. Two mutants generated a stop codon, four mutants had no amino acid changes, Of the 10 mutations, only four mutations were found to have the amino acid-modified pFRPT-mNDT as planned. Thus, 80-90 colonies, 40% of 214 mutations, are expected to have normal pFRPT-mNDT.

Example  3. NDT  Active from random mutations Increased NDT Mutant  Screening

A total of 214 mutant strains obtained in Example 2 were cultured in 20 mL of LB containing kanamycin for 4 hours, and 20 μL of 1 M IPTG (isopropyl β-D-1-thiogalactopyranoside) Lt; / RTI > The culture broth was centrifuged and suspended in 0.5 mL of purified water to complete the reaction library. The reaction conditions for the first screening of 2FDU substrate-specific highly active strains using the mutagen library are shown in Table 5 below.

[Table 5]

A total of 214 mutants were transfected with pFRPT-NDT / DH5α, which was not mutated in the control reaction, and the activity of the mutant was analyzed based on this.

The conversion rate of 2FDA was calculated as follows.

* 2FDA  Reaction conversion = { 2FDA  (mole) x 100} / ({ 2FDA  (mole) + 2FDU  (mole)}

As a result, strains having a conversion ratio of 20% or more were selected, and the plasmid showing the highest action effect was selected from the results. Sequence analysis was performed to confirm changes in the gene and amino acid sequence of NDT.

[Table 6]

[Table 7]

C71, the most effective screening strain among the primary screening strains, was used to detect 2'-deoxy-2'-fluoro-2,6-diaminopurine riboside -diaminopurineriboside (2FDDAP).

The conversion rate of 2FDDAP was calculated as follows.

* 2FDDAP  Reaction conversion = { 2FDDAP  ( mole ) x 100} / ({ 2FDDAP  ( mole ) + 2FDU  (mole)}

Screening was performed at a minimum substrate concentration of 0.2 M, which is capable of actual industrial production. Four kinds of pFRPT-mNDT in which amino acid changes occurred were transformed with E. coli JM109. The specific composition and conditions of the reaction solution are shown in Table 8, and the results are shown in Table 9. [

[Table 8]

[Table 9]

As a result of 2FDDAP reaction, it was confirmed that C71 having 59 Leu → Gln mutation among various mutants had the best 2FDU substrate conversion ability as in 2FDA reaction, and named it pFRPT-FNDT / DH5α. Analysis of the genetic information of FNDT revealed that it had the nucleoside sequence of SEQ ID NO: 9 and analyzed the amino acid sequence to confirm that it had the amino acid sequence of SEQ ID NO: 10.

Further, in order to confirm the difference in the selection of the optimal E. coli host and the 2FDDAP reaction according to the enzyme concentration, the conversion rate over time was measured according to the composition of the reaction solution in Table 8, and the results are shown in Table 10.

[Table 10]

As shown in Table 10, it was confirmed that FNDT-DH5α showed the best activity for 2FDDAP reaction on the 42-hour reaction standard. Thus, the optimal E. coli host of FNDT was determined to be DH5α, and pFRPT-FNDT / DH5α was designated E. coli E206. In addition, FNDT confirmed that the conversion rate to 2FDU substrate was significantly increased (about 3.7 times as compared with the 42-hour reaction) compared to the conventional NDT.

Example  4. FNDT  Identification of the activity of the strain

A potency assay for pFRPT-NDT / JM109, which is an existing NDT-introduced strain, and pFRPT-FNDT / DH5? ( E. coli E206) of Example 3, was performed.

The 1 L fermentation of the strain was carried out by the method of Table 11, and the potency was analyzed using the 2FDA reaction of Table 12 for the produced cells. The results of 5 sets of pFRPT-NDT / JM109 and E. coli E206 cultures are shown in Table 13.

[Table 11]

[Table 12]

[Table 13]

As can be seen in Table 13, pFRPT-NDT / JM109 (E. coli E206) cells showed significantly improved activity (about 290 times) compared to pFRPT-NDT / JM109 cells.

The stomach cells were deposited with the Korean Culture Center of Microorganisms (KCCM) on Aug. 10, 2017, and received the accession number KCCM12092P.

Example  5. FNDT  The 2'- Deoxy -2'- Of fluoroadenosine  Produce

2'-deoxy-2'-fluoroadenosine (2FDA) was prepared using the FNDT of the present invention as a starting material for 2FDU and adenine (ADE) ).

First, 18.0 kg of 2'-deoxy-2'-fluorouridine (2.0 M) and 9.9 kg of adenine (2.0 M) were suspended in 36 L of purified water and 3.6 kg of E. coli E206-dried cells were mixed The reaction was carried out at 48 to 52 ° C and pH 6.1 to 6.5 for 84 hours. The reaction solution was analyzed by HPLC. As a result, it was confirmed that the conversion of 2'-deoxy-2'-fluoroadenosine was 97.6%.

After completion of the reaction, 45 L of purified water and 189 L of methyl alcohol were added, and the pH was adjusted to 1.0-1.3 with 35% hydrochloric acid. Then, 40 kg of celite was added and filtered to produce Gt; uracil < / RTI > was removed in the form of a filtrate. To recover the 2'-deoxy-2'-fluoroadenosine remaining in the filtrate, 27 liters of purified water and 63 L of methyl alcohol were added to the reactor, and the pH was adjusted to 35% using hydrochloric acid 1.0 to 1.3, followed by filtration, and the filtration filtrate was combined twice.

0.36 kg of Acid-charcoal was added to the filtered filtrate from which the cells and uracil were removed, stirred for 1 hour or more, and filtered. The filtrate was concentrated until the total liquid amount reached 63 ~ 81 L while maintaining the external temperature of the reactor at 65 캜 or below. After the concentration was finished, 126 L of purified water was added, and the pH was adjusted to 7.5 to 8.0 using a 10N NaOH aqueous solution.

After the pH was adjusted, the internal temperature of the reactor was raised to 46 ~ 52 캜 and stirred for 1 hour or longer. The internal temperature was gradually cooled to 15 ~ 30 캜 over 2 hours. The resulting filtrate was dried under reduced pressure at an external temperature of 65 DEG C or lower for 12 hours to obtain 16.11 kg of 2'-deoxy-2'-fluoroadenosine (Table 14), and the analysis results of 2FDA produced are shown in Table 15 below.

[Table 14]

[Table 15]

Based on the above results, it was found that high-purity 2'-deoxy-2'-fluoroadenosine having a purity of 99% or more can be effectively produced with a weight yield of about 90% by using the NDT mutant of the present invention or the cells containing the same Respectively.

Example 6. Preparation of 2'-deoxy-2'-fluoroguanosine using FNDT

2FDU and 2,6-Diaminopurine (DAP) were used as substrates for 2'-deoxy-2'-fluoro-2,6-diaminopurine (2'-Deoxy- 2 '-Fluoro-2,6-Diaminopurine; 2FDDAP), and then transformed with the expression strain of adenosine deaminase (SEQ ID NO: 11) derived from lactococcus lactis (pFRPT-LADD (2'-Deoxy-2'-fluoroguanosine (2FDG)) using a method described in Japanese Patent Application Laid-Open No. 10-2016-0033414 (FIG. 2), followed by a subsequent purification step to produce 2'-deoxy-2'-fluoroguanosine.

First, 20.0 g of 2'-deoxy-2'-fluorouridine (1.62 M) and 6.4 g of 2,6-diaminopurine (0.85 M) were suspended in 50 mL of purified water. deoxy-2'-fluoro-2,6-diaminopurine conversion reaction was carried out at 50 ° C and pH 6.0 under the condition of mixing E. coli E206 cells. The conversion of 2FDDAP was calculated as follows.

* 2FDDAP  Reaction conversion = { 2FDDAP (mole) X 100} / { 2FDU (mole) + 2FDDAP (mole)}

After confirming 40% conversion of 2FDDAP, 4.0 g of 2,6-diaminopurine (0.53 M) was added additionally, and after addition of 2.4 g of 2,6-diaminopurine (0.32 M) And 70% conversion was confirmed.

Then, after conversion of 70% to 2'-deoxy-2'-fluoro-2,6-diaminopurine was confirmed, deamination was carried out by adding 0.1 g of pFRPT-LADD / JM109 cells, The reaction proceeded. The pH was maintained at 6.0 during the reaction using a 50% aqueous solution of acetic acid. After the final 80% conversion was confirmed, further 10 g of E. coli E206 cells were added to continue the reaction. The 2'-deoxy-2'-fluoro-2,6-diaminopurine reaction and the 2'-deoxy-2'-fluoroguanosine reaction proceeded as one-pot, and the 2FDG conversion was calculated as Respectively.

* 2FDG  Reaction conversion = { 2FDG (mole) X 100} / { 2FDU (mole) + 2FDDAP (mole) + 2FDG (mole)}

After the final 141 hours, the reaction solution was analyzed by HPLC. As a result, it was confirmed that the conversion of 2'-deoxy-2'-fluoroguanosine was 97.5% (FIG. 3).

After completion of the reaction, 260 mL of purified water and 330 mL of methyl alcohol were added, and the pH was adjusted to 1.3 using 35% hydrochloric acid. Then, 20 g of celite was added and filtered to remove the uracil (uracil) was removed in the form of a filtrate. To recover the remaining 2'-deoxy-2'-fluoroguanosine in the filtrate, 80 mL of purified water and 80 mL of methyl alcohol were added to the filtrate, the pH was adjusted to 1.3 using 35% hydrochloric acid Filtered, and the filtrate was combined.

The filtrate, from which the cells and uracil were removed, was adjusted to pH 7.5 with a 10N NaOH aqueous solution and stirred at 70 ° C for 2 hours. After cooling to 0 ~ 5 ℃ over 3 hours, stirring was continued for 3 hours while maintaining the temperature, and 30.87 g of wet crude cake was recovered by filtration.

300 mL of purified water was added to the crude cake, the pH was adjusted to 10.5 using 10N NaOH, the temperature was raised to 75 ° C, and the mixture was stirred for 1 hour. 2 g of Basic-charcoal was added and further stirred for 1 hour and then filtered.

The filtrate was adjusted to pH 7.5 with a 50% acetic acid aqueous solution, and then heated to 75 ° C, stirred for about 3 hours, slowly cooled to 0 to 5 ° C over 3 hours, and stirred for 3 hours or more while maintaining the temperature. The process product was filtered and the obtained filtrate was dried to obtain 2'-deoxy-2'-fluoroguanosine having 16.52 g (weight yield 82.6%), moisture 7.16% and HPLC purity 99.42% .

From the results of this Example, it was confirmed that high-purity 2'-deoxy-2'-fluoroguanosine can be produced at a high yield by using the NDT mutant strain of the present invention.

Example 7. Preparation of 2'-deoxy-2'-fluoroinosine

The adenosine deaminase (LADD) -expressing strain (pFRPT-LADD / JM109) produced by the present applicant with the 2FDA obtained in the above Example 5 as a substrate was treated with 10 μg / ) Was used to prepare 2'-Deoxy-2'-fluoroinosine.

First, 36.7 g of 2FDA (1.36 M) was suspended in 100 ml of purified water, and 0.1 g of pFRPT-LADD / JM109 cells were mixed and agitated for 1.5 hours at 40 ° C and pH 7.0 to 8.0. The reaction solution was analyzed by HPLC and it was confirmed that 2FDA was converted to 2'-deoxy-2'-fluoroinosine by 100%.

After the completion of the reaction, 300 ml of purified water was added, and the pH was adjusted to 12.0 to 12.5 with 10 N NaOH aqueous solution, and the cells were removed by filtration. The filtrate was neutralized with 35% hydrochloric acid and kept at 0 ~ 5 ℃ for more than 3 hours. After stirring, it was filtered and vacuum dried at 55 ℃ for 12 hours.

As a result, 27 g of 2'-deoxy-2'-fluoroinosine having an HPLC purity of 100.0% was obtained. The weight yield based on the starting amount of 2'-deoxy-2'-fluoroadenine as a starting material was 73.6% , And the molar yield was 73.3%.

From the above results, the NDT variant (FNDT) of the present invention shows excellent substrate specificity for high activity, especially 2'-deoxy-2'-fluoro nucleoside, It was confirmed that it was possible to prepare a compound of the present invention.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Korea Microorganism Conservation Center KCCM12092P 20170810

&Lt; 110 > ST Pharm Co., Ltd. An N-deoxyribosyl transferase mutant, and a method for producing          nucleoside using the same <130> P17-093-STP <160> 11 <170> KoPatentin 3.0 <210> 1 <211> 474 <212> DNA <213> Lactobacillus delbrueckii <400> 1 atgcctaaga aaacgatcta cttcggtgcc ggctggttca ctgaccgcca aaacaaagcc 60 tacaaggaag ccatggaagc cctcaaggaa aacccaacga ttgacctgga aaacagctac 120 gttcccctgg acaaccagta caagggtatc cgggttgatg aacacccgga atacctgcat 180 gacaaggttt gggctacggc cacctacaac aacgacttga acgggatcaa gaccaacgac 240 atcatgctgg gtgtctacat ccctgacgaa gaagacgtcg gcctgggcat ggaactgggt 300 tacgccttga gccaaggcaa gtacgtcctt ttggtcatcc cggacgaaga ctacggcaag 360 ccgatcaacc tcatgagctg gggcgtcagc gacaacgtga tcaagatgag ccagctgaag 420 gacttcaact tcaacaagcc gcgcttcgac ttctacgagg gtgccgttta ttaa 474 <210> 2 <211> 157 <212> PRT <213> Lactobacillus delbrueckii <400> 2 Met Pro Lys Lys Thr Ile Tyr Phe Gly Ala Gly Trp Phe Thr Asp Arg   1 5 10 15 Gln Asn Lys Ala Tyr Lys Glu Ala Met Glu Ala Leu Lys Glu Asn Pro              20 25 30 Thr Ile Asp Leu Glu Asn Ser Tyr Val Pro Leu Asp Asn Gln Tyr Lys          35 40 45 Gly Ile Arg Val Asp Glu His Pro Glu Tyr Leu His Asp Lys Val Trp      50 55 60 Ala Thr Ala Thr Tyr Asn Asn Asp Leu Asn Gly Ile Lys Thr Asn Asp  65 70 75 80 Ile Met Leu Gly Val Tyr Ile Pro Asp Glu Glu Asp Val Gly Leu Gly                  85 90 95 Met Glu Leu Gly Tyr Ala Leu Ser Gln Gly Lys Tyr Val Leu Leu Val             100 105 110 Ile Pro Asp Glu Asp Tyr Gly Lys Pro Ile Asn Leu Met Ser Trp Gly         115 120 125 Val Ser Asp Asn Val Ile Lys Met Ser Gln Leu Lys Asp Phe Asn Phe     130 135 140 Asn Lys Pro Arg Phe Asp Phe Tyr Glu Gly Ala Val Tyr 145 150 155 <210> 3 <211> 6986 <212> DNA <213> Artificial Sequence <220> <223> pFRPT-NDT <400> 3 agatcccatg cctaagaaaa cgatctactt cggtgccggc tggttcactg accgccaaaa 60 caaagcctac aaggaagcca tggaagccct caaggaaaac ccaacgattg acctggaaaa 120 cagctacgtt cccctggaca accagtacaa gggtatccgg gttgatgaac acccggaata 180 cctgcatgac aaggtttggg ctacggccac ctacaacaac gacttgaacg ggatcaagac 240 caacgacatc atgctgggtg tctacatccc tgacgaagaa gacgtcggcc tgggcatgga 300 actgggttac gccttgagcc aaggcaagta cgtccttttg gtcatcccgg acgaagacta 360 cggcaagccg atcaacctca tgagctgggg cgtcagcgac aacgtgatca agatgagcca 420 gctgaaggac ttcaacttca acaagccgcg cttcgacttc tacgagggtg ccgtttatta 480 agggcagatc tttaacgcgg ccgcctctga tcgagataac agagtgttcc tgcaggttgt 540 gcccttcacc acctatatat gaggtgacct cgaaaccgtt ggtcaggcgt acgcggcaaa 600 gcttctgttt tggcggatga gagaagattt tcagcctgat acagattaaa tcagaacgca 660 gaagcggtct gataaaacag aatttgcctg gcggcagtag cgcggtggtc ccacctgacc 720 ccatgccgaa ctcagaagtg aaacgccgta gcgccgatgg tagtgtgggg tctccccatg 780 cgagagtagg gaactgccag gcatcaaata aaacgaaagg ctcagtcgaa agactgggcc 840 tttcgtttta tctgttgttt gtcggtgaac gctctcctga gtaggacaaa tccgccggga 900 gcggatttga acgttgcgaa gcaacggccc ggagggtggc gggcaggacg cccgccataa 960 actgccaggc atcaaattaa gcagaaggcc atcctgacgg atggcctttt tgcgtttcta 1020 caaactcttt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata 1080 accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg 1140 tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac 1200 gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact 1260 ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat 1320 gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtgtttcga gcaccaccac 1380 caccaccact gagatccggc tgctaacaaa gcccgaaagg aagctgagtt ggctgctgcc 1440 accgctgagc aataactagc ataacccctt ggggcctcta aacgggtctt gaggggtttt 1500 ttgctgaaag gaggaactat atccggattg gcgaatggga cgcgccctgt agcggcgcat 1560 taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag 1620 cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc 1680 aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc 1740 ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt 1800 ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa 1860 caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg 1920 cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat 1980 taacgtttac aatttcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt 2040 tatttttcta aatacattca aatatgtatc cgctcatgaa ttaattctta gaaaaactca 2100 tcgagcatca aatgaaactg caatttattc atatcaggat tatcaatacc atatttttga 2160 aaaagccgtt tctgtaatga aggagaaaac tcaccgaggc agttccatag gatggcaaga 2220 tcctggtatc ggtctgcgat tccgactcgt ccaacatcaa tacaacctat taatttcccc 2280 tcgtcaaaaa taaggttatc aagtgagaaa tcaccatgag tgacgactga atccggtgag 2340 aatggcaaaa gtttatgcat ttctttccag acttgttcaa caggccagcc attacgctcg 2400 tcatcaaaat cactcgcatc aaccaaaccg ttattcattc gtgattgcgc ctgagcgaga 2460 cgaaatacgc gatcgctgtt aaaaggacaa ttacaaacag gaatcgaatg caaccggcgc 2520 aggaacactg ccagcgcatc aacaatattt tcacctgaat caggatattc ttctaatacc 2580 tggaatgctg ttttcccggg gatcgcagtg gtgagtaacc atgcatcatc aggagtacgg 2640 ataaaatgct tgatggtcgg aagaggcata aattccgtca gccagtttag tctgaccatc 2700 tcatctgtaa catcattggc aacgctacct ttgccatgtt tcagaaacaa ctctggcgca 2760 tcgggcttcc catacaatcg atagattgtc gcacctgatt gcccgacatt atcgcgagcc 2820 catttatacc catataaatc agcatccatg ttggaattta atcgcggcct agagcaagac 2880 gtttcccgtt gaatatggct cataacaccc cttgtattac tgtttatgta agcagacagt 2940 tttattgttc atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc 3000 cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt 3060 gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac 3120 tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt 3180 gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct 3240 gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga 3300 ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac 3360 acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg 3420 agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt 3480 cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc 3540 tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg 3600 gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc 3660 ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc 3720 ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag 3780 cgaggaagcg gaagagcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc 3840 acaccgcata tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag 3900 tatacactcc gctatcgcta cgtgactggg tcatggctgc gccccgacac ccgccaacac 3960 ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga 4020 ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgaggc 4080 agctgcggta aagctcatca gcgtggtcgt gaagcgattc acagatgtct gcctgttcat 4140 ccgcgtccag ctcgttgagt ttctccagaa gcgttaatgt ctggcttctg ataaagcggg 4200 ccatgttaag ggcggttttt tcctgtttgg tcactgatgc ctccgtgtaa gggggatttc 4260 tgttcatggg ggtaatgata ccgatgaaac gagagaggat gctcacgata cgggttactg 4320 atgatgaaca tgcccggtta ctggaacgtt gtgagggtaa acaactggcg gtatggatgc 4380 ggcgggacca gagaaaaatc actcagggtc aatgccagcg cttcgttaat acagatgtag 4440 gtgttccccg gggtagccag cagcatcctg cgatgcagat ccggaacata atggtgcagg 4500 gcgctgactt ccgcgtttcc agactttacg aaacacggaa accgaagacc attcatgttg 4560 ttgctcaggt cgcagacgtt ttgcagcagc agtcgcttca cgttcgctcg cgtatcggtg 4620 attcattctg ctaaccagta aggcaacccc gccagcctag ccgggtcctc aacgacagga 4680 gcacgatcat gcctgaaatg agctgttgac aattaatcat cggctcgtat aatgtgtgga 4740 attgtgagcg gataacaatt tcacacagga aacagaattc atgttcgaac aacgcgtaaa 4800 ttctgacgta ctgaccgttt ctaccgttaa ctctcaggat caggtaaccc aaaaacccct 4860 gcgtgactcg gttaaacagg cactgaagaa ctattttgct caactgaatg gtcaggatgt 4920 gaatgacctc tatgagctgg tactggctga agtagaacag cccctgttgg acatggtgat 4980 gcaatacacc cgtggtaacc agacccgtgc tgcgctgatg atgggcatca accgtggtac 5040 gctgcgtaaa aaattgaaaa aatacggcat gaactaagct gcacccgtgg ggccgccatg 5100 ccggcgataa tggcctgctt ctcgccgaaa cgtttggtgg cgggaccagt gacgaaggct 5160 tgagcgaggg cgtgcaagat tccgaatacc gcaagcgaca ggccgatcat cgtcgcgctc 5220 cagcgaaagc ggtcctcgcc gaaaatgacc cagagcgctg ccggcacctg tcctacgagt 5280 tgcatgataa agaagacagt cataagtgcg gcgacgatag tcatgccccg cgcccaccgg 5340 aaggagctga ctgggttgaa ggctctcaag ggcatcggtc gagatcccgg tgcctaatga 5400 gtgagctaac ttacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg 5460 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg 5520 cgccagggtg gtttttcttt tcaccagtga gacgggcaac agctgattgc ccttcaccgc 5580 ctggccctga gagagttgca gcaagcggtc cacgctggtt tgccccagca ggcgaaaatc 5640 ctgtttgatg gtggttaacg gcgggatata acatgagctg tcttcggtat cgtcgtatcc 5700 cactaccgag atgtccgcac caacgcgcag cccggactcg gtaatggcgc gcattgcgcc 5760 cagcgccatc tgatcgttgg caaccagcat cgcagtggga acgatgccct cattcagcat 5820 ttgcatggtt tgttgaaaac cggacatggc actccagtcg ccttcccgtt ccgctatcgg 5880 ctgaatttga ttgcgagtga gatatttatg ccagccagcc agacgcagac gcgccgagac 5940 agaacttaat gggcccgcta acagcgcgat ttgctggtga cccaatgcga ccagatgctc 6000 ccgcccagt cgcgtaccgt cttcatggga gaaaataata ctgttgatgg gtgtctggtc 6060 agagacatca agaaataacg ccggaacatt agtgcaggca gcttccacag caatggcatc 6120 ctggtcatcc agcggatagt taatgatcag cccactgacg cgttgcgcga gaagattgtg 6180 caccgccgct ttacaggctt cgacgccgct tcgttctacc atcgacacca ccacgctggc 6240 acccagttga tcggcgcgag atttaatcgc cgcgacaatt tgcgacggcg cgtgcagggc 6300 cagactggag gtggcaacgc caatcagcaa cgactgtttg cccgccagtt gttgtgccac 6360 gcggttggga atgtaattca gctccgccat cgccgcttcc actttttccc gcgttttcgc 6420 agaaacgtgg ctggcctggt tcaccacgcg ggaaacggtc tgataagaga caccggcata 6480 ctctgcgaca tcgtataacg ttactggttt cacattcacc accctgaatt gactctcttc 6540 cgggcgctat catgccatac cgcgaaaggt tttgcgccat tcgatggtgt ccgggatctc 6600 gcgctctcc cttatgcgac tcctgcatta ggaagcagcc cagtagtagg ttgaggccgt 6660 tgagcaccgc cgccgcaagg aatggtgcta gaggatcccc agatcgatct cgatcccgcg 6720 aaataaatgc tgaacaatta ttgcccgttt tacagcgtta cggcttcgaa acgctcgaaa 6780 aactggcagt tttaggctga tttggttgaa tgttgcgcgg tcagaaaatt attttaaatt 6840 tcctcttgtc aattaatcat ccggctcgta taatgcgcgg aattgtgtga gcggataaca 6900 attcccacca tttaacttta agaaggagat atacatatga aagaaaccgc tgctgctaaa 6960 ttcgaacgcc agcacatgga cagccc 6986 <210> 4 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> NDT forward primer <400> 4 gatcatatgc ctaagaaaac gatc 24 <210> 5 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> NDT reverse primer <400> 5 cgttctagat ctgcccttaa taaa 24 <210> 6 <211> 7143 <212> DNA <213> Artificial Sequence <220> Purification of pFRPT-PUNP (purine nucleoside phosphorylase) <400> 6 tatgccgagt gacaagcctc accaccacca ccaccaccac caccaccact cctctgacga 60 cgacgacaag gatccgatga gcgttcatat tggtgcaaaa gaacacgaga ttgcagataa 120 aattttgctt ccaggagatc cacttcgcgc aaaatatatc gctgaaacgt ttttagaggg 180 agctacttgc tataatcaag ttcgcggtat gttaggattt acaggtacat ataaaggcca 240 tcgtatttcc gttcaaggaa caggtatggg tgtaccatct atttctattt atattacaga 300 acttatgcaa agctacaacg ttcaaacatt aattcgcgtc ggaacatgtg gtgctattca 360 aaaagatgta aaagttcgtg atgtcatttt agcgatgaca tcgtcaaccg attcccaaat 420 gaatcgcatg acgttcggag gaattgatta cgctccgaca gctaactttg acttgttaaa 480 aacagcgtac gaaattggaa aagaaaaagg attacaacta aaagttggca gtgtatttac 540 agctgatatg ttttataatg aaaatgcaca atttgaaaaa ctggcacgat acggtgtact 600 ggctgtagag atggaaacaa cagcgcttta tacattagcc gctaaatttg gcagaaaagc 660 attatcggta ttaacagtaa gcgatcacat tttaacaggg gaagagacaa cggctgaaga 720 gcgccaaaca acatttaacg aaatgatcga agtcgctctt gaaacagcga ttcgccaata 780 atctagagct tgcggccgca ctcgacaagc ttctgttttg gcggatgaga gaagattttc 840 agcctgatac agattaaatc agaacgcaga agcggtctga taaaacagaa tttgcctggc 900 ggcagtagcg cggtggtccc acctgacccc atgccgaact cagaagtgaa acgccgtagc 960 gccgatggta gtgtggggtc tccccatgcg agagtaggga actgccaggc atcaaataaa 1020 acgaaaggct cagtcgaaag actgggcctt tcgttttatc tgttgtttgt cggtgaacgc 1080 tctcctgagt aggacaaatc cgccgggagc ggatttgaac gttgcgaagc aacggcccgg 1140 agggtggcgg gcaggacgcc cgccataaac tgccaggcat caaattaagc agaaggccat 1200 cctgacggat ggcctttttg cgtttctaca aactcttttg tttatttttc taaatacatt 1260 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa 1320 ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt 1380 gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt 1440 tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt 1500 ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg 1560 tattatcccg tgtttcgagc accaccacca ccaccactga gatccggctg ctaacaaagc 1620 ccgaaaggaa gctgagttgg ctgctgccac cgctgagcaa taactagcat aaccccttgg 1680 ggcctctaaa cgggtcttga ggggtttttt gctgaaagga ggaactatat ccggattggc 1740 gaatgggacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc 1800 gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt 1860 ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc 1920 cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt 1980 agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt 2040 aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt 2100 gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa 2160 aaatttaacg cgaattttaa caaaatatta acgtttacaa tttcaggtgg cacttttcgg 2220 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg 2280 ctcatgaatt aattcttaga aaaactcatc gagcatcaaa tgaaactgca atttattcat 2340 atcaggatta tcaataccat atttttgaaa aagccgtttc tgtaatgaag gagaaaactc 2400 accgaggcag ttccatagga tggcaagatc ctggtatcgg tctgcgattc cgactcgtcc 2460 aacatcaata caacctatta atttcccctc gtcaaaaata aggttatcaa gtgagaaatc 2520 accatgagtg acgactgaat ccggtgagaa tggcaaaagt ttatgcattt ctttccagac 2580 ttgttcaaca ggccagccat tacgctcgtc atcaaaatca ctcgcatcaa ccaaaccgtt 2640 attcattcgt gattgcgcct gagcgagacg aaatacgcga tcgctgttaa aaggacaatt 2700 acaaacagga atcgaatgca accggcgcag gaacactgcc agcgcatcaa caatattttc 2760 acctgaatca ggatattctt ctaatacctg gaatgctgtt ttcccgggga tcgcagtggt 2820 gagtaaccat gcatcatcag gagtacggat aaaatgcttg atggtcggaa gaggcataaa 2880 ttccgtcagc cagtttagtc tgaccatctc atctgtaaca tcattggcaa cgctaccttt 2940 gccatgtttc agaaacaact ctggcgcatc gggcttccca tacaatcgat agattgtcgc 3000 acctgattgc ccgacattat cgcgagccca tttataccca tataaatcag catccatgtt 3060 ggaatttaat cgcggcctag agcaagacgt ttcccgttga atatggctca taacacccct 3120 tgtattactg tttatgtaag cagacagttt tattgttcat gaccaaaatc ccttaacgtg 3180 agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc 3240 ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg 3300 tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag 3360 cgcagatacc aaatactgtc cttctagtgt agccgtagtt aggccaccac ttcaagaact 3420 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg 3480 gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc 3540 ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg 3600 aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg 3660 cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag 3720 ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc 3780 gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct 3840 ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc 3900 ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc 3960 gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgcctg atgcggtatt 4020 ttctccttac gcatctgtgc ggtatttcac accgcatata tggtgcactc tcagtacaat 4080 ctgctctgat gccgcatagt taagccagta tacactccgc tatcgctacg tgactgggtc 4140 atggctgcgc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc 4200 ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt 4260 tcaccgtcat caccgaaacg cgcgaggcag ctgcggtaaa gctcatcagc gtggtcgtga 4320 agcgattcac agatgtctgc ctgttcatcc gcgtccagct cgttgagttt ctccagaagc 4380 gttaatgtct ggcttctgat aaagcgggcc atgttaaggg cggttttttc ctgtttggtc 4440 actgatgcct ccgtgtaagg gggatttctg ttcatggggg taatgatacc gatgaaacga 4500 gagaggatgc tcacgatacg ggttactgat gatgaacatg cccggttact ggaacgttgt 4560 gagggtaaac aactggcggt atggatgcgg cgggaccaga gaaaaatcac tcagggtcaa 4620 tgccagcgct tcgttaatac agatgtaggt gttccacagg gtagccagca gcatcctgcg 4680 atgcagatcc ggaacataat ggtgcagggc gctgacttcc gcgtttccag actttacgaa 4740 acacggaaac cgaagaccat tcatgttgtt gctcaggtcg cagacgtttt gcagcagcag 4800 tcgcttcacg ttcgctcgcg tatcggtgat tcattctgct aaccagtaag gcaaccccgc 4860 cgcctagcc gggtcctcaa cgacaggagc acgatcatgc ctgaaatgag ctgttgacaa 4920 ttaatcatcg gctcgtataa tgtgtggaat tgtgagcgga taacaatttc acacaggaaa 4980 cagaattcat gttcgaacaa cgcgtaaatt ctgacgtact gaccgtttct accgttaact 5040 ctcaggatca ggtaacccaa aaacccctgc gtgactcggt taaacaggca ctgaagaact 5100 attttgctca actgaatggt caggatgtga atgacctcta tgagctggta ctggctgaag 5160 tagaacagcc cctgttggac atggtgatgc aatacacccg tggtaaccag acccgtgctg 5220 cgctgatgat gggcatcaac cgtggtacgc tgcgtaaaaa attgaaaaaa tacggcatga 5280 actaagctgc acccgtgggg ccgccatgcc ggcgataatg gcctgcttct cgccgaaacg 5340 tttggtggcg ggaccagtga cgaaggcttg agcgagggcg tgcaagattc cgaataccgc 5400 aagcgacagg ccgatcatcg tcgcgctcca gcgaaagcgg tcctcgccga aaatgaccca 5460 gagcgctgcc ggcacctgtc ctacgagttg catgataaag aagacagtca taagtgcggc 5520 gacgatagtc atgccccgcg cccaccggaa ggagctgact gggttgaagg ctctcaaggg 5580 catcggtcga gatcccggtg cctaatgagt gagctaactt acattaattg cgttgcgctc 5640 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg 5700 cgcggggaga ggcggtttgc gtattgggcg ccagggtggt ttttcttttc accagtgaga 5760 cgggcaacag ctgattgccc ttcaccgcct ggccctgaga gagttgcagc aagcggtcca 5820 cgctggtttg ccccagcagg cgaaaatcct gtttgatggt ggttaacggc gggatataac 5880 atgagctgtc ttcggtatcg tcgtatccca ctaccgagat gtccgcacca acgcgcagcc 5940 cggactcggt aatggcgcgc attgcgccca gcgccatctg atcgttggca accagcatcg 6000 cagtgggaac gatgccctca ttcagcattt gcatggtttg ttgaaaaccg gacatggcac 6060 tccagtcgcc ttcccgttcc gctatcggct gaatttgatt gcgagtgaga tatttatgcc 6120 agccagccag acgcagacgc gccgagacag aacttaatgg gcccgctaac agcgcgattt 6180 gctggtgacc caatgcgacc agatgctcca cgcccagtcg cgtaccgtct tcatgggaga 6240 aaataatact gttgatgggt gtctggtcag agacatcaag aaataacgcc ggaacattag 6300 tgcaggcagc ttccacagca atggcatcct ggtcatccag cggatagtta atgatcagcc 6360 cactgacgcg ttgcgcgaga agattgtgca ccgccgcttt acaggcttcg acgccgcttc 6420 gttctaccat cgacaccacc acgctggcac ccagttgatc ggcgcgagat ttaatcgccg 6480 cgacaatttg cgacggcgcg tgcagggcca gactggaggt ggcaacgcca atcagcaacg 6540 actgtttgcc cgccagttgt tgtgccacgc ggttgggaat gtaattcagc tccgccatcg 6600 ccgcttccac tttttcccgc gttttcgcag aaacgtggct ggcctggttc accacgcggg 6660 aaacggtctg ataagagaca ccggcatact ctgcgacatc gtataacgtt actggtttca 6720 cattcaccac cctgaattga ctctcttccg ggcgctatca tgccataccg cgaaaggttt 6780 tgcgccattc gatggtgtcc gggatctcga cgctctccct tatgcgactc ctgcattagg 6840 aagcagccca gtagtaggtt gaggccgttg agcaccgccg ccgcaaggaa tggtgctaga 6900 ggatccccag atcgatctcg atcccgcgaa ataaatgctg aacaattatt gcccgtttta 6960 cagcgttacg gcttcgaaac gctcgaaaaa ctggcagttt taggctgatt tggttgaatg 7020 ttgcgcggtc agaaaattat tttaaatttc ctcttgtcaa ttaatcatcc ggctcgtata 7080 atgcgcggaa ttgtgtgagc ggataacaat tcccaccatt taactttaag aaggagatat 7140 aca 7143 <210> 7 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> pFRPT forward primer <400> 7 gagcggataa caattcccac c 21 <210> 8 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> pFRPT reverse primer <400> 8 tctcatccgc caaaacagaa g 21 <210> 9 <211> 474 <212> DNA <213> Artificial Sequence <220> 2'-Deoxy-2'-fluoronucleoside specific N-deoxyribosyltransferase <400> 9 atgcctaaga aaacgatcta cttcggtgcc ggctggttca ctgaccgcca aaacaaagcc 60 tacaaggaag ccatggaagc cctcaaggaa aacccaacga ttgacctgga aaacagctac 120 gttcccctgg acaaccagta caagggtatc cgggttgatg aacacccgga ataccagcat 180 gacaaggttt gggctacggc cacctacaac aacgacttga acgggatcaa gaccaacgac 240 atcatgctgg gtgtctacat ccctgacgaa gaagacgtcg gcctgggcat ggaactgggt 300 tacgccttga gccaaggcaa gtacgtcctt ttggtcatcc cggacgaaga ctacggcaag 360 ccgatcaacc tcatgagctg gggcgtcagc gacaacgtga tcaagatgag ccagctgaag 420 gacttcaact tcaacaagcc gcgcttcgac ttctacgagg gtgccgttta ttaa 474 <210> 10 <211> 157 <212> PRT <213> Artificial Sequence <220> 2'-Deoxy-2'-fluoronucleoside specific N-deoxyribosyltransferase <400> 10 Met Pro Lys Lys Thr Ile Tyr Phe Gly Ala Gly Trp Phe Thr Asp Arg   1 5 10 15 Gln Asn Lys Ala Tyr Lys Glu Ala Met Glu Ala Leu Lys Glu Asn Pro              20 25 30 Thr Ile Asp Leu Glu Asn Ser Tyr Val Pro Leu Asp Asn Gln Tyr Lys          35 40 45 Gly Ile Arg Val Asp Glu His Pro Glu Tyr Gln His Asp Lys Val Trp      50 55 60 Ala Thr Ala Thr Tyr Asn Asn Asp Leu Asn Gly Ile Lys Thr Asn Asp  65 70 75 80 Ile Met Leu Gly Val Tyr Ile Pro Asp Glu Glu Asp Val Gly Leu Gly                  85 90 95 Met Glu Leu Gly Tyr Ala Leu Ser Gln Gly Lys Tyr Val Leu Leu Val             100 105 110 Ile Pro Asp Glu Asp Tyr Gly Lys Pro Ile Asn Leu Met Ser Trp Gly         115 120 125 Val Ser Asp Asn Val Ile Lys Met Ser Gln Leu Lys Asp Phe Asn Phe     130 135 140 Asn Lys Pro Arg Phe Asp Phe Tyr Glu Gly Ala Val Tyr 145 150 155 <210> 11 <211> 352 <212> PRT <213> Artificial Sequence <220> &Lt; 223 > Adenosine deaminase derived from Lactococcus lactis <400> 11 Met Lys Lys Lys Arg Glu Lys Leu Val Leu Lys Ser Glu Ile Ile Ala   1 5 10 15 Gln Met Pro Lys Val Glu Leu His Cys His Leu Asp Gly Ser Leu Ser              20 25 30 Leu Ser Val Ile Lys Glu Leu Ala Lys Asn Ala Gly Ile His Met Thr          35 40 45 Met Ser Asp Glu Glu Ile Leu Glu Lys Ala Gln Ala Pro Glu Asn Thr      50 55 60 Lys Asn Leu Leu Glu Tyr Leu Gln Arg Phe Asp Phe Val Leu Pro Leu  65 70 75 80 Leu Gln Thr Tyr Lys Asn Leu Glu Leu Ala Ala Tyr Asp Val Val Arg                  85 90 95 Gln Ala Ala Asn Asp Asn Ile Lys Tyr Ile Glu Ile Arg Phe Ala Pro             100 105 110 Ser Gln His Leu Leu Glu Asn Leu Thr Leu Glu Glu Ala Val Glu Ala         115 120 125 Val Ile Ala Gly Leu Ser Arg Ala Glu Asn Asp Phe Asp Ile Arg Ala     130 135 140 Asn Ala Leu Val Cys Gly Leu Lys Gln Glu Pro Ile Gln Lys Leu Gln 145 150 155 160 Lys Leu Leu Pro Leu Phe Asp Lys Ile Pro Asp Glu His Leu Val Gly                 165 170 175 Phe Asp Met Ala Gly Asp Glu Leu Asn Tyr Pro Gln Glu Lys Phe Val             180 185 190 Asp Leu Ile His Asp Ile Lys Ile Lys Gly Val Asn Val Thr Leu His         195 200 205 Ala Gly Glu Cys Pro Ala Cys Glu Lys Asn Ile Leu Asp Ser Ile Ala     210 215 220 Met Gly Ala Ser Arg Ile Gly His Gly Ile Met Thr Lys Asn Leu Ser 225 230 235 240 Glu Ala Glu Gln Lys Met Met Ile Glu Lys Gln Ile Val Leu Glu Met                 245 250 255 Ala Pro Thr Ser Asn Phe Gln Thr Lys Ala Val Thr Glu Leu Ala Gln             260 265 270 Tyr Pro Phe Lys Glu Leu Tyr Asp Lys Gly Ile His Val Thr Leu Asn         275 280 285 Thr Asp Asn Arg Met Val Ser Ala Thr Asn Leu Ser Lys Glu Tyr Glu     290 295 300 Lys Ile Ser Ala Trp Tyr Pro Asp Phe Ser Leu Ser Asp Phe Glu Lys 305 310 315 320 Ile Asn His Tyr Ala Ile Asp Gly Ala Phe Ile Gly Gln Glu Glu Lys                 325 330 335 Glu Glu Leu His Gln Arg Phe Thr Lys Glu Tyr Lys Lys Ile Ser Glu             340 345 350

Claims (18)

The 5'-deoxy-2'-fluoro-nucleoside is characterized by the L59Q mutation in which the 59th leucine in the amino acid sequence of SEQ ID NO: 2 is replaced by glutamine. N-deoxyribosyltransferase variant wherein the substrate reactivity to fluoronucleoside is increased relative to the wild-type. 2. The N-deoxyribosyltransferase mutant according to claim 1, wherein said mutant has an activity of catalyzing a transglycosylation reaction. delete The method of claim 1, wherein the 2'-deoxy-2'-fluoro nucleoside is selected from the group consisting of 2'-Deoxy-2'-fluorouridine, N-deoxy Ribosyltransferase mutant. A polynucleotide encoding the N-deoxyribosyltransferase variant of claim 1. 6. The polynucleotide of claim 5, wherein the polynucleotide comprises the nucleotide sequence of SEQ ID NO: A recombinant vector operatively linked to a regulatory sequence of the polynucleotide of claim 5. A microorganism into which the recombinant vector of claim 7 is introduced. 9. The microorganism according to claim 8, wherein the microorganism is E. coli DH5 ?. 10. The microorganism according to claim 9, wherein the microorganism is KCCM12092P. 11. The microorganism according to any one of claims 8 to 10, wherein the microorganism has an activity of catalyzing a transglycosylation reaction. A method for producing a nucleoside by forming an N-glucoside bond between a base donor and a sugar residue donor using the N-deoxyribosyltransferase mutant of claim 1 or a microorganism expressing the mutant. 13. The method of claim 12, wherein the base donor is selected from the group consisting of adenine, guanine, hypoxanthine, xanthine, 6-mercaptoulinine, 6-thioguanine, N-alkyl or acyladenyl, 2-alkoxyadenine, , 2-thiocytosine, 4-thiouracil, N-acylcytosine, 5-thioguanidine, , 5-halogenovinyluracil, 1,3,4-triazole-3-carboxamide 1,2,4-triazole-3-carboxylic acid, 1,2,4- Imidazol-5-olate, benzimidazole, 1-deazaadenine, 3-deazaadenine, 3-deazaguanine, 7- But are not limited to, deazaadenine, deazaadenine, 7-deazaguanine, 8-azaadenine, 7-deaza-8-azaspiroxan, 5- azathiamine, 5- azasitonin, 6- azauracil, 3- deazauracil, And nicotinic acid amide And any one selected from the group,
Wherein the sugar residue donor is selected from the group consisting of inosine, guanosine, uridine, ribofuranosyl thymine, ribose-1-phosphate, 2'-deoxyinosine, 2'- deoxyguanosine, 2'-deoxyuridine, thymidine, 2 ', 3'-dideoxyinosine, 2', 3'-dioxyguanosine, 2 ', 3'-dideoxyuridine, 3'-deoxythymidine, 2-deoxyribose- 2,3-dideoxyribose-1-phosphate. &Lt; / RTI &gt; 14. The method of claim 13, wherein the base donor is adenine, 2,6-diaminopurine or cytosine. 14. The method of claim 13, wherein the sugar residue donor is selected from the group consisting of Thymidine, 2'-deoxyuridine, or 2'-deoxy- 2'-fluorouridine. &Lt; / RTI &gt; Deoxy-2'-fluoroadenosine is produced by treating the N-deoxyribosyltransferase mutant of claim 1 with 2'-deoxy-2'-fluorouridine and adenine Deoxy-2'-fluoroadenosine. &Lt; / RTI &gt; The N-deoxyribosyltransferase mutant of claim 1 is treated with 2'-deoxy-2'-fluorouridine and 2,6-diaminopurine to produce 2'-deoxy-2'-fluoro- Preparing 2,6-diaminopurine; And
2-deoxy-2'-fluoro-2,6-diaminopurine is treated with adenosine deaminase derived from lactococcus lactis to prepare 2'-deoxy-2'-fluoroguanosine &Lt; RTI ID = 0.0 &gt;2'-deoxy-2'-fluoroguanosine.&Lt; / RTI &gt; Preparing 2'-deoxy-2'-fluoroadenosine by treating the N-deoxyribosyltransferase mutant of claim 1 with 2'-deoxy-2'-fluorouridine and adenine; And
2'-deoxy-2'-fluoroadenosine to produce 2'-deoxy-2'-fluoroinosine by treating the 2'-deoxy-2'-fluoroadenosine with adenosine deaminase derived from lactococcus lactis. Deoxy-2'-fluoroinosine.

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