CN114231522B - Immobilized N-deoxyribotransferase and deoxynucleoside preparation method - Google Patents

Immobilized N-deoxyribotransferase and deoxynucleoside preparation method Download PDF

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CN114231522B
CN114231522B CN202111615269.7A CN202111615269A CN114231522B CN 114231522 B CN114231522 B CN 114231522B CN 202111615269 A CN202111615269 A CN 202111615269A CN 114231522 B CN114231522 B CN 114231522B
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deoxyribotransferase
immobilized
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CN114231522A (en
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谢磊
李�杰
黄月园
张波琨
孙丰来
徐幸福
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Shanghai SynTheAll Pharmaceutical Co Ltd
Shanghai STA Pharmaceutical R&D Ltd
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Shanghai STA Pharmaceutical R&D Ltd
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Abstract

The application discloses a preparation method of immobilized N-deoxyribotransferase and deoxynucleoside. Wherein the method for preparing the immobilized N-deoxyribotransferase comprises the following steps: a. obtaining an N-deoxyribotransferase with an affinity tag; b. contacting the affinity tagged N-deoxyribotransferase with an affinity ligand to immobilize the affinity tagged N-deoxyribotransferase to the affinity ligand; washing the affinity ligand to obtain the immobilized N-deoxyribotransferase.

Description

Immobilized N-deoxyribotransferase and deoxynucleoside preparation method
Technical Field
The application relates to the technical field of recombinase catalytic reaction, in particular to a preparation method of immobilized N-deoxyribotransferase and deoxynucleoside.
Background
2' -Deoxycytidine (2 ' -Deoxycytigine, abbreviated dA), also known as "2' -deoxyribocytosine cytidine", is a natural deoxyribonucleoside as an organic constituent of the genetic material deoxyribonucleic acid (DNA) involved in the transmission of genetic information of cells of a living organism. As an Eg5 kinesin regulator, 2 '-deoxycytidine has antiproliferative and apoptosis induction activities, so that the 2' -deoxycytidine becomes an important medical intermediate, can be used for synthesizing anticancer drugs of gemcitabine (dFdC), anti-HIV drugs of zalcitabine (ddC), a biochemical reagent of deoxycytidine-5 '-monophosphate (5' -dCMP) and the like, and is widely applied to nucleic acid and genetic engineering. With the growing development of the pharmaceutical industry and biotechnology, in order to meet the increasing demand of the raw material market for 2' -deoxycytidine, the demand of nucleoside analogs as an important component of antiviral drugs is also increasing drastically.
In 2006, moosa et al reported in the patent a chemical synthesis of β -L-2' -deoxy-cytidine, which uses protected ribofuranose as starting material, which is condensed with protected pyrimidine and deprotected to give the target product. However, the synthetic methods reported in the literature all have the formation of the isomer alpha-2' -deoxycytidine, and although the authors optimize the synthetic method, the quantity ratio of alpha: beta configuration substances of the products can only reach 1:5 or 1:6, and the selectivity is very low.
In recent years, a technique for preparing deoxynucleosides by enzyme catalysis has appeared. Chinese patent application CN105754899A discloses a method for synthesizing deoxynucleoside by using N-deoxyribotransferase enzyme method, including the induction expression and purification of recombinant N-deoxyribotransferase NDT in recombinant bacterium E.coli BL21 (DE 3), adding ribose donor and acceptor into buffer solution, adding N-deoxyribotransferase crude enzyme solution, placing into a shaking table with rotating speed of 180rpm at 35 ℃ for reaction for 2h, sampling and adding methanol after the reaction is finished to terminate the reaction. However, the crude enzyme solution used in this process contains a large amount of heteroenzyme, which hydrolyzes the product to produce a large amount of impurities, resulting in a lower purity of the final deoxynucleoside product.
There remains a need in the art for a process that can produce deoxynucleosides, particularly 2' -deoxycytidine, in high purity, at high conversion and at low cost, while avoiding the production of isomers.
Disclosure of Invention
In order to solve the above technical problems, an aspect of the present application provides a method for preparing immobilized N-deoxyribotransferase, comprising the steps of:
a. Obtaining an N-deoxyribotransferase with an affinity tag;
b. Contacting the affinity tagged N-deoxyribotransferase with an affinity ligand to immobilize the affinity tagged N-deoxyribotransferase to the affinity ligand; and
C. Washing the affinity ligand to obtain the immobilized N-deoxyribotransferase.
In another aspect, the present invention provides a method for preparing deoxynucleosides, comprising the steps of:
a. Obtaining an N-deoxyribotransferase with an affinity tag;
b. Contacting the affinity tagged N-deoxyribotransferase with an affinity ligand to immobilize the affinity tagged N-deoxyribotransferase to the affinity ligand;
c. Washing the affinity ligand to obtain an immobilized N-deoxyribotransferase;
d. Providing a reaction composition comprising a deoxyribose donor and a base donor;
e. adding the immobilized N-deoxyribotransferase to the reaction composition; and
F. Obtaining the deoxynucleoside.
Drawings
The application is described in more detail below with reference to the attached drawing figures, wherein:
FIG. 1 is a liquid-phase diagram showing the HPLC of a product obtained by catalyzing the formation of 2' -deoxycytidine from a deoxyribose donor and cytosine with a crude enzyme solution of N-deoxyribose transferase;
FIG. 2 is a liquid phase diagram showing the product HPLC of catalyzing the formation of 2' -deoxycytidine from a deoxyribose donor and cytosine with an immobilized N-deoxyribose transferase according to one embodiment of the application;
FIG. 3 is a schematic diagram showing the change in conversion of catalytic reactions with immobilized N-deoxyribotransferase according to one embodiment of the application at different amounts of immobilized enzyme addition and at different times; and
FIG. 4 is a schematic diagram showing the effect of varying molar amounts of cytosine on reaction conversion when catalyzing the formation of 2' -deoxycytidine from a deoxyribose donor and cytosine with an immobilized N-deoxyribose transferase according to one embodiment of the application.
Detailed Description
The application relates to a preparation method of immobilized N-deoxyribotransferase. The method comprises the following steps: a. obtaining the N-deoxyribotransferase with the affinity tag. In the present application, the N-deoxyribotransferase is any suitable enzyme capable of catalyzing the reaction of a deoxyribose donor and a base donor to produce a deoxynucleoside, and the affinity tagged N-deoxyribotransferase may be obtained by any suitable method. In one embodiment of the application, the N-deoxyribotransferase is an N-deoxyribotransferase from Chlorella pyrenoidosa (Chroococcidiopsis thermalis). In one embodiment of the application, the affinity tagged N-deoxyribotransferase is obtained by a fermentation process comprising inoculating a host cell expressing the affinity tagged N-deoxyribotransferase into a fermentation medium, inducing protein expression and extracting to obtain a crude enzyme solution comprising the affinity tagged N-deoxyribotransferase. In one embodiment of the application, the host cell is a eukaryotic cell. In one embodiment of the application, the host cell is a prokaryotic cell. In one embodiment of the application, the host cell is an E.coli cell. In one embodiment of the application, the affinity tag is a histidine tag. In one embodiment of the application, the amino acid sequence of the affinity tagged N-deoxyribotransferase is as set forth in SEQ ID NO: 2. In one embodiment of the application, the nucleotide sequence encoding the affinity tagged N-deoxyribotransferase is set forth in SEQ ID NO: 1. In one embodiment of the application, the fermentation medium may be any suitable medium known in the art. In one embodiment of the application, the fermentation medium comprises TB liquid medium.
The preparation method of the immobilized N-deoxyribotransferase also comprises the following steps: b. contacting the affinity tagged N-deoxyribotransferase with an affinity ligand to immobilize the affinity tagged N-deoxyribotransferase to the affinity ligand. In the present application, an "affinity ligand" refers to a substance that can specifically bind to an affinity tag. Any suitable affinity tag and affinity ligand pair known in the art may be used in the present application. In one embodiment of the application, the affinity tag is a histidine tag and the affinity ligand is a metal affinity ligand. In a preferred embodiment of the application, the metal affinity ligand comprises a nickel ion metal chelating medium or a cobalt ion metal chelating medium. In a preferred embodiment of the application, the metal affinity ligand is selected from nickel ion or cobalt ion activated LX-1000IDA, seplite LX-1000IDA, FP-IDA405/EB or Ni-IDA-Sefinose resin. In one embodiment of the application, the contacting step comprises passing the crude enzyme stream of affinity tagged N-deoxyribotransferase through a chromatography column preloaded with an affinity ligand to adsorb the affinity tagged N-deoxyribotransferase on the affinity ligand.
The preparation method of the immobilized N-deoxyribotransferase also comprises the following steps: c. washing the affinity ligand to obtain the immobilized N-deoxyribotransferase. In one embodiment of the application, the washing step comprises washing the affinity ligand with a detergent to remove unbound N-deoxyribotransferase and other impurities in the crude enzyme solution. In the present application, the detergent may be any suitable agent known in the art that washes the affinity ligand without causing the bound N-deoxyribotransferase to detach. In the present application, the immobilized N-deoxyribotransferase includes an N-deoxyribotransferase and an affinity ligand. In one embodiment of the application, the enzyme load of the immobilized N-deoxyribotransferase is 1-200mg N-deoxyribotransferase/g affinity ligand. In a preferred embodiment of the application, the enzyme load of the immobilized N-deoxyribotransferase is 10-100mg of N-deoxyribotransferase per g of affinity ligand. In a preferred embodiment of the application, the enzyme load of the immobilized N-deoxyribotransferase is 30-80mg of N-deoxyribotransferase per g of affinity ligand. In a preferred embodiment of the application, the enzyme load of the immobilized N-deoxyribotransferase is about 50mg of N-deoxyribotransferase per g of affinity ligand.
The application also relates to a preparation method of the deoxynucleoside. The method comprises the following steps: a. the immobilized N-deoxyribotransferase was obtained according to the preparation method described above.
The preparation method of the deoxynucleoside also comprises the following steps: b. a reaction composition is provided that includes a deoxyribose donor and a base donor. In one embodiment of the application, the deoxynucleoside comprises a 2' -deoxynucleoside. In a preferred embodiment of the application, the deoxynucleoside comprises 2 '-deoxycytidine, the deoxyribose donor comprises 2' -deoxythymidine, 2 '-deoxyuridine or 2' -deoxyadenosine, and the base donor comprises cytosine. In one embodiment of the application, the molar concentration ratio of deoxyribodonor to base donor is from 1:2 to 1:8. In a preferred embodiment of the application, the molar concentration ratio of deoxyribodonor to base donor is from 1:3 to 1:6. In a preferred embodiment of the application, the molar concentration ratio of deoxyribodonor to base donor is about 1:5. In one embodiment of the application, the concentration of deoxyribose donor is 5-100mg/mL. In a preferred embodiment of the application, the concentration of deoxyribose donor is 10-50mg/mL. In a preferred embodiment of the application, the concentration of deoxyribose donor is about 24mg/mL. In one embodiment of the application, the concentration of the base donor is 10-200mg/mL. In a preferred embodiment of the application, the concentration of the base donor is 20-100mg/mL. In a preferred embodiment of the application, the concentration of the base donor is about 55.5mg/mL. In one embodiment of the application, the reaction composition comprises: a deoxyribose donor at a concentration of 5-100mg/mL; a base donor at a concentration of 10-200mg/mL; and a buffer having a pH of 6.0 to 8.0. In a preferred embodiment of the application, the concentration of deoxyribose donor is about 10-50mg/mL. In a preferred embodiment of the application, the concentration of deoxyribose donor is about 100mg/mL. In a preferred embodiment of the application, the concentration of the base donor is 40-200mg/mL. In a preferred embodiment of the application, the concentration of the base donor is about 200mg/mL. In the present application, the buffer is any buffer suitable for the reaction of the deoxyribose donor with the base donor to occur therein. In a preferred embodiment of the application, the pH of the buffer is between 6.5 and 7.5. In a preferred embodiment of the application, the pH of the buffer is about 7.0. In a preferred embodiment of the application, the buffer comprises a phosphate buffer.
The preparation method of the deoxynucleoside also comprises the following steps: c. adding the immobilized N-deoxyribotransferase to the reaction composition. In one embodiment of the application, the amount of the immobilized N-deoxyribotransferase added in this step is 1-100mg/mL. In a preferred embodiment of the present application, the amount of the immobilized N-deoxyribotransferase added in this step is 1.5-70mg/mL. In a preferred embodiment of the application, the amount of said immobilized N-deoxyribotransferase added in this step is about 2.5-60mg/mL. In a preferred embodiment of the present application, the amount of the immobilized N-deoxyribotransferase added in this step is 2.5-50mg/mL. In a preferred embodiment of the present application, the amount of the immobilized N-deoxyribotransferase added in this step is 2.5-40mg/mL. In a preferred embodiment of the present application, the amount of the immobilized N-deoxyribotransferase added in this step is 2.5-10mg/mL. In a preferred embodiment of the application, the amount of the immobilized N-deoxyribotransferase added in this step is about 2.5mg/mL. In one embodiment of the application, the reaction temperature in this step is 25-70 ℃. In a preferred embodiment of the application, the reaction temperature in this step is 50-60 ℃. In a preferred embodiment of the application, the reaction temperature in this step is about 60 ℃. In one embodiment of the application, the reaction time in this step is 1 to 20 hours. In a preferred embodiment of the application, the reaction time in this step is from 4 to 20 hours. In a preferred embodiment of the application, the reaction time in this step is about 20 hours.
The preparation method of the deoxynucleoside also comprises the following steps: d. obtaining the deoxynucleoside. In the present application, this step may employ any suitable separation technique. In one embodiment of the application, this step comprises filtration through a filter membrane to remove insoluble material. In another embodiment of the application, the step comprises centrifugation to remove insoluble material.
The following description of the embodiments of the present application will be made clearly and fully with reference to the accompanying drawings, in which some, but not all embodiments of the application are shown. All other embodiments, which can be made by those skilled in the art based on the embodiments of the application without making any inventive effort, are intended to fall within the scope of the application.
Experimental materials
Recombinant E.coli strains: coli BL21 (DE 3)/pEt-28 a (+), which contains an expression plasmid (purchased from Novagen, product number: 69864-3 CN) comprising a codon-optimized ndt-b2 gene sequence (SEQ ID NO: 1).
Metal affinity ligand chromatographic column: ni ion activated Ni-IDA-Sefinose resin (purchased from Shanghai Biotechnology, product number: C600813).
A TB liquid medium comprising: 12g/L peptone, 24g/L yeast powder, 4g/L glycerol, 2.31g/L KH 2PO4 and 16.43g/L K 2HPO4·3H2 O.
Phosphate buffer (50 mM), its preparation method is: weigh K 2HPO4·3H2O：5.57g,KH2PO4 separately: 2.65g, 800mL of ultrapure water was added to dissolve it sufficiently, HCl was added to adjust the pH of the solution to 7.0, and finally ultrapure water was added to a constant volume of 1L.
Phosphate (2 mM) buffer (pH 7.0): weigh K 2HPO4·3H2O：0.23g,KH2PO4 separately: 0.11g, 800mL of ultrapure water was added to dissolve it sufficiently, HCl was added to adjust the pH of the solution to 7.0, and finally ultrapure water was added to a constant volume of 1L.
HPLC detection method: the mobile phase is 15% acetonitrile/water, the detection wavelength is 254nm, and the detection time is 3min.
Example 1
(1) Preparation of crude enzyme solution of N-deoxyribotransferase
Inoculating recombinant escherichia coli strain into TB liquid culture medium, performing protein expression fermentation by IPTG, centrifuging the cultured bacterial liquid for 30min by using a centrifuge at 4000rpm, collecting fermentation bacterial bodies, namely recombinant escherichia coli bacterial bodies containing N-deoxyribotransferase with histidine tag, re-suspending the collected bacterial bodies by using 2mM pH 7.0 phosphate buffer solution, placing the re-suspended bacterial bodies in an ice-water bath, and performing cell disruption by using an ultrasonic cell disrupter under the conditions of 200w, ultrasonic for 3s at intervals of 5s for 30min. And centrifuging to collect supernatant to obtain crude enzyme solution containing N-deoxyribotransferase.
(2) Preparation of immobilized N-deoxyribotransferase
The crude enzyme solution obtained was passed as a top column liquid through a chromatography column pre-loaded with a metal affinity ligand to immobilize histidine-tagged N-deoxyribotransferase on the metal affinity ligand.
(3) Measurement of enzyme Loading
2' -Deoxythymidine (200 mM) and cytosine (200 mM) were weighed, added to the reaction system, 2mL of phosphate buffer (50 mM) pH7.0 was added thereto, stirred and mixed well, and the temperature of the reaction solution was adjusted to 60 ℃. 1mL of the crude enzyme solution prepared in the step (1) (protein concentration 2.5mg/mL: determined by Bradford protein concentration determination kit according to the product instructions) was added, and after 5 minutes of reaction, the crude enzyme solution was sampled and subjected to HPLC to determine that the conversion rate (Con (crude enzyme solution)) of the crude enzyme solution was 14.3%;
the conversion was calculated as follows:
Con=a/(a+b) ×100, where a=peak area of the product; b = peak area of starting material
In the same manner, 2' -deoxythymidine (200 mM) and cytosine (200 mM) were weighed, the reaction system was added, 2mL of phosphate buffer (50 mM) pH7.0 buffer was added, and the mixture was stirred and stirred well, and the temperature of the reaction solution was adjusted to 60 ℃. 10mg of the immobilized N-deoxyribotransferase prepared in the step (2) was added (weighed by an electronic balance), and after 5 minutes of reaction, the sample was taken by HPLC, and the conversion rate of the immobilized enzyme (Con (immobilized enzyme)) was determined to be 3.2%;
The immobilized enzyme loading was calculated as Δ=con (crude enzyme solution)/Con (immobilized enzyme) ×10=50. Here, the value indicates that 0.5mg of enzyme is loaded per 10mg of affinity ligand, i.e. enzyme load = 50mg of N-deoxyribotransferase per g of affinity ligand.
Example 2
Comparing the product components produced after catalyzing the same substrate with the crude enzyme solution of N-deoxyribotransferase and the immobilized N-deoxyribotransferase
2' -Deoxythymidine (200 mM) and cytosine (200 mM) were weighed, added to the reaction system, 2mL of phosphate buffer (50 mM) of pH7.0 was added, stirred and mixed well, the temperature of the reaction solution was adjusted to 60℃and 2mL of crude N-deoxyribotransferase enzyme solution obtained in step (1) of example 1 was added, and stirred and reacted at 60℃for 20 hours to obtain an N-deoxyribotransferase catalytic reaction solution. The reaction mixture was left at 100℃and after heat treatment for 10min, 1mL of the sample was centrifuged (12000 rpm,2 min). 200. Mu.L of the supernatant was collected, 1mL of phosphate buffer (50 mM) at pH7.0 was added thereto, and after thoroughly mixing, the mixture was filtered through a sample filter, and the components of the reaction mixture were detected by High Performance Liquid Chromatography (HPLC), which showed that uracil and impurity peaks were present in the product after 20 hours of reaction (FIG. 1).
2' -Deoxythymidine (200 mM) and cytosine (200 mM) were weighed, added to the reaction system, 4mL of phosphate buffer (50 mM) pH7.0 was added thereto, stirred and mixed well, the temperature of the reaction solution was adjusted to 60℃and 50mg of the immobilized N-deoxyribotransferase obtained in the step (2) of example 1 was added thereto, and stirred and reacted at 60℃for 20 hours to obtain an N-deoxyribotransferase-catalyzed reaction solution. The reaction mixture was left at 100℃and after heat treatment for 10min, 1mL of the sample was centrifuged (12000 rpm,2 min). 200. Mu.L of the supernatant was collected, 1mL of phosphate buffer (50 mM) pH7.0 was added thereto, and after thoroughly mixing, the mixture was filtered through a sample filter, and the components of the reaction mixture were detected by High Performance Liquid Chromatography (HPLC). The results showed that after 20 hours of reaction, only thymine, uracil and impurity peaks were absent in the product (FIG. 2).
The result shows that the purified immobilized enzyme effectively avoids the thymine from being hydrolyzed into uracil by the enzyme in the crude enzyme liquid in the catalytic reaction process, simplifies the subsequent process steps of further purifying the product and reduces the production cost.
Example 3
Comparative determination of enzyme Activity of immobilized N-deoxyribotransferase catalytic reaction
5Mg, 10mg, 20mg and 40mg of the immobilized N-deoxyribotransferase obtained in the step (2) of example 1 were weighed, 2mL of phosphate buffer (50 mM) of pH7.0 was added, and after thoroughly mixing, 200mg of 2' -deoxythymidine and 138mg of cytosine were added, respectively, the temperature of the reaction solution was adjusted to 60℃and the reaction was carried out by shaking at 1000rpm on a shaking table for 2,4, 6 and 20 hours, and then samples were taken, respectively. 100. Mu.L of the reaction mixture was allowed to stand at 100℃and, after heat treatment for 10 minutes, centrifuged (12000 rpm, 2 minutes). The supernatant was collected, 1mL of phosphate buffer (50 mM) at pH7.0 was added thereto, and after thoroughly mixing, the mixture was filtered through a sample filter, and the components of the reaction mixture were detected by High Performance Liquid Chromatography (HPLC). The results showed (fig. 3): after 20h of reaction, the conversion at equilibrium was about 70%. The more the addition amount of the immobilized enzyme is, the faster the reaction speed is, the conversion rate of the reaction can reach 65% after the reaction of 40mg of the immobilized enzyme for 2 hours, and the catalytic conversion rate of 5mg of the immobilized enzyme is 23%. However, only 5mg of the immobilized enzyme was required to react for 20 hours, and the conversion rate of the reaction was increased to 70%, so that 5mg of the immobilized enzyme (2.5 mg/mL) was used as the optimum amount of the enzyme for the catalytic reaction from the economical point of view. It is further demonstrated that the immobilized N-deoxyribotransferase prepared by the present invention has high activity.
Example 4
Determining the effect of different concentrations of cytosine on the catalytic conversion of immobilized enzyme reactions
50Mg of the immobilized N-deoxyribotransferase obtained in the step (2) of example 1 was weighed, 2mL of a phosphate buffer (50 mM) of pH7.0 was added, and after thoroughly mixing, 48mg of 2' -deoxythymidine was added, and 44mg (0.4M), 67mg (0.6M), 89mg (0.8M), 111mg (1M), 133mg (1.2M), 155mg (1.4M) and 178mg (1.6M) of cytosine were added, respectively, and the reaction solution temperature was adjusted to 60℃and the reaction was shaken on a shaker at 1000rpm for 2,4, 6, 20 hours, followed by sampling, respectively. 100. Mu.L of the reaction mixture was allowed to stand at 100℃and, after heat treatment for 10 minutes, centrifuged (12000 rpm, 2 minutes). The supernatant was collected, 1mL of phosphate buffer (50 mM) at pH7.0 was added thereto, and after thoroughly mixing, the mixture was filtered through a sample filter, and the components of the reaction mixture were detected by High Performance Liquid Chromatography (HPLC). The results showed (fig. 4): after 20h of reaction, the conversion of the reaction may be over 75% when the concentration of cytosine is 0.6-1.2M (where the molar ratio of 2' -deoxythymidine to cytosine=1:3-1:6) and the conversion of the reaction may be up to 81% when the concentration of cytosine is 1M (where the concentration of 2' -deoxythymidine is 0.2M and the molar ratio of 2' -deoxythymidine to cytosine=1:5).
In summary, compared with the prior art, the invention has the following advantages:
1. The crude enzyme liquid of the N-deoxyribotransferase with the affinity tag is contacted with the affinity ligand, so that the N-deoxyribotransferase with the affinity tag can be effectively adsorbed, and the heteroenzyme in the crude enzyme liquid can be effectively removed, thereby achieving high-efficiency purification effect;
2. fixing the N-deoxyribotransferase with the affinity tag on an affinity ligand to realize the effects of high-efficiency enzyme activity and high recycling rate of the N-deoxyribotransferase with the affinity tag;
3. the invention can thoroughly and effectively remove the heteroenzyme in the crude enzyme liquid, and utilizes the immobilized N-deoxyribotransferase with an affinity tag to catalyze and synthesize the deoxynucleoside, thereby effectively solving the problem of product hydrolysis;
4. The invention achieves higher conversion rate and production efficiency in conversion synthesis by using the deoxyribose donor and the base donor with the specific range of molar ratio; and
5. Compared with a chemical method, the method for producing 2' -deoxycytidine by catalysis has the advantages that no isomer is produced by an enzyme catalysis method, and the purity and the utilization rate of the product are improved.
The foregoing is merely a specific application example of the present application, and the protection scope of the present application is not limited in any way. Other variations or modifications of the above teachings will be apparent to those of ordinary skill in the art. It is not necessary or obvious to describe all embodiments. All such technical schemes formed by equivalent transformation or equivalent substitution fall within the protection scope of the application.
Sequence listing
<110> Shanghai full pharmaceutical research and development Co., ltd
Shanghai He full pharmaceutical Co., ltd
<120> Immobilized N-deoxyribotransferase and deoxynucleoside preparation method
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atgcatcatc atcatcatca taagcgcaaa attatctatc tagctagtcc ctatggattt 60
tcgcagcagc aaaagacgct acttttacct cccatcgttc gggctttgga ggcgttgggc 120
atagaagttt gggaaccgtt tgcccgtaac aatcaaatcg atttttctca agctgattgg 180
gcgtatcgcg tggcgcaggc agatttgcag gatgtgaaaa actgcgatgg catttttgcg 240
gttgtcaacg gcacgccacc agatgaagga gtcatggtag agttgggaat ggcgatcgcc 300
ctgaataaag caattttctt attccgagac gacttccggc gctgtagcga taacgaacgg 360
tatcccttaa atctcatgct ttttgctggc ttaccggaaa ttggctggga aaattattac 420
tatacttctg tagacgaaat ccagtctcac gataaagcat tgtacaaatg gttaacagga 480
atgtag 486
<210> 2
<211> 161
<212> PRT
<213> Artificial sequence
<220>
<223> Unknown
<400> 2
Met His His His His His His Lys Arg Lys Ile Ile Tyr Leu Ala Ser
1 5 10 15
Pro Tyr Gly Phe Ser Gln Gln Gln Lys Thr Leu Leu Leu Pro Pro Ile
20 25 30
Val Arg Ala Leu Glu Ala Leu Gly Ile Glu Val Trp Glu Pro Phe Ala
35 40 45
Arg Asn Asn Gln Ile Asp Phe Ser Gln Ala Asp Trp Ala Tyr Arg Val
50 55 60
Ala Gln Ala Asp Leu Gln Asp Val Lys Asn Cys Asp Gly Ile Phe Ala
65 70 75 80
Val Val Asn Gly Thr Pro Pro Asp Glu Gly Val Met Val Glu Leu Gly
85 90 95
Met Ala Ile Ala Leu Asn Lys Ala Ile Phe Leu Phe Arg Asp Asp Phe
100 105 110
Arg Arg Cys Ser Asp Asn Glu Arg Tyr Pro Leu Asn Leu Met Leu Phe
115 120 125
Ala Gly Leu Pro Glu Ile Gly Trp Glu Asn Tyr Tyr Tyr Thr Ser Val
130 135 140
Asp Glu Ile Gln Ser His Asp Lys Ala Leu Tyr Lys Trp Leu Thr Gly
145 150 155 160
Met

Claims (16)

1. A method for preparing deoxynucleosides, said method comprising the steps of:
a. Obtaining N-deoxyribotransferase with an affinity tag, wherein the amino acid sequence of the N-deoxyribotransferase with the affinity tag is shown as SEQ ID NO. 2;
b. Contacting the affinity tagged N-deoxyribotransferase with an affinity ligand to immobilize the affinity tagged N-deoxyribotransferase to the affinity ligand;
c. Washing the affinity ligand to obtain an immobilized N-deoxyribotransferase;
d. providing a reaction composition comprising a deoxyribose donor and a base donor, the base donor being cytosine and the deoxyribose donor being 2' -deoxythymidine, the base donor having a concentration of 0.6-1.2M and the deoxyribose donor having a concentration of 24-100 mg/mL;
e. Adding the immobilized N-deoxyribotransferase to the reaction composition, wherein the amount of the immobilized N-deoxyribotransferase added is 2.5-25 mg/mL and the reaction time is 6-20 hours, or wherein the amount of the immobilized N-deoxyribotransferase added is 5-25 mg/mL and the reaction time is 4-20 hours; and
F. Obtaining the deoxynucleoside.
2. The method of preparation of claim 1, wherein the affinity tag is a histidine tag and the affinity ligand is a metal affinity ligand.
3. The method of making according to claim 2, wherein the metal affinity ligand comprises a nickel ion metal chelating medium or a cobalt ion metal chelating medium.
4. The method of preparation of claim 1, wherein the reaction composition comprises:
a buffer having a pH of 6.0-8.0.
5. The method of claim 1, wherein the concentration of the deoxyribodonor is 24 mg/mL.
6. The method of claim 1, wherein the concentration of the base donor is 55.5 mg/mL.
7. The method of claim 4, wherein the buffer has a pH of 6.5 to 7.5.
8. The method of claim 4, wherein the buffer has a pH of 7.0.
9. The method of preparation of claim 4, wherein the buffer comprises a phosphate buffer.
10. The method of claim 1, wherein the amount of immobilized N-deoxyribotransferase added in step e is 2.5-10 mg/mL.
11. The method of claim 1, wherein the amount of immobilized N-deoxyribotransferase added in step e is 2.5 mg/mL.
12. The process according to claim 1, wherein the reaction temperature in step e is 25 to 70 ℃.
13. The process according to claim 1, wherein the reaction temperature in step e is 50-60 ℃.
14. The process according to claim 1, wherein the reaction temperature in step e is 60 ℃.
15. The process of claim 1, wherein the reaction time in step e is 20 hours.
16. The method of claim 1, wherein step f comprises filtration through a filter membrane and/or centrifugation to remove insoluble materials.
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Publication number Priority date Publication date Assignee Title
CN1429268A (en) * 2000-05-12 2003-07-09 沃尔夫冈·肯特 Novel deoxyribonucleside kinase enzyme multi-substrate variants
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CN105969757A (en) * 2016-06-03 2016-09-28 中国科学院成都生物研究所 Enzyme immobilization method and application
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