CN114231522A - Process for preparing immobilized N-deoxyribotransferase and deoxynucleoside - Google Patents

Process for preparing immobilized N-deoxyribotransferase and deoxynucleoside Download PDF

Info

Publication number
CN114231522A
CN114231522A CN202111615269.7A CN202111615269A CN114231522A CN 114231522 A CN114231522 A CN 114231522A CN 202111615269 A CN202111615269 A CN 202111615269A CN 114231522 A CN114231522 A CN 114231522A
Authority
CN
China
Prior art keywords
deoxyribotransferase
immobilized
affinity
present application
donor
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
CN202111615269.7A
Other languages
Chinese (zh)
Other versions
CN114231522B (en
Inventor
谢磊
李�杰
黄月园
张波琨
孙丰来
徐幸福
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Shanghai SynTheAll Pharmaceutical Co Ltd
Shanghai STA Pharmaceutical R&D Ltd
Original Assignee
Shanghai SynTheAll Pharmaceutical Co Ltd
Shanghai STA Pharmaceutical R&D Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Shanghai SynTheAll Pharmaceutical Co Ltd, Shanghai STA Pharmaceutical R&D Ltd filed Critical Shanghai SynTheAll Pharmaceutical Co Ltd
Priority to CN202111615269.7A priority Critical patent/CN114231522B/en
Publication of CN114231522A publication Critical patent/CN114231522A/en
Application granted granted Critical
Publication of CN114231522B publication Critical patent/CN114231522B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/14—Enzymes or microbial cells immobilised on or in an inorganic carrier
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1048—Glycosyltransferases (2.4)
    • C12N9/1077—Pentosyltransferases (2.4.2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/18—Preparation of compounds containing saccharide radicals produced by the action of a glycosyl transferase, e.g. alpha-, beta- or gamma-cyclodextrins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C12P19/28—N-glycosides
    • C12P19/38—Nucleosides
    • C12P19/385—Pyrimidine nucleosides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y204/00—Glycosyltransferases (2.4)
    • C12Y204/02—Pentosyltransferases (2.4.2)
    • C12Y204/02006—Nucleoside deoxyribosyltransferase (2.4.2.6)

Abstract

Methods for the preparation of immobilized N-deoxyribotransferases and deoxynucleosides are disclosed. The preparation method of the immobilized N-deoxyribotransferase comprises the following steps: a. obtaining N-deoxyribotransferase with affinity label; 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 an immobilized N-deoxyribotransferase.

Description

Process for preparing immobilized N-deoxyribotransferase and deoxynucleoside
Technical Field
The application relates to the technical field of recombinase catalytic reactions, in particular to a preparation method of immobilized N-deoxyribotransferase and deoxynucleoside.
Background
2 ' -deoxycytidine (2 ' -deoxycytidine, abbreviated as dA), also known as "2 ' -deoxyribonucleoside cytidine", is a natural deoxyribonucleoside that is an organic constituent of the genetic material deoxyribonucleic acid (DNA) and is involved in the transmission of genetic information in cells of living organisms. As an Eg5 kinesin regulator, 2 '-deoxycytidine has antiproliferative and apoptosis-inducing activities, so the 2' -deoxycytidine becomes an important medical intermediate, can be used for synthesizing an anti-cancer drug gemcitabine (dFdC), an anti-HIV drug zalcitabine (ddC), a biochemical reagent deoxycytidine-5 '-monophosphate (5' -dCMP) and the like, and is widely applied to nucleic acid and genetic engineering. With the great 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 important components of antiviral drugs is also increasing sharply.
In 2006, Moosa et al reported a chemical synthesis method of β -L-2' -deoxy-cytidine, which uses protected ribofuranose as a starting material, and performs condensation with protected pyrimidine and deprotection to obtain a target product. However, the synthesis methods of 2 '-deoxycytidine reported in the literature all have the formation of the isomer α -2' -deoxycytidine, and although the authors optimize the synthesis method, the amount ratio of the α: β configuration substances in the product can only reach 1: 5 or 1: 6, and the selectivity is very low.
In recent years, techniques for producing deoxynucleosides by enzymatic catalysis have emerged. Chinese patent application CN105754899A discloses a method for synthesizing deoxynucleoside by N-deoxyribotransferase enzyme method, which comprises the induced expression and purification of recombinant N-deoxyribotransferase NDT in recombinant bacteria E.coli BL21(DE3), the addition of ribose donor and acceptor in buffer solution, the addition of N-deoxyribotransferase crude enzyme solution, the reaction in a shaker with the rotation speed of 180rpm at 35 ℃ for 2h, and the sampling after the reaction is finished and the reaction is terminated in methanol. However, the crude enzyme solution used in this method contains a large amount of miscellaneous enzymes, which hydrolyze the product to produce a large amount of impurities, resulting in a low purity of the final deoxynucleoside product.
There remains a need in the art to find a process that can prepare deoxynucleosides, particularly 2' -deoxycytidine, in high purity, high conversion and at low cost, while avoiding the production of isomers.
Disclosure of Invention
In order to solve the above-mentioned problems, one aspect of the present application provides a method for preparing an immobilized N-deoxyribotransferase, comprising the steps of:
a. obtaining N-deoxyribotransferase with affinity label;
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 producing a deoxynucleoside, comprising the steps of:
a. obtaining N-deoxyribotransferase with affinity label;
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 present application is described in more detail below with reference to the attached drawing figures, wherein:
FIG. 1 is a HPLC liquid chromatogram showing the production of 2' -deoxycytidine by catalyzing deoxyribose donor and cytosine with crude enzyme solution of N-deoxyribotransferase;
FIG. 2 is an HPLC liquid chromatogram showing the production of 2' -deoxycytidine from a deoxyribose donor and cytosine catalyzed by an immobilized N-deoxyribotransferase according to one embodiment of the present application;
FIG. 3 is a schematic diagram showing the change in conversion rate of a catalytic reaction with an immobilized N-deoxyribotransferase according to one embodiment of the present application at different amounts of immobilized enzyme added and at different times; and
FIG. 4 is a schematic diagram showing the effect of different molar amounts of cytosine on reaction conversion when a deoxyribose donor and cytosine are catalyzed by an immobilized N-deoxyribotransferase according to one embodiment of the present application to produce 2' -deoxycytidine.
Detailed Description
The application relates to a preparation method of immobilized N-deoxyribose transferase. The method comprises the following steps: a. obtaining the N-deoxyribotransferase with affinity label. In the present application, an N-deoxyribotransferase is any suitable enzyme that catalyzes the reaction of a deoxyribose donor and a base donor to produce a deoxynucleoside, and an affinity-tagged N-deoxyribotransferase can be obtained by any suitable method. In one embodiment of the present application, the N-deoxyribotransferase is an N-deoxyribotransferase from Chroococcus calomelas (Chroococci thermophilus). In one embodiment of the present 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 containing the affinity-tagged N-deoxyribotransferase. In one embodiment of the present application, the host cell is a eukaryotic cell. In one embodiment of the present application, the host cell is a prokaryotic cell. In one embodiment of the present application, the host cell is an E.coli cell. In one embodiment of the present application, the affinity tag is a histidine tag. In one embodiment of the present application, the amino acid sequence of the affinity tagged N-deoxyribotransferase is as set forth in SEQ ID NO: 2, respectively. In one embodiment of the present application, the affinity tagged N-deoxyribotransferase is encoded by a nucleotide sequence set forth in SEQ ID NO: 1 is shown. In one embodiment of the present application, the fermentation medium may be any suitable medium known in the art. In one embodiment of the present application, the fermentation medium comprises TB liquid medium.
The preparation method of the immobilized N-deoxyribotransferase further 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, "affinity ligand" refers to a substance capable of specifically binding to an affinity tag. In the present application, any suitable affinity tag and affinity ligand pair known in the art may be used. In one embodiment of the present application, the affinity tag is a histidine tag and the affinity ligand is a metal affinity ligand. In a preferred embodiment of the present 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 present application, the metal affinity ligand is selected from the group consisting of nickel ion or cobalt ion activated LX-1000IDA, Seplite LX-1000IDA, FP-IDA405/EB or Ni-IDA-Sefiniose resin. In one embodiment of the present application, the contacting step comprises flowing the crude enzyme solution of the affinity-tagged N-deoxyribotransferase through a chromatography column pre-loaded with an affinity ligand to adsorb the affinity-tagged N-deoxyribotransferase to the affinity ligand.
The preparation method of the immobilized N-deoxyribotransferase further comprises the following steps: c. washing the affinity ligand to obtain the immobilized N-deoxyribotransferase. In one embodiment of the present application, the washing step comprises washing the affinity ligand with a detergent to remove unbound N-deoxyribosyltransferase and other impurities from the crude enzyme solution. In the present application, the detergent may be any agent known in the art suitable for washing affinity ligands without causing the detachment of the bound N-deoxyribotransferase. In the present application, the immobilized N-deoxyribotransferase includes N-deoxyribotransferase and affinity ligand. In one embodiment of the present application, the enzyme load of the immobilized N-deoxyribotransferase is 1-200mg N-deoxyribotransferase per g affinity ligand. In a preferred embodiment of the present application, the enzyme load of the immobilized N-deoxyribotransferase is 10-100mg N-deoxyribotransferase per g affinity ligand. In a preferred embodiment of the present application, the enzyme load of the immobilized N-deoxyribotransferase is 30-80mg N-deoxyribotransferase per g affinity ligand. In a preferred embodiment of the present 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 previously.
The preparation method of the deoxynucleoside also comprises the following steps: b. a reaction composition comprising a deoxyribose donor and a base donor is provided. In one embodiment of the present application, the deoxynucleoside comprises a 2' -deoxynucleoside. In a preferred embodiment of the present 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 present application, the molar concentration ratio of the deoxyribose donor and the base donor is 1: 2 to 1: 8. In a preferred embodiment of the present application, the molar concentration ratio of the deoxyribose donor and the base donor is 1: 3 to 1: 6. In a preferred embodiment of the present application, the molar concentration ratio of the deoxyribose donor to the base donor is about 1: 5. In one embodiment of the present application, the concentration of the deoxyribose donor is 5-100 mg/mL. In a preferred embodiment of the present application, the concentration of the deoxyribose donor is 10 to 50 mg/mL. In a preferred embodiment of the present application, the concentration of the deoxyribose donor is about 24 mg/mL. In one embodiment of the present application, the concentration of the base donor is 10-200 mg/mL. In a preferred embodiment of the present application, the concentration of the base donor is 20-100 mg/mL. In a preferred embodiment of the present application, the concentration of base donors is about 55.5 mg/mL. In one embodiment of the present application, a reactive composition comprises: deoxyribose donor with the concentration of 5-100 mg/mL; a base donor, the concentration of which is 10-200 mg/mL; and a buffer having a pH of 6.0 to 8.0. In a preferred embodiment of the present application, the concentration of the deoxyribose donor is about 10-50 mg/mL. In a preferred embodiment of the present application, the concentration of the deoxyribose donor is about 100 mg/mL. In a preferred embodiment of the present application, the concentration of the base donor is 40-200 mg/mL. In a preferred embodiment of the present application, the concentration of the base donor is about 200 mg/mL. In the present application, the buffer is any buffer suitable for the deoxyribose donor to react with the base donor therein. In a preferred embodiment of the present application, the buffer has a pH of 6.5 to 7.5. In a preferred embodiment of the present application, the pH of the buffer is about 7.0. In a preferred embodiment of the present 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 present application, the amount of said immobilized N-deoxyribotransferase added in this step is 1-100 mg/mL. In a preferred embodiment of the present application, the amount of said immobilized N-deoxyribotransferase added in this step is 1.5-70 mg/mL. In a preferred embodiment of the present application, the amount of immobilized N-deoxyribotransferase added in this step is about 2.5-60 mg/mL. In a preferred embodiment of the present application, the amount of said immobilized N-deoxyribotransferase added in this step is 2.5-50 mg/mL. In a preferred embodiment of the present application, the amount of said immobilized N-deoxyribotransferase added in this step is 2.5-40 mg/mL. In a preferred embodiment of the present application, the amount of said immobilized N-deoxyribotransferase added in this step is 2.5-10 mg/mL. In a preferred embodiment of the present application, the amount of immobilized N-deoxyribotransferase added in this step is about 2.5 mg/mL. In one embodiment of the present application, the reaction temperature in this step is 25-70 ℃. In a preferred embodiment of the present application, the reaction temperature in this step is 50-60 ℃. In a preferred embodiment of the present application, the reaction temperature in this step is about 60 ℃. In one embodiment of the present application, the reaction time in this step is 1 to 20 hours. In a preferred embodiment of the present application, the reaction time in this step is 4 to 20 hours. In a preferred embodiment of the present 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 present application, this step comprises filtration through a filter membrane to remove insolubles. In another embodiment of the present application, this step comprises centrifugation to remove insolubles.
The technical solutions of the present application will be described in detail and fully with reference to the accompanying drawings, and it should be understood that the described embodiments are only some embodiments of the present application, but not all embodiments. All other embodiments obtained by a person of ordinary skill in the art based on the embodiments in the present application without any inventive step are within the scope of protection of the present application.
Experimental Material
Recombinant E.coli strains: escherichia coli BL21(DE3)/pEt-28a (+), which contains an expression plasmid (purchased from Novagen, product number: 69864-3CN) comprising a codon-optimized ndt-b2 gene sequence (SEQ ID NO: 1).
Metal affinity ligand chromatographic column: ni ion activated Ni-IDA-Sefiniose resin (purchased from Shanghai Probiotics, product No.: C600813).
TB liquid medium, comprising: 12g/L peptone, 24g/L yeast powder, 4g/L glycerin, 2.31g/L KH2PO4And K of 16.43g/L2HPO4·3H2O。
Phosphate buffer (50mM), which is prepared by the following method: respectively weigh K2HPO4·3H2O：5.57g，KH2PO4: 2.65g, 800mL of ultrapure water was added to dissolve the solution 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 (2mM) buffer (pH 7.0): respectively weigh K2HPO4·3H2O：0.23g，KH2PO4: 0.11g, 800mL of ultrapure water was added to dissolve the solution sufficiently, HCl was added to adjust the pH of the solution to 7.0, and finally ultrapure water was added to make the volume to 1L.
HPLC detection method: the mobile phase is 15% acetonitrile/water, the detection wavelength is 254nm, and the detection time is 3 min.
Figure RE-GDA0003476304820000061
Example 1
(1) Preparation of crude enzyme solution of N-deoxyribotransferase
Inoculating the recombinant escherichia coli strain into a TB liquid culture medium, inducing protein expression and fermentation by IPTG, centrifuging the cultured bacterial liquid for 30min by using a centrifugal machine at 4000rpm, collecting fermented thalli, namely the recombinant escherichia coli thalli containing the N-deoxyribose transferase with the histidine tag, re-suspending the collected thalli by using 2mM phosphate buffer solution with the pH of 7.0, placing the re-suspended thalli in an ice water bath, and carrying out cell disruption by using an ultrasonic cell crusher under the conditions of 200w, 3s of ultrasound and 5s of interval for 30 min. Centrifuging and collecting supernatant fluid to obtain crude enzyme liquid containing N-deoxyribose transferase.
(2) Preparation of immobilized N-deoxyribotransferase
The obtained crude enzyme solution is used as upper column solution to flow through a chromatographic column which is pre-loaded with metal affinity ligand, so that the N-deoxyribose transferase with the histidine tag is fixed on the metal affinity ligand.
(3) Determination of enzyme load
2' -deoxythymidine (200mM) and cytosine (200mM) were weighed, added to the reaction system, 2mL of phosphate buffer (50mM) pH7.0 was added thereto, and mixed with stirring, and the temperature of the reaction mixture was adjusted to 60 ℃. 1mL of the crude enzyme solution prepared in step (1) (protein concentration 2.5 mg/mL: measured by a Bradford protein concentration measurement kit according to the product instruction) was added, and after 5 minutes of reaction, a sample was taken and subjected to HPLC to determine that the conversion rate of the crude enzyme solution (Con (crude enzyme solution)) was 14.3%;
the conversion was calculated as follows:
con ═ a/(a + B) × 100, where a ═ the peak area of the product; b-area of the peak of the starting Material
In the same manner, 2' -deoxythymidine (200mM) and cytosine (200mM) were weighed, added to the reaction system, 2mL of phosphate buffer (50mM) pH7.0 was added thereto, and mixed with stirring, and the temperature of the reaction mixture was adjusted to 60 ℃. 10mg of immobilized N-deoxyribose transferase prepared in the step (2) (weighed by an electronic balance) was added, and after 5 minutes of reaction, a sample was taken and subjected to HPLC, and the conversion of the immobilized enzyme (Con (immobilized enzyme)) was determined to be 3.2%;
the loading of immobilized enzyme was thus calculated as Δ ═ Con (crude enzyme liquid)/Con (immobilized enzyme) × 10 ═ 50. Here, the value indicates that 0.5mg of enzyme was loaded per 10mg of affinity ligand, i.e., 50mg of N-deoxyribotransferase per g of affinity ligand.
Example 2
Comparing the product components generated by the crude enzyme solution of the N-deoxyribotransferase and the immobilized N-deoxyribotransferase catalyzing the same substrate
2' -deoxythymidine (200mM) and cytosine (200mM) were weighed, added to the reaction system, 2mL of a phosphate buffer (50mM) pH7.0 was added thereto, and the mixture was stirred and mixed, the temperature of the reaction solution was adjusted to 60 ℃, 2mL of the crude N-deoxyribotransferase solution obtained in step (1) of example 1 was added thereto, and the reaction mixture was stirred at 60 ℃ for 20 hours to obtain an N-deoxyribotransferase catalyzed reaction solution. The reaction solution was left at 100 ℃ and heat-treated for 10min, and then 1mL of the sample was centrifuged (12000rpm, 2 min). After collecting 200. mu.L of the centrifuged supernatant and adding 1mL of phosphate buffer (50mM) having pH7.0, and mixing them well, the sample was filtered and the components of the reaction mixture were measured by High Performance Liquid Chromatography (HPLC), it was shown that uracil and impurity peaks appeared in the product after 20 hours of the reaction (FIG. 1).
2' -deoxythymidine (200mM) and cytosine (200mM) were weighed, added to the reaction system, 4mL of phosphate buffer (50mM) pH7.0 was added thereto, and the mixture was stirred and mixed, the temperature of the reaction mixture was adjusted to 60 ℃, 50mg of immobilized N-deoxyribotransferase obtained in step (2) of example 1 was added thereto, and the mixture was stirred and reacted at 60 ℃ for 20 hours to obtain an N-deoxyribotransferase catalyzed reaction mixture. The reaction solution was left at 100 ℃ and heat-treated for 10min, and then 1mL of the sample was centrifuged (12000rpm, 2 min). After collecting 200. mu.L of the centrifuged supernatant and adding 1mL of a phosphate buffer solution (50mM) having a pH of 7.0, and mixing them well, the reaction mixture was filtered and the components of the reaction mixture were measured by High Performance Liquid Chromatography (HPLC). The results show that after 20h reaction, only thymine, no uracil and no impurity peaks were present in the product (FIG. 2).
The result shows that the purified immobilized enzyme effectively avoids the condition that the thymine is hydrolyzed into the uracil by the miscellaneous enzyme in the crude enzyme liquid in the catalytic reaction process, simplifies the subsequent process steps for further purifying the product and reduces the production cost.
Example 3
Comparative determination of enzyme Activity in the reaction catalyzed by immobilized N-deoxyribotransferase
5mg, 10mg, 20mg and 40mg of the immobilized N-deoxyribotransferase obtained in the step (2) of example 1 were weighed, 2mL of a phosphate buffer (50mM) having a pH of 7.0 was added thereto, and after mixing the mixture sufficiently, 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 on a shaker at 1000rpm for 2, 4, 6 and 20 hours, followed by sampling. 100. mu.L of the reaction mixture was placed at 100 ℃ and heat-treated for 10min, followed by centrifugation (12000rpm, 2 min). The centrifuged supernatant was added to 1mL of pH7.0 phosphate buffer (50mM), mixed well, and the mixture was filtered and the components of the reaction mixture were measured by High Performance Liquid Chromatography (HPLC). The results show (fig. 3): after 20h of reaction, the conversion at equilibrium was about 70%. The reaction speed is faster as the addition amount of the immobilized enzyme is larger, the reaction conversion rate of 40mg of the immobilized enzyme can reach 65% after the reaction is carried out for 2 hours, and the catalytic conversion rate of 5mg of the immobilized enzyme is 23%. However, only 5mg of immobilized enzyme is needed, and the conversion rate of the reaction can be pushed to 70% after 20 hours of reaction, so from the viewpoint of economy, 5mg of immobilized enzyme (2.5mg/mL) can be used as the optimum amount of the enzyme for the catalytic reaction. Further proves that the immobilized N-deoxyribose transferase prepared by the invention has the characteristic of high activity.
Example 4
Determining the influence of different concentrations of cytosine on the catalytic conversion rate of immobilized enzyme reaction
50mg of the immobilized N-deoxyribotransferase obtained in the step (2) of example 1 was weighed, 2mL of a phosphate buffer solution (50mM) having a pH of 7.0 was added thereto, and after mixing the mixture well, 48mg of 2' -deoxythymidine was added thereto, 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 thereto, respectively, and the temperature of the reaction mixture was adjusted to 60 ℃ and the reaction mixture was shaken on a shaker at 1000rpm for 2, 4, 6 and 20 hours, and then samples were taken. 100. mu.L of the reaction mixture was placed at 100 ℃ and heat-treated for 10min, followed by centrifugation (12000rpm, 2 min). The centrifuged supernatant was added to 1mL of pH7.0 phosphate buffer (50mM), mixed well, and the mixture was filtered and the components of the reaction mixture were measured by High Performance Liquid Chromatography (HPLC). The results show (fig. 4): after 20 hours of reaction, the conversion path of the reaction can exceed 75% when the concentration of cytosine is 0.6-1.2M (in this case, the concentration of 2 '-deoxythymidine is 0.2M, and the molar ratio of the reaction raw materials is 2' -deoxythymidine: cytosine is 1: 3-1: 6), and the conversion rate of the reaction can reach 81% at the maximum when the concentration of cytosine is 1M (in this case, the concentration of 2 '-deoxythymidine is 0.2M, and the molar ratio of the reaction raw materials is 2' -deoxythymidine: cytosine is 1: 5).
In summary, compared with the prior art, the invention has the following advantages:
1. the crude enzyme solution of the N-deoxyribose transferase with the affinity tag is contacted with the affinity ligand, so that the N-deoxyribose transferase with the affinity tag can be effectively adsorbed, the miscellaneous enzymes in the crude enzyme solution can be effectively removed, and the efficient purification effect is achieved;
2. the N-deoxyribose transferase with the affinity tag is fixed on the affinity ligand, so that the effects of realizing high-efficiency enzyme activity and improving the repeated utilization rate of the N-deoxyribose transferase with the affinity tag are achieved;
3. the method can thoroughly and effectively remove miscellaneous enzymes in the crude enzyme solution, and utilizes the immobilized N-deoxyribotransferase with affinity labels to catalyze and synthesize deoxynucleosides, thereby effectively solving the problem of product hydrolysis;
4. the invention achieves higher conversion rate and production efficiency in conversion synthesis by using a specific range of molar ratio of the deoxyribose donor to the base donor; and
5. compared with the chemical method, the method for producing the 2' -deoxycytidine by catalysis has the advantages that the enzyme catalysis method does not produce isomers, and the purity of the product and the utilization rate of the product are improved.
The above are only examples of the present application, and do not limit the scope of the present application. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. All embodiments need not be described or illustrated herein. The technical solutions like this formed by equivalent transformation or equivalent substitution fall within the protection scope of the present application.
Sequence listing
<110> Shanghai Hequan drug development Co Ltd
SHANGHAI SYNTHEALL PHARMACEUTICALS Co.,Ltd.
<120> Process for producing immobilized N-deoxyribotransferase and deoxynucleoside
<130> 218324 1CNCN
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 486
<212> DNA
<213> Artificial sequence
<220>
<223> unknown
<400> 1
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 (10)

1. A method for preparing an immobilized N-deoxyribotransferase, comprising the steps of:
a. obtaining N-deoxyribotransferase with affinity label;
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.
2. The method of claim 1, wherein the affinity tag is a histidine tag and the affinity ligand is a metal affinity ligand; preferably, the metal affinity ligand comprises a nickel ion metal chelating medium or a cobalt ion metal chelating medium.
3. The method of claim 2, wherein the amino acid sequence of the affinity-tagged N-deoxyribotransferase is set forth in SEQ ID NO: 2, respectively.
4. A method for preparing a deoxynucleoside, comprising the steps of:
a. obtaining an immobilized N-deoxyribotransferase according to the production method of any one of claims 1 to 3;
b. providing a reaction composition comprising a deoxyribose donor and a base donor;
c. adding the immobilized N-deoxyribotransferase to the reaction composition; and
d. obtaining the deoxynucleoside.
5. The method of claim 4, wherein the deoxynucleoside comprises a 2' -deoxynucleoside; preferably, the deoxynucleoside comprises 2 '-deoxycytidine, the deoxyribose donor comprises 2' -deoxythymidine, 2 '-deoxyuridine or 2' -deoxyadenosine, and the base donor comprises cytosine.
6. The method according to claim 4, wherein the molar concentration ratio of the deoxyribose donor to the base donor is 1: 2 to 1: 8, preferably 1: 3 to 1: 6, and more preferably 1: 5.
7. The method of claim 4, wherein the reactive composition comprises:
a deoxyribose donor at a concentration of 5-100mg/mL, preferably 10-50mg/mL, more preferably 24 mg/mL;
a base donor at a concentration of 10-200mg/mL, preferably 20-100mg/mL, more preferably 55.5 mg/mL; and
a buffer having a pH of 6.0 to 8.0, preferably 6.5 to 7.5, more preferably 7.0, said buffer preferably comprising a phosphate buffer.
8. The method according to claim 4, wherein the amount of the immobilized N-deoxyribotransferase added in step c is 1 to 100mg/mL, preferably 1.5 to 70mg/mL, more preferably 2.5 to 60mg/mL, more preferably 2.5 to 50mg/mL, more preferably 2.5 to 40mg/mL, more preferably 2.5 to 10mg/mL, more preferably 2.5 mg/mL.
9. The process according to claim 4, wherein the reaction temperature in step c is 25 to 70 ℃, preferably 50 to 60 ℃, more preferably 60 ℃, and the reaction time is 1 to 20 hours, preferably 4 to 20 hours, more preferably 20 hours.
10. The method of claim 4, wherein step d comprises filtration through a filter membrane and/or centrifugation to remove insolubles.
CN202111615269.7A 2021-12-27 2021-12-27 Immobilized N-deoxyribotransferase and deoxynucleoside preparation method Active CN114231522B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202111615269.7A CN114231522B (en) 2021-12-27 2021-12-27 Immobilized N-deoxyribotransferase and deoxynucleoside preparation method

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202111615269.7A CN114231522B (en) 2021-12-27 2021-12-27 Immobilized N-deoxyribotransferase and deoxynucleoside preparation method

Publications (2)

Publication Number Publication Date
CN114231522A true CN114231522A (en) 2022-03-25
CN114231522B CN114231522B (en) 2024-04-26

Family

ID=80763583

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202111615269.7A Active CN114231522B (en) 2021-12-27 2021-12-27 Immobilized N-deoxyribotransferase and deoxynucleoside preparation method

Country Status (1)

Country Link
CN (1) CN114231522B (en)

Citations (5)

* Cited by examiner, † Cited by third party
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
CN105754899A (en) * 2016-04-08 2016-07-13 南京工业大学 N-deoxyribose transferase, coding gene as well as high-yield bacterial strain thereof and application
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
CN111454934A (en) * 2020-04-10 2020-07-28 台州学院 Preparation method and application of EDDS lyase immobilized enzyme
CN111549012A (en) * 2020-06-08 2020-08-18 石家庄创组生物科技有限公司 Ribose kinase mutant and application thereof

Patent Citations (5)

* Cited by examiner, † Cited by third party
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
CN105754899A (en) * 2016-04-08 2016-07-13 南京工业大学 N-deoxyribose transferase, coding gene as well as high-yield bacterial strain thereof and application
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
CN111454934A (en) * 2020-04-10 2020-07-28 台州学院 Preparation method and application of EDDS lyase immobilized enzyme
CN111549012A (en) * 2020-06-08 2020-08-18 石家庄创组生物科技有限公司 Ribose kinase mutant and application thereof

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
"nucleoside 2-deoxyribosyltransferase [Chroococcidiopsis thermalis]", 《NCBI》, pages 015153263 *
赵临襄等: "化学制药工艺学", 中国医药科技出版社, pages: 83 *

Also Published As

Publication number Publication date
CN114231522B (en) 2024-04-26

Similar Documents

Publication Publication Date Title
US10683525B2 (en) Method for producing 2-O-glyceryl-alpha-D-glucopyranoside
Erdmann et al. The involvement of 5S RNA in the binding of tRNA to ribosomes
JP4915917B2 (en) Method for producing lacto-N-biose I and galacto-N-biose
Li et al. Cloning and characterization of a sucrose isomerase from Erwinia rhapontici NX-5 for isomaltulose hyperproduction
CN105473728A (en) Method for preparing sialic acid derivative
EP3395952B1 (en) Composition for producing d-psicose comprising d-psicose 3-epimerase and salt and method for producing d-psicose using same
CN109576239B (en) Heat-resistant phosphorylase and application thereof
CN110791488A (en) Lipase for splitting chiral compound and preparation method and application thereof
CN113481189A (en) Sucrose isomerase mutant and application thereof
CN112760316A (en) Method for producing tagatose by artificial oil body immobilized multienzyme
CN114231522B (en) Immobilized N-deoxyribotransferase and deoxynucleoside preparation method
Bae et al. A unique biochemical reaction pathway towards trehalulose synthesis by an amylosucrase isolated from Deinococcus deserti
CN116240249A (en) Method for hydrolyzing nucleosides by biological enzyme method
CN112831532B (en) Method for enzymatic synthesis of D-leucine
Eis et al. Efficient downstream processing of maltodextrin phosphorylase from Escherichia coli and stabilization of the enzyme by immobilization onto hydroxyapatite
US11760988B2 (en) L-aspartate alpha-decarboxylase mutant and application thereof
CN110791483B (en) Short-chain reductase and preparation method and application thereof
CN114703158A (en) Sucrose phosphorylase mutant, coding gene and application thereof
CN115011622A (en) Screening method and application of D-psicose 3-epimerase mutant
CN107988131B (en) Method for high-yield production of α -ketone-gamma-methylthiobutyric acid
EP2186904B1 (en) Method for producing glucose derivatives
CN114940985B (en) Protein with deoxyadenosine diphosphate kinase and acetate kinase activities and application thereof
JP2724825B2 (en) Method for producing choline cytidine diphosphate
CN117050965A (en) Glycosyltransferase and application thereof in preparing rebaudioside M
CN116622664A (en) Method for generating C-glycoside through biocatalysis

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant