CN109706204B - Method for preparing thymidine from immobilized escherichia coli - Google Patents
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- CN109706204B CN109706204B CN201910053888.8A CN201910053888A CN109706204B CN 109706204 B CN109706204 B CN 109706204B CN 201910053888 A CN201910053888 A CN 201910053888A CN 109706204 B CN109706204 B CN 109706204B
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- IQFYYKKMVGJFEH-XLPZGREQSA-N Thymidine Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](CO)[C@@H](O)C1 IQFYYKKMVGJFEH-XLPZGREQSA-N 0.000 title claims abstract description 94
- 241000588724 Escherichia coli Species 0.000 title claims abstract description 60
- DWRXFEITVBNRMK-UHFFFAOYSA-N Beta-D-1-Arabinofuranosylthymine Natural products O=C1NC(=O)C(C)=CN1C1C(O)C(O)C(CO)O1 DWRXFEITVBNRMK-UHFFFAOYSA-N 0.000 title claims abstract description 47
- IQFYYKKMVGJFEH-UHFFFAOYSA-N beta-L-thymidine Natural products O=C1NC(=O)C(C)=CN1C1OC(CO)C(O)C1 IQFYYKKMVGJFEH-UHFFFAOYSA-N 0.000 title claims abstract description 47
- 229940104230 thymidine Drugs 0.000 title claims abstract description 47
- 238000000034 method Methods 0.000 title claims abstract description 33
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims abstract description 24
- RWQNBRDOKXIBIV-UHFFFAOYSA-N thymine Chemical compound CC1=CNC(=O)NC1=O RWQNBRDOKXIBIV-UHFFFAOYSA-N 0.000 claims abstract description 24
- 238000006243 chemical reaction Methods 0.000 claims abstract description 23
- 239000000758 substrate Substances 0.000 claims abstract description 16
- 229940113082 thymine Drugs 0.000 claims abstract description 12
- 239000008055 phosphate buffer solution Substances 0.000 claims abstract description 10
- 239000000679 carrageenan Substances 0.000 claims abstract description 8
- 229920001525 carrageenan Polymers 0.000 claims abstract description 8
- 229940113118 carrageenan Drugs 0.000 claims abstract description 8
- 235000010418 carrageenan Nutrition 0.000 claims abstract description 8
- UHVMMEOXYDMDKI-JKYCWFKZSA-L zinc;1-(5-cyanopyridin-2-yl)-3-[(1s,2s)-2-(6-fluoro-2-hydroxy-3-propanoylphenyl)cyclopropyl]urea;diacetate Chemical compound [Zn+2].CC([O-])=O.CC([O-])=O.CCC(=O)C1=CC=C(F)C([C@H]2[C@H](C2)NC(=O)NC=2N=CC(=CC=2)C#N)=C1O UHVMMEOXYDMDKI-JKYCWFKZSA-L 0.000 claims abstract description 8
- 239000000463 material Substances 0.000 claims abstract description 5
- 239000000654 additive Substances 0.000 claims abstract description 3
- 230000000996 additive effect Effects 0.000 claims abstract description 3
- 239000003054 catalyst Substances 0.000 claims abstract description 3
- 239000002904 solvent Substances 0.000 claims abstract description 3
- 239000000243 solution Substances 0.000 claims description 20
- 239000011259 mixed solution Substances 0.000 claims description 8
- -1 dicyclohexylamine 2-deoxy- α -D-ribose 1-phosphate Chemical compound 0.000 claims description 7
- 239000003795 chemical substances by application Substances 0.000 claims description 6
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical class OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 claims description 5
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims description 4
- 238000005119 centrifugation Methods 0.000 claims description 4
- 238000001914 filtration Methods 0.000 claims description 4
- NBIIXXVUZAFLBC-UHFFFAOYSA-N phosphoric acid Substances OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 4
- 238000001704 evaporation Methods 0.000 claims description 3
- 239000001963 growth medium Substances 0.000 claims description 3
- 238000009630 liquid culture Methods 0.000 claims description 3
- 238000002156 mixing Methods 0.000 claims description 3
- 238000005406 washing Methods 0.000 claims description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 3
- 238000010438 heat treatment Methods 0.000 claims description 2
- 230000035484 reaction time Effects 0.000 claims description 2
- 238000002791 soaking Methods 0.000 claims description 2
- 239000008363 phosphate buffer Substances 0.000 claims 4
- 239000000834 fixative Substances 0.000 claims 2
- 241001052560 Thallis Species 0.000 abstract description 5
- 230000000694 effects Effects 0.000 abstract description 2
- 238000000746 purification Methods 0.000 abstract description 2
- 102000009097 Phosphorylases Human genes 0.000 abstract 1
- 108010073135 Phosphorylases Proteins 0.000 abstract 1
- 239000004372 Polyvinyl alcohol Substances 0.000 description 7
- 229920002451 polyvinyl alcohol Polymers 0.000 description 7
- 239000000126 substance Substances 0.000 description 7
- 102000004190 Enzymes Human genes 0.000 description 6
- 108090000790 Enzymes Proteins 0.000 description 6
- 230000003197 catalytic effect Effects 0.000 description 4
- 230000000052 comparative effect Effects 0.000 description 4
- 238000009792 diffusion process Methods 0.000 description 4
- 230000002194 synthesizing effect Effects 0.000 description 4
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 239000002994 raw material Substances 0.000 description 3
- 238000003786 synthesis reaction Methods 0.000 description 3
- 230000009286 beneficial effect Effects 0.000 description 2
- 229910052799 carbon Inorganic materials 0.000 description 2
- 238000000855 fermentation Methods 0.000 description 2
- 230000004151 fermentation Effects 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- HBOMLICNUCNMMY-XLPZGREQSA-N zidovudine Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](CO)[C@@H](N=[N+]=[N-])C1 HBOMLICNUCNMMY-XLPZGREQSA-N 0.000 description 2
- 229960002555 zidovudine Drugs 0.000 description 2
- UHDGCWIWMRVCDJ-UHFFFAOYSA-N 1-beta-D-Xylofuranosyl-NH-Cytosine Natural products O=C1N=C(N)C=CN1C1C(O)C(O)C(CO)O1 UHDGCWIWMRVCDJ-UHFFFAOYSA-N 0.000 description 1
- OQRXBXNATIHDQO-UHFFFAOYSA-N 6-chloropyridine-3,4-diamine Chemical compound NC1=CN=C(Cl)C=C1N OQRXBXNATIHDQO-UHFFFAOYSA-N 0.000 description 1
- UHDGCWIWMRVCDJ-PSQAKQOGSA-N Cytidine Natural products O=C1N=C(N)C=CN1[C@@H]1[C@@H](O)[C@@H](O)[C@H](CO)O1 UHDGCWIWMRVCDJ-PSQAKQOGSA-N 0.000 description 1
- 102100036286 Purine nucleoside phosphorylase Human genes 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- 239000002259 anti human immunodeficiency virus agent Substances 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 230000002155 anti-virotic effect Effects 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 238000006555 catalytic reaction Methods 0.000 description 1
- 230000003833 cell viability Effects 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- UHDGCWIWMRVCDJ-ZAKLUEHWSA-N cytidine Chemical compound O=C1N=C(N)C=CN1[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O1 UHDGCWIWMRVCDJ-ZAKLUEHWSA-N 0.000 description 1
- 238000004042 decolorization Methods 0.000 description 1
- YMWUJEATGCHHMB-UHFFFAOYSA-N dichloromethane Substances ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 238000003912 environmental pollution Methods 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 238000011049 filling Methods 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 238000011031 large-scale manufacturing process Methods 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 108010009099 nucleoside phosphorylase Proteins 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 239000000741 silica gel Substances 0.000 description 1
- 229910002027 silica gel Inorganic materials 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 239000002351 wastewater Substances 0.000 description 1
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention belongs to the field of biocatalysis, and particularly relates to a method for preparing thymidine by immobilized escherichia coli. According to the method, PVA and carrageenan are used as embedding base materials, activated carbon is used as an additive, and escherichia coli producing phosphorylase is immobilized to obtain immobilized escherichia coli; in the reaction for preparing thymidine, immobilized colibacillus is used as catalyst, phosphate buffer solution is used as solvent, and 2-deoxy-alpha-D-ribose-1-dicyclohexylamine phosphate and thymine are used as reaction substrates to produce thymidine. According to the invention, the thymidine is prepared by adopting immobilized escherichia coli, so that the tolerance of thalli to a substrate is better improved, the stability of the thalli is improved, and the yield of the prepared thymidine is improved; the result shows that the purity of the thymidine can reach more than 97 percent after purification, and the yield reaches more than 90 percent; after 6 batches of reactions, no obvious activity loss of immobilized escherichia coli exists.
Description
Technical Field
The invention belongs to the field of biocatalysis, and particularly relates to a method for preparing thymidine by immobilized escherichia coli.
Background
Thymidine is an important precursor substance of anti-AIDS drugs zidovudine and azidothymidine, and plays an irreplaceable role in the aspects of antivirus and antitumor. The current thymidine production methods mainly include four methods: chemical synthesis, fermentation, enzymatic and chemical enzymatic methods.
There are various approaches to synthesizing thymidine using chemical synthesis, but there are several disadvantages to synthesizing thymidine using chemical synthesis: the glycosidic bond of the formed thymidine is lack of stereospecificity, the raw material is not easy to obtain, the yield of the thymidine is very low, and a large amount of waste water is generated in the production process, so that the environmental pollution is large (Chinese patent invention, CN 103450302B; CN 102863493B). Although thymidine can be produced singly in a large scale by the fermentation method, the productivity is low due to low cell viability (Chinese patent application, CN 105950689A). The enzyme method is to take thymine base and 2' -deoxynucleoside as raw materials, and thallus or enzyme as a catalytic medium to catalyze and obtain thymidine, but thallus or enzyme is easily inhibited by a substrate, so that the yield of the thymidine is limited (Xuyuan, research on processes for biologically synthesizing thymidine and cytidine by the enzyme method, Shuoshi academic paper, Zhejiang university, 2009). The chemical enzyme method for synthesizing thymidine is to synthesize an intermediate 2-deoxy-alpha-D-ribose-1-phosphoric acid dicyclohexylamine salt by a chemical method, then to synthesize thymidine by taking the thymidine and thymine as substrates and utilizing the catalysis of escherichia coli nucleoside phosphorylase (Meijiafeng, Roliqin, Wanghong, etc., Xue Jiang university of Zhejiang industry, 2014, 42 (2): 172-177).
Disclosure of Invention
In order to solve the problem that the yield of thymidine is low because thalli is easily inhibited by reaction substrates in a chemical enzyme method in the prior art, the invention aims to provide a method for preparing thymidine by immobilized escherichia coli.
The invention is realized by the following scheme:
immobilized escherichia coli is used as a catalyst, 2-deoxy-alpha-D-ribose-1-phosphate dicyclohexylamine salt and thymine are used as substrates, a phosphate buffer solution is used as a solvent, and thymidine is synthesized through reaction, and the method specifically comprises the following steps:
(1) dissolving 2-deoxy-alpha-D-ribose-1-phosphoric acid dicyclohexylamine salt and thymine in a phosphate buffer solution, adding immobilized escherichia coli for reaction, and separating the immobilized escherichia coli to obtain a reaction solution;
(2) and (3) refrigerating, filtering, decompressing, evaporating and purifying the reaction solution in sequence to obtain the thymidine.
Preferably, the molar concentration of the 2-deoxy-alpha-D-ribose 1-phosphate dicyclohexylamine salt after being dissolved in a phosphate buffer solution is 100-150 mM, the molar concentration of the thymine after being dissolved in the phosphate buffer solution is 400-600 mM, and the mass concentration of the immobilized escherichia coli is 200-600 g/L.
Preferably, the pH value of the phosphate buffer solution is 7.5-9.5, and more preferably 8.
Preferably, the reaction temperature is 50-70 ℃, and the reaction time is 10 h.
Preferably, the immobilized escherichia coli is prepared by an embedding method by taking PVA and carrageenan as embedding base materials, superfine activated carbon as an additive and a saturated boric acid and KCI solution as a fixing agent solution, and the method specifically comprises the following steps:
(1) soaking PVA in water, adding carrageenan, and heating to dissolve PVA to obtain a mixed solution for later use;
(2) inoculating the escherichia coli seeds into a liquid culture medium, and sequentially carrying out culture, centrifugation, washing and re-centrifugation to obtain escherichia coli cells;
(3) and (2) adding the escherichia coli cells and the sterilized superfine activated carbon into the mixed solution obtained in the step (1), uniformly mixing, and then dropwise adding a sterilized fixing agent solution for forming to obtain the immobilized escherichia coli.
Preferably, the mass concentration of PVA in the mixed solution is 7-12%, and the mass concentration of carrageenan is 0.4-1.2%; the mass ratio of the escherichia coli cells to the superfine activated carbon is 5: 1, the mass concentration of the Escherichia coli cells is 1-2 g/L.
Preferably, the fixing agent solution is a saturated boric acid and KCl solution, wherein the mass fraction of KCl in the fixing agent solution is 4%.
Has the advantages that:
(1) the invention adopts the immobilized escherichia coli to prepare the thymidine, better improves the tolerance of thalli to a substrate, improves the stability of the thalli, and improves the yield of the prepared thymidine, and the result shows that the purity of the thymidine can reach more than 97 percent and the yield reaches more than 90 percent after purification.
(2) The active carbon is added into the embedding material, so that the reaction yield is greatly improved, and the active carbon reduces the internal diffusion resistance of the immobilized escherichia coli, is beneficial to the diffusion of substances in the immobilized escherichia coli, improves the reaction rate and greatly improves the yield of thymidine.
(3) The immobilized escherichia coli prepared by the method has good operation stability and high reusability, and is beneficial to saving the cost for culturing the escherichia coli.
(4) The thymidine preparation method has the advantages of simple process, low raw material cost, mild reaction conditions and large-scale production.
Detailed Description
Example 1
The method for preparing thymidine by immobilized Escherichia coli in this example has the following steps:
(1) firstly, 3g of polyvinyl alcohol (PVA) is soaked in 50ml of distilled water for 24 hours, then 0.3g of carrageenan is added, the temperature is kept at 110 ℃ for 30min for sterilization and the PVA is fully dissolved, and the mixed solution is cooled to 45 ℃ and kept warm for standby.
(2) A ring of mature E.coli seeds (approximately 1X 10) was grown6Respectively) inoculating the mixture into 30mL of liquid culture medium, carrying out shaking culture at 30 ℃ and 100r/min for 28h, and filling the culture solution into a sterile centrifuge tube; centrifuging at 3000r/min for 10min, washing with sterile physiological saline, and centrifuging again to obtain Escherichia coli cells.
(3) Adding 50mg of escherichia coli cells obtained in the step (II) and 10mg of sterilized ultrafine activated carbon into the mixed solution in the step (I), uniformly mixing, dripping the mixture into sterilized saturated boric acid and KCI solution (wherein the mass fraction of KCl is 7%) by using a needle tube, forming for 36 hours to obtain immobilized escherichia coli cells, placing the immobilized escherichia coli cells into phosphate buffer solution with the pH value of 7.0, and storing at 4 ℃ for later use.
(4) 4.12g of 2-deoxy-alpha-D-ribose 1-phosphate dicyclohexylamine salt and 5.04g of thymine were dissolved in 100ml of a phosphate buffer solution having a pH of 8.0, 20g of the immobilized E.coli cells prepared in the third step were added, and the reaction was carried out at 50 ℃ for 10 hours to separate the immobilized E.coli cells.
(5) And (3) refrigerating the reaction solution obtained in the step (4) in a refrigerator at 4 ℃ for 2-5 days, filtering, removing filter residues, adding a proper amount of activated carbon for decolorization, filtering to obtain a transparent liquid, and evaporating to dryness at 60 ℃ under reduced pressure to obtain yellowish solid powder. Then separating by using a silica gel column, selecting methanol with the volume ratio: 1-dichloromethane: 15, and the thymidine fractions were combined to give a sample of 2.03g as white crystals with a yield of 85% and a purity of 97%.
Examples 2 to 7
The procedures of examples 2 to 7 were substantially the same as those of example 1 except that the following procedure was conducted in Table 1, wherein substrate 1 was dicyclohexylamine 2-deoxy- α -D-ribose 1-phosphate, and substrate 2 was thymine:
TABLE 1
As can be seen from Table 1, example 6, in which the concentration of dicyclohexylamine 2-deoxy- α -D-ribose 1-phosphate was 150mM, the concentration of thymine was 600mM, the reaction temperature was 65 ℃, the thymidine yield was 90% and the purity was 97.0%, was the most effective under the same conditions.
Comparative examples 1 to 2
The procedures of comparative examples 1 to 2 were substantially the same as those of example 6 except that the following are shown in Table 2, wherein the substrate 1 was dicyclohexylamine 2-deoxy- α -D-ribose 1-phosphate, and the substrate 2 was thymine:
TABLE 2
As can be seen from Table 2, the concentration of the immobilized Escherichia coli catalytic substrate in example 6 is obviously higher than that of free Escherichia coli, and the immobilized Escherichia coli catalytic substrate has good substrate tolerance and good operation stability and reusability, and the result shows that the immobilized Escherichia coli still shows good catalytic activity after being reused for 6 times without obvious inactivation; the activity of the free escherichia coli in the comparative example 2 is limited due to the inhibition of the reaction substrate, so that the yield of thymidine is lower and is about 30% lower than that of thymidine prepared by immobilized escherichia coli; as can be seen from the comparison between example 6 and comparative example 1, the addition of activated carbon to the embedding material greatly improves the yield of the reaction, which indicates that the activated carbon reduces the internal diffusion resistance of the immobilized Escherichia coli, facilitates the diffusion of substances in the immobilized Escherichia coli, improves the reaction rate, and greatly improves the yield of thymidine.
The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention, and any minor modifications, equivalent replacements and improvements made to the above embodiment according to the technical spirit of the present invention should be included in the protection scope of the technical solution of the present invention.
Claims (7)
1. A method for preparing thymidine by immobilized escherichia coli is characterized in that the immobilized escherichia coli is used as a catalyst, 2-deoxy-alpha-D-ribose-1-phosphoric acid dicyclohexylamine salt and thymine are used as substrates, a phosphate buffer solution is used as a solvent, and thymidine is synthesized through reaction, and the method specifically comprises the following steps:
(1) dissolving 2-deoxy-alpha-D-ribose-1-phosphoric acid dicyclohexylamine salt and thymine in a phosphate buffer solution, adding immobilized escherichia coli for reaction, and separating the immobilized escherichia coli to obtain a reaction solution;
(2) refrigerating, filtering, decompressing, evaporating and purifying the reaction solution in sequence to obtain thymidine;
the immobilized escherichia coli is prepared by adopting an embedding method and taking PVA and carrageenan as embedding base materials, superfine activated carbon as an additive and saturated boric acid and KCI solution as a fixing agent solution, and the method specifically comprises the following steps:
(1) soaking PVA in water, adding carrageenan, and heating to dissolve PVA to obtain a mixed solution for later use;
(2) inoculating the escherichia coli seeds into a liquid culture medium, and sequentially carrying out culture, centrifugation, washing and re-centrifugation to obtain escherichia coli cells;
(3) adding the escherichia coli cells and the sterilized superfine activated carbon into the mixed solution obtained in the step (1), uniformly mixing, and then dropwise adding a sterilized fixing agent solution for forming to obtain immobilized escherichia coli;
the mass ratio of the escherichia coli cells to the superfine activated carbon is 5: 1, the mass concentration of the Escherichia coli cells is 1-2 g/L.
2. The method for preparing thymidine by immobilized Escherichia coli according to claim 1, wherein the molar concentration of said dicyclohexylamine 2-deoxy- α -D-ribose 1-phosphate in phosphate buffer is 100-150 mM, the molar concentration of said thymidine in phosphate buffer is 400-600 mM, and the mass concentration of said immobilized Escherichia coli is 200-600 g/L.
3. The method for preparing thymidine by immobilized Escherichia coli as claimed in claim 1, wherein the pH of said phosphate buffer is 7.5-9.5.
4. The method for preparing thymidine by immobilized E.coli as claimed in claim 1, wherein the pH of said phosphate buffer is 8.
5. The method for preparing thymidine by immobilized Escherichia coli as claimed in claim 1, wherein the reaction temperature is 50-70 deg.C and the reaction time is 10 h.
6. The method for preparing thymidine by immobilized escherichia coli according to claim 1, wherein the mass concentration of PVA in the mixed solution is 7-12%, and the mass concentration of carrageenan is 0.4-1.2%.
7. The method for preparing thymidine by immobilized Escherichia coli as claimed in claim 1, wherein the fixative solution is a saturated boric acid and KCl solution, and the mass fraction of KCl in the fixative solution is 4%.
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