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CN118931997A - A method for synthesizing adenosine and/or uridine by three-enzyme cascade catalysis - Google Patents

A method for synthesizing adenosine and/or uridine by three-enzyme cascade catalysis Download PDF

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CN118931997A
CN118931997A CN202310516643.0A CN202310516643A CN118931997A CN 118931997 A CN118931997 A CN 118931997A CN 202310516643 A CN202310516643 A CN 202310516643A CN 118931997 A CN118931997 A CN 118931997A
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purine
adenosine
uridine
amino
seq
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马学洋
钱雄锋
刘超
张跃
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Shanghai Feiteng Pharmaceutical Technology Co ltd
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Shanghai Feiteng Pharmaceutical Technology Co ltd
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Abstract

本发明公开了一种三酶级联催化合成腺苷和/或尿苷的方法。所述方法包括：以核糖化合物和核酸碱基为底物，以核糖激酶、磷酸戊糖变位酶和核苷磷酸化酶为催化剂，进行反应，合成腺苷和/或尿苷；所述核糖化合物包括D‑核糖和/或其衍生物；所述核酸碱基包括嘌呤碱基和/或嘧啶碱基；所述核苷磷酸化酶包括嘌呤核苷磷酸化酶和/或嘧啶核苷磷酸化酶。本发明整合RK、PPM和NP，构建三酶级联合成腺/尿苷的方法，并挖掘RK、PPM和NP进行组合，实现高效合成腺苷和/或尿苷。

The present invention discloses a method for synthesizing adenosine and/or uridine by three-enzyme cascade catalysis. The method comprises: using a ribose compound and a nucleic acid base as substrates, using ribokinase, phosphopentose mutase and nucleoside phosphorylase as catalysts, reacting to synthesize adenosine and/or uridine; the ribose compound comprises D-ribose and/or its derivatives; the nucleic acid base comprises a purine base and/or a pyrimidine base; the nucleoside phosphorylase comprises a purine nucleoside phosphorylase and/or a pyrimidine nucleoside phosphorylase. The present invention integrates RK, PPM and NP, constructs a method for synthesizing adenosine/uridine by three-enzyme cascade, and explores the combination of RK, PPM and NP to achieve efficient synthesis of adenosine and/or uridine.

Description

Method for synthesizing adenosine and/or uridine by three-enzyme cascade catalysis
Technical Field
The invention belongs to the technical field of biology, and relates to a method for synthesizing adenosine and/or uridine by three-enzyme cascade catalysis.
Background
Adenosine is a naturally occurring endogenous purine nucleoside that is spread throughout human cells and is a degradation product of Adenosine Triphosphate (ATP). Adenosine is known for its vascular dilation effect, and has a broad range of cardiac effects and rapid and pronounced coronary dilation effects, as well as cardioprotective effects in triggering or mediating ischemic preconditioning, alleviating reperfusion injury, etc. Uridine is a pyrimidine nucleoside essential for RNA and DNA synthesis, has various biological functions, and can play a role in the metabolic processes of substances such as sugar, fat, protein and the like by being converted into intermediate bioactive molecules such as uridine diphosphate glucose, uridine diphosphate N-K-acetylglucosamine and the like.
The synthesis of adenosine and uridine can be divided into a chemical synthesis method, a biological fermentation method and an enzyme engineering synthesis method, as disclosed in patent CN105884846A, the adenosine is esterified, an acylating agent and an acid binding agent are acylated to obtain an acylate, the obtained acylate is reduced and purified to obtain 2 '-deoxyadenosine, the final HPLC purity can reach more than 99%, but the chemical synthesis method for preparing 2' -deoxyadenosine has long steps, the reaction condition is severe, the final yield is low, various organic reagents are needed in the reaction process, and the environmental pollution is obvious; for example, CN101575630a discloses a method for producing deoxyadenosine by bioconversion, which catalyzes the synthesis of 2' -deoxyadenosine by culturing lactobacillus helveticus as an enzyme source; CN104178541a also discloses a method for producing 2 '-deoxyadenosine by using escherichia coli transformation, which directly adopts escherichia coli thallus with high nucleoside phosphorylase activity as an enzyme source, and mixes the escherichia coli thallus with deoxythymidine and adenine as a transformation substrate to perform transformation reaction to synthesize the 2' -deoxyadenosine, but the microbial fermentation process is complex, high in cost, long in period and low in production efficiency.
In summary, the development of a method for synthesizing adenosine and/or uridine by high-efficiency enzyme catalysis has important significance for the application fields of adenosine and uridine.
Disclosure of Invention
Aiming at the defects and the actual demands of the prior art, the invention provides a method for synthesizing adenosine and/or uridine by three-enzyme cascade catalysis, the invention designs a process for synthesizing adenosine and/or uridine by three-enzyme cascade catalysis, and digs out specific enzyme combinations to realize high-efficiency synthesis of adenosine and/or uridine.
In order to achieve the above purpose, the invention adopts the following technical scheme:
In a first aspect, the present invention provides a method for the catalytic synthesis of adenosine and/or uridine by a three enzyme cascade, said method comprising:
Reacting a riboside compound and a nucleobase as substrates, and using ribokinase, pentose phosphate mutase and nucleoside phosphorylase as catalysts to synthesize adenosine and/or uridine, wherein the riboside compound comprises D-ribose and/or derivatives thereof, the nucleobase comprises a purine base and/or a pyrimidine base, and the nucleoside phosphorylase comprises a purine nucleoside phosphorylase and/or a pyrimidine nucleoside phosphorylase.
In the invention, a method for synthesizing adenosine/uridine by three enzyme cascade is constructed by integrating RK (ribose kinase, ribokinase), PPM (pentose phosphate mutase, phosphopentomutase) and NP (nucleoside phosphorylase ), and RK, PPM and NP are mined and combined to realize high-efficiency synthesis of adenosine and/or uridine.
In the invention, RK, PPM and NP are mined and combined, and the enzyme and the sources thereof are as follows: ribose kinase (ribokinase, RK) from Acanthamoeba castellanii (AcRK) and ESCHERICHIA COLI (EcRK), respectively; pentose phosphate mutases (phosphopentomutase, PPM) from ESCHERICHIA COLI (EcPPM), bacillus cereus (BcPPM), streptococcus mutans (SmPPM) and Thermotoga maritima (TmPPM), respectively; purine nucleoside phosphorylase (purine nucleoside phosphorylase, PNP) from Deinococcus geothermalis (DgPNP), parageobacillus thermoglucosidasius (PtPNP) and Geobacillus stearothermophilus (GsPNP), respectively; pyrimidine nucleoside phosphorylase (PYRIMIDINE NUCLEOSIDE PHOSPHORYLASE, pyNP) from Parageobacillus thermoglucosidasius (PtPyNP), geobacillus stearothermophilus (GsPyNP) and Brevibacillus borstelensis (BbPyNP), respectively.
Preferably, the combination of RK, PPM and NP comprises: a combination of AcRK, tmPPM and GsPNP, a combination of EcRK, tmPPM and GsPNP, a combination of EcRK, ecPPM and GsPNP, a combination of EcRK, bcPPM and GsPNP, a combination of EcRK, smPPM and GsPNP, a combination of EcRK, tmPPM and PtPNP, a combination of EcRK, tmPPM and DgPNP, a combination of EcRK, tmPPM and PtPyNP, a combination of EcRK, tmPPM and GsPyNP, a combination of EcRK, tmPPM and BbPyNP, a combination of EcRK, tmPPM and PtPyNP, or a combination of EcRK, tmPPM and GsPyNP.
Preferably, the amino acid sequence of the ribokinase comprises the sequence shown in SEQ ID NO.1 (AcRK) or SEQ ID NO.2 (EcRK).
Preferably, the amino acid sequence of the pentose phosphate mutase comprises the sequence shown in SEQ ID NO.3 (EcPPM), SEQ ID NO.4 (BcPPM), SEQ ID NO.5 (SmPPM) or SEQ ID NO.6 (TmPPM).
Preferably, the amino acid sequence of the purine nucleoside phosphorylase comprises the sequence shown in SEQ ID NO.7 (DgPNP), SEQ ID NO.8 (PtPNP) or SEQ ID NO.9 (GsPNP).
Preferably, the amino acid sequence of the pyrimidine nucleoside phosphorylase comprises the sequence shown in SEQ ID NO.10 (PtPyNP), SEQ ID NO.11 (GsPyNP) or SEQ ID NO.12 (BbPyNP).
It will be appreciated that enzymes of the invention engineered to have specific RK, PPM and NP, substituted, deleted or added with one or at least two amino acid residues, function identically or similarly to the original protein, are also contemplated to achieve the effects of the invention.
SEQ ID NO.1:AcRK
MEENQQQCPVRHIWEPQQGQVQQQASTGSECPVRNTWDTSSLYHTNGQFQHPPISQTNGQARCPMGYFSISASSGAPASSSDGAPKCPFASLSLGSSTDRRAPPEQLRLTILGHVTNDINIFVGKETRAQGGGVLFSGVAASNLGMNVEVVTKCSAEDKPVFQKIFAPSGTKVQFLPSQETTCCENNYPKPNSDERVQRFHAVGAPFTVDDLKHIESTIVHINPLSYGEFPDELIPKIKELNPSVQYLVADAQGFIRHIDMQNGGKISHKDWAAKEQYLKYFDLFKVDDKEATVLTGEKDMKRAMQILHEKGAKMVLGTYNAGVLLFDGNIFYQASFGPWKVEGRTGRGDTVTASFLAAAGLSGGPWKRNVALEFAARVTTTKMQYPGPYRRPTASL.
SEQ ID NO.2:EcRK
MQNAGSLVVLGSINADHILNLQSFPTPGETVTGNHYQVAFGGKGANQAVAAGRSGANIAFIACTGDDSIGESVRQQLATDNIDITPVSVIKGESTGVALIFVNGEGENVIGIHAGANAALSPALVEAQRERIANASALLMQLESPLESVMAAAKIAHQNKTIVALNPAPARELPDELLALVDIITPNETEAEKLTGIRVENDEDAAKAAQVLHEKGIRTVLITLGSRGVWASVNGEGQRVPGFRVQAVDTIAAGDTFNGALITALLEEKPLPEAIRFAHAAAAIAVTRKGAQPSVPWREEIDAFLDRQR.
SEQ ID NO.3:EcPPM
MKRAFIMVLDSFGIGATEDAERFGDVGADTLGHIAEACAKGEADNGRKGPLNLPNLTRLGLAKAHEGSTGFIPAGMDGNAEVIGAYAWAHEMSSGKDSVSGHWEIAGVPVLFEWGYFSDHENSFPQELLDKLVERANLPGYLGNCRSSGTVILDQLGEEHMKTGKPIFYTSAASVFQIACHEETFGLDKLYELCEIAREELTNGGYNIGRVIARPFIGDKAGNFQRTGNRRDLAVEPPAPTVLQKLVDEKHGQVVSVGKIADIYANCGITKKVKATGLDALFDATIKEMKEAGDNTIVFTNFVDFDSSWGHRRDVAGYAAGLELFDRRLPELMSLLRDDDILILTADHGCDPTWTGTDHTREHIPVLVYGPKVKPGSLGHRETFADIGQTLAKYFGTSDMEYGKAMF.
SEQ ID NO.4:BcPPM
MNKYKRIFLVVMDSVGIGEAPDAEQFGDLGSDTIGHIAEHMNGLQMPNMVKLGLGNIREMKGISKVEKPLGYYTKMQEKSTGKDTMTGHWEIMGLYIDTPFQVFPEGFPKELLDELEEKTGRKIIGNKPASGTEILDELGQEQMETGSLIVYTSADSVLQIAAHEEVVPLDELYKICKIARELTLDEKYMVGRVIARPFVGEPGNFTRTPNRHDYALKPFGRTVMNELKDSDYDVIAIGKISDIYDGEGVTESLRTKSNMDGMDKLVDTLNMDFTGLSFLNLVDFDALFGHRRDPQGYGEALQEYDARLPEVFAKLKEDDLLLITADHGNDPIHPGTDHTREYVPLLAYSPSMKEGGQELPLRQTFADIGATVAENFGVKMPEYGTSFLNELKK.
SEQ ID NO.5:SmPPM
MSLSTFNRIHLVVLDSVGIGAAPDANNFSNAGVPDGASDTLGHISKTVGLNVPNMAKIGLGNIPRDTPLKTVPAENHPTGYVTKLEEVSLGKDTMTGHWEIMGLNITEPFDTFWNGFPEEIISKIEKFSGRKVIREANKPYSGTAVIDDFGPRQMETGELIIYTSADPVLQIAAHEDVIPLDELYRICEYARSITLERPALLGRIIARPYVGKPRNFTRTANRHDYALSPFAPTVLNKLADAGVSTYAVGKINDIFNGSGITNDMGHNKSNSHGVDTLIKTMGLSAFTKGFSFTNLVDFDALYGHRRNAHGYRDCLHEFDERLPEIIAAMKVDDLLLITADHGNDPTYAGTDHTREYVPLLAYSPSFTGNGVLPVGHYADISATIADNFGVDTAMIGESFLDKLI.
SEQ ID NO.6:TmPPM
MRVVLIVLDSVGIGEMPDAHLYGDEGSNTIVNTAKAVSGLHLPNMAKLGLGNLDDIPGVEPVKPAEGIYGKMMEKSPGKDTTTGHWEIAGVILKKPFDLFPEGFPKELIEEFERRTGRKVIGNKPASGTEIIKELGPIHEKTGALIVYTSADSVFQIAAKKEIVPLEELYRYCEIARELLNEMGYKVARVIARPFTGEWPNYVRTPERKDFSLEPEGKTLLDVLTENGIPVYGVGKIADIFAGRGVTENYKTKDNNDGIDKTISLMKEKNHDCLIFTNLVDFDTKYGHRNDPVSYAKALEEFDARLPEIMHNLNEDDVLFITADHGCDPTTPSTDHSREMVPLLGYGGRLKKDVYVGIRETFADLGQTIADIFGVPPLENGTSFKNLIWE.
SEQ ID NO.7:DgPNP
MVARVPARPFASPPATLDRVSVHLNARPGEIAETVLLPGDPLRAQHIAETFFENPVQHNSVRGMLGFTGTYRGVPVSVQGTGMGIASSMIYVNELIRDYGCQTLIRVGTAGSYQPDVHVRDLVLAQAACTDSNINNIRFGLRNFAPIADFELLLRAYQMARDRGFATHVGNILSSDTFYQDDPESYKLWAQYGVLAVEMEAAGLYTLAAKYGVRALTILTISDHLVTREETTAEERQTTFNGMIEVALDAALGLAVPSNSM.
SEQ ID NO.8:PtPNP
MSIHIEAKQQEIAEKILLPGDPLRAQYIAETFLEGATCYNRVRGMLGFTGTYKGHRISVQGTGMGVPSISIYVNELIQSYHVQTLIRVGTCGAIQKDVNVRDVILAMSASTDSNMNRLTFRGRDYAPTANFALLRTAYEVGAEKGLPLKVGSVFTADMFYNDEPDWETWARYGVLAVEMETAALYTLAAKFGRKALSVLTVSDHILTGEETTAQERQTTFNDMIEVALETAIRVE.
SEQ ID NO.9:GsPNP
MSVHIGAKEHEIADKILLPGDPLRAKYIAETFLEGATCYNQVRGMLGFTGTYKGHRISVQGTGMGVPSISIYITELMQSYNVQTLIRVGTCGAIQKDVKVRDVILAMTSSTDSQMNRMTFGGIDYAPTANFDLLKTAYEIGKEKGLQLKVGSVFTADMFYNENAQFEKLARYGVLAVEMETTALYTLAAKFGRKALSVLTVSDHILTGEETTAEERQTTFNEMIEVALETAIRQ.
SEQ ID NO.10:PtPyNP
MVDLIAKKRDGYELSKEEIDFIIRGYTNGDIPDYQMSAFAMAVFFRGMTEEETAALTMAMVRSGDVIDLSKIEGMKVDKHSTGGVGDTTTLVLGPLVASVGVPVAKMSGRGLGHTGGTIDKLESVPGFHVEIDNEQFIELVNKNKIAIIGQTGNLTPADKKLYALRDVTATVDSIPLIASSIMSKKIAAGADAIVLDVKTGAGAFMKDFAGAKRLATAMVEIGKRVGRKTMAVISDMSQPLGYAVGNALEVKEAIDTLKGKGPEDLQELCLTLGSYMVYLAEKASSLEEARALLEASIREGKALETFKVFLSAQGGDASVVDDPTKLPQAKYRWELEAPEDGYVAEIVADEVGTAAMLLGAGRATKEATIDLSVGLVLHKKVGDAVKKGESLVTIYSNTENIEEVKQKLAKSIRLSSIPVAKPTLIYETIS.
SEQ ID NO.11:GsPyNP
MRMVDLIEKKRDGHALTKEEIQFIIEGYTKGDIPDYQMSALAMAIFFRGMNEEETAELTMAMVHSGDTIDLSRIEGIKVDKHSTGGVGDTTTLVLGPLVASVGVPVAKMSGRGLGHTGGTIDKLESVPGFHVEITNDEFIDLVNKNKIAVVGQSGNLTPADKKLYALRDVTATVNSIPLIASSIMSKKIAAGADAIVLDVKTGVGAFMKDLNDAKALAKAMVDIGNRVGRKTMAIISDMSQPLGYAIGNALEVKEAIDTLKGEGPEDFQELCLVLGSHMVYLAEKASSLEEARHMLEKAMKDGSALQTFKTFLAAQGGDASVVDDPSKLPQAKYIIELEAKEDGYVSEIVADAVGTAAMWLGAGRATKESTIDLAVGLVLRKKVGDAVKKGESLVTIYSNREQVDDVKQKLYENIRISATPVQAPTLIYDKIS.
SEQ ID NO.12:BbPyNP
MRMVDIIAKKRDGLELSSEEIQFLVSGYTDGSIPDYQMSAWAMAVLLRGMTPRETGDLTLAMAGSGEQLDLSSLKGIKVDKHSTGGVGDKTTLVVAPLVAAAGIPVAKMSGRGLGHSGGTIDKLESFAGFQVERTREQFLQQVREIGVSVIGQSGNLTPADKKLYALRDVTATVEAVPLIASSIMSKKIAAGADAILLDVKVGKGAFMKTLEQAETLASAMAQIGTQVGRRTVAVISDMNQPLGFAVGNALEVKEAIDTLAGRGPKDLTELALAIGAHMLVLGELVADVEEGRKRLEEIMDSGKAVEKLAQMIEAQGGNKEDVYNPDRLPKASLTAEVKASQDGYISAIDAETVGHASVVLGAGRLTKEMPIDLAVGIVLAKKRGDQVRKGDVLATVHANDEILLKQAVEELKGAYTYDSESLIDQPLIYKIITE.
Preferably, the derivatives include aldopentoses such as D-arabinose, L-arabinose, D-xylose, L-lyxose and D-ribose; deoxy sugars such as 2' -deoxy-D-ribose, 2',3' -dideoxy-D-ribose, D-fucose, L-fucose, D-rhamnose, L-rhamnose, and the like.
Preferably, the method comprises the steps of, the purine bases include purine, adenine, guanine, 6-hydroxy purine, 6-fluoropurine, 6-chloropurine, 6-methylaminopurine, 6-dimethylaminopurine, 6-trifluoromethylaminopurine, 6-benzoylaminopurine, 6-acetylaminopurine, 6-hydroxyaminopurine, 6-methoxypurine, 6-benzoyloxypurine, 6-methylpurine, 6-ethylpurine, 6-trifluoromethylpurine, 6-phenylpurine, 6-mercaptopurine, 6-methylthiopurine, 2, 6-diaminopurine, 2-amino-6-chloropurine, 2-amino-6-iodopurine, 2-aminopurine 2-amino-6-mercaptopurine, 2-amino-6-methylthiopurine, 2-amino-6-hydroxyaminopurine, 2-amino-6-methoxypurine, 2-amino-6-methylpurine, 2-amino-6-cyclopropylaminomethylpurine, 2-amino-6-phenylpurine, 2-amino-8-bromopurine, 6-cyanopurine, 2-chloroadenine, 2-fluoroadenine, 2-propylguanine, 2-aminopropyladenine, 3-deazaadenine, 3-deazaguanine, 9-methylinosine, xanthine, hypoxanthine, 6-methyladenine, 6-thioguanine, 7-methyladenine, 7-methylguanine, 7-deazaguanine, 7-deaza-8-azaguanine, 8-nitroadenine, 8-nitroguanine, 8-fluoroadenine, 8-chloroadenine, 8-bromoadenine, 8-aminoadenine, 8-hydroxyadenine, 8-fluoroguanine, 8-chloroguanine, 8-bromoguanine, 8-aminoguanine or 8-hydroxyguanine, and the like.
Preferably, the pyrimidine base includes cytosine, uracil, 5-fluorocytosine, 5-fluorouracil, 5-chlorocytosine, 5-chlorouracil, 5-bromocytosine, 5-bromouracil, 5-iodocytosine, 5-iodouracil, 5-methylcytosine, 5-methyluracil, 5-ethylcytosine, 5-ethyluracil, 5-fluoromethylcytosine, 5-fluoromethyluracil, 5-trifluoromethyl cytosine, 5-trifluoromethyluracil, 5-vinyluracil, 5-bromovinyluracil, 5-chlorovinyluracil, 5-acetylenyl cytosine, 5-acetylenyl uracil, 5-propynyluracil, 5-hydroxycytosine, 5-methoxyuracil, 2-thiocytosine, 2-thiouracil, 3-methylcytosine, 4-thiouracil, 4-thio, 5, 6-dihydro cytosine, 5-nitrocytosine, N4-ethylcytosine, N4-acetylcytosine, or the like.
Preferably, the molar ratio of ribose compound to nucleobase is 1 (1.1-1.3).
Preferably, the ribose compound, ribose kinase, pentose phosphate mutase, and nucleic acid phosphorylase are added in a ratio of 1:0.1:0.1:0.1.
Preferably, the temperature of the reaction is 30 to 40 ℃, including but not limited to 31 ℃, 32 ℃, 35 ℃, 36 ℃, 37 ℃, 38 ℃ or 39 ℃, for a period of time of 0.5 to 2 hours, including but not limited to 0.6 hours, 0.7 hours, 0.8 hours, 0.9 hours, 1 hour, 1.2 hours, 1.5 hours, 1.6 hours, 1.8 hours or 1.9 hours.
Compared with the prior art, the invention has the following beneficial effects:
the invention integrates RK (ribose kinase, ribokinase), PPM (pentose phosphate mutase, phosphopentomutase) and NP (nucleoside phosphorylase ) to construct a method for synthesizing adenosine/uridine by three enzyme cascade, and excavates RK, PPM and NP for combination, thereby realizing high-efficiency synthesis of adenosine and/or uridine.
Drawings
FIG. 1 is a diagram of SDS-PAGE of enzyme purification;
FIG. 2 is a diagram of an adenosine synthesis HPLC assay;
FIG. 3 is a diagram of 2-aminoadenosine synthetic HPLC detection;
FIG. 4 is a diagram of 2-fluoroadenosine synthetic HPLC detection;
FIG. 5 is a diagram of 2' -deoxyadenosine synthesis HPLC assay;
FIG. 6 is a synthetic HPLC detection view of 2-amino-2' -deoxyadenosine;
FIG. 7 is a diagram of uridine synthetic HPLC detection;
FIG. 8 is a diagram of a 5-fluorouridine synthetic HPLC assay;
FIG. 9 is a diagram of 2' -deoxyuridine synthetic HPLC detection;
FIG. 10 is a synthetic HPLC detection of 5-fluoro-2' -deoxyuridine.
Detailed Description
The technical means adopted by the invention and the effects thereof are further described below with reference to the examples and the attached drawings. It is to be understood that the specific embodiments described herein are merely illustrative of the invention and are not limiting thereof.
The specific techniques or conditions are not identified in the examples and are described in the literature in this field or are carried out in accordance with the product specifications. The reagents or equipment used were conventional products available for purchase through regular channels, with no manufacturer noted.
The enzymes involved in the specific examples of the present invention and their sources are as follows:
Ribose kinase (ribokinase, RK) from Acanthamoeba castellanii (named enzyme AcRK) and ESCHERICHIA COLI (named enzyme EcRK), respectively;
Pentose phosphate mutases (phosphopentomutase, PPM) from ESCHERICHIA COLI (named enzyme EcPPM), bacillus cereus (named enzyme BcPPM), streptococcus mutans (named enzyme SmPPM) and Thermotoga maritima (named enzyme TmPPM), respectively;
Purine nucleoside phosphorylase (purine nucleoside phosphorylase, PNP) from Deinococcus geothermalis (named enzyme DgPNP), parageobacillus thermoglucosidasius (named enzyme PtPNP) and Geobacillus stearothermophilus (named enzyme GsPNP), respectively;
Pyrimidine nucleoside phosphorylase (PYRIMIDINE NUCLEOSIDE PHOSPHORYLASE, pyNP) from Parageobacillus thermoglucosidasius (named enzyme PtPyNP), geobacillus stearothermophilus (named enzyme GsPyNP) and Brevibacillus borstelensis (named enzyme BbPyNP), respectively.
Example 1
Ribokinase (RK), pentose Phosphate Mutase (PPM), purine Nucleoside Phosphorylase (PNP), pyrimidine nucleoside phosphorylase (PyNP) gene synthesis, clone expression and purification
Recombinant plasmids (the plasmid vector is pET-28a (+)) containing genes shown by sequences SEQ ID NO.13-24 (corresponding amino acid sequences shown by SEQ ID NO. 1-12) are respectively synthesized through complete genes, and the recombinant plasmids are introduced into competent cells of escherichia coli BL21 (DE 3) by adopting a chemical transformation method to obtain corresponding recombinant strains.
Each recombinant strain was cultured by shaking overnight at 37℃in 10mL of LB medium containing 50. Mu.g/mL of kanamycin, and the overnight cultured broth was transferred to 100mL of LB medium containing 50. Mu.g/mL of kanamycin at a ratio of 1%, and shaking cultured at 37℃until OD 600 reached between 0.6 and 0.8. IPTG (isopropyl-. Beta. -D-thiogalactoside) is added into a shaking flask to a final concentration of 0.4mM, the mixture is induced for 18h at 25 ℃, the mixture is centrifuged and collected for 10min at 18h, the thalli is washed once by buffer A (100 mM Tris-HCl, 100mM NaCl,10% glycerol (pH 7.5)), the thalli is resuspended by 10mL of buffer A after centrifugation and collected, the obtained cell homogenate is centrifuged at 10000rpm for 30min, the supernatant is filtered by a filter membrane of 0.45 μm after centrifugation, the supernatant obtained by filtration is purified by nickel ion affinity chromatography, the obtained protein solution is subjected to removal of imidazole by a PD-10 desalting column, the obtained protein solution is concentrated to about 10mg/mL by an ultrafiltration tube, the obtained enzyme solution is frozen at-80 ℃ for later use, and the SDS-PAGE detection results of the purified protein solution are shown in FIG. 1, which show that the required enzymes are successfully prepared, lane 1: acRK; lane 2: ecRK; lane 3: ecPPM; lane 4: bcPPM; lane 5: smPPM; lane 6: tmPPM; lane 7: dgPNP; lane 8: ptPNP; lane 9: gsPNP; lane 10: ptPyNP; lane 11: gsPyNP; lane 12: bbPyNP.
SEQ ID NO.13:AcRK
atggaagaaaaccagcagcagtgcccggttcgtcacatctgggaaccgcagcagggtcaggttcagcagcaggcttctaccggttctgaatgcccggttcgtaacacctgggacacctcttctctgtaccacaccaacggtcagttccagcacccgccgatctctcagaccaacggtcaggctcgttgcccgatgggttacttctctatctctgcttcttctggtgctccggcttcttcttctgacggtgctccgaaatgcccgttcgcttctctgtctctgggttcttctaccgaccgtcgtgctccgccggaacagctgcgtctgaccatcctgggtcacgttaccaacgacatcaacatcttcgttggtaaagaaacccgtgctcagggtggtggtgttctgttctctggtgttgctgcttctaacctgggtatgaacgttgaagttgttaccaaatgctctgctgaagacaaaccggttttccagaaaatcttcgctccgtctggtaccaaagttcagttcctgccgtctcaggaaaccacctgctgcgaaaacaactacccgaaaccgaactctgacgaacgtgttcagcgtttccacgctgttggtgctccgttcaccgttgacgacctgaaacacatcgaatctaccatcgttcacatcaacccgctgtcttacggtgaatttccggacgaactgatcccgaaaatcaaagaactgaacccgtctgttcagtacctggttgcggacgctcagggcttcatccgtcacatagacatgcagaacggtggtaaaatctctcacaaagactgggctgctaaagaacagtacctgaaatacttcgacctgttcaaagttgacgacaaagaagctaccgttctgaccggtgaaaaagacatgaaacgtgctatgcagatcctgcacgaaaaaggtgctaaaatggttctgggtacctacaacgctggtgttctgctgttcgacggtaacatcttctaccaggcttctttcggtccgtggaaagttgaaggtcgtaccggtcgtggtgacaccgtaactgctagcttcctggctgctgcgggtctgtctggtggtccgtggaaacgtaacgttgctctggaatttgctgctcgtgttaccaccaccaaaatgcagtacccgggtccgtaccgtcgtccgaccgcttctctg.
SEQ ID NO.14:EcRK
atgcagaacgctggttctctggttgttctgggttctatcaacgctgaccacatcctgaacctgcagtctttcccgaccccgggtgaaaccgttaccggtaaccactaccaggtagcgttcggtggtaaaggcgctaaccaggctgttgctgctggtcgttctggtgctaacatcgctttcatcgcttgcaccggtgacgactctatcggtgaatctgttcgtcagcagctggctaccgacaacatcgacatcaccccggtttctgttatcaaaggtgaatctaccggtgttgctctgatcttcgttaacggtgaaggtgaaaacgttatcggtatccacgctggtgctaacgctgctctgtctccggctctggttgaagctcagcgtgaacgtatcgctaacgcttctgctctgctgatgcagctggaatctccgctggaatctgttatggctgctgctaaaatcgctcaccagaacaaaaccatcgttgctctgaacccggctccggctcgtgaactgccggacgaactgctggctctggttgacatcatcaccccgaacgaaaccgaagctgaaaaactgaccggtatccgtgttgaaaacgacgaagacgctgctaaagctgctcaggttctgcacgaaaaaggtatccgtaccgttctgatcaccctgggttctcgtggtgtttgggcttctgttaacggtgaaggtcagcgtgttccgggtttccgtgttcaggctgttgacaccatcgctgctggtgacaccttcaacggtgctctgatcaccgctctgctggaagaaaaaccgctgccggaagcgataaggttcgctcacgctgcggctgctatcgctgttacccgtaaaggtgctcagccgtctgttccgtggcgtgaagaaatcgacgctttcctggaccgtcagcgt.
SEQ ID NO.15:EcPPM
atgaaacgcgcgtttattatggtgctggatagctttggcattggcgcgaccgaagatgcggaacgctttggcgatgtgggcgcggataccctgggccatattgcggaagcgtgcgcgaaaggcgaagcggataacggccgcaaaggcccgctgaacctgccgaacctgacccgcctgggcctggcgaaagcgcatgaaggcagcaccggctttattccggcgggcatggatggcaacgcggaagtgattggcgcgtatgcgtgggcgcatgaaatgagcagcggcaaagatagcgtgagcggccattgggaaattgcgggcgtgccggtgctgtttgaatggggctattttagcgatcatgaaaacagctttccgcaagaactgttagataaactggtggagcgcgcgaacctgccgggctatctgggcaactgccgcagcagcggcaccgtgattctggatcagctgggcgaagaacacatgaaaaccggcaaaccgattttttatacgagcgcggcgagcgtgtttcagattgcgtgccatgaagaaacctttggcctggataaactgtatgaactgtgcgaaattgcgcgcgaagaactgaccaacggcggctataacattggccgcgtgattgcgcgcccgtttattggcgataaagcgggcaactttcagcgcaccggcaaccgccgcgatctggcggtggaaccgccggcgccgaccgtgctgcagaaactggtggatgaaaaacatggccaagtggtgagcgtgggcaaaattgcggatatttatgcgaactgcggcattaccaaaaaagtgaaagcgaccggcctggatgcgctgtttgatgcgaccattaaagaaatgaaagaagcgggcgataacaccattgtgtttaccaactttgtggattttgatagcagctggggccatcgccgcgatgtggcgggctatgcggcgggcctggaactgtttgatcgccgcctgccggaactgatgagcctgctgcgcgatgacgatattctgattctgaccgcggatcatggctgcgatccgacctggaccggcaccgatcatacccgcgaacatattccggtgctggtgtatggcccgaaagtgaaaccgggcagcctgggccatcgcgaaacctttgcggatattggtcagaccctggcgaaatattttggcacgagcgatatggaatatggcaaagcgatgtttctcgag.
SEQ ID NO.16:BcPPM
atgaacaaatacaaacgtatcttcctggttgttatggactctgttggtatcggtgaagctccggacgctgaacagttcggtgacctgggttctgacaccatcggtcacatcgctgaacacatgaacggtctgcagatgccgaacatggttaaactgggtctgggtaacatccgtgaaatgaaaggtatctctaaagttgaaaaaccgctgggttactacaccaaaatgcaggaaaaatctaccggtaaagacaccatgaccggtcactgggaaatcatgggtctgtacatcgacaccccatttcaggtattcccggagggcttcccgaaagaactcctggacgaactggaagaaaaaaccggtcgtaaaatcatcggtaacaaaccggcttctggtaccgaaatcctggacgaactgggtcaggaacagatggaaaccggttctctgatcgtttacacctctgctgactctgttctgcagatcgctgctcacgaagaagttgttccgctggacgaactgtacaaaatctgcaaaatcgctcgtgaactgaccctggacgaaaaatacatggttggtcgtgttatcgctcgtccgttcgttggtgaaccgggtaacttcacccgtaccccgaaccgtcacgactacgctctgaaaccgttcggtcgtaccgttatgaacgaactgaaagactctgactacgacgttatcgctatcggtaaaatctctgacatctacgacggtgaaggtgttaccgaatctctgcgtaccaaatctaacatggacggtatggacaaactggttgacaccctgaacatggacttcaccggtctgtctttcctgaacctggttgacttcgacgctctgttcggtcaccgtcgtgacccgcagggttacggtgaagctctgcaggaatacgacgctaggctgccagaagtgttcgcaaaactgaaagaagacgacctgctgctgatcaccgctgaccacggtaacgacccgatccacccgggtaccgaccacacccgtgaatacgttccgctgctggcttactctccgtctatgaaagaaggtggtcaggaactgccgctgcgtcagaccttcgctgacatcggtgctaccgttgctgaaaacttcggtgttaaaatgccggaatacggtacctctttcctgaacgaactgaagaaa.
SEQ ID NO.17:SmPPM
atgtctctgtctaccttcaaccgtatccacctggttgttctggactctgttggtatcggtgctgctccggacgctaacaacttctctaacgctggtgttccggacggtgcttctgacaccctgggtcacatctctaaaaccgttggtctgaacgttccgaacatggctaaaatcggtctgggtaacatcccgcgtgacaccccgctgaaaaccgttccggctgaaaaccacccgaccggttacgttaccaaactggaagaagtttctctgggtaaagacaccatgaccggtcactgggaaatcatgggtctgaacatcaccgaaccgttcgacaccttctggaatggcttcccggaagaaataatctctaaaatcgaaaaattctctggtcgtaaagttatccgtgaagctaacaaaccgtactctggtaccgctgttatcgacgacttcggtccgcgtcagatggaaaccggtgaactgatcatctacacctctgctgacccggttctgcagatcgctgctcacgaagacgttatcccgctggacgaactgtaccgtatctgcgaatacgctcgttctatcaccctggaacgtccggctctgctgggtcgtatcatcgctcgtccgtacgttggtaaaccgcgtaacttcacccgtaccgctaaccgtcacgactacgctctgtctccgttcgctccgaccgttctgaacaaactggctgacgctggtgtttctacctacgctgttggtaaaatcaacgacatcttcaacggttctggtatcaccaacgacatgggtcacaacaaatctaactctcacggtgttgacaccctgatcaaaactatgggtctgtctgcgttcactaaaggcttctctttcaccaatctggttgacttcgacgctctgtacggtcaccgtcgtaacgctcacggttaccgtgactgcctgcacgaatttgacgaacgtctgccggaaatcatcgctgctatgaaagttgacgacctgctgctgatcaccgctgaccacggtaacgacccgacctacgctggtaccgaccacacccgtgaatacgttccgctgctggcttactctccgtctttcaccggtaacggtgttctgccggttggtcactacgctgacatctctgctaccatcgctgacaacttcggtgttgacaccgctatgataggcgaatcgttcctggacaaactcatc.
SEQ ID NO.18:TmPPM
atgcgtgttgttctgatcgttctggactctgttggtatcggtgaaatgccggacgctcacctgtacggtgacgaaggttctaacaccatcgttaacaccgctaaagctgtttctggtctgcacctgccgaacatggctaaactgggtctgggtaacctggacgacatcccgggtgttgaaccggttaaaccggctgaaggtatctacggtaaaatgatggaaaaatctccgggtaaagacaccaccaccggtcactgggaaatcgctggtgttatcctgaaaaaaccgttcgacctgtttccagaaggcttcccgaaagaactcatcgaagaatttgaacgtcgtaccggtcgtaaagttatcggtaacaaaccggcttctggtaccgaaatcatcaaagaactgggtccgatccacgaaaaaaccggtgctctgatcgtttacacctctgctgactctgttttccagatcgctgctaaaaaagaaatcgttccgctggaagaactgtaccgttactgcgaaatcgctcgtgaactgctgaacgaaatgggttacaaagttgctcgtgttatcgctcgtccgttcaccggtgaatggccgaactacgttcgtaccccggaacgtaaagacttctctctggaaccggaaggtaaaaccctgctggacgttctgaccgaaaacggtatcccggtttacggtgttggtaaaatcgctgacatcttcgctggtcgtggtgttaccgaaaactacaaaaccaaagacaacaacgacggtatcgacaaaaccatctctctgatgaaagaaaaaaaccacgactgcctgatcttcaccaacctggttgacttcgacaccaaatacggtcaccgtaacgacccggtttcttacgctaaagctctggaagaatttgacgctcgtctgccggaaatcatgcacaacctgaacgaagacgacgttctgttcatcaccgctgaccacggttgcgacccgaccaccccgtctaccgaccactctcgtgaaatggttccgctgctgggttacggtggtcgtttaaaaaaggacgtgtacgtgggtatccgtgaaaccttcgctgacctgggtcagaccatcgctgacatcttcggtgttccgccgctggaaaacggtacctctttcaaaaacctgatctgggaa.
SEQ ID NO.19:DgPNP
atggttgctcgtgttccggctcgtccgttcgcttctccgccggctaccctggaccgtgtttctgttcacctgaacgctcgtccgggtgaaatcgctgaaaccgttctgctgccgggtgacccgctgcgtgctcagcacatcgctgaaaccttcttcgaaaacccggttcagcacaactctgttcgtggtatgctgggtttcaccggtacctaccgtggtgttccggtttctgttcagggtaccggtatgggtatcgcttcttctatgatctacgttaacgaactgatccgtgactacggttgccagaccctgatccgtgttggtaccgctggttcttaccagccggacgttcacgttcgtgacctggttctggctcaggctgcttgcaccgactctaacatcaacaacatccgtttcggtctgcgtaacttcgctccgatcgctgacttcgaactgctgctgcgtgcttaccagatggctcgtgaccgtggtttcgctacccacgttggtaacatcctgtcttctgacaccttctaccaggacgacccggaatcttacaaactgtgggctcagtacggtgttctggctgttgaaatggaagctgctggtctgtacaccctggctgctaaatacggtgttcgtgctctgaccatcctgaccatctctgaccacctggttacccgtgaagaaaccaccgctgaagaacgtcagaccaccttcaacggtatgatcgaagttgctctggacgctgctctgggtctggctgttccgtctaactctatgtaaggatcc.
SEQ ID NO.20:PtPNP
atgtctatccacatcgaagctaaacagcaggaaatcgctgaaaaaatcctgctgccgggtgacccgctgcgtgctcagtacatcgctgaaaccttcctggaaggtgctacctgctacaaccgtgttcgtggtatgctgggtttcaccggtacctacaaaggtcaccgtatctctgttcagggtaccggtatgggtgttccgtctatctctatctacgttaacgaactgatccagtcttaccacgttcagaccctgatccgtgttggtacctgcggtgctatccagaaagacgttaacgttcgtgacgttatcctggctatgtctgcttctaccgactctaacatgaaccgtctgaccttccgtggtcgtgactacgctccgaccgctaacttcgctctgctgcgtaccgcttacgaagttggtgctgaaaaaggtctgccgctgaaagttggttctgttttcaccgctgacatgttctacaacgacgaaccggactgggaaacctgggctcgttacggtgttctggctgttgaaatggaaaccgctgctctgtacaccctggctgctaaattcggtcgtaaagctctgtctgttctgaccgtttctgaccacatcctgaccggtgaagaaaccaccgctcaggaacgtcagaccaccttcaacgacatgatcgaagttgctctggaaaccgctatccgtgttgaataaggatcc.
SEQ ID NO.21:GsPNP
atgtctgttcacatcggtgctaaagaacacgaaatcgctgacaaaatcctgctgccgggtgacccgctgcgtgctaaatacatcgctgaaaccttcctggaaggtgctacctgctacaaccaggttcgtggtatgctgggtttcaccggtacctacaaaggtcaccgtatctctgttcagggtaccggtatgggtgttccgtctatctctatctacatcaccgaactgatgcagtcttacaacgttcagaccctgatccgtgttggtacctgcggtgctatccagaaagacgttaaagttcgtgacgttatcctggctatgacctcttctaccgactctcagatgaaccgtatgaccttcggtggtatcgactacgctccgaccgctaacttcgacctgctgaaaaccgcttacgaaatcggtaaagaaaaaggtctgcagctgaaagttggttctgttttcaccgctgacatgttctacaacgaaaacgctcagttcgaaaaactggctcgttacggtgttctggctgttgaaatggaaaccaccgctctgtacaccctggctgctaaattcggtcgtaaagctctgtctgttctgaccgtttctgaccacatcctgaccggtgaagaaaccaccgctgaagaacgtcagaccaccttcaacgaaatgatcgaagttgctctggaaaccgctatccgtcagtaaggatcc.
SEQ ID NO.22:PtPyNP
atggttgacctgatcgctaaaaaacgtgacggttacgaactgtctaaagaagaaatcgacttcatcatccgtggttacaccaacggtgacatcccggactaccagatgtctgctttcgctatggctgttttcttccgtggtatgaccgaagaagaaaccgctgctctgaccatggctatggttcgttctggtgacgttatcgacctgtctaaaatcgaaggtatgaaagttgacaaacactctaccggtggtgttggtgacaccaccaccctggttctgggtccgctggttgcttctgttggtgttccggttgctaaaatgtctggtcgtggtctgggtcacaccggtggtaccatcgacaaactcgaatctgttccgggcttccacgttgaaatagacaacgaacagttcatcgaactggttaacaaaaacaaaatcgctatcatcggtcagaccggtaacctgaccccggctgacaaaaaactgtacgctctgcgtgacgttaccgctaccgttgactctatcccgctgatcgcttcttctatcatgtctaaaaaaatcgctgctggtgctgacgctatcgttctggacgttaaaaccggtgctggtgctttcatgaaagacttcgctggtgctaaacgtctggctaccgctatggttgaaatcggtaaacgtgttggtcgtaaaaccatggctgttatctctgacatgtctcagccgctgggttacgctgttggtaacgctctggaagttaaagaagctatcgacaccctgaaaggtaaaggtccggaagacctgcaggaactgtgcctgaccctgggttcttacatggtttacctggctgaaaaagctagttctctggaagaagctcgtgctctgctggaagctagtatccgtgaaggtaaagctctggaaaccttcaaagttttcctgtctgctcagggtggtgacgcttctgttgttgacgacccgaccaaactgccgcaggctaaataccgttgggaactggaagctccggaagacggttacgttgctgaaatcgttgctgacgaagttggtaccgctgctatgctgctgggtgctggtcgtgctaccaaagaagctaccatcgacctgtctgttggtctggttctgcacaaaaaagttggtgacgctgttaaaaaaggtgaatctctggttaccatctactctaacaccgaaaacatcgaagaagttaaacagaaactggctaaatctatccgtctgtcttctatcccggttgctaaaccgaccctgatctacgaaaccatctcttaaggatcc.
SEQ ID NO.23:GsPyNP
atgcgtatggttgacctgatcgaaaaaaaacgtgacggtcacgctctgaccaaagaagaaatccagttcatcatcgaaggttacaccaaaggtgacatcccggactaccagatgtctgctctggctatggctatcttcttccgtggtatgaacgaagaagaaaccgctgaactgaccatggctatggttcactctggtgacaccatcgacctgtctcgtatcgaaggtatcaaagttgacaaacactctaccggtggtgttggtgacaccaccaccctggttctgggtccgctggttgcttctgttggtgttccggttgctaaaatgtctggtcgtggtctgggtcacaccggtggtaccatcgacaaactcgaatctgttccgggcttccacgttgaaataaccaacgacgaatttatcgacctggttaacaaaaacaaaatcgctgttgttggtcagtctggtaacctgaccccggctgacaaaaaactgtacgctctgcgtgacgttaccgctaccgttaactctatcccgctgatcgcttcttctatcatgtctaaaaaaatcgctgctggtgctgacgctatcgttctggacgttaaaaccggtgttggtgctttcatgaaagacctgaacgacgctaaagctctggctaaagctatggttgacatcggtaaccgtgttggtcgtaaaaccatggctatcatctctgacatgtctcagccgctgggttacgctatcggtaacgctctggaagttaaagaagctatcgacaccctgaaaggtgaaggtccggaagacttccaggaactgtgcctggttctgggttctcacatggtttacctggctgaaaaagctagttctctggaagaagctcgtcacatgctggaaaaagctatgaaagacggttctgctctgcagaccttcaaaaccttcctggctgctcagggtggtgacgcttctgttgttgacgacccgtctaaactgccgcaggctaaatacatcatcgaactggaagctaaagaagacggttacgtttctgaaatcgttgctgacgctgttggtaccgctgctatgtggctgggtgctggtcgtgctaccaaagaatctaccatcgacctggctgttggtctggttctgcgtaaaaaagttggtgacgctgttaaaaaaggtgaatctctggttaccatctactctaaccgtgaacaggttgacgacgttaaacagaaactgtacgaaaacatccgtatctctgctaccccggttcaggctccgaccctgatctacgacaaaatctcttaaggatcc.
SEQ ID NO.24:BbPyNP
atgcgtatggttgacatcatcgctaaaaaacgtgacggtctggaactgtcttctgaagaaatccaatttctggtaagcggttacaccgacggttctatcccggactaccagatgtctgcttgggctatggctgttctgctgcgtggtatgaccccgcgtgaaaccggtgacctgaccctggctatggctggttctggtgaacagctggacctgtcttctctgaaaggtatcaaagttgacaaacactctaccggtggtgttggtgacaaaaccaccctggttgttgctccgctggttgctgctgctggtatcccggttgctaaaatgtctggtcgtggtctgggtcactctggtggtaccatcgacaaactggaatctttcgctggtttccaggttgaacgtacccgtgaacagttcctgcagcaggttcgtgaaatcggtgtttctgttatcggtcagtctggtaacctgaccccggctgacaaaaaactgtacgctctgcgtgacgttaccgctaccgttgaagctgttccgctgatcgcttcttctatcatgtctaaaaaaatcgctgctggtgctgacgctatcctgctggacgttaaagttggtaaaggtgctttcatgaaaaccctggaacaggctgaaaccctggcttctgctatggctcagatcggtacccaggttggtcgtcgtaccgttgctgttatctctgacatgaaccaaccactgggcttcgctgttggtaatgctctggaagttaaagaagctatcgacaccctggctggtcgtggtccgaaagacctgaccgaactggctctggctatcggtgctcacatgctggttctgggtgaactggttgctgacgttgaagaaggtcgtaaacgtctggaagaaatcatggactctggtaaagctgttgaaaaactggctcagatgatcgaagctcagggtggtaacaaagaagacgtttacaacccggaccgtctgccgaaagctagtctgaccgctgaagttaaagctagtcaggacggttacatctctgctatcgacgctgaaaccgttggtcacgcttctgttgttctgggtgctggtcgtctgaccaaagaaatgccgatcgacctggctgttggtatcgttctggctaaaaaacgtggtgaccaggttcgtaaaggtgacgttctggctaccgttcacgctaacgacgaaatcctgctgaaacaggctgttgaagaactgaaaggtgcttacacctacgactctgaatctctgatcgaccagccgctgatctacaaaatcatcaccgaataaggatcc.
Example 2
In this example, acRK, tmPPM and GsPNP were used to synthesize adenosine in a one-pot method.
1ML of a reaction solution consisting of 1g/L of 5mM D-ribose (Adamas-beta, reagent grade), 1g/L of adenine hydrochloride (Adamas-beta, reagent grade), 5mM of 5' -adenine nucleotide disodium salt (Adamas-beta, reagent grade), 5mM of magnesium chloride (Adamas-beta, reagent grade), 0.1mM of manganese chloride (Adamas-beta, reagent grade), 0.1g/L AcRK,0.1g/L TmPPM,0.1g/LGsPNP and 50mM of Tris-HCl buffer (pH 8.0) was reacted at 37℃for 1 hour, and 0.1mL of diluted hydrochloric acid (0.1 mol/L) was added to terminate the reaction. HPLC analysis of the reacted solution, the results of which are shown in FIG. 2, a is an adenosine label; b is a blank control without enzyme; c is a catalytic solution containing EcRK, tmPPM and GsPNP, one of the solutions after the reaction is identical to the peak of adenosine (Adamas-beta, reagent grade), indicating successful preparation of adenosine, and the adenosine synthesis efficiency is 19.6% calculated by area percentage method.
HPLC analysis conditions:
Column: ATLANTIS T3,3um,3.0 x 150mm;
column temperature: 30 ℃;
pump flow rate: 1.0mL/min;
And (3) detection: UV 250nm;
mobile phase: mobile phase a was acetonitrile (containing 0.05% formic acid) and mobile phase B was ultrapure water (containing 0.05% formic acid);
Elution procedure: 0-2min 100% B;2-4min 0-30% A;4-10min 30-90% A;10-10.1min 90-0% A;10.1-13min 100% B.
Example 3
The present example performed the one pot synthesis of adenosine by EcRK, tmPPM and GsPNP.
The difference compared with example 2 is that AcRK was replaced with EcRK equivalent amount, and HPLC analysis as described in example 2 was performed after the completion of the reaction, and the result showed that one of the peaks in the post-reaction solution was identical to the peak of adenosine (Adamas-beta, reagent grade) and the adenosine synthesis efficiency was 29.4% in terms of area%.
Example 4
In this example, ecRK, ecPPM and GsPNP one pot synthesis of adenosine were performed.
The only difference compared to example 2 is that AcRK is replaced with an equivalent EcRK and TmPPM is replaced with an equivalent EcPPM. After the completion of the reaction, HPLC analysis as in example 2 was performed, and the result showed that one of the peaks in the post-reaction solution was identical to the peak of adenosine (Adamas-beta, reagent grade), and the adenosine synthesis efficiency was 28.4% in terms of area%.
Example 5
In this example, the one pot synthesis of adenosine by EcRK, bcPPM and GsPNP was performed.
The only difference compared to example 2 is that AcRK is replaced with an equivalent EcRK and TmPPM is replaced with an equivalent BcPPM. After the completion of the reaction, HPLC analysis as in example 2 was performed, and the result showed that one of the peaks in the post-reaction solution was identical to the peak of adenosine (Adamas-beta, reagent grade), and the adenosine synthesis efficiency was 27.3% in terms of area%.
Example 6
In this example, the one pot synthesis of adenosine was performed on EcRK, smPPM and GsPNP.
The only difference compared to example 2 is that AcRK is replaced with an equivalent EcRK and TmPPM is replaced with an equivalent SmPPM. After the completion of the reaction, HPLC analysis as in example 2 was performed, and the result showed that one of the peaks in the post-reaction solution was identical to the peak of adenosine (Adamas-beta, reagent grade), and the adenosine synthesis efficiency was 31.3% in terms of area%.
Example 7
The present example performed the one pot synthesis of adenosine by EcRK, tmPPM and DgPNP.
The only difference compared to example 2 is that AcRK is replaced with an equivalent EcRK and GsPNP is replaced with an equivalent DgPNP. After the completion of the reaction, HPLC analysis as in example 2 was performed, and the result showed that one of the peaks in the post-reaction solution was identical to the peak of adenosine (Adamas-beta, reagent grade), and the adenosine synthesis efficiency was 41.0% in terms of area%.
Example 8
The present example performed the one pot synthesis of adenosine by EcRK, tmPPM and PtPNP.
The only difference compared to example 2 is that AcRK is replaced with an equivalent EcRK and GsPNP is replaced with an equivalent PtPNP. After the completion of the reaction, HPLC analysis as in example 2 was performed, and the result showed that one of the peaks in the post-reaction solution was identical to the peak of adenosine (Adamas-beta, reagent grade), and the adenosine synthesis efficiency was 42.2% in terms of area%.
Example 9
In this example, ecRK, tmPPM and GsPNP one pot method were performed to synthesize 2-aminoadenosine.
The difference compared to example 2 is only that AcRK is replaced by an equivalent amount EcRK and adenine hydrochloride is replaced by an equivalent amount of 2-amino-adenine. After the reaction was completed, HPLC analysis as in example 2 was performed, and the results are shown in FIG. 3, wherein a is 2-aminoadenosine standard; b is a blank control without enzyme; c is a catalytic solution containing EcRK, tmPPM and GsPNP, one of the solutions after the reaction is identical to the peak of 2-aminoadenosine (Adamas-beta, reagent grade), and the 2-aminoadenosine synthesis efficiency is 22.0% in terms of area percent.
Example 10
In this example, ecRK, tmPPM and GsPNP one pot method were performed to synthesize 2-fluoroadenosine.
The difference compared to example 2 is only that AcRK is replaced by an equivalent amount EcRK and adenine hydrochloride is replaced by an equivalent amount of 2-fluoroadenine. HPLC analysis as in example 2 after the end of the reaction indicated that one of the peaks in the post-reaction solution was identical to the peak of 2-fluoroadenosine (Adamas-beta, reagent grade) (FIG. 4), a being a 2-fluoroadenosine standard; b is a blank control without enzyme; c is a catalytic solution containing EcRK, tmPPM and GsPNP, and the 2-fluoroadenosine synthesis efficiency is 10.7 percent calculated by area percent.
Example 11
In this example, ecRK, tmPPM and GsPNP one pot method were performed to synthesize 2' -deoxyadenosine.
The difference compared to example 2 is only that AcRK is replaced by an equivalent amount EcRK and D-ribose is replaced by an equivalent amount of 2' -deoxy-D-ribose. HPLC analysis as described in example 2 after the end of the reaction indicated that one of the peaks in the post-reaction solution was identical to the peak of 2 '-deoxyadenosine (Adamas-beta, reagent grade) (fig. 5), a being a 2' -deoxyadenosine standard; b is a blank control without enzyme; c is a catalytic solution containing EcRK, tmPPM and GsPNP, and the 2' -deoxyadenosine synthesis efficiency is 25.6 percent in terms of area percent.
Example 12
In this example, ecRK, ecPPM and GsPNP were used to synthesize 2' -deoxyadenosine in one pot.
The difference compared to example 2 is only that AcRK is replaced by an equivalent amount EcRK, tmPPM is replaced by an equivalent amount EcPPM and D-ribose is replaced by an equivalent amount of 2' -deoxy-D-ribose. HPLC analysis as in example 2 after the end of the reaction indicated that one of the peaks in the post-reaction solution was exactly the same as the peak of 2 '-deoxyadenosine (Adamas-beta, reagent grade) in terms of area percent, with a 2' -deoxyadenosine synthesis efficiency of 28.2%.
Example 13
In this example, 2' -deoxyadenosine was synthesized by the EcRK, bcPPM and GsPNP one-pot method.
The difference compared to example 2 is only that AcRK is replaced by an equivalent amount EcRK, tmPPM is replaced by an equivalent amount BcPPM and D-ribose is replaced by an equivalent amount of 2' -deoxy-D-ribose. HPLC analysis as in example 2 after the end of the reaction indicated that one of the peaks in the post-reaction solution was identical to the peak of 2 '-deoxyadenosine (Adamas-beta, reagent grade), and the 2' -deoxyadenosine synthesis efficiency was 26.3% as an area percentage.
Example 14
In this example, 2' -deoxyadenosine was synthesized by the EcRK, smPPM and GsPNP one pot method.
The difference compared to example 2 is only that AcRK is replaced by an equivalent amount EcRK, tmPPM is replaced by an equivalent amount SmPPM and D-ribose is replaced by an equivalent amount of 2' -deoxy-D-ribose. HPLC analysis as described in example 2 after the end of the reaction indicated exactly the same peak as the peak of 2' -deoxyadenosine (Adamas-beta, reagent grade) in the post-reaction solution. The 2' -deoxyadenosine synthesis efficiency was 33.1% calculated as area percent.
Example 15
In this example, 2' -deoxyadenosine was synthesized by the EcRK, bcPPM and DgPNP one-pot method.
The difference compared to example 2 is only that AcRK is replaced by an equivalent amount EcRK, tmPPM is replaced by an equivalent amount BcPPM, gsPNP is replaced by an equivalent amount DgPNP, and D-ribose is replaced by an equivalent amount of 2' -deoxy-D-ribose. HPLC analysis as described in example 2 after the end of the reaction indicated exactly the same peak as the peak of 2' -deoxyadenosine (Adamas-beta, reagent grade) in the post-reaction solution. The 2' -deoxyadenosine synthesis efficiency was 33.1% calculated as area percent.
Example 16
In this example, 2' -deoxyadenosine was synthesized by the EcRK, bcPPM and PtPNP one-pot method.
The difference compared to example 2 is only that AcRK is replaced by an equivalent amount EcRK, tmPPM is replaced by an equivalent amount BcPPM, gsPNP is replaced by an equivalent amount PtPNP, and D-ribose is replaced by an equivalent amount of 2' -deoxy-D-ribose. HPLC analysis as described in example 2 after the end of the reaction indicated exactly the same peak as the peak of 2' -deoxyadenosine (Adamas-beta, reagent grade) in the post-reaction solution. The 2' -deoxyadenosine synthesis efficiency was 32.5% calculated as area percent.
Example 17
In this example, ecRK, tmPPM and DgPNP one pot method were used to synthesize 2-amino-2' -deoxyadenosine.
The difference compared to example 2 is only that AcRK is replaced by an equivalent EcRK, gsPNP is replaced by an equivalent DgPNP, D-ribose is replaced by an equivalent 2' -deoxy-D-ribose, and adenine hydrochloride is replaced by an equivalent 2-aminoadenine. HPLC analysis as described in example 2 after the end of the reaction indicated exactly the same peak as the peak of 2-amino-2' -deoxyadenosine (Adamas-beta, reagent grade) in the post-reaction solution. The 2-amino-2' -deoxyadenosine synthesis efficiency was 25.1% as calculated by area percent.
Example 18
This example is conducted to synthesize 2-amino-2' -deoxyadenosine using the EcRK, tmPPM and PtPNP one pot method.
The difference compared to example 2 is only that AcRK is replaced by an equivalent EcRK, gsPNP is replaced by an equivalent PtPNP, D-ribose is replaced by an equivalent 2' -deoxy-D-ribose, and adenine hydrochloride is replaced by an equivalent 2-aminoadenine. HPLC analysis as described in example 2 after the end of the reaction indicated exactly the same peak as the peak of 2-amino-2' -deoxyadenosine (Adamas-beta, reagent grade) in the post-reaction solution. The 2-amino-2' -deoxyadenosine synthesis efficiency was 24.4% as calculated as area percentage.
Example 19
This example is conducted to synthesize 2-amino-2' -deoxyadenosine using the EcRK, tmPPM and GsPNP one pot method.
The difference compared to example 2 is only that AcRK is replaced by an equivalent amount EcRK, D-ribose is replaced by an equivalent amount of 2' -deoxy-D-ribose, adenine hydrochloride is replaced by an equivalent amount of 2-aminoadenine. HPLC analysis as in example 2 after the end of the reaction indicated that one of the peaks in the post-reaction solution was identical to the peak of 2-amino-2 '-deoxyadenosine (Adamas-beta, reagent grade) (FIG. 6), a being a 2-amino-2' -deoxyadenosine standard; b is a blank control without enzyme; c is a catalytic solution containing EcRK, tmPPM and GsPNP, and the 2-amino-2' -deoxyadenosine synthesis efficiency is 23.7 percent calculated by area percent.
Example 20
This example performed the one pot synthesis of uridine by EcRK, tmPPM and PtPyNP.
1ML of a reaction solution consisting of 1g/L D-ribose (Adamas-beta, reagent grade), 1g/L uracil (Adamas-beta, reagent grade), 5mM 5' -adenine nucleotide disodium salt (Adamas-beta, reagent grade), 5mM magnesium chloride (Adamas-beta, reagent grade), 0.1mM manganese chloride (Adamas-beta, reagent grade), 0.1g/L EcRK,0.1g/L TmPPM,0.1g/L PtPyNP and 50mM Tris-HCl buffer (pH 8.0) was reacted at 37℃for 1 hour, and 0.1mL of diluted hydrochloric acid (0.1 mol/L) was added to terminate the reaction. HPLC analysis as described in example 2 after the reaction indicated exactly the same peak in the post-reaction solution as the peak of uridine (Adamas-beta, reagent grade). The uridine synthesis efficiency was 12.5% calculated as area percent.
Example 21
This example performed the one pot synthesis of uridine by EcRK, tmPPM and GsPyNP.
The only difference compared to example 20 is that PtPyNP is replaced by an equivalent amount GsPyNP. HPLC analysis as in example 2 after the end of the reaction indicated that one of the peaks in the post-reaction solution was identical to the peak of uridine (Adamas-beta, reagent grade), a being a uridine standard; b is a blank control without enzyme; c is a catalytic solution containing EcRK, tmPPM and GsPyNP, and the uridine synthesis efficiency is 11.9% calculated by area percent.
Example 22
This example performed the one pot synthesis of uridine by EcRK, tmPPM and BbPyNP.
The only difference compared to example 20 is that PtPyNP is replaced by an equivalent amount BbPyNP. HPLC analysis as described in example 2 after the end of the reaction indicated exactly the same peak in the post-reaction solution as the peak of uridine (Adamas-beta, reagent grade). The uridine synthesis efficiency was 12.1% calculated as area percent.
Example 23
In this example, ecRK, tmPPM and PtPyNP were used to synthesize 5-fluorouridine in a one-pot method.
The only difference compared to example 20 is that the uracil is replaced by an equivalent amount of 5-fluorouracil. HPLC analysis as described in example 2 after the end of the reaction indicated exactly the same peak as the peak of 5-fluorouridine (Adamas-beta, reagent grade) in the post-reaction solution. The 5-fluorouridine synthesis efficiency was 9.5% as calculated as area percentage.
Example 24
In this example, one pot synthesis of 5-fluorouridine was performed with EcRK, tmPPM and GsPyNP
The only difference compared to example 20 is that PtPyNP is replaced by an equivalent amount GsPyNP and uracil is replaced by 5-fluorouracil. HPLC analysis as described in example 2 after the end of the reaction indicated that one of the peaks in the post-reaction solution was identical to the peak of 5-fluorouridine (Adamas-beta, reagent grade) (fig. 9), a being a 5-fluorouridine standard; b is a blank control without enzyme; c is a catalytic solution containing EcRK, tmPPM and GsPyNP, and the 5-fluorouridine synthesis efficiency is 9.6 percent in terms of area percent.
Example 25
In this example, ecRK, tmPPM and BbPyNP were used to synthesize 5-fluorouridine in a one-pot method.
The only difference compared to example 20 is that PtPyNP is replaced by an equivalent amount BbPyNP and uracil is replaced by 5-fluorouracil. HPLC analysis as described in example 2 after the end of the reaction indicated exactly the same peak as the peak of 5-fluorouridine (Adamas-beta, reagent grade) in the post-reaction solution. The 5-fluorouridine synthesis efficiency was 9.5% as calculated as area percentage.
Example 26
This example is conducted for the one pot synthesis of 2' -deoxyuridine by EcRK, tmPPM and PtPyNP.
The difference compared to example 20 is only that the equivalent amount of 2' -deoxy-D-ribose is used instead of D-ribose. HPLC analysis as described in example 2 after the end of the reaction indicated exactly the same peak as the peak of 2' -deoxyuridine (Adamas-beta, reagent grade) in the post-reaction solution. The 2' -deoxyuridine synthesis efficiency was 10.8% calculated as area percentage.
Example 27
This example is conducted for the one pot synthesis of 2' -deoxyuridine by EcRK, tmPPM and GsPyNP.
The difference compared to example 20 is only that PtPyNP is replaced by an equivalent amount GsPyNP and D-ribose is replaced by 2' -deoxy-D-ribose. HPLC analysis as described in example 2 after the end of the reaction indicated that one of the peaks in the post-reaction solution was identical to the peak of 2 '-deoxyuridine (Adamas-beta, reagent grade) (fig. 10), a being a 2' -deoxyuridine standard; b is a blank control without enzyme; c is a catalytic solution containing EcRK, tmPPM and GsPyNP, and the 2' -deoxyuridine synthesis efficiency is 11.6% calculated as area percentage.
Example 28
This example is conducted for the one pot synthesis of 2' -deoxyuridine by EcRK, tmPPM and BbPyNP.
The difference compared to example 20 is only that PtPyNP is replaced by an equivalent amount BbPyNP and D-ribose is replaced by 2' -deoxy-D-ribose. HPLC analysis as described in example 2 after the end of the reaction indicated exactly the same peak as the peak of 2' -deoxyuridine (Adamas-beta, reagent grade) in the post-reaction solution. The 2' -deoxyuridine synthesis efficiency was 11.8% calculated as area percentage.
Example 29
This example is conducted for the one pot synthesis of 5-fluoro-2' -deoxyuridine using EcRK, tmPPM and PtPyNP.
The difference compared to example 20 is only that the equivalent amount of 2' -deoxy-D-ribose is used instead of D-ribose and that 5-fluorouracil is used instead of uracil. HPLC analysis as described in example 2 after the end of the reaction indicated exactly the same peak in the post-reaction solution as the peak of 5-fluoro-2' -deoxyuridine (Adamas-beta, reagent grade). The 5-fluoro-2' -deoxyuridine synthesis efficiency was 13.0% as calculated in area percent.
Example 30
This example is conducted for the one pot synthesis of 5-fluoro-2' -deoxyuridine using EcRK, tmPPM and GsPyNP.
The difference compared to example 20 is only that PtPyNP is replaced by an equivalent amount GsPyNP, D-ribose is replaced by 2' -deoxy-D-ribose, and uracil is replaced by 5-fluorouracil. HPLC analysis as in example 2 after the end of the reaction indicated that one of the peaks in the post-reaction solution was identical to the peak of 5-fluoro-2 '-deoxyuridine (Adamas-beta, reagent grade), a being a 5-fluoro-2' -deoxyuridine standard; b is a blank control without enzyme; c is a catalytic solution containing EcRK, tmPPM and GsPyNP. The 5-fluoro-2' -deoxyuridine synthesis efficiency was 13.1% as calculated in area percent.
Example 31
This example is conducted for the one pot synthesis of 5-fluoro-2' -deoxyuridine using EcRK, tmPPM and BbPyNP.
The difference compared to example 20 is only that PtPyNP is replaced by an equivalent amount BbPyNP, D-ribose is replaced by 2' -deoxy-D-ribose, and uracil is replaced by 5-fluorouracil. HPLC analysis as described in example 2 after the end of the reaction indicated exactly the same peak in the post-reaction solution as the peak of 5-fluoro-2' -deoxyuridine (Adamas-beta, reagent grade). The 5-fluoro-2' -deoxyuridine synthesis efficiency was 12.1% as calculated as area percentage.
The statistical analysis results are shown in table 1, and show that the invention can efficiently prepare adenosine and uridine and corresponding derivatives by specific ribose-excavating kinase, pentose phosphate mutase, purine nucleoside phosphorylase and pyrimidine nucleoside phosphorylase and designing specific combinations.
TABLE 1
In summary, the invention integrates RK (ribokinase, sugarkinase), PPM (pentose phosphate mutase, phosphopentomutase) and NP (nucleoside phosphorylase ) to construct a method for synthesizing adenosine/uridine by three enzyme cascade, and excavates RK, PPM and NP to combine, thereby providing a new idea for efficiently synthesizing adenosine and/or uridine, and increasing enzyme dosage according to the existing biochemical knowledge and enzymatic knowledge, the conversion rate of the reaction route of the invention to the substrate can be greatly improved by optimizing the feeding proportion of the three enzymes and the feeding proportion of the substrate, optimizing the reaction conditions (pH, temperature, divalent cation type) and other measures, the conversion rate of the substrate can be expected to reach or exceed the conversion rate of the substrate of the existing route, and the nucleoside derivative product with diversified reaction routes has obvious advantages compared with the single products of other routes.
The applicant states that the detailed method of the present invention is illustrated by the above examples, but the present invention is not limited to the detailed method described above, i.e. it does not mean that the present invention must be practiced in dependence upon the detailed method described above. It should be apparent to those skilled in the art that any modification of the present invention, equivalent substitution of raw materials for the product of the present invention, addition of auxiliary components, selection of specific modes, etc., falls within the scope of the present invention and the scope of disclosure.

Claims (10)

1. A method for the catalytic synthesis of adenosine and/or uridine by a three enzyme cascade, said method comprising:
Reacting ribose compound and nucleobase as substrate and ribose kinase, pentose phosphate mutase and nucleoside phosphorylase as catalyst to synthesize adenosine and/or uridine;
The ribose compound comprises D-ribose and/or derivatives thereof;
The nucleobases include purine bases and/or pyrimidine bases;
the nucleoside phosphorylase includes purine nucleoside phosphorylase and/or pyrimidine nucleoside phosphorylase.
2. The method for the catalytic synthesis of adenosine and/or uridine by three enzyme cascade according to claim 1, wherein the amino acid sequence of ribokinase comprises the sequence shown in SEQ ID No.1 or SEQ ID No. 2.
3. The method for the catalytic synthesis of adenosine and/or uridine by three enzyme cascade according to claim 1 or 2, wherein the amino acid sequence of pentose phosphate mutase comprises the sequence shown in SEQ ID No.3, SEQ ID No.4, SEQ ID No.5 or SEQ ID No. 6.
4. A method of catalytic synthesis of adenosine and/or uridine by a three enzyme cascade according to any of claims 1-3, wherein the amino acid sequence of the purine nucleoside phosphorylase comprises the sequence shown in SEQ ID No.7, SEQ ID No.8 or SEQ ID No. 9.
5. The method for the catalytic synthesis of adenosine and/or uridine by three enzyme cascade according to any of claims 1-4, wherein the amino acid sequence of pyrimidine nucleoside phosphorylase comprises the sequence shown in SEQ ID No.10, SEQ ID No.11 or SEQ ID No. 12.
6. The method for the catalytic synthesis of adenosine and/or uridine by the three enzyme cascade according to any of the claims 1-5, wherein the derivatives comprise aldopentoses and deoxysugars;
preferably, the aldopentose comprises any one of D-arabinose, L-arabinose, D-xylose, L-lyxose or D-ribose;
Preferably, the deoxy sugar comprises any one of 2' -deoxy-D-ribose, 2',3' -dideoxy-D-ribose, D-fucose, L-fucose, D-rhamnose or L-rhamnose.
7. The method for the catalytic synthesis of adenosine and/or uridine by three enzyme cascade according to any of the claims 1-6, wherein, the purine bases include purine, adenine, guanine, 6-hydroxy purine, 6-fluoro purine, 6-chloro purine, 6-methylaminopurine, 6-dimethylamino purine, 6-trifluoromethyl aminopurine, 6-benzoylamino purine, 6-acetylaminopurine, 6-hydroxyaminopurine, 6-methoxy purine, 6-benzoyloxy purine, 6-methyl purine, 6-ethyl purine, 6-trifluoromethyl purine, 6-phenyl purine, 6-mercapto purine, 6-methylthiopurine, 2, 6-diamino purine, 2-amino-6-chloro purine, 2-amino-6-iodo purine, 2-amino-6-mercapto purine, 2-amino-6-methylthiopurine, 2-amino-6-hydroxyaminopurine, 2-amino-6-methoxy purine, 2-amino-6-methyl purine, 2-amino-6-cyclopropylaminomethyl purine, 2-amino-6-phenyl purine, 2-amino-8-bromo purine, 6-cyano purine, 2-chloro adenine, 2-amino-propyl adenine, 2-fluoro-3-amino-3-deaza, 2-methyl purine, 2-amino-propyl adenine, 3-deaza, xanthine, hypoxanthine, 6-methyladenine, 6-thioguanine, 7-methyladenine, 7-methylguanine, 7-deazaguanine, 7-deaza-8-azaguanine, 8-nitroadenine, 8-nitroguanine, 8-fluoroadenine, 8-chloroadenine, 8-bromoadenine, 8-aminoadenine, 8-hydroxyadenine, 8-fluoroguanine, 8-chloroguanine, 8-bromoguanine, 8-aminoguanine or 8-hydroxyguanine.
8. The method of claim 1-7, wherein the pyrimidine base comprises cytosine, uracil, 5-fluorocytosine, 5-fluorouracil, 5-chlorocytosine, 5-chlorouracil, 5-bromocytosine, 5-bromouracil, 5-iodocytosine, 5-iodouracil, 5-methylcytosine, 5-methyluracil, 5-ethylcytosine, 5-ethyluracil, 5-fluoromethylcytosine, 5-fluoromethyluracil, 5-trifluoromethyl cytosine, 5-trifluoromethyl uracil, 5-vinyluracil, 5-bromovinyluracil, 5-chlorovinyluracil, 5-ethynylcytosine, 5-ethynyluracil, 5-hydroxycytosine, 5-methoxyuracil, 2-thiocytosine, 2-thiouracil, 3-methylcytosine, 4-thiouracil, thymine, 4-thiouracil, 5-dihydro-cytosine, 5-acetylcytosine, 4-acetyl-cytosine, or 4-acetyl-cytosine.
9. The method for synthesizing adenosine and/or uridine by three enzyme cascade catalysis according to any of claims 1-8, wherein the molar ratio of ribose compound and nucleobase is 1 (1.1-1.3);
Preferably, the ribose compound, ribose kinase, pentose phosphate mutase and nucleoside phosphorylase are added in a mass ratio of 1:0.1:0.1:0.1.
10. The method for synthesizing adenosine and/or uridine by three enzyme cascade catalysis according to any of claims 1-8, wherein the reaction temperature is 30-40 ℃ for 0.5-2 h.
CN202310516643.0A 2023-05-09 2023-05-09 A method for synthesizing adenosine and/or uridine by three-enzyme cascade catalysis Pending CN118931997A (en)

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