CN101503724B - New method for preparing nucleotide by using biological phosphorylation technology - Google Patents
New method for preparing nucleotide by using biological phosphorylation technology Download PDFInfo
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- CN101503724B CN101503724B CN2009100259814A CN200910025981A CN101503724B CN 101503724 B CN101503724 B CN 101503724B CN 2009100259814 A CN2009100259814 A CN 2009100259814A CN 200910025981 A CN200910025981 A CN 200910025981A CN 101503724 B CN101503724 B CN 101503724B
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Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention discloses a novel method for preparing nucleotide by using a biological phosphorylation technology, which uses purine or pyrimidine nucleotide precursor substances and phosphate ions as substrates, uses glucose as a ribose group donor and an energy donor, uses permeable microbial cells, and realizes efficient preparation of the nucleotide by regulating and controlling a cofactor regeneration system, a substance energy coupling regeneration system and metabolic pathway key enzyme activity through chemical small molecule effect substances. According to the invention, the metabolic flow is regulated and controlled by adopting the micromolecular chemical effect substances, so that the regeneration and utilization conditions of energy and coenzyme are improved, the metabolic flow is transferred to a target path, the metabolic byproducts are reduced, and the product yield is improved; the method for changing the permeability of the cell membrane accelerates the permeation of reaction components to microbial cells, shortens the reaction time and improves the utilization rate of the substrate.
Description
Technical field
The invention belongs to the biocatalysis technology field, be specifically related to a kind of novel method of utilizing biological phosphorylation technology to prepare Nucleotide from the nucleotide precursor material.
Background technology
Nucleotide (NMP) is a kind of important phosphoryl compound, and it participates in body metabolism, promotes internal organs to improve and recovery, improves hemopoietic function of bone marrow, and can be used as the ancillary drug of treatment cancer virus.It can make the excessive hyperplasia of white corpuscle, for symptoms such as various radioactive substances or drug-induced leukopenia, non-specific thrombopenia good curative effect is arranged, and also is used for the treatment of acute and chronic hepatitis.Four kinds of 5 '-Nucleotide not only can be as drug use, but also is the raw materials for production of many antiviral antitumor drugs, as being used for synthetic antiviral Famciclovir, lamivudine, Adefovir, Entecavir, emtricitabine etc.These new synthetic medicines are expected to become the sulfa drugs that continues, novel antiviral of after the microbiotic another type, antitumor drug.In food service industry, by initial food fragrance adding agent, expand to functional food additives with raising organism immunologic function, can be added in the food such as bread, biscuit.Especially the result of use of Nucleotide in infant or baby food is very obvious, can effectively strengthen the ability that the infant resists bacillary dysentery, reduces the generation of diarrhoea.
At present, the compound method of Nucleotide mainly contains following three kinds: chemical synthesis, RNA enzymolysis process and biological catalysis.
Chemical method is produced Nucleotide, and normally the reactive derivative with phosphoric acid or tetra-sodium carries out phosphating reaction to nucleosides.The reactive derivative of the general tetra-sodium of using always has trihalophosporus oxide, pyrophosphoryl chloride, two-right-oil of mirbane tetra-sodium etc.In addition, obtain 5 '-Nucleotide, must be before phosphorylation reaction with 2 ', 3 ' hydroxyl of ribose on the proper protection base protection nucleosides.General ethanoyl, Bian Ji, isopropylidene or the benzylidene functionalized chemical radical protection of adopting.The method that extensively adopts nucleosides and phosphoryl chloride in trialkylphosphate, to react in the industry.The step of chemical method synthesizing ribonucleotide experience is many, route is long, and stereoselectivity is poor, and related reagent is expensive, and certain toxicity is arranged, and production cost is higher.
It is to be raw material with the yeast rna that the RNA enzymolysis process is produced Nucleotide, the 5 '-phosphodiesterase degradation of rna that utilizes Penicillium citrinum and streptomyces aureus to produce, production Nucleotide.Its technical process be with yeast after diluted alkaline or dense salt extracting; In the pH2.5 settle, obtain RNA, again through 5 '-phosphodiesterase enzymolysis; Obtain the mixed solution of four kinds of Nucleotide, this mixture can be obtained the pure article of four kinds of Nucleotide through the ion exchange resin separation and purification.Wherein phosphodiesterase mainly is the nuclease P 1 of producing through the Penicillium citrinum solid fermentation.It is an extracellular enzyme, can directly be used for hydrolysed nucleic acid, and its percent hydrolysis is generally between 70%-85%), this production technique is simple, and raw material sources are abundant, and with low cost, so for a long time, China all is the industrial production of carrying out Nucleotide with this method.But four kinds of Nucleotide purposes and consumption are different, and it is bigger that the market requirement differs, but this method almost is four kinds of Nucleotide of generation of equivalent, have brought certain difficulty for the sale of product.
The biological catalysis synthesizing ribonucleotide utilizes mikrobe as the enzyme source exactly, and the precursor substance of catalysis Nucleotide is converted into Nucleotide.Japan consonance fermentation company utilizes uridylic acid precursor substance vitamin B13 to transform UMP through bacterial classification is improved, and the semi-invariant of UMP is up to 28g/L (Fujio T at present; Maruyama A..Biosci Biotech Biochem, 1997,61 (6): 956-959) domestic have only Shanghai Normal University and Shandong University that the synthetic report of such compound is arranged; (the Qian Xiuping of Shanghai Normal University; Chinese Journal of Pharmaceuticals, 2006,37 (12)) adopted streptomycin resistance, kalamycin resistance and product structure analogue resistance etc. as the screening means; Brevibacterium ammoniagenes is carried out ionic fluid and ultraviolet mutagenesis; Select the stronger bacterial strain of a strain UMP generative capacity, the conversion capability of UMP has reached 2g/L, but differs greatly with international most advanced level.Shandong University (Appl Microbiol Biotechnol 2007,76 (2) 321-328) makes the UMP cumulative concentration reach 10g/L through the optimization to the Brevibacterium ammoniagenes conversion condition.
But have only uridylic acid that relevant report is arranged at present, and other several kinds important ucleotides materials, such as adenylic acid(AMP), cytidylic acid etc. all do not utilize biocatalysis technology synthetic report.The present inventor thinks and is difficult at present realize that Nucleotide biocatalysis synthetic reason mainly is: 1. cellular metabolism approach is complicated, and under normal physiological conditions, cell distributes metabolism stream by the inherent mode, can not excess produce the ucleotides phosphoryl compound; 2. in the process of utilizing precursor substance synthesizing ribonucleotides such as VITAMIN B4, cytosine(Cyt), uridylic, vitamin B13, the phosphorus acylation reaction that relates to is a lot, but can't set up energy regeneration coupling system efficiently; 3. can't set up regenerating coenzyme system efficiently in the building-up process of important intermediate phosphoribosyl pyrophosphate (PRPP), cause the biosynthesis block of PRPP; 4. reaction process relates to the distribution of the pathways metabolism metabolism stream that EMP (glycolytic pathway) and two of HMP (phosphopentose pathway) vie each other, and can't realize the optimum allocation of metabolism stream.The existence of above problem makes that the technology of biocatalysis synthesizing ribonucleotide is not used on a large scale.
Summary of the invention
Problem to above biosynthesizing Nucleotide; The invention provides a kind of novel method of utilizing biological phosphorylation technology to prepare Nucleotide; To have from the microbial culture medium of nucleotide precursor material and sugared product nucleus thuja acid ability or the handled thing of this nutrient solution is the enzyme source; In the aqueous medium that contains this enzyme source, nucleotide precursor material and saccharide compound, carry out enzyme reaction, produce and accumulation Nucleotide.
Key of the present invention is:
1, the present invention has adopted the whole-cell catalytic technology; The own enzyme that has directly utilized cell is the precursor substance synthesizing ribonucleotide of catalysis Nucleotide; Precursor substance comprises vitamin B13, VITAMIN B4, uridylic, cytosine(Cyt), can produce Nucleotide arbitrarily according to demand, and reaction system is simple; Nontoxic, low production cost.
2, utilized small-molecule substances such as azophenlyene Methylsulfate, flavin derivatives to change intracellular coenzyme (NAD/NADH, NADP/NADPH) regeneration balance; Impelling metabolism to flow to destination path shifts; Substrate utilization ratio gets a promotion; By-products content reduces, and this makes the back extraction process easily simple relatively.
3, through adding organic acid and metals ions such as mg ion, potassium ion and ammonium ion such as chemical small molecules effector substance such as oxysuccinic acid, pyruvic acid, fumaric acid; The regeneration rate of control ATP homenergic; ATP regeneration and phosphorus acylation reaction coupling are got up, and the yield of Nucleotide significantly rises.
4, the present invention carries out pre-treatment through the permeability of pair cell, has improved the permeability of cell walls, and the accelerated reaction component makes the reaction times significantly shorten to the infiltration of microorganism cells.
The concrete technical scheme that the present invention adopts is following:
A kind of novel method of utilizing biological phosphorylation technology to prepare Nucleotide; Nucleotide precursor material and phosphate anion with purine or miazines are substrate; With glucose as ribose groups donor and energy donor; The microorganism cells of having property of utilization is lived through chemical small molecules effector substance regulation and control cofactor regeneration system, matter energy link-coupled regeneration system and pathways metabolism key enzyme enzyme, realizes efficient production Nucleotide.
Wherein, described Nucleotide (NMP) is meant amp (AMP), uridylic acid(UMP) (UMP), cytidine monophosphate (CMP), and its structural formula is following:
Wherein, the nucleotide precursor material of described purine or miazines is vitamin B13, VITAMIN B4, uridylic or cytosine(Cyt).
Wherein, being reflected in the aqueous solution of Nucleotide of preparation carried out, and reaction conditions is pH5～10, preferred pH6～8, and temperature is 20～50 ℃, preferred 25～40 ℃, the reaction times is 10～40 hours, preferred 2～12 hours.
Wherein, the add-on of nucleotide precursor material is 1～100mM, PO
4 3-Add-on be 0.01～2M, preferred 0.02～0.5M, the add-on of glucose is 0.1～1M, the add-on of microorganism cells is for pressing wet thallus 100～800g/L.
Wherein, described chemical small molecules effector substance is: the chemical small-molecule substance relevant with cofactor regeneration, with the chemical small-molecule substance relevant with the energy coupling regeneration system, with regulate the pathways metabolism key enzyme enzyme relevant chemical small-molecule substance of living; Wherein, The described chemical small-molecule substance relevant with cofactor regeneration is one or more in azophenlyene Methylsulfate and the flavin derivatives; The described chemical small-molecule substance relevant with the energy coupling regeneration system is one or more in mg ion, potassium ion, the ammonium ion, and described is in oxysuccinic acid, pyruvic acid, the fumaric acid one or more with regulating the pathways metabolism key enzyme enzyme relevant chemical small-molecule substance of living.Wherein, the chemical small-molecule substance working concentration relevant with cofactor regeneration is 0.01～20mM, preferred 0.05～5mM; The chemical small-molecule substance working concentration relevant with the energy coupling regeneration system is 1～200mM, and preferred 1～50mM is 0.001mM～1mM with regulating pathways metabolism key enzyme enzyme relevant chemical small-molecule substance working concentration alive, preferred 0.005～0.5mM.
Wherein, described microorganism cells comprises the bacterium of aerobacter, Escherichia, Serratia, micrococcus sp for utilizing the mikrobe of nucleotide precursor material synthesizing ribonucleotide; The yeast that yeast belong, mycocandida, Pichia, torulopsis, Debaryomyces, zygosaccharomyces genus, genus kluyveromyces, Hansenula and Brettanomyces belong to.
Wherein, described microorganism cells preferably has the bacterial strain of strong ATP regeneration activity and PRPP composite reactive, comprises Brevibacterium ammoniagenes, subtilis, yeast saccharomyces cerevisiae and Torulopsis candida.
Wherein, The microorganism cells of described having property is to adopt following method to carry out the thalline broken wall treatment and get microorganism cells; Thereby the permeability that changes cytolemma specifically comprises surfactant method, organic solvent method, freeze-thaw method, ultrasonication method, aeration drying, freeze-drying or bacteriolyze method.Preferred surfactant method or organic solvent method; Described tensio-active agent is non-ionics (like polyethylene oxide amines, triton x-100), cationic surfactant (like the hexadecyl trimethylamine bromide) or AS (Sarkosyl L salt); Working concentration is 0.1～50g/L, and the concentration of preferred 1～20g/L is used, when promptly surfactant method is handled microorganism cells; Tensio-active agent is directly added reaction solution; For TV is the reaction solution of 1L, adds 0.1～50g, preferably adds 1～20g; Described organic solvent is YLENE, toluene, Fatty Alcohol(C12-C14 and C12-C18), acetone or ETHYLE ACETATE; Working concentration is 0.1～50mL/L, and the concentration of preferred 1～20mL/L is used, when promptly organic solvent method is handled microorganism cells; Organic solvent is directly added reaction solution; For TV is the reaction solution of 1L, adds 0.1～50mL, preferably adds 1～20mL.Other handles the method for cell permeability, like freeze-thaw method, ultrasonication method, aeration drying etc., after employing is handled strain cell earlier, the bacterial strain of handling well is added the mode of reaction solution again.
Wherein, the form of utilizing of above-mentioned production bacterial strain is to produce the dry thing of strain cell, the centrifugal cell that obtains of culture of isolated, the lyophilized products of cell, commercially available yeast powder, air-dry bacterial strain or waste yeast mud by fermentation.
Wherein, phosphate ion can be enumerated Tripyrophosphoric acid such as ortho-phosphoric acid, tetra-sodium, tripolyphosphate, potassium primary phosphate, SODIUM PHOSPHATE, MONOBASIC, inorganic phosphates such as Sodium phosphate, dibasic.
Beneficial effect of the present invention is:
Reaction system involved in the present invention is simple, generally only needs pair cell to carry out certain pre-treatment, adds substrate then, reaches a certain amount of effector substance, and reaction is carried out smoothly, and this makes back extraction process with respect to easily simple; The present invention not only reaction conditions is gentle, pollution-free, has avoided chemical method synthetic shortcoming, can also be according to the Nucleotide of demand production any kind, and four kinds of Nucleotide having avoided the enzymolysis working system to bring are sold unbalanced shortcoming; The added series of effects material of the present invention has improved the regeneration of energy and coenzyme and has utilized situation, makes metabolism flow to destination path migration has taken place, so metabolic by-prods significantly reduces, efficiency of pcr product also improves a lot; The method of the change cell leakage that the present invention adopts has been quickened the infiltration of reactive component to microorganism cells, makes the reaction times significantly shorten, and the utilization ratio of substrate and other raw materials (like glucose etc.) also increases.
Embodiment:
According to following embodiment, can understand the present invention better.Yet, those skilled in the art will readily understand that the described concrete material proportion of embodiment, processing condition and result thereof only are used to explain the present invention, and the present invention that should also can not limit in claims to be described in detail.
Embodiment 1: utilize VITAMIN B4 to synthesize AMP.
Capacity be in the reactive tank of 15L modulation by VITAMIN B4 300mMol, glucose 1.5Mol, sal epsom 12mMol, micrococci cell 2800 grams, air-dry processing, supersound process is 20 minutes again; Ammonium sulfate 15mMol, Sodium phosphate, dibasic 0.3Mol, azophenlyene Methylsulfate 7.5mMol; The reaction solution 10L that pyruvic acid 0.2mMol and water are formed, using sodium hydroxide to transfer pH was 7, in 30 ℃ of stirring at low speed reactions 4 hours; After reaction finishes; Use the perchloric acid precipitation, AMP is carried out quantitative analysis, contain AMP287mM (112g) in the conversion fluid with HPLC.
Embodiment 2: utilize uridylic to synthesize UMP.
Capacity be in the reactive tank of 15L modulation by uridylic 740mMol, glucose 6Mol, sal epsom 18mMol, Brevibacterium ammoniagenes cell 2700 grams, ammonium sulfate 18mMol, vitamin G 7.6mMol; SODIUM PHOSPHATE, MONOBASIC 0.26Mol, fumaric acid 0.8mMol, 10 milliliters of reaction solution 10L that form with water of toluene; Using sodium hydroxide to transfer pH is 7, in 30 ℃ of stirring at low speed reactions 4 hours, after reaction finishes; Use the perchloric acid precipitation, UMP is carried out quantitative analysis, contain UMP711mM (323g) in the conversion fluid with HPLC.
Embodiment 3: utilize vitamin B13 to synthesize UMP.
Capacity be in the reactive tank of 15L modulation by vitamin B13 900mMol, glucose 8.6Mol, bacillus subtilis mycetocyte 3000 grams; Ammonium chloride 20mMol, magnesium chloride 15mMol, SODIUM PHOSPHATE, MONOBASIC 0.2Mol; Azophenlyene Methylsulfate 0.8mMol, oxysuccinic acid 3mMol, the reaction solution 10L that Sarkosyl L salt 15g and water are formed; Transfer pH to 6.5 with sodium hydroxide, stirring at low speed reaction 8h under 37 ℃ of conditions is after reaction finishes; Use the perchloric acid precipitation, UMP is carried out quantitative analysis, contain UMP787mM (289g) in the conversion fluid with HPLC.
Embodiment 4: utilize cytosine(Cyt) to synthesize CMP.
Capacity be in the reactive tank of 300L modulation by cytosine(Cyt) 22.5Mol, glucose 240Mol, 20 kilograms of paddy ammonia rod bacilli-cells; Repone K 350mMol, magnesium chloride 400mMol, Sodium phosphate, dibasic 6.1Mol; Vitamin G 16mMol, fumaric acid 50mMol, the reaction solution 300L that polyethylene oxide amines 350g and water are formed; Transfer pH to 6.5 with sodium hydroxide, stirring at low speed reaction 10h under 37 ℃ of conditions is after reaction finishes; Use the perchloric acid precipitation, UMP is carried out quantitative analysis, contain CMP21Mol (6783g) in the conversion fluid with HPLC.
Claims (8)
1. method of utilizing biological phosphorylation technology to prepare Nucleotide; It is characterized in that nucleotide precursor material and phosphate anion with purine or miazines are substrate; With glucose as ribose groups donor and energy donor; The microorganism cells of having property of utilization is lived through chemical small molecules effector substance regulation and control cofactor regeneration system, matter energy link-coupled regeneration system and pathways metabolism key enzyme enzyme, realizes preparation Nucleotide;
Wherein, described Nucleotide NMP is meant amp AMP, uridylic acid(UMP) UMP, cytidine monophosphate CMP, and its structural formula is following:
Wherein, the nucleotide precursor material of described purine or miazines is vitamin B13, VITAMIN B4, uridylic or cytosine(Cyt);
Wherein, described chemical small molecules effector substance is: the chemical small-molecule substance relevant with cofactor regeneration, with the chemical small-molecule substance relevant with the energy coupling regeneration system, with regulate the pathways metabolism key enzyme enzyme relevant chemical small-molecule substance of living; Wherein, The described chemical small-molecule substance relevant with cofactor regeneration is the azophenlyene Methylsulfate; The described chemical small-molecule substance relevant with the energy coupling regeneration system is one or more in mg ion, potassium ion, the ammonium ion, and described is in oxysuccinic acid, pyruvic acid, the fumaric acid one or more with regulating the pathways metabolism key enzyme enzyme relevant chemical small-molecule substance of living; The chemical small-molecule substance working concentration relevant with cofactor regeneration is 0.01～20mM; The chemical small-molecule substance working concentration relevant with the energy coupling regeneration system is 1～200mM; With regulating pathways metabolism key enzyme enzyme relevant chemical small-molecule substance working concentration alive is 0.001～1mM.
2. the method for utilizing biological phosphorylation technology to prepare Nucleotide according to claim 1 is characterized in that preparing being reflected in the aqueous solution of Nucleotide and carries out, pH5～10, and temperature of reaction is 20～50 ℃, the reaction times is 10～40 hours.
3. the method for utilizing biological phosphorylation technology to prepare Nucleotide according to claim 1, the add-on that it is characterized in that the nucleotide precursor material is 1～100mM, PO
4 3-Add-on be 0.01～2M, the add-on of glucose is 0.1～1M, the add-on of microorganism cells is for pressing wet thallus 100～800g/L.
4. the method for utilizing biological phosphorylation technology to prepare Nucleotide according to claim 1 is characterized in that the chemical small-molecule substance working concentration relevant with cofactor regeneration is 0.05～5mM; The chemical small-molecule substance working concentration relevant with the energy coupling regeneration system is 1～50mM; With regulating pathways metabolism key enzyme enzyme relevant chemical small-molecule substance working concentration alive is 0.005～0.5mM.
5. the method for utilizing biological phosphorylation technology to prepare Nucleotide according to claim 1; It is characterized in that described microorganism cells for utilizing the mikrobe of nucleotide precursor material synthesizing ribonucleotide, comprises the bacterium of aerobacter, Escherichia, Serratia, micrococcus sp; The yeast that yeast belong, mycocandida, Pichia, torulopsis, Debaryomyces, zygosaccharomyces genus, genus kluyveromyces, Hansenula and Brettanomyces belong to.
6. the method for utilizing biological phosphorylation technology to prepare Nucleotide according to claim 5 is characterized in that described microorganism cells is the bacterial strain with strong ATP regeneration activity and PRPP composite reactive, comprises yeast saccharomyces cerevisiae and Torulopsis candida.
7. the method for utilizing biological phosphorylation technology to prepare Nucleotide according to claim 1; The microorganism cells that it is characterized in that described having property is to adopt following method to handle and get microorganism cells: surfactant method, organic solvent method, freeze-thaw method, ultrasonication method, aeration drying, freeze-drying or bacteriolyze method.
8. the method for utilizing biological phosphorylation technology to prepare Nucleotide according to claim 7, the microorganism cells that it is characterized in that described having property are to adopt surfactant method or organic solvent method to handle and get microorganism cells; Described tensio-active agent is non-ionics, cationic surfactant or AS, and working concentration is 0.1～50g/L; Described organic solvent is YLENE, toluene, Fatty Alcohol(C12-C14 and C12-C18), acetone or ETHYLE ACETATE, and working concentration is 0.1～50mL/L.
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| CN1207135A (en) * | 1996-09-17 | 1999-02-03 | 协和发酵工业株式会社 | Processes for producing sugar nucleotides and complex carbohydrates |
| CN1267735A (en) * | 1999-02-08 | 2000-09-27 | 协和发酵工业株式会社 | Process for producing purine nucleotide |
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| CN1207135A (en) * | 1996-09-17 | 1999-02-03 | 协和发酵工业株式会社 | Processes for producing sugar nucleotides and complex carbohydrates |
| CN1267735A (en) * | 1999-02-08 | 2000-09-27 | 协和发酵工业株式会社 | Process for producing purine nucleotide |
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| 张志军等.核苷酸生产技术现状及展望.《现代化工》.2004,第24卷(第11期),19－23. * |
| 钱秀萍等.尿苷酸微生物转化菌株的选育.《中国医药工业杂志》.2006,第37卷(第12期),814-816. * |
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