CN101555509B - Method for catalyzing and synthesizing uridine phosphinylidyne compound in an oriented way - Google Patents
Method for catalyzing and synthesizing uridine phosphinylidyne compound in an oriented way Download PDFInfo
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Abstract
The invention discloses a method for catalyzing and synthesizing uridine phosphinylidyne compound in an oriented way; the method takes uridine or saratin and phosphate radical ion as substrate as wellas glucose as ribose radical donor and energy donor, utilizes microbial cells with permeability as enzyme source, and promotes the reaction system to biologically catalyzing and synthesizing the urid ine phosphinylidyne compound in an oriented way by means of changing reaction temperature and dissolved oxygen periodically. According to the demand, the method can be used for producing any of three matters, and the reaction system is simple, nontoxic, production cost is low and control method is easy, so that the production control in industrialization is facilitated. When the uridine is used assubstrate, the yields of UMP, UTP and UDPG are respectively 77%, 67% and 61%; when the saratin is used as substrate, the yields of UMP, UTP and UDPG are respectively 70%, 61% and 53%.
Description
Technical field
The invention belongs to the biocatalysis technology field, be specifically related to utilize the control technique method of the synthetic uridine phosphoryl compound of directional catalyzing respectively.
Background technology
Uridylic acid (UMP) is as the verivate of uridylic; Be important biochemical reagents; Also be the very big medicine intermediate of consumption, milk formula additive and fodder additives simultaneously; It also is synthetic precursor such as UTP (UTP), Uridine pyrophosphate-glucose (UDPG), cytidine triphosphate(CTP) (CTP), cytidine diphosphate uridines such as (CDPC) and cytidine phosphinylidyne compounds; A large amount of pharmacology and clinical medicine research show that this compounds has good clinical effectiveness at aspects such as the treatment of cardiovascular and cerebrovascular, hepatitis, mellitus, antiviral (SARS) and nerve degeneration difficult diseases such as (parkinsonism, Alzheimer's diseases) and assisting therapy, are the basic material of synthesis of oligose and other physiologically active substances simultaneously.
UTP (UTP) be termolecular phosphoric acid be combined in uridine ribose 5 '-Nucleotide on the OH base, distribute extensively, be the direct precursor of RNA synthetic.With sugar metabolism substantial connection is arranged also, can generate UDP-glucose and tetra-sodium through enzyme catalysis by UTP and 1-phosphoglucose.UTP can prevent and treat pneumonia, diseases such as treatment sinusitis, ciliary dyskinesia, otitis media and bronchitis.In addition; (ATP) is the same with atp; Can also be as the P2 acceptor on extracellular informational molecule (first messenger) activating cells surface, and regulate multiple physiological response through second messenger system in the cell, especially significant to the physiology and the pathologic, physiologic activity of cardiovascular systems.
Uridine diphosphoglucose (UDPG) is a most important glycosyl donor in the body, can promote the liver detoxification effect, and treatment toxic and infectious hepatitis have important drug development and be worth.UDPG be for the first time with the coenzyme form in nineteen fifty, in the conversion of glucose process, find at the research semi-lactosi by Leloir and his colleague.UDPG not only can be used as the glucone donor; Multiple oligosaccharides and polysaccharide is synthetic in the participation organism; Biosynthesizing like trehalose, sucrose, starch, VITAMINs; But also be synthetic other nucleoside diphosphate monose, like the precursor of Uridinediphosphogalactose, uridine diphosphoglucose acid, uridine diphosphate xylose etc.
The uridine phosphoryl compound promptly is the general name to above-mentioned UMP, UTP, UDPG.
The production of UMP has that chemical method is synthetic, enzymolysis process synthetic, fermentation method is synthetic and biological catalysis is synthetic.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; MaruyamaA..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.
UTP mainly utilizes microorganism biological catalysis synthetic at present.(Takeda I., Watarabe S.Stabilizing Nucleotides Derivatives.Japan.7,237,036, Sep 18.1972.) such as Takeda I is with the synthetic UTP of yeast bio catalysis UMP.The synthetic of UDPG mainly is that chemical method is synthetic synthetic with enzyme process at present.
In yeast cell, uridine can synthesize UMP under the catalysis of uridine kinase; Vitamin B13 can and phosphoribosyl pyrophosphate (PRPP) under the effect of OPRT, generate orotidylic acid (OMP), OMP generates UMP through the catalysis of orotidylic decarboxylase then.UMP and ATP shift a phosphate through the uridylic acid kinases then, are converted into UDP and ADP; UDP consumes ATP then through nucleoside diphosphokinase, continues phosphoric acid and turns to UTP.UTP and 1-phosphoglucose can generate UDPG and tetra-sodium through enzyme catalysis.There is the complete enzyme of difference catalysis uridine and vitamin B13 synthetic UMP, UTP and UDPG to be thus in the yeast cell.But the at present domestic relevant report that does not also have biocatalysis uridine or synthetic UTP of vitamin B13 and UDPG is not seen yet and is utilized the report of same reaction system through the synthetic above-mentioned three kinds of uridine phosphoryl compounds of fine setting reaction conditions directional catalyzing.
Summary of the invention
Technical problem to be solved by this invention provides the method for the synthetic above-mentioned three kinds of uridine phosphoryl compounds of a kind of microorganism cells directional catalyzing.
In order to solve the problems of the technologies described above, thinking of the present invention is:
With uridine or vitamin B13 is substrate, and microorganism cells enzyme system is the enzyme source, synthetic uridine phosphoryl compound under certain reaction system.Generally speaking; Reaction through after a while may exist UMP, UTP and three kinds of reaction product of UDPG simultaneously in the system, but can make reaction leading to a certain product direction through control measures; Thereby obtain the higher relatively a certain product of concentration, to reduce segregational load, to reduce cost.This is a key point of the present invention.Through long-term experiment, we find that above-mentioned control measures mainly comprise interim adjustment temperature of reaction and dissolved oxygen amount.
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 synthetic three kinds of uridine phosphoryl compounds of catalysis uridine or vitamin B13, can produce any a kind of of three kinds of materials according to demand, and reaction system is simple; Nontoxic, low production cost.
2. the present invention as means, influences the activity of relevant enzyme in the uridine compound phosphorylated process through interim control reaction temperature and oxygen-supply quantity.Under the Different control condition, the enzymic activity that helps synthetic a kind of material is strengthened, the enzymic activity that helps generating other materials simultaneously then weakens, and the active selectivity of this kind of enzyme has determined the final product that reacts different.Control device is simple, has greatly made things convenient for the production control in the industriallization.
The concrete technical scheme that the present invention adopts is following:
The method of the synthetic uridine phosphoryl compound of a kind of directional catalyzing; With uridine or vitamin B13 and phosphate anion is substrate; With glucose as ribose groups donor and energy donor; The microorganism cells of having property of utilization is the enzyme source, is means through stage change temperature of reaction and dissolved oxygen amount, impels the synthetic uridine phosphoryl compound of the directed biocatalysis of reaction system.
Wherein, described uridine phosphoryl compound is uridylic acid (UMP), UTP (UTP) or uridine diphosphoglucose (UDPG), and its structural formula is following:
Wherein, uridine, i.e. UR, structural formula is following:
Uridine
Wherein, vitamin B13, i.e. OA, structural formula is following:
Vitamin B13
If with the uridine is substrate, one of following three kinds of reactions, orientable synthetic uridine phosphoryl compound are carried out in (preferred pH6～8) in the aqueous solution of pH5～10:
(a) under 25～35 ℃, dissolved oxygen amount 60～120% (preferred 70～100%), reaction was to 8～10 hours, and primary product is UMP;
(b) under 25～35 ℃, dissolved oxygen amount 60～120% (preferred 70～100%), reaction was to 8～10 hours; Adjust temperature to 35～45 ℃ again, the adjustment dissolved oxygen amount is 15～50% (preferred 20～30%), and reaction was to 4～6 hours, and primary product is UTP;
(c) under 25～35 ℃, dissolved oxygen amount 60～120% (preferred 70～100%), reaction was to 8～10 hours; Adjust temperature to 35～45 ℃ again, the adjustment dissolved oxygen amount is 15～50% (preferred 20～30%), and reaction was to 4～6 hours; Adjust temperature to 20～30 ℃ again, the adjustment dissolved oxygen amount is 0～30% (preferred 5～20%), and reaction was to 4～8 hours, and primary product is UDPG.
If with the vitamin B13 is substrate, in the aqueous solution of pH5～10 (preferred pH6～8), carry out one of following three kinds of reactions, orientable synthetic uridine phosphoryl compound:
(a) under 25～35 ℃, dissolved oxygen amount 60～120% (preferred 70～100%), reaction was to 16～20 hours, and primary product is UMP;
(b) under 25～35 ℃, dissolved oxygen amount 60～120% (preferred 70～100%), reaction was to 16～20 hours; Adjust temperature to 35～45 ℃ again, the adjustment dissolved oxygen amount is 15～50% (preferred 20～30%), and reaction was to 8～10 hours, and primary product is UTP;
(c) under 25～35 ℃, dissolved oxygen amount 60～120% (preferred 70～100%), reaction was to 16～20 hours; Adjust temperature to 35～45 ℃ again, the adjustment dissolved oxygen amount is 15～50% (preferred 20～30%), and reaction was to 8～10 hours; Adjust temperature to 20～30 ℃ again, the adjustment dissolved oxygen amount is 0～30% (preferred 5～20%), and reaction was to 6～8 hours, and primary product is UDPG..
In the above-mentioned reaction, the add-on of substrate uridine or vitamin B13 is 5～200mM, preferred 50～100mM; Phosphatic add-on is 0.1～2M, preferred 0.2～0.8M; The add-on of glucose is 0.1～1M, preferred 0.5～1M; The add-on of microorganism cells is for pressing wet thallus 100～800g/L.
Described reaction system also adds any one or a few the compsn in ammonium ion, mg ion and the potassium ion; Mg
2+Optional from inorganic salt such as sal epsom, magnesium nitrate, magnesium chlorides, its initial action concentration is 1～200mM, preferred 5～50mM; K
+Initial action concentration is 1～200mM, preferred 10～40mM; NH
4 +Initial action concentration is 1～200mM, preferred 2～40mM.
Described microorganism cells is the mikrobe that can utilize uridine or vitamin B13 to synthesize uridylic acid and continue phosphorylated, 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.Described microorganism cells preferably has the bacterial strain of strong ATP regeneration activity, 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.
Wherein, dissolved oxygen amount is regulated through regulating methods such as stir speed (S.S.), air flow, regulates according to the detection data of dissolved oxygen survey meter.With the saturated air dissolved oxygen amount is 100%.
Beneficial effect of the present invention is:
It is substrate that the present inventor has studied with uridine or vitamin B13 and phosphate anion; With glucose as ribose groups donor and energy donor; The microorganism cells of having property of utilization is the enzyme source, is the directionally synthetic three kinds of specific uridine phosphoryl compounds of biocatalysis of means through stage adjustment temperature of reaction and dissolved oxygen amount.Can produce any a kind of of three kinds of materials according to demand, reaction system is simple, and is nontoxic, low production cost.And control device is simple, has greatly made things convenient for the production control in the industriallization.
Can directedly produce UMP through interim conditioned reaction condition, UTP and UDPG, the yield of three kinds of products can reach 77%, 67%, 61% respectively when UR did substrate; The yield of three kinds of products can reach 70%, 61% and 53% respectively when OA did substrate.
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 the directed UMP of production of uridine.
Modulation is by uridine 600mMol, glucose 5Mol, sal epsom 50mMol, Brevibacterium ammoniagenes 2400 grams, ammonium chloride 20mMol, Repone K 300mMol, SODIUM PHOSPHATE, MONOBASIC 2.0Mol in the reactive tank of capacity 15L; 50 milliliters of reaction solution 10L that form with water of toluene, using sodium hydroxide to transfer pH is 7.0, dissolved oxygen is controlled at 70%; Temperature is 30 ℃, reacts and finishes reaction after 8 hours, uses the perchloric acid precipitation; With HPLC product is carried out quantitative analysis, primary product is UMP in the conversion fluid, and its content is 457mMol (208g); Yield is 76.1%, and this moment, UTP content was 68.3mMol (33.1g), and UDPG content is 24.6mMol (15g).
Embodiment 2: utilize the directed UTP of production of uridine.
Modulation is by uridine 600mMol, glucose 5Mol, sal epsom 50mMol, Brevibacterium ammoniagenes 2400 grams, ammonium chloride 20mMol, Repone K 300mMol, SODIUM PHOSPHATE, MONOBASIC 2.0Mol in the reactive tank of capacity 15L; 50 milliliters of reaction solution 10L that form with water of toluene, using sodium hydroxide to transfer pH is 7.0, and dissolved oxygen is controlled at 70%, and temperature is 30 ℃; React after 8 hours, dissolved oxygen is adjusted into 25%, and temperature is adjusted into 37 ℃; Continue reaction 6 hours, finish reaction, use the perchloric acid precipitation; With HPLC product is carried out quantitative analysis, primary product is UTP in the conversion fluid, and its content is 398mMol (193g); Yield is 66.3%, and this moment, UMP content was 57.3mMol (18.6g), and UDPG content is 80.2mMol (48.9g).
Embodiment 3: utilize the directed UDPG of production of uridine.
Modulation is by uridine 600mMol, glucose 5Mol, sal epsom 50mMol, Brevibacterium ammoniagenes 2400 grams, ammonium chloride 20mMol, Repone K 300mMol, SODIUM PHOSPHATE, MONOBASIC 2.0Mol in the reactive tank of capacity 15L; 50 milliliters of reaction solution 10L that form with water of toluene, using sodium hydroxide to transfer pH is 7.0, and dissolved oxygen is controlled at 70%, and temperature is 30 ℃; React after 8 hours, dissolved oxygen is adjusted into 25%, and temperature is adjusted into 37 ℃, continues reaction after 6 hours; Dissolved oxygen is adjusted into 5%, and temperature is adjusted into 25 ℃, continues reaction 4 hours, finishes reaction; Use the perchloric acid precipitation, with HPLC product is carried out quantitative analysis, primary product is UDPG in the conversion fluid, and its content is 361mMol (220g); Yield is 60.2%, and this moment, UMP content was 17.9mMol (6.16g), and the content of UTP is 81.2mMol (38.7g).
Embodiment 4: utilize the directed UMP of production of uridine.
Modulation is by uridine 900mMol, glucose 8Mol, magnesium chloride 125mMol, pichia spp 3200 grams, ammonium chloride 35mMol, saltpetre 400mMol, Sodium phosphate, dibasic 2.5Mol in the reactive tank of capacity 15L; 75 milliliters of reaction solution 10L that form with water of YLENE, using sodium hydroxide to transfer pH is 7.0, dissolved oxygen is controlled at 80%; Temperature is 32 ℃, reacts and finishes reaction after 9 hours, uses the perchloric acid precipitation; With HPLC product is carried out quantitative analysis, primary product is UMP in the conversion fluid, and its content is 634mMol (208g); Yield is 70.4%, and this moment, UTP content was 98.3mMol (47.2g), and UDPG content is 44.6mMol (27.2g).
Embodiment 5: utilize the directed UTP of production of uridine.
Modulation is by uridine 900mMol, glucose 8Mol, magnesium chloride 85mMol, pichia spp 3200 grams, ammonium chloride 35mMol, saltpetre 400mMol, Sodium phosphate, dibasic 2.5Mol in the reactive tank of capacity 15L; 75 milliliters of reaction solution 10L that form with water of YLENE, using sodium hydroxide to transfer pH is 7.0, and dissolved oxygen is controlled at 80%, and temperature is 32 ℃; React after 9 hours, dissolved oxygen is adjusted into 25%, and temperature is adjusted into 38 ℃; Continue reaction 6 hours, finish reaction, use the perchloric acid precipitation; With HPLC product is carried out quantitative analysis, primary product is UTP in the conversion fluid, and its content is 574mMol (276g); Yield is 63.8%, and this moment, UMP content was 77.3mMol (25.1g), and UDPG content is 104mMol (63.4g).
Embodiment 6: utilize the directed UDPG of production of uridine.
Modulation is by uridine 900mMol, glucose 8Mol, magnesium chloride 85mMol, pichia spp 3200 grams, ammonium chloride 35mMol, saltpetre 400mMol, Sodium phosphate, dibasic 2.5Mol in the reactive tank of capacity 15L; 75 milliliters of reaction solution 10L that form with water of YLENE, using sodium hydroxide to transfer pH is 7.0, and dissolved oxygen is controlled at 80%, and temperature is 32 ℃; React after 9 hours, dissolved oxygen is adjusted into 25%, and temperature is adjusted into 38 ℃, continues reaction after 6 hours; Dissolved oxygen is adjusted into 10%, and temperature is adjusted into 27 ℃, continues reaction 5 hours, finishes reaction; Use the perchloric acid precipitation, with HPLC product is carried out quantitative analysis, primary product is UDPG in the conversion fluid, and its content is 521mMol (317g); Yield is 57.9%, and this moment, UMP content was 26.9mMol (8.72g), and the content of UTP is 121mMol (58.6g).
Embodiment 7: utilize the directed UMP of production of vitamin B13.
Modulation is by vitamin B13 600mMol, glucose 7.5Mol, sal epsom 300mMol, yeast saccharomyces cerevisiae 3000 grams, ammonium chloride 24mMol, vitriolate of tartar 210mMol, SODIUM PHOSPHATE, MONOBASIC 4Mol in the reactive tank of capacity 15L; The reaction solution 10L that triton x-100 50 grams and water are formed, using sodium hydroxide to transfer pH is 8.0, and dissolved oxygen is controlled at 80%, and temperature is 30 ℃; Reacted 18 hours, reaction is used the perchloric acid precipitation after finishing; With HPLC reaction product is carried out quantitative analysis, primary product is UMP in the conversion fluid, and its content is 411mMol (133g); Yield is 68.5%, and this moment, the concentration of UTP was 58.1mMol (28.1g), and the concentration of UDPG is 32.8mMol (20.0g).
Embodiment 8: utilize the directed UTP of production of vitamin B13.
Modulation is by vitamin B13 600mMol, glucose 7.5Mol, sal epsom 300mMol, yeast saccharomyces cerevisiae 3000 grams, ammonium chloride 24mMol, vitriolate of tartar 210mMol, SODIUM PHOSPHATE, MONOBASIC 4Mol in the reactive tank of capacity 15L; The reaction solution 10L that triton x-100 50 grams and water are formed, using sodium hydroxide to transfer pH is 8.0, and dissolved oxygen is controlled at 80%, and temperature is 30 ℃; React after 18 hours, dissolved oxygen is adjusted into 30%, and temperature is adjusted into 37 ℃; Continue reaction 10 hours, finish reaction, use the perchloric acid precipitation; With HPLC reaction product is carried out quantitative analysis, primary product is UTP in the conversion fluid, and its content is 358.2mMol (173g); Yield is 59.7%, and this moment, the content of UMP was 84.2mMol (27.3g), and the content of UDPG is 66.5mMol (40.6g).
Embodiment 9: utilize the directed UDPG of production of vitamin B13.
Modulation is by vitamin B13 600mMol, glucose 7.5Mol, sal epsom 300mMol, yeast saccharomyces cerevisiae 3000 grams, ammonium chloride 24mMol, vitriolate of tartar 210mMol, SODIUM PHOSPHATE, MONOBASIC 4Mol in the reactive tank of capacity 15L; The reaction solution 10L that triton x-100 50 grams and water are formed, using sodium hydroxide to transfer pH is 8.0, and dissolved oxygen is controlled at 80%, and temperature is 30 ℃; React after 18 hours, dissolved oxygen is adjusted into 30%, and temperature is adjusted into 37 ℃, continues reaction after 10 hours; Dissolved oxygen is adjusted into 10%, and temperature is adjusted into 27 ℃, reacts 8 hours, finishes reaction; Use the perchloric acid precipitation, with HPLC reaction product is carried out quantitative analysis, primary product is UDPG in the conversion fluid, and its content is 303mMol (185g); Yield is 50.5%, and this moment, the content of UMP was 49.0mMol (15.9g), and the content of UTP is 62.3mMol (30.2g).
Embodiment 10: utilize the directed UMP of production of vitamin B13.
Modulation is by vitamin B13 800mMol, glucose 9Mol, magnesium nitrate 500mMol, subtilis 4000 grams (through air-dry), volatile salt 30mMol, Repone K 300mMol, the reaction solution 10L that SODIUM PHOSPHATE, MONOBASIC 5Mol and water are formed in the reactive tank of capacity 15L; Using sodium hydroxide to transfer pH is 8.0, and dissolved oxygen is controlled at 90%, and temperature is 28 ℃; Reaction to 20 hour, reaction finishes, and uses the perchloric acid precipitation; With HPLC reaction product is carried out quantitative analysis, primary product is UMP in the conversion fluid, and its content is 532mMol (172g); Yield is 66.5%, and this moment, the concentration of UTP was 59.2mMol (28.7g), and the concentration of UDPG is 34.8mMol (21.2g).
Embodiment 11: utilize the directed UTP of production of vitamin B13.
Modulation is by vitamin B13 800mMol, glucose 9Mol, magnesium nitrate 500mMol, subtilis 4000 grams (through air-dry), volatile salt 30mMol, Repone K 300mMol, the reaction solution 10L that SODIUM PHOSPHATE, MONOBASIC 5Mol and water are formed in the reactive tank of capacity 15L; Using sodium hydroxide to transfer pH is 8.0, and dissolved oxygen is controlled at 90%, and temperature is 28 ℃; React after 20 hours, dissolved oxygen is adjusted into 40%, and temperature is adjusted into 35 ℃; Continue reaction to 10 hours, finish reaction, use the perchloric acid precipitation; With HPLC reaction product is carried out quantitative analysis, primary product is UTP in the conversion fluid, and its content is 454mMol (220g); Yield is 56.7%, and this moment, the content of UMP was 87.2mMol (28.3g), and the content of UDPG is 86.5mMol (52.8g).
Embodiment 12: utilize the directed UDPG of production of vitamin B13.
Modulation is by vitamin B13 800mMol, glucose 9Mol, magnesium nitrate 500mMol, subtilis 4000 grams (through air-dry), volatile salt 30mMol, Repone K 300mMol, the reaction solution 10L that SODIUM PHOSPHATE, MONOBASIC 5Mol and water are formed in the reactive tank of capacity 15L; Using sodium hydroxide to transfer pH is 8.0, and dissolved oxygen is controlled at 90%, and temperature is 28 ℃, reacts after 20 hours; Dissolved oxygen is adjusted into 40%, and temperature is adjusted into 35 ℃, continues reaction to 10 hours, and dissolved oxygen is adjusted into 10%; Temperature is adjusted into 25 ℃, and reaction finished reaction to 6 hours, used the perchloric acid precipitation; With HPLC reaction product is carried out quantitative analysis, primary product is UDPG in the conversion fluid, and its content is 388mMol (237g); Yield is 48.5%, and this moment, the content of UMP was 59.2mMol (19.2g), and the content of UTP is 72.3mMol (35.1g).
Claims (2)
1. the method for the synthetic uridine phosphoryl compound of a directional catalyzing; It is characterized in that with uridine or vitamin B13 and phosphate anion be substrate; With glucose as ribose groups donor and energy donor; The microorganism cells of having property of utilization is the enzyme source, is means through stage change temperature of reaction and dissolved oxygen amount, impels the synthetic uridine phosphoryl compound of the directed biocatalysis of reaction system;
Wherein, described uridine phosphoryl compound is uridylic acid (UMP), UTP (UTP) or uridine diphosphoglucose (UDPG), and its structural formula is following:
Wherein, described mikrobe is Brevibacterium ammoniagenes, pichia spp, yeast saccharomyces cerevisiae or subtilis;
Wherein, the microorganism cells of described having property is to adopt following method to handle and get microorganism cells: surfactant method, organic solvent method, freeze-thaw method, aeration drying or freeze-drying; Described tensio-active agent is polyethylene oxide amines, triton x-100, hexadecyl trimethylamine bromide or Sarkosyl L salt, and working concentration is 0.1～20g/L; Described organic solvent is YLENE, toluene, Fatty Alcohol(C12-C14 and C12-C18), acetone or ETHYLE ACETATE, and working concentration is 0.1～20mL/L;
Wherein, the add-on of substrate uridine or vitamin B13 is 5～200mM, and phosphatic add-on is 0.1～2M, and the add-on of glucose is 0.1～1M, and the add-on of microorganism cells is for pressing wet thallus 100～800g/L;
With uridine and phosphate anion is substrate, in the aqueous solution of pH6～8, carries out one of following three kinds of reactions, directed synthetic uridine phosphoryl compound:
(a) under 25～35 ℃, dissolved oxygen amount 70～100% reacted 8～10 hours, and primary product is UMP;
(b) under 25～35 ℃, dissolved oxygen amount 70～100% reacted 8～10 hours; Adjust temperature to 35～45 ℃ again, the adjustment dissolved oxygen amount is 20～30%, reacts 4～6 hours, and primary product is UTP;
(c) under 25～35 ℃, dissolved oxygen amount 70～100% reacted 8～10 hours; Adjust temperature to 35～45 ℃ again, the adjustment dissolved oxygen amount is 20～30%, reacts 4～6 hours; Adjust temperature to 20～30 ℃ again, the adjustment dissolved oxygen amount is 5～20%, reacts 4～8 hours, and primary product is UDPG.;
With vitamin B13 and phosphate anion is substrate, in the aqueous solution of pH6～8, carries out one of following three kinds of reactions, directed synthetic uridine phosphoryl compound:
(a) under 25～35 ℃, dissolved oxygen amount 70～100% reacted 16～20 hours, and primary product is UMP;
(b) under 25～35 ℃, dissolved oxygen amount 70～100% reacted 16～20 hours; Adjust temperature to 35～45 ℃ again, the adjustment dissolved oxygen amount is 20～30%, reacts 8～10 hours, and primary product is UTP;
(c) under 25～35 ℃, dissolved oxygen amount 70～100% reacted 16～20 hours; Adjust temperature to 35～45 ℃ again, the adjustment dissolved oxygen amount is 20～30%, reacts 8～10 hours; Adjust temperature to 20～30 ℃ again, the adjustment dissolved oxygen amount is 5～20%, reacts 6～8 hours, and primary product is UDPG..
2. the method for the synthetic uridine phosphoryl compound of directional catalyzing according to claim 1 is characterized in that described reaction system also adds any one or a few the compsn in ammonium ion, mg ion and the potassium ion; Mg
2+Initial action concentration is 1～200mM; K
+Initial action concentration is 1～200mM; NH
4 +Initial action concentration is 1～200mM.
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| CN101724670B (en) * | 2009-11-17 | 2012-02-01 | 南京工业大学 | Method for combined production of chiral hydroxy ester and uridine phosphinylideyne compounds |
| CN101792786B (en) * | 2010-03-04 | 2012-07-11 | 南京工业大学 | Method for synthesizing cytidine phosphinylidyne compounds through oriented catalysis |
| CN101805769B (en) * | 2010-03-15 | 2012-05-23 | 南京工业大学 | Novel method for producing uracil nucleotide |
| CN108441532A (en) * | 2018-02-26 | 2018-08-24 | 安徽翠鸟生物技术有限公司 | A kind of preparation method of uridine 5'-diphosphate |
| CN110846361A (en) * | 2019-12-04 | 2020-02-28 | 美亚药业海安有限公司 | Method for preparing uridine diphosphate glucose by immobilized enzyme method |
| CN112126603B (en) * | 2020-09-30 | 2022-05-10 | 中国科学院天津工业生物技术研究所 | Brevibacterium ammoniagenes mutant strain for high yield of uridine diphosphate and application thereof |
| CN114805459A (en) * | 2022-05-20 | 2022-07-29 | 江苏集萃工业生物技术研究所有限公司 | Separation and extraction method of UMP sodium salt |
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