CN101555509A - Method for synthesizing uridine phosphoryl compound by directional catalysis - Google Patents
Method for synthesizing uridine phosphoryl compound by directional catalysis Download PDFInfo
- Publication number
- CN101555509A CN101555509A CNA2009100308384A CN200910030838A CN101555509A CN 101555509 A CN101555509 A CN 101555509A CN A2009100308384 A CNA2009100308384 A CN A2009100308384A CN 200910030838 A CN200910030838 A CN 200910030838A CN 101555509 A CN101555509 A CN 101555509A
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- Prior art keywords
- uridine
- dissolved oxygen
- hours
- oxygen amount
- synthetic
- Prior art date
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Links
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- DRTQHJPVMGBUCF-PSQAKQOGSA-N beta-L-uridine Natural products O[C@H]1[C@@H](O)[C@H](CO)O[C@@H]1N1C(=O)NC(=O)C=C1 DRTQHJPVMGBUCF-PSQAKQOGSA-N 0.000 title claims abstract description 62
- DRTQHJPVMGBUCF-UHFFFAOYSA-N uracil arabinoside Natural products OC1C(O)C(CO)OC1N1C(=O)NC(=O)C=C1 DRTQHJPVMGBUCF-UHFFFAOYSA-N 0.000 title claims abstract description 62
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- 238000006243 chemical reaction Methods 0.000 claims abstract description 93
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- DJJCXFVJDGTHFX-XVFCMESISA-N uridine 5'-monophosphate Chemical compound O[C@@H]1[C@H](O)[C@@H](COP(O)(O)=O)O[C@H]1N1C(=O)NC(=O)C=C1 DJJCXFVJDGTHFX-XVFCMESISA-N 0.000 claims description 44
- PGAVKCOVUIYSFO-UHFFFAOYSA-N [[5-(2,4-dioxopyrimidin-1-yl)-3,4-dihydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl] phosphono hydrogen phosphate Chemical compound OC1C(O)C(COP(O)(=O)OP(O)(=O)OP(O)(O)=O)OC1N1C(=O)NC(=O)C=C1 PGAVKCOVUIYSFO-UHFFFAOYSA-N 0.000 claims description 35
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- 229920001282 polysaccharide Polymers 0.000 description 1
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- 230000008929 regeneration Effects 0.000 description 1
- 238000011069 regeneration method Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
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- 229920006395 saturated elastomer Polymers 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
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- 230000000087 stabilizing effect Effects 0.000 description 1
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- UNXRWKVEANCORM-UHFFFAOYSA-I triphosphate(5-) Chemical compound [O-]P([O-])(=O)OP([O-])(=O)OP([O-])([O-])=O UNXRWKVEANCORM-UHFFFAOYSA-I 0.000 description 1
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Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention discloses a method for directionally catalyzing and synthesizing uridine phosphoryl compounds, which uses uridine or orotic acid and phosphate radical ions as substrates, glucose as a ribose group donor and an energy donor, uses permeable microbial cells as an enzyme source, and promotes a reaction system to directionally and biologically catalyze and synthesize the uridine phosphoryl compounds by taking the stepwise change of reaction temperature and dissolved oxygen as means. The invention can produce any one of three substances according to the requirement, has simple reaction system, no toxicity, low production cost and simple and easy control means, and greatly facilitates the production control in industrialization. When uridine is used as a substrate, the yield of UMP, UTP and UDPG can reach 77%, 67% and 61% respectively; when the orotic acid is used as a substrate, the yield of UMP, UTP and UDPG can reach 70%, 61% and 53% respectively.
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 derivative of uridylic; be important biochemical reagents; also be the very big medicine intermediate of consumption simultaneously; infant food additive and fodder additives; also be such as uridine triphosphate (UTP); Uridine pyrophosphate-glucose (UDPG); cytidine triphosphate(CTP) (CTP); the synthetic precursor of cytidine diphosphate uridines such as (CDPC) and cytidine phosphinylidyne compounds; a large amount of pharmacology and clinical medicine studies show that this compounds is at cardiovascular and cerebrovascular; hepatitis; diabetes; antiviral (SARS) and nerve degeneration (parkinsonism; Alzheimer's disease) etc. there is good clinical effectiveness aspect such as the treatment of difficult diseases and assisting therapy, is the basic material of synthesis of oligose and other physiologically active substances simultaneously.
Uridine triphosphate (UTP) be termolecular phosphoric acid be combined in uridine ribose 5 '-Nucleotide on the OH base, widely distributed, 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 adenosine triphosphate, can also be as the P2 acceptor on extracellular informational molecule (first messenger) activating cells surface, and regulate multiple physiological response by 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 he's colleague.UDPG not only can be used as the glucosyl group donor, participate in the synthetic of interior multiple oligosaccharides of organism and polysaccharide, biosynthesizing as trehalose, sucrose, starch, VITAMIN, but also be synthetic other nucleoside diphosphate monose, as the precursor of uridine diphosphate galactose, 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 microorganism as the enzyme source exactly, and the precursor substance of catalysis Nucleotide is converted into Nucleotide.Japan consonance fermentation company is by improveing bacterial classification, utilize uridylic acid precursor substance vitamin B13 to transform UMP, 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. are 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 by 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 synthesizes UTP with yeast bio catalysis UMP.The synthetic of UDPG mainly is that chemical method is synthetic and enzyme process is synthetic at present.
In yeast cell, uridine can synthesize UMP under the catalysis of uridine kinase; Vitamin B13 can and phosphoribosyl pyrophosphate (PRPP) (PRPP) under the effect of orotate phosphoribosyl transferase, generate orotidylic acid (OMP), OMP generates UMP through the catalysis of orotidylic decarboxylase then.UMP and ATP shift a phosphate by the uridylic acid kinases then, are converted into UDP and ADP; UDP consumes ATP then by 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 by 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 the microorganism cells enzyme is the enzyme source, synthetic uridine phosphoryl compound under certain reaction system.Generally speaking, through reaction after a while, may there be UMP, UTP and three kinds of reaction product of UDPG in the system simultaneously, but can make reaction leading by control measures to a certain product direction, 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.By long-term experiment, we find that above-mentioned control measures mainly comprise interim temperature of reaction and the dissolved oxygen amount adjusted.
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 by interim control reaction temperature and oxygen-supply quantity.Under 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
React different final products.Control device is simple, has greatly made things convenient for the production control in the industrialization.
The concrete technical scheme that the present invention adopts is as follows:
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; by stage change temperature of reaction and dissolved oxygen amount is means, impels the synthetic uridine phosphoryl compound of the directed biocatalysis of reaction system.
Wherein, described uridine phosphoryl compound is uridylic acid (UMP), uridine triphosphate (UTP) or uridine diphosphoglucose (UDPG), and its structural formula is as follows:
Wherein, uridine, i.e. UR, structural formula is as follows:
Uridine
Wherein, vitamin B13, i.e. OA, structural formula is as follows:
Vitamin B13
If with the uridine is substrate, (preferred pH6~8) carry out one of following three kinds of reactions, orientable synthetic uridine phosphoryl compound 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, adjusting 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, adjusting dissolved oxygen amount is 15~50% (preferred 20~30%), and reaction was to 4~6 hours; Adjust temperature to 20~30 ℃ again, adjusting 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, adjusting 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, adjusting dissolved oxygen amount is 15~50% (preferred 20~30%), and reaction was to 8~10 hours; Adjust temperature to 20~30 ℃ again, adjusting 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 composition in ammonium ion, magnesium 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 microorganism 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 nonionic surface active agent (as polyethylene oxide amines, triton x-100), cationic surfactant (as the hexadecyl trimethylamine bromide) or anion surfactant (Sarkosyl L salt), working concentration is 0.1~50g/L, the concentration of preferred 1~20g/L is used, when being surfactant method processing microorganism cells, tensio-active agent is directly added reaction solution, for cumulative volume is the reaction solution of 1L, add 0.1~50g, preferably add 1~20g; Described organic solvent is dimethylbenzene, toluene, Fatty Alcohol(C12-C14 and C12-C18), acetone or ethyl acetate, working concentration is 0.1~50mL/L, the concentration of preferred 1~20mL/L is used, when being organic solvent method processing microorganism cells, organic solvent is directly added reaction solution, for cumulative volume is the reaction solution of 1L, adds 0.1~50mL, preferably adds 1~20mL.Other handles the method for cell permeability, as 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 by 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 by 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 industrialization.
Can directedly produce UMP by 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, the present invention may be better understood.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 illustrate the present invention, and should also can not limit the present invention described in detail in claims.
Embodiment 1: utilize the directed UMP of production of uridine.
Modulation is by uridine 600mMol in the reactive tank of capacity 15L, glucose 5Mol, sal epsom 50mMol, Brevibacterium ammoniagenes 2400 grams, ammonium chloride 20mMol, Repone K 300mMol, SODIUM PHOSPHATE, MONOBASIC 2.0Mol, the reaction solution 10L that 50 milliliters of toluene and water are formed, transferring pH with sodium hydroxide is 7.0, and dissolved oxygen is controlled at 70%, and temperature is 30 ℃, react and finish reaction after 8 hours, use the perchloric acid precipitation, with HPLC product is carried out quantitative analysis, primary product is UMP in the conversion fluid, 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 in the reactive tank of capacity 15L, glucose 5Mol, sal epsom 50mMol, Brevibacterium ammoniagenes 2400 grams, ammonium chloride 20mMol, Repone K 300mMol, SODIUM PHOSPHATE, MONOBASIC 2.0Mol, the reaction solution 10L that 50 milliliters of toluene and water are formed, transferring pH with sodium hydroxide 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 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, 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 in the reactive tank of capacity 15L, glucose 5Mol, sal epsom 50mMol, Brevibacterium ammoniagenes 2400 grams, ammonium chloride 20mMol, Repone K 300mMol, SODIUM PHOSPHATE, MONOBASIC 2.0Mol, the reaction solution 10L that 50 milliliters of toluene and water are formed, transferring pH with sodium hydroxide is 7.0, dissolved oxygen is controlled at 70%, and temperature is 30 ℃, reacts after 8 hours, dissolved oxygen is adjusted into 25%, temperature is adjusted into 37 ℃, continues reaction after 6 hours, and dissolved oxygen is adjusted into 5%, temperature is adjusted into 25 ℃, continue reaction 4 hours, finish 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), and yield is 60.2%, 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 in the reactive tank of capacity 15L, glucose 8Mol, magnesium chloride 125mMol, pichia spp 3200 grams, ammonium chloride 35mMol, saltpetre 400mMol, Sodium phosphate dibasic 2.5Mol, the reaction solution 10L that 75 milliliters of dimethylbenzene and water are formed, transferring pH with sodium hydroxide is 7.0, and dissolved oxygen is controlled at 80%, and temperature is 32 ℃, react and finish reaction after 9 hours, use the perchloric acid precipitation, with HPLC product is carried out quantitative analysis, primary product is UMP in the conversion fluid, 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 in the reactive tank of capacity 15L, glucose 8Mol, magnesium chloride 85mMol, pichia spp 3200 grams, ammonium chloride 35mMol, saltpetre 400mMol, Sodium phosphate dibasic 2.5Mol, the reaction solution 10L that 75 milliliters of dimethylbenzene and water are formed, transferring pH with sodium hydroxide 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 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, 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 in the reactive tank of capacity 15L, glucose 8Mol, magnesium chloride 85mMol, pichia spp 3200 grams, ammonium chloride 35mMol, saltpetre 400mMol, Sodium phosphate dibasic 2.5Mol, the reaction solution 10L that 75 milliliters of dimethylbenzene and water are formed, transferring pH with sodium hydroxide is 7.0, dissolved oxygen is controlled at 80%, and temperature is 32 ℃, reacts after 9 hours, dissolved oxygen is adjusted into 25%, temperature is adjusted into 38 ℃, continues reaction after 6 hours, and dissolved oxygen is adjusted into 10%, temperature is adjusted into 27 ℃, continue reaction 5 hours, finish 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), and yield is 57.9%, 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 in the reactive tank of capacity 15L, glucose 7.5Mol, sal epsom 300mMol, yeast saccharomyces cerevisiae 3000 grams, ammonium chloride 24mMol, vitriolate of tartar 210mMol, SODIUM PHOSPHATE, MONOBASIC 4Mol, the reaction solution 10L that triton x-100 50 grams and water are formed, transferring pH with sodium hydroxide is 8.0, dissolved oxygen is controlled at 80%, 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), and yield is 68.5%, 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 in the reactive tank of capacity 15L, glucose 7.5Mol, sal epsom 300mMol, yeast saccharomyces cerevisiae 3000 grams, ammonium chloride 24mMol, vitriolate of tartar 210mMol, SODIUM PHOSPHATE, MONOBASIC 4Mol, the reaction solution 10L that triton x-100 50 grams and water are formed, transferring pH with sodium hydroxide 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 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, 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 in the reactive tank of capacity 15L, glucose 7.5Mol, sal epsom 300mMol, yeast saccharomyces cerevisiae 3000 grams, ammonium chloride 24mMol, vitriolate of tartar 210mMol, SODIUM PHOSPHATE, MONOBASIC 4Mol, the reaction solution 10L that triton x-100 50 grams and water are formed, transferring pH with sodium hydroxide is 8.0, dissolved oxygen is controlled at 80%, and temperature is 30 ℃, reacts after 18 hours, dissolved oxygen is adjusted into 30%, temperature is adjusted into 37 ℃, continues reaction after 10 hours, and dissolved oxygen is adjusted into 10%, temperature is adjusted into 27 ℃, reacted 8 hours, and finished 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), and yield is 50.5%, 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 in the reactive tank of capacity 15L, 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, transferring pH with sodium hydroxide is 8.0, dissolved oxygen is controlled at 90%, and temperature is 28 ℃, and reaction was to 20 hours, reaction finishes, use the perchloric acid precipitation, with HPLC reaction product is carried out quantitative analysis, primary product is UMP in the conversion fluid, 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 in the reactive tank of capacity 15L, 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, transferring pH with sodium hydroxide is 8.0, dissolved oxygen is controlled at 90%, temperature is 28 ℃, reacts after 20 hours, and dissolved oxygen is adjusted into 40%, 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), and yield is 56.7%, 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 in the reactive tank of capacity 15L, 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, transferring pH with sodium hydroxide is 8.0, dissolved oxygen is controlled at 90%, temperature is 28 ℃, react after 20 hours, dissolved oxygen is adjusted into 40%, and temperature is adjusted into 35 ℃, continue reaction to 10 hours, dissolved oxygen is adjusted into 10%, and temperature is adjusted into 25 ℃, and reaction was to 6 hours, finish reaction, use the perchloric acid precipitation, with HPLC reaction product is carried out quantitative analysis, primary product is UDPG in the conversion fluid, 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 (10)
1, the method for the synthetic uridine phosphoryl compound of a kind of 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; by stage change temperature of reaction and dissolved oxygen amount is means, impels the synthetic uridine phosphoryl compound of the directed biocatalysis of reaction system.
3, the method for the synthetic uridine phosphoryl compound of directional catalyzing according to claim 2 is characterized in that with the uridine being substrate, carries out one of following three kinds of reactions in the aqueous solution of pH5~10, directed synthetic uridine phosphoryl compound:
(a) under 25~35 ℃, dissolved oxygen amount 60~120% reacted 8~10 hours, and primary product is UMP;
(b) under 25~35 ℃, dissolved oxygen amount 60~120% reacted 8~10 hours; Adjust temperature to 35~45 ℃ again, adjusting dissolved oxygen amount is 15~50%, reacts 4~6 hours, and primary product is UTP;
(c) under 25~35 ℃, dissolved oxygen amount 60~120% reacted 8~10 hours; Adjust temperature to 35~45 ℃ again, adjusting dissolved oxygen amount is 15~50%, reacts 4~6 hours; Adjust temperature to 20~30 ℃ again, adjusting dissolved oxygen amount is 0~30%, reacts 4~8 hours, and primary product is UDPG..
4, the method for the synthetic uridine phosphoryl compound of directional catalyzing according to claim 2 is characterized in that with the vitamin B13 being substrate, carries out one of following three kinds of reactions in the aqueous solution of pH5~10, directed synthetic uridine phosphoryl compound:
(a) under 25~35 ℃, dissolved oxygen amount 60~120% reacted 16~20 hours, and primary product is UMP;
(b) under 25~35 ℃, dissolved oxygen amount 60~120% reacted 16~20 hours; Adjust temperature to 35~45 ℃ again, adjusting dissolved oxygen amount is 15~50%, reacts 8~10 hours, and primary product is UTP;
(c) under 25~35 ℃, dissolved oxygen amount 60~120% reacted 16~20 hours; Adjust temperature to 35~45 ℃ again, adjusting dissolved oxygen amount is 15~50%, reacts 8~10 hours; Adjust temperature to 20~30 ℃ again, adjusting dissolved oxygen amount is 0~30%, reacts 6~8 hours, and primary product is UDPG..
The method of 5, synthesizing the uridine phosphoryl compound according to any described directional catalyzing in the claim 1~4; the add-on that it is characterized in that substrate uridine or vitamin B13 is 5~200mM; phosphatic add-on is 0.1~2M; the add-on of glucose is 0.1~1M, and the add-on of microorganism cells is for pressing wet thallus 100~800g/L.
6,, it is characterized in that described reaction system also adds any one or a few the composition in ammonium ion, magnesium ion and the potassium ion according to the method for the synthetic uridine phosphoryl compound of any described directional catalyzing in the claim 1~4; Mg
2+Initial action concentration is 1~200mM; K
+Initial action concentration is 1~200mM; NH
4 +Initial action concentration is 1~200mM.
The method of 7, synthesizing the uridine phosphoryl compound according to any described directional catalyzing in the claim 1~4, it is characterized in that the microorganism of described microorganism cells, comprise the bacterium of aerobacter, Escherichia, Serratia, micrococcus sp for utilizing the synthetic uridylic acid of uridine or vitamin B13 and continuing phosphorylated; The yeast that yeast belong, mycocandida, Pichia, torulopsis, Debaryomyces, zygosaccharomyces genus, genus kluyveromyces, Hansenula and Brettanomyces belong to.
8, the method for the synthetic uridine phosphoryl compound of directional catalyzing according to claim 7 is characterized in that described microorganism cells is Brevibacterium ammoniagenes, subtilis, yeast saccharomyces cerevisiae or Torulopsis candida.
9, the method for the synthetic uridine phosphoryl compound of directional catalyzing 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.
10, the method for the synthetic uridine phosphoryl compound of directional catalyzing according to claim 1, 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 nonionic surface active agent, cationic surfactant or anion surfactant, and working concentration is 0.1~50g/L; Described organic solvent is dimethylbenzene, toluene, Fatty Alcohol(C12-C14 and C12-C18), acetone or ethyl acetate, and working concentration is 0.1~50mL/L.
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