CN105603025B - A kind of fermentation method for producing of coproduction uridine and 3-hydroxy-2-butanone - Google Patents
A kind of fermentation method for producing of coproduction uridine and 3-hydroxy-2-butanone Download PDFInfo
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- CN105603025B CN105603025B CN201610137185.XA CN201610137185A CN105603025B CN 105603025 B CN105603025 B CN 105603025B CN 201610137185 A CN201610137185 A CN 201610137185A CN 105603025 B CN105603025 B CN 105603025B
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Abstract
The invention belongs to fermentation engineering fields, and in particular to the fermentation method for producing of a kind of coproduction uridine and 3-hydroxy-2-butanone.Existing technology individually produces uridine or 3-hydroxy-2-butanone using bacillus subtilis mostly, and yield level is not high, it is low that there are raw material availabilities, problem at high cost, the bacillus subtilis with industrial applications potentiality and its corresponding zymotechnique that the present invention provides one plant to be come out by mutagenic and breeding, uridine 25-32g/L, 3-hydroxy-2-butanone 50-65g/L can be respectively reached with coproduction uridine and 3-hydroxy-2-butanone, yield.The utilization rate for greatly improving raw material, reduces cost, to alleviate resource and environmental pressure.
Description
Technical field:
The invention belongs to fermentation engineering fields, and in particular to the fermenting and producing side of a kind of coproduction uridine and 3-hydroxy-2-butanone
Method.
Background technique:
Uridine is the precursor for constituting uridylic acid, and the latter can synthesize liver detoxification substance glucuronic acid.Sodium uridilate
Flavouring and pharmaceutical raw material are acted not only as, food additives is alternatively arranged as and is added in milk powder, increase substantially baby's
Immunity.In addition to this, uridine can be used for huge red blood cell anaemia, can also share with other nucleosides, base in treatment liver, brain blood
The illness such as pipe and angiocarpy.Animal experiment, which shows that it is shared with inosine, can promote metabolism of myocardium, accelerate protein, core
The biosynthesis and energy production of acid improve and promote brain cell metabolic cycles.Uridine is used for field of medicaments, and can repair causes
The damaged gene of disease improves body antibody level.An its prior purposes is produced as medicine intermediate
Antiviral anti-tumor drug, such as iodoxuridine, bromine glycosides, fluorine glycosides can block the gene chemical synthesis of various cancer cells and virus, treat cancer
Disease caused by disease or virus.
The production method of uridine has chemical synthesis, hydrolyzes RNA method and microbe fermentation method, microbe fermentation method is because of its week
The advantages that phase is short, easy to control, yield is high, pollution is small, the potentiality with heavy industrialization, therefore it is increasingly subject to the weight of people
Depending on.The research of fermentation method production uridine starts from the 1960s, Wu Tian chemical company of Japan is since last century the eighties,
Fermentation strain screen and fermentation technology optimization to uridine have carried out a series of research and have achieved great successes, report
Research achievement represent the highest level of fermentation method production uridine, the research process of Wu Tian company uridine fermenting and producing is as follows:
First using wild-type B. subtilis as starting strain during high yield uridine strain improvement, by NTG mutagenesis, select
One plant of uracil-deficient bacterial strain No.122, the bacterial strain, being capable of accumulated whey acid when providing 50mg/L uracil
8.58g/L (50mM) orotic acid and 7.78g/L (27mM) orotidine；On the basis of No.122, again passes by NTG and lure
Become, bacterial strain No.258 is screened in the culture medium containing 300mg/L 6- azauracil, this strain for accumulating uridine 10g/L,
Uracil 7g/L.Continue NTG mutagenesis, screens bacterial strain No.508 in the 6- azauracil culture medium containing 5g/L, the bacterium
Strain accumulation uridine 46g/L, uracil 6g/L.Finally, continuing mutagenesis to reduce the accumulation of by-product uracil, sieve
The bacterial strain No.556 for choosing one plant of uridine phosphorylase almost loss of activity, can accumulate uridine 55g/L, uracil accumulation <
1g/L；Finally by the optimization to condition of culture and medium component, stablize accumulation uridine 65g/L in 6000L fermentor.
There are two types of carbamyl phosphate synthetase (EC 6.3.5.5, carbamoyl phosphate by B.subtilis
Synthetase, CPSase): first is that pyrAA/pyrAB gene encodes, the first step reaction of catalysis UMP de novo synthesis；Two
It is carA/carB gene coding, participates in Arginine biosynthesis.Although both carbamyl phosphate synthetases are all catalyzed paddy ammonia
Amide and CO2Reaction, 2 molecule ATP of consumption generate carbamyl phosphate.But the two of B.subtilis carbamyl phosphate synthesis
Enzyme physiological function is different, their expression and enzymatic activity adjustment mechanism is also entirely different.
PyrAA/pyrAB is located at the 5th and 6 of pyr operon, encodes carbamyl phosphate synthesis relevant to UMP synthesis
Enzyme.Wherein pyrAA mrna length is 1095bp, and the small subunit of encoding carbamoyl phosphate synthetase is catalyzed ε-ammonia of glutamine
Group-transfer is to large subunit.PyrAB mrna length be 3216bp, the large subunit of encoding carbamoyl phosphate synthetase, catalysis MgATP,
CO2With NH3Synthesize carbamyl phosphate.PyrAA/pyrAB belongs to pyr operon, and expression is also activated by PRPP, by UMP, UDP and
The feedback repression of UTP.The carbamyl phosphate synthetase or an allosteric enzymes, enzyme activity of pyrAA/pyrAB coding are fed back by UMP
Inhibit, is activated by PRPP, and need Mg2+Presence.
The difficult point of pyrimidine nucleoside superior strain breeding is the feedback repression and feedback inhibition of releasing final product, especially
It is the releasing of feedback inhibition, although researcher has been discovered that the specific regulatory mechanism of pyrimidine nucleoside anabolism, and
Pyrimidine operon is elaborated from molecular level by the mechanism of end-production repression, but for the ammonia first by final product feedback inhibition
The specific regulatory site of acyl phosphate synthase is not known but.
The precedent of the production uridine of fermentation method not yet domestic at present, the research in relation to uridine fermenting and producing are started late, into
Also relatively slowly, there is also very big gaps compared with external for exhibition.
The entitled 3-Hydroxybutanone of 3-hydroxy-2-butanone chemistry is naturally present in grape, cocoa, banana, corn, cheese and meat etc. and is permitted
In more food.It is a kind of widely applied flavorant, is mainly used for the production of the fragrance such as cream, dairy products, Yoghourt.As one
Kind flavorant, China GB2760-86 are defined as allowing to eat, and U.S.'s food and extraction association's (FEMA) security number are 2008.
Meanwhile 3-hydroxy-2-butanone is as a kind of four important carbon platform chemicals, is classified as 30 kinds by U.S. Department of Energy and is preferentially exploited
One of platform chemicals, especially chiral 3-hydroxy-2-butanone can be widely applied to chiral drug and chemistry as high valuable chemicals
The synthesis of intermediate.In addition, 3-hydroxy-2-butanone can also be used to modify the antibiotics such as penicillin, ampicillin, to improve medicine
Effect reduces side effect etc., so as to cause the attention of medical industry.
The production method of 3-hydroxy-2-butanone mainly includes chemical synthesis, enzyme catalysis method and microbe fermentation method.The change of 3-hydroxy-2-butanone
Learning synthetic method is mainly the reduction of diacetyl partial hydrogenation and the production of 2,3- butanediol selective oxidation；The enzyme transforming process master of 3-hydroxy-2-butanone
If catalysis diacetyl reduction obtains 3-hydroxy-2-butanone by isolating and purifying diacetyl reductase, can also by 3-hydroxy-2-butanone reductase with
Nadh oxidase is catalyzed the oxidation of 2,3- butanediol and obtains.But diacetyl and 2,3-butanediol are not large chemical products, they
The non-renewable fossil resources such as petroleum are derived from, therefore raw material sources are insufficient, environmental pollution is serious, complex process, product matter
Amount requirement etc. limits the extensive development of chemical synthesis, does not meet the trend of sustainable development, therefore microbe fermentation method
Producing 3-hydroxy-2-butanone is the hot topic studied at present.
Existing multiple-microorganism is natural at present or has the ability for producing 3-hydroxy-2-butanone, the grandson of Donghua University after Metabolically engineered
Peace etc. is built using serratia marcescens, the 3-hydroxy-2-butanone of 75g/L is produced using sucrose as carbon source through fermentation, the fermentation method to report at present produces
The maximum output of 3-hydroxy-2-butanone.3-hydroxy-2-butanone is in food, the extensive use of medicine and other fields, but also producing opening for the safe strain of 3-hydroxy-2-butanone
Hair becomes the hot topic of 3-hydroxy-2-butanone strain improvement, and bacillus subtilis naturally has the ability for producing 3-hydroxy-2-butanone, be as safe bacterial strain
Fermentation method production 3-hydroxy-2-butanone often selects bacterial strain.
Existing technology individually produces uridine or 3-hydroxy-2-butanone using bacillus subtilis mostly, and yield level is not
Height, it is low that there are raw material availabilities, problem at high cost, has industrialization by what mutagenic and breeding came out the present invention provides one plant
The bacillus subtilis of application potential and its corresponding zymotechnique can greatly improve raw material with coproduction uridine and 3-hydroxy-2-butanone
Utilization rate reduces cost, to alleviate resource and environmental pressure.
Summary of the invention:
The object of the present invention is to provide one kind to overcome the prior art with the fermentation method for producing of coproduction uridine and 3-hydroxy-2-butanone
Middle uridine and 3-hydroxy-2-butanone individually produce, and raw material availability is low, low output and complex process defect.
To achieve the goals above, the technical solution adopted in the present invention is as follows:
A kind of fermentation method for producing of coproduction uridine and 3-hydroxy-2-butanone, production strain enter after inclined-plane culture and seed culture
Fermented and cultured, fermented and cultured are 34-38 DEG C using flow feeding technique in batches, inoculum concentration 10%-15%, cultivation temperature, pH
Glycerol adding yeast powder mixed aqueous solution, the content of glycerol are flowed in 6.7-7.0, opposite dissolved oxygen control in 10%-30%, fermentation process
In 5-15g/L, fermentation period is 52-60 hours for control；
The glycerol yeast powder mixed aqueous solution is the mixed aqueous solution of glycerol containing 800g/L and 60g/L yeast powder；
The pH is adjusted by 0.5M sodium hydroxide solution and soybean meal hydrolysate；
The culture medium of the fermented and cultured forms are as follows: yeast powder 5g/L, (NH4)2SO45g/L, KH2PO41g/L, K2HPO4
5.2g/L, sodium citrate 10g/L, sodium glutamate 20g/L, corn pulp 20mL/L, CaCl21g/L, urea 20g/L, MgSO4·
7H2O 5g/L, MnSO40.02g/L, ZnSO40.02g/L；PH6.7~7.0,115 DEG C, sterilize 15min；
The production strain is bacillus subtilis (Bacillus subtilis) A260, and the bacterial strain is in 2015 12
The moon is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on 2nd, address: Chaoyang District, Beijing City North Star west
The institute 3 of road 1, Institute of Microorganism, Academia Sinica, postcode 100101, deposit number are CGMCC No.11775.
The bacillus subtilis A260 is passed through using the bacillus subtilis of one plant of accumulation pyrimidine nucleoside as starting strain
The mutagenesis of atmospheric pressure at room plasma and pyrimidine nucleoside analogue resistant strain are screened and are selected, the specific steps are as follows:
Using the withered grass gemma TD131 for producing pyrimidine nucleoside as starting strain, the mutagenesis of atmospheric pressure at room plasma is carried out, is then containing 300mg/L
Bacterial strain T823 is filtered out on 2- thiouracil minimal medium, then using T823 as starting strain, passes through atmospheric pressure at room plasma
After mutagenesis, strains A 260 is filtered out on containing 100mg/L 6- azauracil minimal medium.
The pyrimidine nucleoside operon of mutant strain A260 is sequenced, and carries out alignment, hair with starting strain TD131
The encoding gene of the carbamyl phosphate synthetase of existing mutant strain A260 mutates, and mutational site is in large subunit (regulatory site institute
In subunit, encoded by pyrAB gene) on, pyrAB gene the 2846th, 2847 and 2848 bit bases missing, eventually lead in A260
949 glutamic acid of carbamyl phosphate synthetase large subunit lacks (E949*), and the bacterial strain bacterium pyrimidine nucleoside operon is other
Gene does not find to be mutated, and gene relevant to 3-hydroxy-2-butanone synthesis does not also find to be mutated.
The nucleotide sequence of original pyrAB gene is as shown in sequence table SEQ ID No.1；
Original carbamyl phosphate synthetase large subunit amino acid sequence is as shown in sequence table SEQ ID No.3；
The nucleotide sequence of pyrAB gene is mutated as shown in sequence table SEQ ID No.2；
Carbamyl phosphate synthetase large subunit variant amino acid sequence is as shown in sequence table SEQ ID No.4；
Nucleotide sequence described in the Position Number corresponding sequence table SEQ ID No.1 of nucleotide of the present invention；
Amino acid sequence described in the Position Number corresponding sequence table SEQ ID No.3 of amino acid in the present invention.
The utility model has the advantages that
1, the present invention provides the fermentation manufacturing techniques of a kind of coproduction uridine and 3-hydroxy-2-butanone, finally using provided by the present invention
Zymotechnique after fermentation, uridine 25-32g/L, 3-hydroxy-2-butanone 50-65g/L can be accumulated in fermentation liquid.
2, the bacillus subtilis A260, A260 for capableing of coproduction uridine and 3-hydroxy-2-butanone the present invention provides one plant are with one plant
The bacillus subtilis for accumulating pyrimidine nucleoside is that starting strain carries out mutagenesis, but obtains the windfall effect of high yield 3-hydroxy-2-butanone,
And 3-hydroxy-2-butanone fermentation output of fluid is up to 50-65g/L, is better than the prior art.
3, the present invention by mutagenic obtained strains A 260 after fermentation condition optimization uridine yield up to 30-32g/
L is 7.2 times of starting strain TD131 uridine yield (4.1 ± 0.5g/L).
Detailed description of the invention:
96 screening micropore plate result of Fig. 1
Fig. 2 shaking flask the selection result
Fig. 3 A260 fermentation coproduction uridine and 3-hydroxy-2-butanone conditional curve
Specific embodiment:
The screening of 1. bacillus subtilis A260 of embodiment
The present invention sieves initial strains TD131 through atmospheric pressure at room plasma mutagenesis-analogue resistance screening-high throughput
Choosing-shaking flask secondary screening obtains strains A 260, and detailed process is as follows:
1. starting strain: B.subtilis TD131
2. atmospheric pressure at room plasma mutagenesis
(1) it thallus cultural method: protects tube and meets LB plate, three rides, 37 DEG C of culture 12h；Single colonie is chosen from plate
It meets LB and shakes pipe, 37 DEG C, 200rpm, cultivate 12h；Pipe switching LB shaking flask is shaken, inoculum concentration 1%, cultivates 4-6h by 37 DEG C, 200rpm.
(2) preparation method of bacteria suspension: cultured thallus is collected by centrifugation, after sterile saline is washed 2-3 times, then uses
Sterile saline dilutes in right amount is made OD600It is worth the bacteria suspension in 0.6-0.8, takes 10 μ L bacteria suspensions to be coated in be processed on slide glass.
(3) ARTP mutagenesis: ARTP processing parameter are as follows: slide glass is at flow ports 2mm；Power is 120W；Throughput is
10SLM；Action time is 25s.
3. the screening of Analogue resistant mutant
Bacteria suspension after ARTP mutagenic treatment is coated on the minimal medium containing a certain concentration analogue
On plate, after 37 DEG C of cultures for 24 hours, the biggish bacterial strain of bacterium colony is possible targeted mutagenesis bacterial strain.
4. high flux screening pyrimidine nucleoside Producing Strain
It is connected on the LB plate marked, and is transferred into from picking macrocolony point in analogue resistant panel
In the deep hole for sterilizing and filling 400 microlitres of sterile LB mediums, after 37 DEG C of LB plate are incubated overnight, 4 DEG C of preservations, as further
Screening use.36 DEG C of plate, 200rpm, after cultivating 10h, with 10% inoculum concentration, turns to be inoculated into one by one and fill 400 microlitres of orifice plates
In the sterile new orifice plate of fermentation medium, 36 DEG C, 200rmp, 30h is cultivated.After culture, it is centrifuged fermented sample, it will be upper thin
After releasing 100 times, 200 microlitres are taken respectively into ELISA Plate, with light absorption value OD of the microplate reader test sample at 270nm270, will
OD270Higher bacterial strain carries out preservation, carries out further shaking flask secondary screening.Fig. 1 is screening micropore plate result.
5. shaking flask secondary screening
(1) culture medium:
Activated inclined plane culture medium (g/L): glucose 1, peptone 10, beef extract 10, yeast powder 5, NaCl 2.5, agar
20, pH 6.7~7.0,0.1MPa, 20min.
Seed culture medium (g/L): glucose 40, yeast powder 5, peptone 10, sodium nitrate 15, potassium dihydrogen phosphate 1, phosphoric acid hydrogen
Dipotassium 5.2, magnesium sulfate 1, sodium citrate 1, pH 6.7~7.0,0.075MPa, 15min.
Fermentation medium (g/L): glucose 100, yeast powder 5, peptone 10, sodium nitrate 15, potassium dihydrogen phosphate 1, phosphoric acid
Hydrogen dipotassium 5.2, magnesium sulfate 1, sodium citrate 1, pH 6.7~7.0,0.075MPa, 15min.Grape sugar disappears.
(2) cultural method:
Inclined-plane culture: taking -80 DEG C of preservation of bacteria strain streak inoculations in activated inclined plane, 37 DEG C of culture 12h, and passes on primary.
Seed culture: a ring inclined-plane seed is scraped with oese and is inoculated in the 500mL triangle equipped with 30mL seed culture medium
In bottle, nine layers of gauze sealing, 37 DEG C, 200r/min shaken cultivation 6h.
Fermented and cultured: drawing 3mL seed liquor and be inoculated in the 500mL baffle flask equipped with 27mL fermentation medium, and nine
Layer gauze sealing, 37 DEG C, 200r/min shaken cultivation 30h.
(3) analysis method:
PH measurement: it is measured using pH 6.4-8.0 accurate pH test paper.
Biomass estimation: in fermentation process, with the extinction at spectrophotometric determination 600nm after fermentation liquid is suitably diluted
Angle value OD600, to characterize thalli growth situation.
Residual glucose: fermentation liquid is centrifuged 2min in 13000r/min at room temperature, takes 10 μ L of supernatant to be added to 990 μ L and goes
It is mixed well in ionized water, then takes 25 μ L to measure its residual sugar amount using SBA-40E series of biologic sensing analysis instrument, institute's measured value is
For the residual sugar amount in fermentation liquid, unit g/L.
Pyrimidine nucleoside concentration mensuration: the 100 μ L of fermented liquid supernatant liquid after taking centrifugation is added in 900 μ L deionized waters, with
1g/L standard sample is reference and makees identical dilution processing, and whole samples are measured after 0.45 μm of membrane filtration using HPLC
Pyrimidine nucleoside yield in fermentation liquid.The chromatographic column used is Phenomenex Gemini 5u C18 110A 150*4.6mm, column
30 DEG C of temperature, mobile phase is acetonitrile: water=2:98, flow velocity 1mL/min, each 10 μ L of sample feeding, analysis time 12min.
Fig. 2 is the result of shaking flask screening.
Is verified in the fermentation of 6.5L fermentor
2 mutant strain A260 of embodiment detects the tolerance of different structure analog
(1) detection method: taking 10 microlitres of bacterium solutions to meet LB from guarantor's tube and shake pipe, 37 DEG C, 200rpm, cultivate 10-12 hours,
It is collected by centrifugation cultured thallus, after sterile saline is washed 2-3 times, with 100,000 times of sterile saline gradient dilution,
Then 100 microlitres of bacterium solutions are taken to be respectively coated on containing on various concentration analogue minimal medium plate, each concentration three
It is a parallel, it compares as minimal medium plate (without analogue).37 DEG C culture for 24 hours after, to plate chief clump count into
Row statistics, the height of how much reflection tolerances of clump count.It can be grown in high concentration resistant panel in certain time a large amount of
Bacterium colony illustrates that this bacterial strain is strong to the tolerance of the analogue.
Minimal medium (g/L): ammonium sulfate 3, dipotassium hydrogen phosphate 1, hydrolyzed casein 3, VB1、VB3、VB5、VB13, VH it is each
0.1mg, MgSO4·7H2O 0.1, ZnSO4·7H2O、MnSO4·7H2Each 2mg of O, it is tryptophan, threonine, methionine, different bright
Each 0.1g of propylhomoserin, glucose 10, agar 20, wherein glucose is configured to the solution of 800g/L, and 0.075MPa, 15min sterilize,
Remaining 0.1MPa, 20min sterilizing.
(2) testing result:
Tolerance testing result of 1: the two plant of bacterium of table to different pyrimidine nucleoside analogues
A260 is respectively increased the tolerance of 2- thiouracil compared with starting strain TD131 from the point of view of testing result
About 23.3 times；About 30 times are improved to the tolerance of 6- azauracil；The tolerance of 5 FU 5 fluorouracil is improved
About 200 times.
The zymotechnique of embodiment 3 mutant strain A260 coproduction uridine and 3-hydroxy-2-butanone
(1) culture medium:
Activated inclined plane culture medium (g/L): glucose 1, peptone 10, beef extract 10, yeast powder 5, NaCl 2.5, agar
20, pH 6.7~7.0, sterilising conditions: 121 DEG C, 20min
Seed culture medium (g/L): glucose 40, yeast powder 5, peptone 10, sodium nitrate 15, potassium dihydrogen phosphate 1, phosphoric acid hydrogen
Dipotassium 5.2, magnesium sulfate 1, sodium citrate 1, pH 6.7~7.0, sterilising conditions: 115 DEG C, 15min.
Fermentation medium: yeast powder 5g/L, (NH4)2SO45g/L, KH2PO41g/L, K2HPO45.2g/L, sodium citrate
10g/L, sodium glutamate 20g/L, corn pulp 20mL/L, CaCl21g/L, urea 20g/L, MgSO4·7H2O 5g/L, MnSO4
0.02g/L, ZnSO40.02g/L；6.7~7.0,115 DEG C of pH, sterilize 15min；
The another mixed aqueous solution and 200mL soybean meal hydrolysate for preparing 1L and containing 80% glycerol and 6% yeast powder, dregs of beans hydrolysis
Liquid is commercial product, sterilising conditions: 115 DEG C, 15min, is added for stream in fermentation process, sterilising conditions: 115 DEG C, 15min.
(2) cultural method:
Inclined-plane culture: taking -80 DEG C of preservation of bacteria strain streak inoculations in activated inclined plane, 37 DEG C of culture 12h, and passes on primary.
Seed culture: a ring inclined-plane seed is scraped with oese and is inoculated in the 1L triangular flask equipped with 100mL seed culture medium
In, nine layers of gauze sealing, 37 DEG C, 200r/min shaken cultivation 6h.
Fermented and cultured: using flow feeding technique in batches, just constant volume 3L, inoculum concentration 10%, cultivation temperature are fermentor
36 DEG C, pH 7.0, glycerol adding yeast powder mixed aqueous solution is flowed in opposite dissolved oxygen control in 10-30%, fermentation process, and glycerol contains
Amount control was fermented in 5-15g/L with (115 DEG C, the 15min sterilization treatment) adjustment of 0.5M sodium hydroxide solution and soybean meal hydrolysate
Journey pH value, fermentation period are 60 hours.
(3) analysis method:
PH measurement: pH 6.4-8.0 accurate pH test paper and fermentor pH determination of electrode are used；
Biomass estimation: in fermentation process, with the extinction at spectrophotometric determination 600nm after fermentation liquid is suitably diluted
Angle value OD600, to characterize thalli growth situation；
Uridine assay: the fermented liquid supernatant liquid after taking centrifugation, after diluting suitable multiple with deionized water, with 0.22 μm
Membrane filtration, using pyrimidine nucleoside yield in HPLC method measurement fermentation liquid.The chromatographic column used is Phenomenex Gemini
5u C18 110A 150*4.6mm, 30 DEG C of column temperature, mobile phase is 2% (v/v) acetonitrile solution, flow velocity 1mL/min, each sample
10 μ L of sample introduction, the detection of detector UV detector, Detection wavelength 270nm.
3-hydroxy-2-butanone assay: the fermented liquid supernatant liquid after taking centrifugation, after diluting suitable multiple with deionized water, with 0.22
μm membrane filtration, using 3-hydroxy-2-butanone content in HPLC method measurement fermentation liquid.The chromatographic column used for Bole Aminex HPX-87H,
30 DEG C of column temperature, mobile phase is 5mM sulfuric acid solution, and flow velocity 0.5mL/min, sample injection volume is 20 μ L, and detector is to show poor inspection
Survey device.
(4) fermentation results are shown in attached drawing 3
After fermentation, the uridine yield in fermentation liquid reaches 32g/L, and 3-hydroxy-2-butanone yield reaches 65g/L.
The zymotechnique of embodiment 4 mutant strain A260 coproduction uridine and 3-hydroxy-2-butanone
Other conditions with embodiment 3,
Fermented and cultured: using flow feeding technique in batches, just constant volume 3L, inoculum concentration 15%, cultivation temperature are fermentor
34 DEG C, pH 7.0, glycerol adding yeast powder mixed aqueous solution is flowed in opposite dissolved oxygen control in 10-30%, fermentation process, and glycerol contains
Amount control was fermented in 5-15g/L with (115 DEG C, the 15min sterilization treatment) adjustment of 0.5M sodium hydroxide solution and soybean meal hydrolysate
Journey pH value, fermentation period are 52 hours.
After fermentation, the uridine yield in fermentation liquid reaches 27g/L, and 3-hydroxy-2-butanone yield reaches 55g/L.
Claims (5)
1. a kind of fermentation method for producing of coproduction uridine and 3-hydroxy-2-butanone, which is characterized in that production strain is through inclined-plane culture and seed
Enter fermented and cultured after culture, fermented and cultured is using flow feeding technique, inoculum concentration 10%-15%, cultivation temperature are in batches
34-38 DEG C, pH 6.7-7.0, it is water-soluble that the mixing of glycerol adding yeast powder is flowed in opposite dissolved oxygen control in 10%-30%, fermentation process
Liquid, in 5-15g/L, fermentation period is 52-60 hours for the content control of glycerol；
The production strain is bacillus subtilis (Bacillus subtilis) A260, deposit number CGMCC
No.11775。
2. a kind of fermentation method for producing of coproduction uridine and 3-hydroxy-2-butanone as described in claim 1, which is characterized in that the glycerol
Yeast powder mixed aqueous solution is the mixed aqueous solution of glycerol containing 800g/L and 60g/L yeast powder.
3. a kind of fermentation method for producing of coproduction uridine and 3-hydroxy-2-butanone as described in claim 1, which is characterized in that the pH is logical
It crosses 0.5M sodium hydroxide solution and soybean meal hydrolysate is adjusted.
4. a kind of fermentation method for producing of coproduction uridine and 3-hydroxy-2-butanone as described in claim 1, which is characterized in that the fermentation
The culture medium of culture forms are as follows: yeast powder 5g/L, (NH4)2SO45g/L, KH2PO41g/L, K2HPO45.2g/L, sodium citrate
10g/L, sodium glutamate 20g/L, corn pulp 20mL/L, CaCl21g/L, urea 20g/L, MgSO4·7H2O 5g/L, MnSO4
0.02g/L, ZnSO40.02g/L；PH 6.7~7.0 sterilizes 115 DEG C, 15min.
5. a kind of fermentation method for producing of coproduction uridine and 3-hydroxy-2-butanone as described in claim 1, which is characterized in that fermentation ends
Uridine 25-32g/L, 3-hydroxy-2-butanone 50-65g/L can be accumulated in post-fermentation liquid；Specific production method is as follows:
It producing strain and enters fermented and cultured after inclined-plane culture and seed culture, fermented and cultured uses flow feeding technique in batches,
Inoculum concentration is 10%-15%, and cultivation temperature is 34-38 DEG C, pH 6.7-7.0, and opposite dissolved oxygen is controlled in 10%-30%, is fermented
Glycerol adding yeast powder mixed aqueous solution is flowed in journey, in 5-15g/L, fermentation period is 52-60 hours for the content control of glycerol；
The glycerol yeast powder mixed aqueous solution is the mixed aqueous solution of glycerol containing 800g/L and 60g/L yeast powder；
The pH is adjusted by 0.5M sodium hydroxide solution and soybean meal hydrolysate；
The culture medium of the fermented and cultured forms are as follows: yeast powder 5g/L, (NH4)2SO45g/L, KH2PO41g/L, K2HPO4
5.2g/L, sodium citrate 10g/L, sodium glutamate 20g/L, corn pulp 20mL/L, CaCl21g/L, urea 20g/L, MgSO4·
7H2O 5g/L, MnSO40.02g/L, ZnSO40.02g/L；6.7~7.0,115 DEG C of pH, sterilize 15min.
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| CN101974444A (en) * | 2010-05-13 | 2011-02-16 | 南京工业大学 | Bacillus subtilis capable of producing D-ribose with high yield and co-producing acetoin |
| CN102634563A (en) * | 2011-10-19 | 2012-08-15 | 江南大学 | Screening method of strain capable of producing acetoin and acetoin production method based on strain fermenting method |
| CN102876760A (en) * | 2012-10-22 | 2013-01-16 | 南京工业大学 | Application of Bacillus subtilis in high-yield adenosine and acetoin co-production |
| CN104630200A (en) * | 2013-11-06 | 2015-05-20 | 洛阳德胜生物科技有限公司 | Bacterial strain mutagenesis cultivation in membrane bioreactor and adenosine fermentation method |
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| CN101974444A (en) * | 2010-05-13 | 2011-02-16 | 南京工业大学 | Bacillus subtilis capable of producing D-ribose with high yield and co-producing acetoin |
| CN102634563A (en) * | 2011-10-19 | 2012-08-15 | 江南大学 | Screening method of strain capable of producing acetoin and acetoin production method based on strain fermenting method |
| CN102876760A (en) * | 2012-10-22 | 2013-01-16 | 南京工业大学 | Application of Bacillus subtilis in high-yield adenosine and acetoin co-production |
| CN104630200A (en) * | 2013-11-06 | 2015-05-20 | 洛阳德胜生物科技有限公司 | Bacterial strain mutagenesis cultivation in membrane bioreactor and adenosine fermentation method |
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| 高产乙偶姻枯草芽孢杆菌的代谢工程改造;张显;《中国博士学位论文全文数据库（电子期刊）基础科学辑》;20140515;A006-72 * |
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