CN104372036B - A kind of method that fermenting and producing thick grass acid yield is improved using sorbierite as carbon source - Google Patents
A kind of method that fermenting and producing thick grass acid yield is improved using sorbierite as carbon source Download PDFInfo
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Abstract
The invention discloses a kind of method that fermenting and producing thick grass acid yield is improved using sorbierite as carbon source.It discloses a kind of fermentation medium for producing shikimic acid, the culture medium includes carbon source, and described carbon source is 0.1~5% sorbierite, and described percentage is the grams of contained sorbierite in every 100 milliliters of fermentation mediums.Using sorbierite as carbon source in the fermentation medium for producing shikimic acid of the present invention, it significantly improves the yield that strain fermentation produces shikimic acid, and its yield is more traditional to be improved using glucose as carbon source to 750%.The technique of the present invention is easy and effective, low in raw material price, in the industrialization production that can be applied to biosynthesis shikimic acid, and the process optimization for producing metabolite to others with Escherichia coli fermentation has positive directive function.
Description
Technical field
The present invention relates to a kind of method that fermenting and producing thick grass acid yield is improved using sorbierite as carbon source.
Background technology
Shikimic acid（Shikimic acid）, chemical entitled [3R- (3 α, 4 α, 5 β)] -3,4,5- trihydroxy -1- cyclohexene -
1- carboxylic acids, molecular formula is C7H10O5, relative molecular weight is 174.15.
The H7N9 hypotypes found in first Chinese with influenza virus H 5 N 1 and H1N1 hypotypes and 2013 it is continuous quick-fried
Hair, the patent medicine " Tamiflu " of Roche Holding Ag（Oseltamivir phosphate, Oseltamivir）Fall over each other the first choice of reserve as countries in the world
Anti-influenza virus medicament.The crucial chiral starting materials synthesized as " Tamiflu ", shikimic acid also causes the extensive concern of people.Mesh
Before, it is anistree that the shikimic acid needed for synthesis " Tamiflu " largely comes from Magnoliacea plant（Illicium verum）, but utilize eight
Shikimic acid is extracted at angle, and complex manufacturing, yield is relatively low, and raw material is easily limited by factors such as seasonal climates, it is difficult to meet extensive
Needed for production, and largely felling plant also result in significant damage to environment.And there is pole using micro-organisms shikimic acid
Wide application prospect, in research in recent years, focus is increasingly becoming using Escherichia coli fermentation production shikimic acid.
However, the efficiency that this area produces shikimic acid currently with Escherichia coli is not still high, it is difficult to meet market to big
The demand of oxalic acid, therefore, it is necessary to be improved by certain technology to fermentation process, to improve fermenting and producing shikimic acid
Yield.
The content of the invention
The technical problems to be solved by the invention are the inefficient present situations for current fermenting and producing shikimic acid, and are carried
For a kind of method that fermenting and producing thick grass acid yield is improved using sorbierite as carbon source.Current this area, which yet there are no, utilizes sorb
Alcohol improves the report of fermenting and producing thick grass acid yield as carbon source, the invention belongs to pioneering, the present invention by by carbon source by common
Glucose be changed to sorbierite, the growth performance of bacterial strain is improved, finally making the yield of shikimic acid has unexpectedly
Greatly improve.
One of technical scheme that the present invention is provided is：A kind of fermentation medium for producing shikimic acid, it includes carbon source, described
Carbon source be 0.1~5% sorbierite, described percentage is gram of contained sorbierite in every 100 milliliters of fermentation mediums
Number, such as 1% represents the quality of sorbierite in every 100 milliliters of fermentation mediums for 1 gram.
In the present invention, the fermentation medium that described fermentation medium also includes the conventional production shikimic acid in this area is wrapped
The component contained, i.e. described fermentation medium also include the nitrogen that the fermentation medium of the conventional production shikimic acid in this area is included
The components such as source, inorganic salts.
It is preferred that described fermentation medium also includes 1~5% nitrogen source and 0.1~5% inorganic salts, described nitrogen source is selected from
Soy peptone, tryptone, dusty yeast, (NH4)2SO4、NH4NO3And KNO3In one or more, described inorganic salts choosing
From MgSO4、K2HPO4、KH2PO4、Na2HPO4、MnCl2、CuSO4With the one or more in NaCl, described percentage is every
The grams of contained each component in 100 milliliters of fermentation mediums.
Wherein it is preferred to, the addition of the soy peptone is 0.1~2%, and the addition of the tryptone is 0.1
~2%, the addition of the dusty yeast is 0.1~the 2%, (NH4)2SO4Addition be 0.1~3%, the NH4NO3Add
Dosage is 0.1~3%, the KNO3Addition be 0.1~3%, the MgSO4Addition be 0.1~0.6%, it is described
K2HPO4Addition be 0.1~0.7%, the KH2PO4Addition be 0.05~0.7%, the Na2HPO4Addition be
0.05~0.6%, the MnCl2Addition be 0.05~0.6%, the CuSO4Addition be 0.1~0.6%, the NaCl
Addition be 0.05~0.6%, described percentage is the grams of contained each component in every 100 milliliters of fermentation mediums.
Described nitrogen source more preferably soy peptone and/or NH4NO3, most preferably soy peptone；Described inorganic salts are more
It is preferred that MgSO4、K2HPO4、KH2PO4With NaCl combination.
In the present invention, described fermentation medium can also making an addition in fermentation medium including this area routine
Organic acid.
It is preferred that described fermentation medium also includes one kind in tyrosine, citric acid, phenylalanine and sodium glutamate
Or it is a variety of, tyrosine and citric acid are preferably included, the addition of the tyrosine is 0.05~2%, the addition of the citric acid
For 0.01~0.2%, the addition of the phenylalanine is 0.05~0.2%, the addition of the sodium glutamate for 0.05~
0.5%, described percentage is the grams of contained each component in every 100 milliliters of fermentation mediums.
Preferably, described fermentation medium includes 0.1~5% sorbierite, 0.1~2% soy peptone, 0.1 to the present invention
~3%NH4NO3, 0.1~0.6%MgSO4, 0.05~0.5% tyrosine, 0.05~0.15% citric acid, 0.1~0.7%K2HPO4、
0.05~0.7%KH2PO4With 0.05~0.6%NaCl, described percentage is each contained by every 100 milliliters of fermentation mediums
The grams of component.
It is further preferred that described fermentation medium includes 0.1~5% sorbierite, 0.5% soy peptone, 0.6%
NH4NO3、0.2%MgSO4, 0.1% tyrosine, 0.1% citric acid, 0.3%K2HPO4、0.1%KH2PO4And 0.1%NaCl, described hundred
Divide than the grams for each component contained in every 100 milliliters of fermentation mediums.
Most preferably, described fermentation medium includes 2% sorbierite, 0.5% soy peptone, 0.6%NH4NO3、0.2%
MgSO4, 0.1% tyrosine, 0.1% citric acid, 0.3%K2HPO4、0.1%KH2PO4And 0.1%NaCl, described percentage is every 100
The grams of contained each component in milliliter fermentation medium.
The two of the technical scheme that the present invention is provided are：It is a kind of to add the side that sorbierite improves fermenting and producing thick grass acid yield
Method, it includes step：With foregoing fermentation medium culture Escherichia coli, you can.
In the present invention, described Escherichia coli are conventional described for this area, if its can fermenting and producing shikimic acid,
Such as E.coli BL21, E.coli JW0379-1, the present invention preferably E. coli JW0379-1.
On the basis of common sense in the field is met, above-mentioned each optimum condition can be combined, and produce each preferable reality of the present invention
Example.
Agents useful for same and raw material of the present invention are commercially available in addition to special instruction.
The positive effect of the present invention is：
The fermentation medium for being used to produce shikimic acid of the present invention is using sorbierite as carbon source, and it significantly improves bacterial strain hair
Ferment produces the yield of shikimic acid, and its yield is more traditional to be improved using glucose as carbon source to 750%.The technique of the present invention is simple
Effectively, in low in raw material price, the industrialization production that can be applied to biosynthesis shikimic acid, and others are sent out with Escherichia coli
The process optimization of ferment production metabolite has positive directive function.
Brief description of the drawings
Fig. 1 is the measurement result of thick grass acid content in comparative example 1 and the tunning of embodiment 1~7, wherein 0 is comparative example
1,1~7 be respectively embodiment 1~7.
Embodiment
The present invention is further illustrated below by the mode of embodiment, but does not therefore limit the present invention to described reality
Apply among a scope.The experimental method of unreceipted actual conditions in the following example, conventionally and condition, or according to business
Product specification is selected.
Bacterial strain used is recombination bacillus coli JW0379-1 in following embodiments（Baba T,Ara T,Hasegawa M,
et al.Construction of Escherichia coli K-12in-frame,single-gene knockout
mutants:the Keio collection.Mol Syst Biol2006.2:1-11）, its genotype is F-, Δ (araD-
AraB) 567, Δ lacZ4787（::rrnB-3）, Δ aroL753::Kan, λ-, rph-1, Δ (rhaD-rhaB) 568,
hsdR514.Recombination bacillus coli JW0379-1 bacterial strains are by the present inventor in January, 2010 from E.coli Genetic Stock
Center（New Haven,USA）Purchase is obtained.
In following embodiments：
Fermentation medium：Specifically as described in each embodiment and comparative example 1, in addition to fermentation medium is different, each embodiment
With remaining condition all same of comparative example 1.
Seed culture medium：Each embodiment and comparative example all same.Consisting of：Yeast extract 0.5%, tryptone 1%,
NaCl1%（Luria-Bertani culture mediums, reference《Molecular Cloning:A Laboratory guide》The third edition,（J. Pehanorm Brooker D.W. draws plug
You write））.
Condition of culture：Each embodiment and comparative example all same.The strain of glycerol tube preservation is inoculated into equipped with 20mL/
250mL seed culture mediums（Contain 25mg/L kanamycins）Triangular flask in, will in 37 DEG C, 220rpm shaker overnight cultures
Seed liquor is pressed（Volume ratio）3% inoculum concentration is seeded to 20mL/250mL fermentation mediums（Contain 25mg/L kanamycins）In, 37
DEG C, on 220rpm shaking tables culture 1mL is sampled after 24 hours, 5000rpm centrifuges the survey for taking supernatant to carry out thick grass acid content for 5 minutes
It is fixed.
The assay method of thick grass acid content：Each embodiment and comparative example all same.Using what is disclosed in patent CN1351711
Periodate oxidation method is measured to the content of shikimic acid.0.25mL fermented supernatant fluids are taken to add 0.25mL periodate reagents
（0.5%H5IO6：0.5%NaIO4=1：1）, 37 DEG C are incubated addition 0.5mL Na after 0.5h2SO3With NaOH mixed solutions（0.056M
Na2SO3：1MNaOH=2：3）Terminating reaction, absorbance is measured after mixing under 382nm wavelength.According to standard items absorbance curve
Calculate the yield of contained shikimic acid in sample.
Embodiment 1
Fermentation medium：Sorbierite 0.1%, soy peptone 0.5%, NH4NO30.6%、MgSO40.2%th, tyrosine 0.1%,
Citric acid 0.1%, K2HPO40.3%、KH2PO40.1%、NaCl0.1%.Described percentage is in every 100 milliliters of fermentation medium
The grams of contained each component.
Embodiment 2
Fermentation medium：Sorbierite 0.5%, soy peptone 0.5%, NH4NO30.6%、MgSO40.2%th, tyrosine 0.1%,
Citric acid 0.1%, K2HPO40.3%、KH2PO40.1%、NaCl0.1%.Described percentage is in every 100 milliliters of fermentation medium
The grams of contained each component.
Embodiment 3
Fermentation medium：Sorbierite 1%, soy peptone 0.5%, NH4NO30.6%、MgSO40.2%th, tyrosine 0.1%, lemon
Lemon acid 0.1%, K2HPO40.3%、KH2PO40.1%、NaCl0.1%.Described percentage is institute in every 100 milliliters of fermentation mediums
The grams of each component contained.
Embodiment 4
Fermentation medium：Sorbierite 2%, soy peptone 0.5%, NH4NO30.6%、MgSO40.2%th, tyrosine 0.1%, lemon
Lemon acid 0.1%, K2HPO40.3%、KH2PO40.1%、NaCl0.1%.Described percentage is institute in every 100 milliliters of fermentation mediums
The grams of each component contained.
Embodiment 5
Fermentation medium：Sorbierite 3%, soy peptone 0.5%, NH4NO30.6%、MgSO40.2%th, tyrosine 0.1%, lemon
Lemon acid 0.1%, K2HPO40.3%、KH2PO40.1%、NaCl0.1%.Described percentage is institute in every 100 milliliters of fermentation mediums
The grams of each component contained.
Embodiment 6
Fermentation medium：Sorbierite 4%, soy peptone 0.5%, NH4NO30.6%、MgSO40.2%th, tyrosine 0.1%, lemon
Lemon acid 0.1%, K2HPO40.3%、KH2PO40.1%、NaCl0.1%.Described percentage is institute in every 100 milliliters of fermentation mediums
The grams of each component contained.
Embodiment 7
Fermentation medium：Sorbierite 5%, soy peptone 0.5%, NH4NO30.6%、MgSO40.2%th, tyrosine 0.1%, lemon
Lemon acid 0.1%, K2HPO40.3%、KH2PO40.1%、NaCl0.1%.Described percentage is institute in every 100 milliliters of fermentation mediums
The grams of each component contained.
Embodiment 8
Fermentation medium：Sorbierite 2%, soy peptone 0.5%, NH4NO30.6%、MgSO40.05%th, tyrosine 1.0%, lemon
Lemon acid 0.1%, K2HPO40.02%、NaCl0.03%.Described percentage is each group contained in every 100 milliliters of fermentation mediums
The grams divided.
Embodiment 9
Fermentation medium：Sorbierite 2%, soy peptone 0.5%, NH4NO30.6%、MgSO40.2%th, tyrosine 0.05%, lemon
Lemon acid 0.01%, K2HPO40.3%、KH2PO40.1%、NaCl0.1%.Described percentage is institute in every 100 milliliters of fermentation mediums
The grams of each component contained.
Embodiment 10
Fermentation medium：Sorbierite 2%, soy peptone 0.5%, NH4NO30.6%、MgSO40.2%th, tyrosine 2.0%, lemon
Lemon acid 0.2%, K2HPO40.3%、KH2PO40.1%、NaCl0.1%.Described percentage is institute in every 100 milliliters of fermentation mediums
The grams of each component contained.
Embodiment 11
Fermentation medium：Sorbierite 2%, soy peptone 0.5%, NH4NO30.6%、MgSO40.2%th, tyrosine 0.05%, lemon
Lemon acid 0.1%, K2HPO40.3%、KH2PO40.1%、NaCl0.05%.Described percentage is institute in every 100 milliliters of fermentation mediums
The grams of each component contained.
Embodiment 12
Fermentation medium：Sorbierite 2%, soy peptone 0.5%, NH4NO30.6%、MgSO40.2%th, tyrosine 0.1%, lemon
Lemon acid 0.1%, K2HPO40.3%、KH2PO40.1%、NaCl0.6%.Described percentage is institute in every 100 milliliters of fermentation mediums
The grams of each component contained.
Embodiment 13
Fermentation medium：Sorbierite 2%, soy peptone 0.1%, NH4NO33%、MgSO40.2%th, tyrosine 0.5%, lemon
Acid 0.1%, K2HPO40.7%、KH2PO40.7%、NaCl0.1%.Described percentage is contained in every 100 milliliters of fermentation mediums
The grams of some each components.
Embodiment 14
Fermentation medium：Sorbierite 2%, soy peptone 2%, NH4NO30.1%、MgSO40.2%th, tyrosine 0.1%, lemon
Acid 0.15%, K2HPO40.3%、KH2PO40.05%、NaCl0.1%.Described percentage is institute in every 100 milliliters of fermentation mediums
The grams of each component contained.
Embodiment 15
Fermentation medium：Sorbierite 2%, soy peptone 0.5%, NH4NO30.5%、MgSO40.2%th, tyrosine 0.1%, lemon
Lemon acid 0.05%, K2HPO40.1%、KH2PO40.1%、NaCl0.1%.Described percentage is institute in every 100 milliliters of fermentation mediums
The grams of each component contained.
Embodiment 16
Fermentation medium：Sorbierite 2%, soy peptone 0.5%, NH4NO30.6%、MgSO41%th, tyrosine 0.1%, lemon
Acid 0.1%, K2HPO41%、KH2PO41%、NaCl2%.Described percentage is each contained by every 100 milliliters of fermentation mediums
The grams of component.
Embodiment 17
Fermentation medium：Sorbierite 2%, soy peptone 0.5%, MgSO40.2%th, tyrosine 0.1%, citric acid 0.1%,
K2HPO40.3%、KH2PO40.1%、NaCl0.1%.Described percentage is each group contained in every 100 milliliters of fermentation mediums
The grams divided.
Embodiment 18
Fermentation medium：Sorbierite 2%, tryptone 0.5%, NH4NO30.6%、MgSO40.2%th, tyrosine 0.1%, lemon
Acid 0.1%, K2HPO40.3%、KH2PO40.1%、NaCl0.1%.Described percentage is contained in every 100 milliliters of fermentation mediums
The grams of some each components.
Embodiment 19
Fermentation medium：Sorbierite 2%, tryptone 0.1%, NH4NO30.6%、MgSO40.2%th, tyrosine 0.1%, lemon
Acid 0.1%, K2HPO40.3%、KH2PO40.1%、NaCl0.1%.Described percentage is contained in every 100 milliliters of fermentation mediums
The grams of some each components.
Embodiment 20
Fermentation medium：Sorbierite 2%, tryptone 2%, NH4NO30.6%、MgSO40.2%th, tyrosine 0.1%, citric acid
0.1%、K2HPO40.3%、KH2PO40.1%、NaCl0.1%.Described percentage is contained in every 100 milliliters of fermentation mediums
Each component grams.
Embodiment 21
Fermentation medium：Sorbierite 2%, dusty yeast 0.5%, NH4NO30.6%、MgSO40.2%th, tyrosine 0.1%, citric acid
0.1%、K2HPO40.3%、KH2PO40.1%、NaCl0.1%.Described percentage is contained in every 100 milliliters of fermentation mediums
Each component grams.
Embodiment 22
Fermentation medium：Sorbierite 2%, dusty yeast 0.1%, NH4NO30.6%、MgSO40.2%th, tyrosine 0.1%, citric acid
0.1%、K2HPO40.3%、KH2PO40.1%、NaCl0.1%.Described percentage is contained in every 100 milliliters of fermentation mediums
Each component grams.
Embodiment 23
Fermentation medium：Sorbierite 2%, dusty yeast 2%, NH4NO30.6%、MgSO40.2%th, tyrosine 0.1%, citric acid
0.1%、K2HPO40.3%、KH2PO40.1%、NaCl0.1%.Described percentage is contained in every 100 milliliters of fermentation mediums
Each component grams.
Embodiment 24
Fermentation medium：Sorbierite 2%, NH4NO30.6%、MgSO40.2%th, tyrosine 0.1%, citric acid 0.1%,
K2HPO40.3%、KH2PO40.1%、NaCl0.1%.Described percentage is each group contained in every 100 milliliters of fermentation mediums
The grams divided.
Embodiment 25
Fermentation medium：Sorbierite 2%, (NH4)2SO40.6%、MgSO40.2%th, tyrosine 0.1%, citric acid 0.1%,
K2HPO40.3%、KH2PO40.1%、NaCl0.1%.Described percentage is each group contained in every 100 milliliters of fermentation mediums
The grams divided.
Embodiment 26
Fermentation medium：Sorbierite 2%, KNO30.1%、MgSO40.2%th, tyrosine 0.1%, citric acid 0.1%,
K2HPO40.3%、KH2PO40.1%、NaCl0.1%.Described percentage is each group contained in every 100 milliliters of fermentation mediums
The grams divided.
Embodiment 27
Fermentation medium：Sorbierite 2%, (NH4)2SO43%、MgSO40.2%th, tyrosine 0.1%, citric acid 0.1%,
K2HPO40.3%、KH2PO40.1%、NaCl0.1%.Described percentage is each group contained in every 100 milliliters of fermentation mediums
The grams divided.
Embodiment 28
Fermentation medium：Sorbierite 2%, soy peptone 0.5%, NH4NO30.6%、MgSO40.2%th, citric acid 0.1%,
K2HPO40.3%、KH2PO40.1%、NaCl0.1%.Described percentage is each group contained in every 100 milliliters of fermentation mediums
The grams divided.
Embodiment 29
Fermentation medium：Sorbierite 2%, soy peptone 0.5%, NH4NO30.6%、MgSO40.2%th, tyrosine 0.1%,
K2HPO40.3%、KH2PO40.1%、NaCl0.1%.Described percentage is each group contained in every 100 milliliters of fermentation mediums
The grams divided.
Embodiment 30
Fermentation medium：Sorbierite 2%, soy peptone 0.5%, NH4NO30.6%、MgSO40.2%、K2HPO40.3%、
KH2PO40.1%、NaCl0.1%.Described percentage is the grams of contained each component in every 100 milliliters of fermentation mediums.
Embodiment 31
Fermentation medium：Sorbierite 2%, soy peptone 0.5%, NH4NO30.6%、MgSO40.2%th, tyrosine 0.1%, benzene
Alanine 0.1%, K2HPO40.3%、KH2PO40.1%、NaCl0.1%.Described percentage is in every 100 milliliters of fermentation medium
The grams of contained each component.
Embodiment 32
Fermentation medium：Sorbierite 2%, soy peptone 0.5%, NH4NO30.6%、MgSO40.2%th, tyrosine 0.1%, paddy
Propylhomoserin sodium 0.05%, K2HPO40.3%、KH2PO40.1%、NaCl0.1%.Described percentage is in every 100 milliliters of fermentation medium
The grams of contained each component.
Embodiment 33
Fermentation medium：Sorbierite 2%, soy peptone 0.5%, NH4NO30.6%、MgSO40.2%th, tyrosine 0.1%, paddy
Propylhomoserin sodium 0.2%, K2HPO40.3%、KH2PO40.1%、NaCl0.1%.Described percentage is in every 100 milliliters of fermentation medium
The grams of contained each component.
Embodiment 34
Fermentation medium：Sorbierite 2%, soy peptone 0.5%, NH4NO30.6%、MgSO40.2%th, tyrosine 0.1%, lemon
Lemon acid 0.1%, Na2HPO40.3%、KH2PO40.1%.Described percentage is each contained by every 100 milliliters of fermentation mediums
The grams of component.
Embodiment 35
Fermentation medium：Sorbierite 2%, soy peptone 0.5%, NH4NO30.6%、CuSO40.2%th, tyrosine 0.1%, lemon
Lemon acid 0.1%, K2HPO40.3%、KH2PO40.1%、NaCl0.1%.Described percentage is institute in every 100 milliliters of fermentation mediums
The grams of each component contained.
Embodiment 36
Fermentation medium：Sorbierite 2%, soy peptone 0.5%, NH4NO30.6%、MgSO40.2%th, tyrosine 0.1%, lemon
Lemon acid 0.1%, K2HPO40.3%、MnCl20.1%、NaCl0.1%.Described percentage is contained in every 100 milliliters of fermentation mediums
The grams of some each components.
Embodiment 37
Fermentation medium：Sorbierite 2%, soy peptone 0.5%, NH4NO30.6%、MgSO40.2%th, tyrosine 0.1%, lemon
Lemon acid 0.1%, K2HPO40.3%、CuSO40.05%、NaCl0.1%.Described percentage is institute in every 100 milliliters of fermentation mediums
The grams of each component contained.Embodiment 28
Embodiment 38
Fermentation medium：Sorbierite 2%, soy peptone 0.5%, NH4NO30.6%、MgSO40.2%th, tyrosine 0.1%, lemon
Lemon acid 0.1%, K2HPO40.3%、CuSO40.6%、NaCl0.1%.Described percentage is contained in every 100 milliliters of fermentation mediums
The grams of some each components.Embodiment 29
Embodiment 39
Fermentation medium：Sorbierite 2%, soy peptone 0.5%, NH4NO30.6%th, tyrosine 0.1%, citric acid 0.1%,
NaCl0.1%.Described percentage is the grams of contained each component in every 100 milliliters of fermentation mediums.
Embodiment 40
Fermentation medium：Sorbierite 2%, soy peptone 0.5%, NH4NO30.6%、MgSO40.2%th, tyrosine 0.1%, lemon
Lemon acid 0.1%.Described percentage is the grams of contained each component in every 100 milliliters of fermentation mediums.
Embodiment 41
Fermentation medium：Sorbierite 2%, soy peptone 0.5%, NH4NO30.6%、MgSO40.2%th, tyrosine 0.1%, lemon
Lemon acid 0.1%, NaCl0.1%.Described percentage is the grams of contained each component in every 100 milliliters of fermentation mediums.
Comparative example 1（In addition to carbon source is glucose, remaining component is same as Example 4）
Fermentation medium：Glucose 2%, soy peptone 0.5%, NH4NO30.6%、MgSO40.2%th, tyrosine 0.1%, lemon
Lemon acid 0.1%, K2HPO40.3%, KH2PO40.1%, NaCl0.1%.Described percentage is institute in every 100 milliliters of fermentation mediums
The grams of each component contained.
Yield based on the fermentation method production shikimic acid under the technique of comparative example 1 is set to 100%, other embodiment is used as
Reference.
The detection of thick grass acid content is carried out to the tunning of embodiment 1~31 and comparative example 1, as a result such as table 1 and Fig. 1 institutes
Show.It will be seen from figure 1 that fermentation medium is using sorbierite as carbon source, compared to comparative example 1（Traditional cultural method is with grape
Sugar is used as carbon source）The ability of bacterial strain uses therefor production shikimic acid, which has, to be obviously improved.When sorbitol content is 2%, its yield is compared with Portugal
Grape sugar is improved to 750% as carbon source, and when sorbierite addition is more than 2%, thick grass acid yield no longer increases.If in view of culture
The Cost Problems of base, then 2% sorbierite is the optimum addition of culture medium of the present invention.
The measurement result of shikimic acid relative amount in each embodiment of table 1 and comparative example tunning
| Numbering | Shikimic acid relative amount | Numbering | Shikimic acid relative amount |
| Embodiment 1 | 148% | Embodiment 22 | 114% |
| Embodiment 2 | 232% | Embodiment 23 | 336% |
| Embodiment 3 | 397% | Embodiment 24 | 134% |
| Embodiment 4 | 750% | Embodiment 25 | 132% |
| Embodiment 5 | 747% | Embodiment 26 | 107% |
| Embodiment 6 | 745% | Embodiment 27 | 129% |
| Embodiment 7 | 746% | Embodiment 28 | 703% |
| Embodiment 8 | 740% | Embodiment 29 | 708% |
| Embodiment 9 | 733% | Embodiment 30 | 643% |
| Embodiment 10 | 645% | Embodiment 31 | 584% |
| Embodiment 11 | 725% | Embodiment 32 | 528% |
| Embodiment 12 | 746% | Embodiment 33 | 575% |
| Embodiment 13 | 258% | Embodiment 34 | 732% |
| Embodiment 14 | 374% | Embodiment 35 | 728% |
| Embodiment 15 | 637% | Embodiment 36 | 730% |
| Embodiment 16 | 677% | Embodiment 37 | 723% |
| Embodiment 17 | 702% | Embodiment 38 | 724% |
| Embodiment 18 | 573% | Embodiment 39 | 683% |
| Embodiment 19 | 232% | Embodiment 40 | 645% |
| Embodiment 20 | 315% | Embodiment 41 | 704% |
| Embodiment 21 | 732% | Comparative example 1 | 100% |
Claims (9)
1. a kind of fermentation medium for producing shikimic acid, it includes carbon source, it is characterised in that described carbon source is 0.1~2% mountain
Pears alcohol, described percentage is the grams of contained sorbierite in every 100 milliliters of fermentation mediums.
2. fermentation medium as claimed in claim 1, it is characterised in that described fermentation medium also includes 1~5% nitrogen source
With 0.1~5% inorganic salts, described nitrogen source is selected from soy peptone, tryptone, dusty yeast, (NH4)2SO4、NH4NO3With
KNO3In one or more, described inorganic salts are selected from MgSO4、K2HPO4、KH2PO4、Na2HPO4、MnCl2、CuSO4And NaCl
In one or more, described percentage is the grams of contained each component in every 100 milliliters of fermentation mediums.
3. fermentation medium as claimed in claim 2, it is characterised in that the addition of the soy peptone is 0.1~
2%, the addition of the tryptone is 0.1~2%, and the addition of the dusty yeast is 0.1~the 2%, (NH4)2SO4
Addition be 0.1~3%, the NH4NO3Addition be 0.1~3%, the KNO3Addition be 0.1~3%, institute
State MgSO4Addition be 0.1~0.6%, the K2HPO4Addition be 0.1~0.7%, the KH2PO4Addition be
0.05~0.7%, the Na2HPO4Addition be 0.05~0.6%, the MnCl2Addition be 0.05~0.6%, institute
State CuSO4Addition be 0.1~0.6%, the addition of the NaCl is 0.05~0.6%, and described percentage is every 100
The grams of contained each component in milliliter fermentation medium.
4. fermentation medium as claimed in claim 3, it is characterised in that described nitrogen source be soy peptone and/or
NH4NO3, described inorganic salts are MgSO4、K2HPO4、KH2PO4With NaCl combination.
5. fermentation medium as claimed in claim 3, it is characterised in that described fermentation medium also includes tyrosine, lemon
One or more in lemon acid, phenylalanine and sodium glutamate, the addition of the tyrosine is 0.05~2%, the lemon
The addition of acid is 0.01~0.2%, and the addition of the phenylalanine is 0.05~2%, the addition of the sodium glutamate
For 0.05~0.5%, described percentage is the grams of contained each component in every 100 milliliters of fermentation mediums.
6. fermentation medium as claimed in claim 5, it is characterised in that described fermentation medium includes 0.1~2% sorb
Alcohol, 0.1~2% soy peptone, 0.1~3%NH4NO3, 0.1~0.6%MgSO4, 0.05~0.5% tyrosine, 0.05~
0.15% citric acid, 0.1~0.7%K2HPO4, 0.05~0.7%KH2PO4With 0.05~0.6%NaCl, described percentage
For the grams of each component contained in every 100 milliliters of fermentation mediums.
7. fermentation medium as claimed in claim 6, it is characterised in that described fermentation medium includes 0.1~2% sorb
Alcohol, 0.5% soy peptone, 0.6%NH4NO3, 0.2%MgSO4, 0.1% tyrosine, 0.1% citric acid, 0.3%K2HPO4、
0.1%KH2PO4And 0.1%NaCl, described percentage is gram of contained each component in every 100 milliliters of fermentation mediums
Number.
8. a kind of method that fermenting and producing thick grass acid yield is improved using sorbierite as carbon source, it includes step：Will with such as right
Ask the fermentation medium culture Escherichia coli described in 1~7 any one, you can.
9. improving the method for fermenting and producing thick grass acid yield using sorbierite as carbon source as claimed in claim 8, its feature exists
In described Escherichia coli are E.coli JW0379-1.
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| CN101245357A (en) * | 2008-03-28 | 2008-08-20 | 北京化工大学 | Method for improving shikimic acid volume of production with zymotechnics |
| CN102994439A (en) * | 2012-12-12 | 2013-03-27 | 江南大学 | Escherichia coli recombinant strain producing shikimic acid, and construction method and application thereof |
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| CN101245357A (en) * | 2008-03-28 | 2008-08-20 | 北京化工大学 | Method for improving shikimic acid volume of production with zymotechnics |
| CN102994439A (en) * | 2012-12-12 | 2013-03-27 | 江南大学 | Escherichia coli recombinant strain producing shikimic acid, and construction method and application thereof |
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