CN102757999A - Fermentation medium for production of 7-aminocephalosporanic acid and fermentation method of fermentation medium - Google Patents
Fermentation medium for production of 7-aminocephalosporanic acid and fermentation method of fermentation medium Download PDFInfo
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Abstract
The invention provides a fermentation medium for production of 7-ACA (7-aminocephalosporanic acid) shown as a formula I. The fermentation medium contains, by the total volume of the fermentation medium, 4.2-1.8% (g/100mL) of carbon sources, 10-12% (g/100mL) of nitrogen sources and trace element solution; the fermentation medium contains, by the trace element solution volume, 0.2-13.2 (g/100mL) of ZnSO4 7H2O and 0.2-0 (g/100mL) of CuSO4 5H2O; metal ions include potassium, magnesium, calcium, iron and manganese, and CaCO3 accounts for 0.1-1% (g/100mL) according to the total volume of the fermentation medium; and pH (potential of hydrogen) of the fermentation medium ranges from 5.0 to 10.0.
Description
Technical field
The present invention relates to biological technical field, relate in particular to a kind of fermention medium and fermentation process thereof that is used to produce 7-amino-cephalosporanic acid (7-ACA).
Background technology
Cephalosporins has that has a broad antifungal spectrum, anti-microbial activity are strong, penicillin resistant enzyme, high, the low toxin of curative effect, is widely used clinically.Cephalosporins greatly all is to connect different side chains and the semisynthetic antibiotics that obtains from its parent nucleus 7-amino-cephalosporanic acid (7-ACA).The molecular formula of 7-ACA is C
10H
12N
2O
5S, molecular weight are 272.28, and chemical name is 3-acetyl-o-methyl-5-sulphur-7-amino-8-oxygen-1-azabicyclic oct-2-ene-2-carboxylic acid, 300 ℃ of fusing points, iso-electric point pI3.5, white crystals or crystalline powder.
Present known 7-ACA preparation method is mainly obtained by cephalosporin (CPC) with chemical method and catalyzed by biological enzyme in the industry.CPC is produced by cephalosporium acremonium (Acremonium chrysogenum).Because the environmental pollution of chemical cracking method is big, raw materials cost is high, so the production of 7-ACA is main with enzymatic conversion method.Existing catalyzed by biological enzyme comprises two step enzyme methods and a step enzyme method.Two step enzyme methods have been used for suitability for industrialized production, and it relates to the catalysis of two enzymes (D-amino-acid oxidase and glutaryl-transferring enzyme) substep.One step enzyme method forms 7-ACA with the direct catalysis CPC of cephalosporin C acylase, does not also have industriallization at present, technical being further improved.
Disclose the strain reorganization bacterium that makes up in the one Chinese patent application 200810205094.0, the cephalosporin C acylase gene has been imported in the cephalosporium acremonium, and one-step fermentation generation 7-ACA, but its fermentation level is lower, and bigger with the production level gap.Thereby the present invention mainly studies the fermentation level that how to utilize this reorganization bacterium to improve 7-ACA through the improvement zymotechnique.
The reorganization bacterium is owing to import the cephalosporin C acylase of an external source; And the fermentation condition of original cephalosporin some and be not suitable for cephalosporin C acylase and bring into play maximum enzyme work during the fermentation the cephalosporin that former fermentation produces is changed into 7-ACA; Therefore; Need carry out the optimization of fermentation condition and medium component to this bacterium of recombinating, the reorganization bacterium is fermented under the condition that is suitable for 7-ACA production, improve the fermentation level of 7-ACA.
Summary of the invention
The present invention aims to provide the fermention medium of a kind of extensive, high efficiency production suc as formula the 7-ACA shown in the I.
Another object of the present invention provides a kind of method of using above-mentioned fermention medium to produce 7-ACA.
In first aspect of the present invention, a kind of fermention medium suc as formula the 7-amino-cephalosporanic acid shown in the I (7-ACA) that is used to produce is provided, in the TV of said fermention medium, it contains:
4.2-18% (g/100mL) carbon source;
10-12% (g/100mL) nitrogenous source; With
Trace element solution;
With the volumeter of said trace element solution, wherein contain ZnSO
47H
2O 0.2-13.2 (g/100mL), CuSO
45H
2O 0.2-0 (g/100mL);
Said metals ion comprises potassium, magnesium, calcium, iron and manganese, in the TV of said fermention medium, wherein CaCO
30.1-1% (g/100mL);
Said fermention medium pH 5.0-10.0;
In the above-mentioned fermention medium, preferably, wherein contain CaCO
30.1-0.8 (g/100mL).
In the above-mentioned fermention medium, preferably, in said trace element solution, contain ZnSO
47H
2O 0.2-13.2 (g/100mL), CuSO
45H
2O 0.1-0 (g/100mL); More preferably, in said trace element solution, contain ZnSO
47H
2O 0.2-3.3 (g/100mL).
The pH 7.0-10.0 of above-mentioned fermention medium.
Above-mentioned fermention medium provided by the invention, in its TV, it contains:
4.2-18% (g/100mL) carbon source;
10-12% (g/100mL) nitrogenous source; With
Trace element solution;
With the volumeter of said trace element solution, wherein contain ZnSO
47H
2O 0.2-3.3 (g/100mL), CuSO
45H
2O 0.1-0 (g/100mL);
Said metals ion comprises potassium, magnesium, calcium, iron and manganese, in the TV of said fermention medium, wherein CaCO
30.1-0.8% (g/100mL);
Said fermention medium pH 7.0-9.6;
Said carbon source comprises starch, dextrin, glucose;
Said nitrogenous source comprises steeping water, methionine(Met), urea and (NH
4)
2SO
4
In another preference, contain 1-6% (g/100mL) starch, 3-10% (g/100mL) dextrin and 0.2-2.0% (g/100mL) glucose in the said fermention medium; Also contain 9-10% (g/100mL) steeping water and 1.0-2.0% (g/100mL) (NH in the said fermention medium
4)
2SO
4
In another preference, in the TV of said fermention medium, it contains steeping water 10% (g/100mL); Starch 3% (g/100mL), dextrin 6% (g/100mL), glucose 0.5% (g/100mL); Methionine(Met) 0.6% (g/100mL), urea 0.3% (g/100mL), KH
2PO
40.9% (g/100mL), MgSO
47H
2O 0.3% (g/100mL), (NH
4)
2SO
41.3% (g/100mL), CaCO
30.1-1% (g/100mL), trace element solution 1ml and soya-bean oil 0.4ml/20ml, pH 5.0-10.0;
With the volumeter of said trace element solution, wherein contain FeSO
47H
2O 0.8 (g/100mL), MnSO
4H
2O 0.2 (g/100mL), ZnSO
47H
2O 0.2-13.2 (g/100mL), CuSO
45H
2O 0.2-0 (g/100mL).
In another preference, in the TV of said fermention medium, it contains steeping water 10% (g/100mL); Starch 3% (g/100mL), dextrin 6% (g/100mL), glucose 0.5% (g/100mL); Methionine(Met) 0.6% (g/100mL), urea 0.3% (g/100mL), KH
2PO
40.9% (g/100mL), MgSO
47H
2O 0.3% (g/100mL), (NH
4)
2SO
41.3% (g/100mL), CaCO
30.1-0.8% (g/100mL), trace element solution 1ml and soya-bean oil 0.4ml/20ml, pH 7.0-9.6;
With the volumeter of said trace element solution, wherein contain FeSO
47H
2O 0.8w/L% (g/100mL), MnSO
4H
2O 0.2 (g/100mL), ZnSO
47H
2O 0.2-3.3 (g/100mL), CuSO
45H
2O 0-0.1 (g/100mL).
In second aspect of the present invention, a kind of purposes of aforesaid fermention medium provided by the invention is provided, be used for fermentation culture bacterial classification Acremonium chrysogenum/pYG233 and/or production suc as formula the 7-amino-cephalosporanic acid shown in the I (7-ACA).
In the third aspect of the invention, the method for a kind of production suc as formula the 7-amino-cephalosporanic acid shown in the I (7-ACA) is provided, described method comprises step:
(1) in aforesaid fermention medium provided by the invention, bacterial classification Acremonium chrysogenum/pYG233 is carried out fermentation culture; Obtain structure suc as formula the 7-amino-cephalosporanic acid shown in the I (7-ACA).
In aforesaid method, the fermentation culture temperature is 24-30 ℃, and pH 5.0-10.0, incubation time are 3-8 days; Preferably, the fermentation culture temperature is 24-28 ℃, pH 7.0-9.6, incubation time 5-8 days.
In another preference, fermentation culture is carried out in shaking table, and shaking table vibration rotating speed is 220rpm.
In view of the above, the present invention carries out the optimization of fermentation condition and medium component to the reorganization bacterium, and the reorganization bacterium is fermented under the condition that is suitable for 7-ACA production, improves the fermentation level of 7-ACA.
Embodiment
The contriver is through a series of tests and contrast; Found that disclosed bacterial classification Acremonium chrysogenum/pYG233 in a kind of available one Chinese patent application 200810205094.0 is extensive, high efficiency production is suc as formula the fermention medium of the 7-ACA shown in the I; The output of tunning 7-ACA is high; Reach 1701 μ g/mL, the substratum more of the prior art of tiring has improved 750%.
Particularly, the present invention is the bacterium that sets out with disclosed reorganization bacterial classification Acremonium chrysogenum/pYG233 among the one Chinese patent application CN200810205094.0, and said substratum is in g/100mL, wherein,
Basic medium: wort 20, SANMALT-S 4.0 and gather peptone 1, pH 7.0, add purified agar powder 2%
Seed culture medium: steeping water 6, sucrose 3.5, glucose 0.5, methionine(Met) 0.05, (NH
4)
2SO
40.8, CaCO
30.5 and soya-bean oil 0.2ml/20ml, pH 6.5
Fermention medium: steeping water 10, starch 3, dextrin 6, glucose 0.5, methionine(Met) 0.6, urea 0.3, KH
2PO
40.9, MgSO
47H
2O 0.3, (NH
4)
2SO
41.3, CaCO
3(0.1-1 preferably being 0.1-0.8), trace element solution (FeSO
47H
2O, 0.8%, MnSO
4H
2O 0.2, ZnSO
47H
2O0.2-13.2 (preferably being 0.2-3.3), CuSO
45H
2O 0.2-0 (preferably being 0.1-0)) 1ml and soya-bean oil 0.4ml/20ml, pH 6.2
In the present invention, above-mentionedly set out the bacterium culture presevation on basic medium, 4 ℃ of preservations, a switching in month is once.
Then, will preserving the inclined-plane, to be inoculated in loading amount be the 20ml seed culture medium, and 250ml shakes in the bottle, cultivated 3 days in rotary shaking table, and rotating speed is 230r/min, 28 ℃ of temperature.Being forwarded to loading amount with 10% (v/v) inoculum size again is that the 250ml of 20ml fermention medium shakes in the bottle, and 26 ℃, 220rpm cultivated 3-8 days.Regulated the fermented liquid that pH6.5-9.6 obtains containing 7-ACA in the fermenting process from 3-5 days.It is 1701 μ g/ml that HPLC detect the 7th day can obtain the concentration of 7-ACA product in fermented liquid.
The present invention also provides the method for a kind of production suc as formula the 7-ACA shown in the I, comprising step:
In above-mentioned fermention medium provided by the invention, Acremonium chrysogenum/pYG233 seed is carried out fermentation culture, obtain 7-ACA.
The above-mentioned characteristic that the present invention mentions, or the characteristic that embodiment mentions can arbitrary combination.All characteristics that this case specification sheets is disclosed can with any composition forms and usefulness, each characteristic that is disclosed in the specification sheets can anyly provide the alternative characteristics of identical, impartial or similar purpose to replace.Therefore removing has special instruction, the characteristic that is disclosed to be merely the general example of equalization or similar features.
Major advantage of the present invention is:
1, the present invention is according to the external activity characteristics of CPC acyltransferase; Optimize medium component and fermentation condition control in the reorganization bacterium fermenting process, make cephalosporin C acylase under suitable catalytic condition, CPC directly changed into the 7-ACA product greatly.
2,7-ACA fermentation method for producing technology provided by the invention is simple, with low cost, output is high.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in the restriction scope of the present invention.The experimental technique of unreceipted actual conditions in the following example is usually according to the normal condition or the condition of advising according to manufacturer.Unless otherwise indicated, otherwise all percentage ratio, ratio, ratio or umber by weight.
Unit in the percent weight in volume among the present invention is well-known to those skilled in the art, for example is meant the weight of solute in 100 milliliters solution.
Unless otherwise defined, employed all specialties are identical with the meaning that scientific words and one skilled in the art are familiar with in the literary composition.In addition, any with the institute similar content of putting down in writing or the equalization method and material all can be applicable in the inventive method.The usefulness that preferable implementation method described in the literary composition and material only present a demonstration.
The performance liquid chromatography that uses among the present invention (HPLC) method is:
Fermented liquid is collected filtrating and is directly carried out the HPLC analysis through common filter paper filtering, measures with external standard method.Employing C18 (moving phase is methyl alcohol for 5 μ m, 4.6 * 150mm) posts: 0.2% SODIUM PHOSPHATE, MONOBASIC (5: 95), and the detection wavelength is 254nm, and flow velocity 1ml/min, column temperature are 40 ℃, and sample size is 10 μ l.
In embodiments of the present invention, fermention medium is in g/100mL.
Embodiment 1
Adopt the fermentation condition that is fit to CPC production to carry out fermentation culture and produce 7-ACA
The reorganization Acremonium chrysogenum/pYG233 thalline of preserving is carried out single bacterium colony to be separated; 30 single bacterium colonies of picking carry out the inclined-plane cultivation of going down to posterity, and cultivate 7-10d for 28 ℃, and will growing good inclined-plane again, to be inoculated in loading amount be the 20ml seed culture medium; 250ml shakes in the bottle; Cultivate 3d in rotary shaking table, rotating speed is 230r/min, 28 ℃ of temperature.Being forwarded to loading amount with 10% (v/v) inoculum size again is 20ml fermention medium (steeping water 10 (g/100mL), starch 3 (g/100mL), dextrin 6 (g/100mL), glucose 0.5 (g/100mL), methionine(Met) 0.6 (g/100mL), urea 0.3 (g/100mL), KH
2PO
40.9 (g/100mL), MgSO
47H
2O 0.3 (g/100mL), (NH
4)
2SO
41.3 (g/100mL), CaCO
31 (g/100mL), trace element solution (FeSO
47H
2O, 0.8 (g/100mL), MnSO
4H
2O 0.2 (g/100mL), ZnSO
47H
2O, 0.2 (g/100mL), CuSO
45H
2O, 0.2 (g/100mL)) 1ml, soya-bean oil, 0.4ml/20ml, pH, 6.2) 250ml shake in the bottle, 25 ℃, 230rpm cultivates 7d,, obtain a strain 7-ACA fermentation yield be up to about 200 μ g/ml reorganization Acremonium chrysogenum/pYG233 bacterial strain.Afterwards this plant height is produced bacterium and carry out the pH in medium component optimization and the control fermenting process.
Embodiment 2
The CaCO of different concns in the fermention medium
3Influence to the 7-ACA fermentative prodn
It is the 20ml seed culture medium that the high yield bacterium inclined-plane of preserving is inoculated in loading amount, and the CaCO in the former fermention medium is carried out in fermentation condition and control by method in the instance 1
3Add by different concns, fermenting, HPLC detects 7-ACA concentration in the fermented liquid after seven days.The result sees table 1.
Different Ca CO in table 1 fermention medium
3Concentration is to the influence of 7-ACA output
| Instance number | CaCO 3Concentration (g/100mL) | 7-ACA output (μ g/ml) | Increase rate (%) |
| 1 | 1.0 | 200 | 100 |
| 2 | 0.8 | 243 | 121.5 |
| 3 | 0.5 | 332 | 166 |
| 4 | 0.4 | 294 | 147 |
| 5 | 0.1 | 287 | 143.5 |
Annotate: increase rate (%) is meant the per-cent of comparing raising with instance for No. 1.
Can find out that from table 1 concentration of 7-ACA in fermented liquid is along with CaCO
3CaCO is worked as in the reduction of concentration and increasing
3Concentration be reduced to 0.5g/L the time, the highest 332 μ g/ml of the output of 7-ACA work as CaCO
3Concentration be lower than 0.5g/L the time, the output of 7-ACA decreases.
Embodiment 3
The contained different Cu of trace element solution in the fermention medium
2+Ionic concn is to the influence of 7-ACA output
It is the 20ml seed culture medium that the high yield bacterium inclined-plane of preserving is inoculated in loading amount, and the CuSO of trace element solution in the former fermention medium is carried out in fermentation condition and control by method in the instance 1
45H
2O adds by different concns, and fermenting, HPLC detects 7-ACA concentration in the fermented liquid after seven days.The result sees table 2.
Different Cu in table 2 fermention medium
2+Ionic concn is to the influence of 7-ACA output
Annotate: increase rate (%) is meant the per-cent of comparing raising with instance for No. 6, and No. 6 implementation condition is identical with embodiment 1
Can find out that from table 2 concentration of 7-ACA in fermented liquid is along with Cu
2+Cu is removed in the reduction of ionic concn and increasing in fermention medium
2+During ion, the highest 293 μ g/ml of the output of 7-ACA explain CuSO
45H
2O has hindered the production of 7-ACA in fermention medium.
Embodiment 4
Different Zn in the fermention medium
2+Ionic concn is to the influence of 7-ACA output
It is the 20ml seed culture medium that the high yield bacterium inclined-plane of preserving is inoculated in loading amount, and the ZnSO of trace element solution in the former fermention medium is carried out in fermentation condition and control by method in the instance 1
47H
2O adds by different concns, and fermenting, HPLC detects 7-ACA concentration in the fermented liquid after seven days.The result sees table 3.
The different Zn of trace element solution in table 3 fermention medium
2+Ionic concn is to the influence of 7-ACA output
Annotate: increase rate (%) is meant the per-cent of comparing raising with instance for No. 9, and No. 9 implementation condition is identical with embodiment 1
The result finds out from table 3, and the concentration of 7-ACA in fermented liquid is along with Zn in the fermention medium
2+The variation of ionic concn ionic concn and changing, the Zn that adds when fermention medium
2+Ionic concn is 0.33 o'clock, the highest 328 μ g/ml of the output of 7-ACA.
Embodiment 5
Fermention medium behind the complex optimum is to the influence of 7-ACA fermentative prodn
To preserve the inclined-plane, to be inoculated in loading amount be the 20ml seed culture medium, and 250ml shakes in the bottle, cultivates 3d in rotary shaking table, and rotating speed is 230r/min, 28 ℃ of temperature.Being forwarded to loading amount with 10% (v/v) inoculum size again is 20ml fermention medium (steeping water 10, starch 3, dextrin 6, glucose 0.5, methionine(Met) 0.6, urea 0.3, KH
2PO
40.9, MgSO
47H
2O 0.3, (NH
4)
2SO
41.3, CaCO
30.5, trace element solution (FeSO
47H
2O, 0.8, MnSO
4H
2O 0.2, ZnSO
47H
2O, 0.33) 1ml, soya-bean oil, 0.4ml/20ml, pH, 6.2) 250ml shake in the bottle, 25 ℃, 230rpm cultivated 7 days,, obtain the 7-ACA fermentation yield and be respectively about 458 μ g/ml.
Find out the configuration metal ions Zn of comprehensive optimum concn by embodiment 5
2+, Cu
2+, and CaCO
3The output of reorganization bacterium fermentative prodn 7-ACA is obviously increased.
Embodiment 6
PH value in the fermenting process is to the influence of 7-ACA fermentative prodn
It is the 20ml seed culture medium that the high yield bacterium inclined-plane of preserving is inoculated in loading amount, the fermention medium composition, and fermentation condition and control are undertaken by method in the instance 5, utilize autoclaved 2M NaOH respectively the pH of fermention medium to be adjusted to 5.0 in fermentation in the time of the 3rd day; 6.0,7.0,8.0; 9.0,9.6,9.8; 10.0 the HPLC that ferments after seven days detects 7-ACA concentration in the fermented liquid, to find out the pH value of the most suitable 7-ACA fermentative prodn.The result sees table 4.
PH value in table 4 fermenting process is to the influence of 7-ACA fermentative prodn
| Instance number | The pH value | 7-ACA output (μ g/ml) | Increase rate (%) |
| 17 | Do not transfer (4.6-4.8) | 218 | 100 |
| 18 | 5.0 | 237 | 109 |
| 19 | 6.0 | 356 | 163 |
| 20 | 7.0 | 592 | 272 |
| 21 | 8.0 | 849 | 389 |
| 22 | 9.0 | 1526 | 700 |
| 23 | 9.6 | 1685 | 773 |
| 24 | 9.8 | 1205 | 553 |
| 25 | 10.0 | 781 | 358 |
Annotate: No. 17 examples are that No. 12 routine conditions in the table 3 are identical, and raising ratio (%) is meant the percentage ratio of comparing with No. 17 routine 7-ACA fermentation yields
The ability of this bacterium product 7-ACA under neutral and meta-alkalescence condition is stronger as can be seen from Table 4, and the meta-alkalescence condition is fit to the production of 7-ACA.
Embodiment 7
The fermentation culture temperature is to the influence of reorganization bacterium fermentative prodn 7-ACA
Like the substratum proportioning components preparing culture medium among the embodiment 5; Behind the autoclaving; Fermentation condition by embodiment 5 ferments, and the shake flask fermentation temperature is controlled, and is separately positioned on 20 ℃, 23 ℃, 24 ℃, 25 ℃, 26 ℃, 28 ℃, 30 ℃ following shaking culture 7 days; The vibration rotating speed is 230rpm, and HPLC detects 7-ACA concentration in the fermented liquid.The result sees table 5.
Table 5 different fermentations culture temperature is to the influence of reorganization bacterium fermentative prodn 7-ACA
| Instance number | Culture temperature (℃) | 7-ACA output (μ g/ml) | Increase rate (%) |
| 26 | 20 | 154 | 36 |
| 27 | 23 | 162 | 37.8 |
| 28 | 24 | 368 | 86.9 |
| 29 | 25 | 428 | 100 |
| 30 | 26 | 504 | 118 |
| 31 | 28 | 356 | 83.2 |
| 32 | 30 | 327 | 76.4 |
Annotate: raising ratio (%) be meant compare with the number of enforcement 29 that is to say with embodiment 5 in condition compare.
Can find out that from table 5 temperature of the righttest fermentative prodn 7-ACA is in 24-30 ℃ of scope for the reorganization bacterium, 26 ℃ is optimum temperuture.
Embodiment 8
Shaking table vibration rotating speed is to the influence of reorganization bacterium fermentative prodn 7-ACA in the fermentation culture process
Like the substratum proportioning components preparing culture medium among the embodiment 5; Behind the autoclaving; Fermentation condition by embodiment 5 ferments; The vibration rotating speed of cultivating shaking table was set to 150rpm, 180rpm, 200rpm, 230rpm shaking culture respectively 7 days, and temperature is set to 25 ℃, and HPLC detects 7-ACA concentration in the fermented liquid.The result sees table 6.
Shaking speed is to the influence of reorganization bacterium fermentative prodn 7-ACA in the table 6 fermentation culture process
| Instance number | Rotating speed (rpm) | 7-ACA output (μ g/ml) | Increase rate (%) |
| 33 | 150 | 308 | 69.5 |
| 34 | 180 | 315 | 71.1 |
| 35 | 200 | 371 | 83.7 |
| 36 | 220 | 468 | 106 |
| 37 | 230 | 443 | 100 |
| 38 | 240 | 417 | 94.1 |
Annotate: increase rate (%) be meant compare with the number of enforcement 37 that is to say with embodiment 5 in condition compare.
From the process of the visible reorganization of table 6 bacterium fermentative prodn 7-ACA, cultivate shaking speed 7-ACA output in the 200-240rpm scope and improve obviously, when cultivating rotating speed and be set to 220rpm, the output of the fermentative prodn 7-ACA of reorganization bacterium is the highest.
Embodiment 8 substratum proportioning components are according to embodiment 5 preparation autoclavings; Fermentation culture temperature and shaking speed are controlled at 26 ℃ and 220rpm respectively; Using the pH value in the autoclaved 2M NaOH regulation and control fermentation shake flask on the 3rd day in fermentation is about 9.6; Fermented the 7th day, 7-ACA concentration is 1701 μ g/ml in HPLC detection fermented liquid.
Embodiment 9
The different fermentations time is to the influence of reorganization bacterium fermentative prodn 7-ACA in the fermentation culture process
The substratum proportioning components is according to embodiment 5 preparation autoclavings; Fermentation culture temperature and shaking speed are controlled at 26 ℃ and 220rpm respectively; Using the pH value in the autoclaved 2M NaOH regulation and control fermentation shake flask on the 3rd day in fermentation is about 9.6; Fermentation was since sampling in the 3rd day, and HPLC detects the different fermentations time 7-ACA condition of production, and the result sees table 7.
The different fermentations time is to the influence of reorganization bacterium fermentative prodn 7-ACA in the table 7 fermentation culture process
| Instance number | Fermentation time | 7-ACA output (μ g/ml) |
| 39 | 3d | 486 |
| 40 | 4d | 681 |
| 41 | 5d | 1024 |
| 42 | 6d | 1694 |
| 43 | 7d | 1701 |
| 44 | 8d | 1471 |
Just had 7-ACA to produce from the visible reorganization of table 7 bacterium fermenting process on the 3rd day in fermentation, along with the prolongation of fermentation time, 7-ACA output constantly adds up, and reaches maximum to the 6th day output during with the 7th day, but the output reduction the 8th day time of fermenting.
The above is merely preferred embodiment of the present invention; Be not in order to limit essence technology contents scope of the present invention; Essence technology contents of the present invention is broadly to be defined in the claim scope of application, and if any technological entity or method that other people accomplish are defined identical with the claim scope of application; Also or a kind of change of equivalence, all will be regarded as and be covered by among this claim scope.
Claims (14)
1. one kind is used to produce the fermention medium suc as formula the 7-amino-cephalosporanic acid shown in the I (7-ACA), it is characterized in that in the TV of said fermention medium, it contains:
4.2-18% (g/100mL) carbon source;
10-12% (g/100mL) nitrogenous source; With
Trace element solution;
With the volumeter of said trace element solution, wherein contain ZnSO
47H
2O 0.2-13.2 (g/100mL), CuSO
45H
2O 0.2-0 (g/100mL);
Said metals ion comprises potassium, magnesium, calcium, iron and manganese, in the TV of said fermention medium, wherein CaCO
30.1-1% (g/100mL);
Said fermention medium pH 5.0-10.0;
2. fermention medium as claimed in claim 1 is characterized in that, wherein contains CaCO
30.1-0.8 (g/100mL).
3. fermention medium as claimed in claim 1 is characterized in that, in said trace element solution, contains ZnSO
47H
2O 0.2-13.2 (g/100mL), CuSO
45H
2O 0.1-0 (g/100mL).
4. fermention medium as claimed in claim 3 is characterized in that, in said trace element solution, contains ZnSO
47H
2O 0.2-3.3 (g/100mL).
5. like the arbitrary described fermention medium of claim 1-4, it is characterized in that the pH 7.0-10.0 of said substratum.
6. fermention medium as claimed in claim 5 is characterized in that, in the TV of said fermention medium, it contains:
4.2-18% (g/100mL) carbon source;
10-12% (g/100mL) nitrogenous source; With
Trace element solution;
With the volumeter of said trace element solution, wherein contain ZnSO
47H
2O 0.2-3.3 (g/100mL), CuSO
45H
2O 0.1-0 (g/100mL);
Said metals ion comprises potassium, magnesium, calcium, iron and manganese, in the TV of said fermention medium, wherein CaCO
30.1-0.8% (g/100mL);
Said fermention medium pH 7.0-9.6;
Said carbon source comprises starch, dextrin, glucose;
Said nitrogenous source comprises steeping water, methionine(Met), urea and (NH
4)
2SO
4
7. fermention medium as claimed in claim 6 is characterized in that, contains 1-6% (g/100mL) starch, 3-10% (g/100mL) dextrin and 0.2-2.0% (g/100mL) glucose in the said fermention medium; Also contain 9-10% (g/100mL) steeping water and 1.0-2.0% (g/100mL) (NH in the said fermention medium
4)
2SO
4
8. fermention medium as claimed in claim 1 is characterized in that, in the TV of said fermention medium; It contains steeping water 10% (g/100mL), starch 3% (g/100mL), dextrin 6% (g/100mL); Glucose 0.5% (g/100mL); Methionine(Met) 0.6% (g/100mL), urea 0.3% (g/100mL), KH
2PO
40.9% (g/100mL), MgSO
47H
2O 0.3% (g/100mL), (NH
4)
2SO
41.3% (g/100mL), CaCO
30.1-1% (g/100mL), trace element solution 1ml and soya-bean oil 0.4ml/20ml, pH 5.0-10.0;
With the volumeter of said trace element solution, wherein contain FeSO
47H
2O 0.8 (g/100mL), MnSO
4H
2O 0.2 (g/100mL), ZnSO
47H
2O 0.2-13.2 (g/100mL), CuSO
45H
2O 0.2-0 (g/100mL).
9. fermention medium as claimed in claim 8 is characterized in that, in the TV of said fermention medium; It contains steeping water 10% (g/100mL), starch 3% (g/100mL), dextrin 6% (g/100mL); Glucose 0.5% (g/100mL); Methionine(Met) 0.6% (g/100mL), urea 0.3% (g/100mL), KH
2PO
40.9% (g/100mL), MgSO
47H
2O 0.3% (g/100mL), (NH
4)
2SO
41.3% (g/100mL), CaCO
30.1-0.8% (g/100mL), trace element solution 1ml and soya-bean oil 0.4ml/20ml, pH 7.0-9.6;
With the volumeter of said trace element solution, wherein contain FeSO
47H
2O 0.8w/L% (g/100mL), MnSO
4H
2O 0.2 (g/100mL), ZnSO
47H
2O 0.2-3.3 (g/100mL), CuSO
45H
2O 0-0.1 (g/100mL).
10. the purposes like the arbitrary described fermention medium of claim 1-9 is characterized in that, is used for fermentation culture bacterial classification Acremonium chrysogenum/pYG233 and/or production suc as formula the 7-amino-cephalosporanic acid shown in the I (7-ACA).
11. a production is characterized in that suc as formula the method for the 7-amino-cephalosporanic acid shown in the I (7-ACA) described method comprises step:
(1) in arbitrary described fermention medium, bacterial classification Acremonium chrysogenum/pYG233 is carried out fermentation culture like claim 1-9; Obtain structure suc as formula the 7-amino-cephalosporanic acid shown in the I (7-ACA).
12. method as claimed in claim 11 is characterized in that, the fermentation culture temperature is 24-30 ℃, and pH 5.0-10.0, incubation time are 3-8 days.
13. method as claimed in claim 12 is characterized in that, the fermentation culture temperature is 24-28 ℃, pH 7.0-9.6, incubation time 5-8 days.
14. like the arbitrary described method of claim 11-13, it is characterized in that fermentation culture is carried out in shaking table, shaking table vibration rotating speed is 220rpm.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1224469A (en) * | 1997-04-22 | 1999-07-28 | 吉斯特-布罗卡迪斯有限公司 | Process for fermentative production of deacylated cephaloporins |
| WO2000061767A1 (en) * | 1999-04-09 | 2000-10-19 | Antibioticos, S.A.U. | Extracellular protease from $i(acremonium chrysogenum) with cpc-acetyl hydrolase activity and its utilization in the synthesis of deacetylated derivatives of cephalosporin c and inactivation of the gene for increasing production of cephalosporin |
| CN101389635A (en) * | 2006-02-23 | 2009-03-18 | 帝斯曼知识产权资产管理有限公司 | Improved cephalosporin production |
| CN101768594A (en) * | 2008-12-30 | 2010-07-07 | 上海医药工业研究院 | Nucleotide sequence for coding CPC acyltransferase and method for producing 7-ACA |
-
2011
- 2011-04-29 CN CN2011101096597A patent/CN102757999A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1224469A (en) * | 1997-04-22 | 1999-07-28 | 吉斯特-布罗卡迪斯有限公司 | Process for fermentative production of deacylated cephaloporins |
| WO2000061767A1 (en) * | 1999-04-09 | 2000-10-19 | Antibioticos, S.A.U. | Extracellular protease from $i(acremonium chrysogenum) with cpc-acetyl hydrolase activity and its utilization in the synthesis of deacetylated derivatives of cephalosporin c and inactivation of the gene for increasing production of cephalosporin |
| CN101389635A (en) * | 2006-02-23 | 2009-03-18 | 帝斯曼知识产权资产管理有限公司 | Improved cephalosporin production |
| CN101768594A (en) * | 2008-12-30 | 2010-07-07 | 上海医药工业研究院 | Nucleotide sequence for coding CPC acyltransferase and method for producing 7-ACA |
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