CN103382445B - Microbial strain for preparing androstenedione and application thereof - Google Patents
Microbial strain for preparing androstenedione and application thereof Download PDFInfo
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- CN103382445B CN103382445B CN201310181803.7A CN201310181803A CN103382445B CN 103382445 B CN103382445 B CN 103382445B CN 201310181803 A CN201310181803 A CN 201310181803A CN 103382445 B CN103382445 B CN 103382445B
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- AEMFNILZOJDQLW-QAGGRKNESA-N androst-4-ene-3,17-dione Chemical compound O=C1CC[C@]2(C)[C@H]3CC[C@](C)(C(CC4)=O)[C@@H]4[C@@H]3CCC2=C1 AEMFNILZOJDQLW-QAGGRKNESA-N 0.000 title claims abstract description 43
- 229960005471 androstenedione Drugs 0.000 title claims abstract description 43
- AEMFNILZOJDQLW-UHFFFAOYSA-N androstenedione Natural products O=C1CCC2(C)C3CCC(C)(C(CC4)=O)C4C3CCC2=C1 AEMFNILZOJDQLW-UHFFFAOYSA-N 0.000 title claims abstract description 43
- 230000000813 microbial effect Effects 0.000 title abstract description 8
- 238000003756 stirring Methods 0.000 claims abstract description 31
- 238000000855 fermentation Methods 0.000 claims abstract description 15
- 230000004151 fermentation Effects 0.000 claims abstract description 15
- 229930182558 Sterol Natural products 0.000 claims abstract description 14
- 235000003702 sterols Nutrition 0.000 claims abstract description 14
- 150000003432 sterols Chemical class 0.000 claims abstract description 14
- 238000002360 preparation method Methods 0.000 claims abstract description 13
- 238000010438 heat treatment Methods 0.000 claims abstract description 10
- 244000005700 microbiome Species 0.000 claims abstract description 9
- 238000006243 chemical reaction Methods 0.000 claims abstract description 8
- 238000001816 cooling Methods 0.000 claims abstract description 7
- 241000186359 Mycobacterium Species 0.000 claims abstract description 5
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 108
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 74
- 238000000034 method Methods 0.000 claims description 40
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 27
- 239000000243 solution Substances 0.000 claims description 27
- 239000012452 mother liquor Substances 0.000 claims description 24
- 239000012043 crude product Substances 0.000 claims description 21
- 239000000203 mixture Substances 0.000 claims description 21
- 238000003860 storage Methods 0.000 claims description 21
- 238000000605 extraction Methods 0.000 claims description 19
- 239000007788 liquid Substances 0.000 claims description 19
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 15
- 239000000047 product Substances 0.000 claims description 15
- 238000000967 suction filtration Methods 0.000 claims description 15
- 230000001954 sterilising effect Effects 0.000 claims description 13
- 230000001580 bacterial effect Effects 0.000 claims description 12
- 238000009833 condensation Methods 0.000 claims description 12
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- 238000002425 crystallisation Methods 0.000 claims description 12
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- 239000002054 inoculum Substances 0.000 claims description 9
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- 238000010992 reflux Methods 0.000 claims description 9
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- 238000012360 testing method Methods 0.000 claims description 9
- 230000000694 effects Effects 0.000 claims description 7
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- 238000009413 insulation Methods 0.000 claims description 7
- 238000004321 preservation Methods 0.000 claims description 7
- 238000000926 separation method Methods 0.000 claims description 7
- 238000005406 washing Methods 0.000 claims description 7
- 238000009826 distribution Methods 0.000 claims description 6
- 239000012065 filter cake Substances 0.000 claims description 6
- 239000000706 filtrate Substances 0.000 claims description 6
- 239000008235 industrial water Substances 0.000 claims description 6
- 239000008399 tap water Substances 0.000 claims description 6
- 235000020679 tap water Nutrition 0.000 claims description 6
- 241000187488 Mycobacterium sp. Species 0.000 claims description 5
- 238000004809 thin layer chromatography Methods 0.000 claims description 5
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 claims description 4
- 238000004128 high performance liquid chromatography Methods 0.000 claims description 4
- 238000007670 refining Methods 0.000 claims description 4
- 239000012267 brine Substances 0.000 claims description 3
- 230000008859 change Effects 0.000 claims description 3
- 239000003610 charcoal Substances 0.000 claims description 3
- 238000005352 clarification Methods 0.000 claims description 3
- 239000012141 concentrate Substances 0.000 claims description 3
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- 238000007689 inspection Methods 0.000 claims description 3
- 238000002156 mixing Methods 0.000 claims description 3
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- 235000002378 plant sterols Nutrition 0.000 claims description 3
- 230000001105 regulatory effect Effects 0.000 claims description 3
- HPALAKNZSZLMCH-UHFFFAOYSA-M sodium;chloride;hydrate Chemical compound O.[Na+].[Cl-] HPALAKNZSZLMCH-UHFFFAOYSA-M 0.000 claims description 3
- 230000001131 transforming effect Effects 0.000 claims description 3
- 238000010792 warming Methods 0.000 claims description 3
- 239000002351 wastewater Substances 0.000 claims description 3
- 108090000790 Enzymes Proteins 0.000 claims description 2
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- 238000009654 indole test Methods 0.000 claims description 2
- CEQFOVLGLXCDCX-WUKNDPDISA-N methyl red Chemical compound C1=CC(N(C)C)=CC=C1\N=N\C1=CC=CC=C1C(O)=O CEQFOVLGLXCDCX-WUKNDPDISA-N 0.000 claims description 2
- 244000068988 Glycine max Species 0.000 abstract description 6
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- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 6
- 229960001701 chloroform Drugs 0.000 description 6
- 238000001514 detection method Methods 0.000 description 6
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- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 4
- 229940079593 drug Drugs 0.000 description 4
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- 231100000350 mutagenesis Toxicity 0.000 description 4
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- WTLKTXIHIHFSGU-UHFFFAOYSA-N 2-nitrosoguanidine Chemical compound NC(N)=NN=O WTLKTXIHIHFSGU-UHFFFAOYSA-N 0.000 description 3
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- 241000186361 Actinobacteria <class> Species 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- MUMGGOZAMZWBJJ-DYKIIFRCSA-N Testostosterone Chemical compound O=C1CC[C@]2(C)[C@H]3CC[C@](C)([C@H](CC4)O)[C@@H]4[C@@H]3CCC2=C1 MUMGGOZAMZWBJJ-DYKIIFRCSA-N 0.000 description 2
- DWCSNWXARWMZTG-UHFFFAOYSA-N Trigonegenin A Natural products CC1C(C2(CCC3C4(C)CCC(O)C=C4CCC3C2C2)C)C2OC11CCC(C)CO1 DWCSNWXARWMZTG-UHFFFAOYSA-N 0.000 description 2
- 238000009825 accumulation Methods 0.000 description 2
- 230000002146 bilateral effect Effects 0.000 description 2
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
- WQLVFSAGQJTQCK-VKROHFNGSA-N diosgenin Chemical compound O([C@@H]1[C@@H]([C@]2(CC[C@@H]3[C@@]4(C)CC[C@H](O)CC4=CC[C@H]3[C@@H]2C1)C)[C@@H]1C)[C@]11CC[C@@H](C)CO1 WQLVFSAGQJTQCK-VKROHFNGSA-N 0.000 description 2
- WQLVFSAGQJTQCK-UHFFFAOYSA-N diosgenin Natural products CC1C(C2(CCC3C4(C)CCC(O)CC4=CCC3C2C2)C)C2OC11CCC(C)CO1 WQLVFSAGQJTQCK-UHFFFAOYSA-N 0.000 description 2
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- 150000003431 steroids Chemical class 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- GCKMFJBGXUYNAG-UHFFFAOYSA-N 17alpha-methyltestosterone Natural products C1CC2=CC(=O)CCC2(C)C2C1C1CCC(C)(O)C1(C)CC2 GCKMFJBGXUYNAG-UHFFFAOYSA-N 0.000 description 1
- QZLYKIGBANMMBK-UGCZWRCOSA-N 5α-Androstane Chemical compound C([C@@H]1CC2)CCC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CCC[C@@]2(C)CC1 QZLYKIGBANMMBK-UGCZWRCOSA-N 0.000 description 1
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- 241000196324 Embryophyta Species 0.000 description 1
- DNXHEGUUPJUMQT-CBZIJGRNSA-N Estrone Chemical compound OC1=CC=C2[C@H]3CC[C@](C)(C(CC4)=O)[C@@H]4[C@@H]3CCC2=C1 DNXHEGUUPJUMQT-CBZIJGRNSA-N 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- GCKMFJBGXUYNAG-HLXURNFRSA-N Methyltestosterone Chemical compound C1CC2=CC(=O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@](C)(O)[C@@]1(C)CC2 GCKMFJBGXUYNAG-HLXURNFRSA-N 0.000 description 1
- MKPDWECBUAZOHP-AFYJWTTESA-N Paramethasone Chemical compound C1([C@@H](F)C2)=CC(=O)C=C[C@]1(C)[C@@H]1[C@@H]2[C@@H]2C[C@@H](C)[C@@](C(=O)CO)(O)[C@@]2(C)C[C@@H]1O MKPDWECBUAZOHP-AFYJWTTESA-N 0.000 description 1
- 240000008042 Zea mays Species 0.000 description 1
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 1
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- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
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- JEIPFZHSYJVQDO-UHFFFAOYSA-N ferric oxide Chemical compound O=[Fe]O[Fe]=O JEIPFZHSYJVQDO-UHFFFAOYSA-N 0.000 description 1
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- GBMDVOWEEQVZKZ-UHFFFAOYSA-N methanol;hydrate Chemical compound O.OC GBMDVOWEEQVZKZ-UHFFFAOYSA-N 0.000 description 1
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
Abstract
The invention discloses a microbial strain for preparing androstenedione. The collection number of the microbial strain is CCTCC NO:M2012522. The preparation of androstenedione by the utilization of the strain comprises the following steps: A, primary seeding tank fermentation: inoculation amount of the strain is 0.5-1.5%, and cultivation is carried out at 160-200rpm and at 0.03-0.07MPa for 30-42h; B, secondary seeding tank fermentation: inoculation amount of the strain is 8-12%, and cultivation is carried out under the same conditions for 20-30h; C, fermentation tank fermentation for conversion to generate androstenedione: inoculation amount of the strain is 10-14%, and cultivation is carried out under the conditions of 27-35 DEG C and 0.03-0.07MPa; and D, termination of conversion: sterol is lower than 0.5%, pH is 8.9-9.0, discharging, heating to 90-100 DEG C and keeping for 30-50min, cooling to 30-50 DEG C, stopping stirring and standing for 4-6h. The invention provides a high-efficiency fermentation strain of mycobacterium. Meanwhile, a surfactant, soya-bean oil and a broth are used to form a two-way system. The fermentation technology is improved, and feeding amount of raw materials and conversion rate of microorganisms are greatly raised.
Description
Technical field
The present invention relates to a kind of technical field of biological fermentation.
Background technology
(androstane-14-alkene-3,20-diketone there is two main approach, the one, diosgenin approach, the 2nd, microbial transformation approach in preparation AD) to steroidal drug intermediate Androstenedione at present.Diosgenin is generally to extract from the plants such as wild Chinese medicinal materials " Dioscorea nipponica Mak. Ningpo Yam Rhizome ", then prepares Androstenedione through chemosynthesis, its complex process, and cost is high, and environmental pollution is serious.The work of microbiological deterioration sterol starts from the sixties in 20th century, found that there is the multiple-microorganism sterol of can degrading, its product Androstenedione (AD) and androstane l, 4-diene-3, l7-diketone (ADD) can be used as the intermediate of many steroid hormone medicines such as synthetic testosterone, Methyltestosterone, Wynestron and paramethasone.Early 1970s, the horse monarch using microbe degraded cholesterol of Japan is produced ADD and is succeeded.After this, rapidly, wherein majority is to using AD, ADD as the synthetic intermediate of steroid drugs in the research work development that natural animal and plant sterol is raw material production steroid drugs.
Research to sterol degradation process shows, AD (D) is the intermediate product in degradation pathway just, under the effect of 9-hydroxylase, can further be degraded into CO2 and H2O.If destroy the gene of 9-hydroxylase, AD (D) just cannot further degrade, thereby can in fermented liquid, be accumulated.The present invention has obtained 9-hydroxylase defect bacterial strain by composite mutagenesis method, have the ability of accumulation AD (D), and this mutant strain also has high concentration substrate tolerance.
In addition, substrate sterol solubleness in water is low is the bottleneck that Androstenedione is produced in bio-transformation.In single water fermenting process, add a small amount of organic solvent or tensio-active agent improves concentration of substrate, effect is unsatisfactory; Superpolymer/superpolymer aqueous two-phase system is limited and price is more expensive to the solubilising of substrate, is not suitable for large-scale commercial production.Organic solvent/water two liquid phase systems solubilisings increase, but shortcoming is the volatility of organic solvent to the toxicity of cell and organic solvent, have increased the complicacy of environment, safety etc.
Summary of the invention
The technical problem to be solved in the present invention is the problems such as concentration is low, low conversion rate that feed intake in, fermenting process unstable for existing 4AD fermented bacterium, the high-efficiency fermenting bacterial strain of a kind of mycobacterium is provided, utilize tensio-active agent, soya-bean oil and fermented liquid to form bilateral system simultaneously, improve zymotechnique, promoted greatly charging capacity and the microbial transformation rate of raw material.
For solving the problems of the technologies described above, the technical solution used in the present invention is as follows.
Microorganism strains for the preparation of Androstenedione, range the Mycobacterium mycobacterium kind in actinomycetes, Latin is called Mycobacteriumsp, called after Mycobacteriumsp.GTF-69, depositary institution is Chinese Typical Representative culture collection center, preservation address: Wuhan, China Wuhan University, preservation date on December 11st, 2012, preserving number CCTCCNO:M2012522; Its principal character is as follows: Gram-positive G+, and cell is shaft-like, hydrogen peroxide enzyme positive, V.P. reaction negative, methyl red test is negative, and indole test is negative, and fermenting plant sterol generates 4AD Androstenedione and a small amount of ADD androsadiendione.
Utilize above-mentioned bacterial strains to prepare the method for Androstenedione, the method comprises the steps:
A, first class seed pot fermentation: according to following table, carry out the preparation of substratum:
| Composition | Concentration, g/L | Effective volume is the amount of 400L, Kg |
| Corn steep liquor | 50.0 | 20.00 |
| Glucose/brown sugar | 6.0 | 2.40 |
| NaNO 3 | 5.4 | 2.16 |
| (NH 4) 2HPO 4 | 0.6 | 0.24 |
| Bubble enemy | 1.0 | 0.40 |
The inoculum size of bacterial strain is 0.5-1.5%, and at 27-35 ℃, 160-200rpm, cultivates 30-42h under 0.03-0.07MPa condition;
B, the fermentation of secondary seed tank: according to following table, carry out the preparation of substratum:
| Composition | Concentration, g/L | Effective volume is 2.5M 3,Kg |
| Corn steep liquor | 50.0 | 125.0 |
| Glucose/brown sugar r | 6.0 | 15.0 |
| NaNO 3 | 5.4 | 13.5 |
| (NH 4) 2HPO 4 | 0.6 | 1.5 |
| Bubble enemy | 1.0 | 2.5 |
The inoculum size of bacterial strain is 8-12%, and at 27-35 ℃, 160-200rpm, cultivates 20-30h under 0.03-0.07MPa condition;
C, ferment tank transform and generate Androstenedione: according to following table, carry out the preparation of substratum:
| Composition | Concentration, g/L | Effective volume is 21m 3Amount, Kg |
| Corn steep liquor | 50.0 | 1050.0 |
| NaNO 3 | 5.4 | 113.4 |
| (NH 4) 2PO 4 | 0.6 | 12.6 |
| Soya-bean cake oil | 160.0 | 3360.0 |
| Sterol | 40 | 735 |
The inoculum size of bacterial strain is 10-14%; At 27-35 ℃, under 0.03-0.07MPa condition, cultivate;
D, stop transforming: requiring sterol lower than 0.5%, is weight/volume, TLC thin-layer chromatography immaculate, PH8.9-9.0, puts tank, is warmed up to 90-100 ℃ and maintains 30-50min, is then cooled to 30-50 ℃, stops stirring standing 4-6 hour.
As a preferred technical solution of the present invention, in steps A, the distribution of substratum is: according to described formula, accurately take each composition, and other medium components beyond de-bubble enemy are all dropped into fermentor tank, and constant volume is to 360L; After constant volume, the PH of substratum is between 3.5-4.3, with NaOH, adjusts between PH to 8.3-8.5, then adds bubble enemy, and sterilizing 30 minutes under 124 degree.
As a preferred technical solution of the present invention, in step B, the distribution of substratum is: according to described formula, accurately take each composition, and other medium components beyond de-bubble enemy are all dropped into fermentor tank, and constant volume is to 2000L; After constant volume, the PH of substratum is between 3.5-4.3, with NaOH, adjusts between PH to 8.3-8.5, then adds bubble enemy, and sterilizing 30 minutes under 124 degree.
As a preferred technical solution of the present invention, in step C, the distribution of substratum is: first accurately take each composition of substratum, oil and sterol are put into fermentor tank and be warming up to 80-90 ℃, and start stirring arm and maintain 20 minutes, and then other compositions are added and use process water constant volume to 16.5m
3; Now medium pH is between 3.5-4.0, uses NaOH to regulate PH to 8.3-8.5, according to following steps sterilizing: first at 100 ℃ of insulation 40min, be then incubated 30min at 125 ℃ of sterilising temps.
As a preferred technical solution of the present invention, after step D, also comprise following subsequent technique:
The extraction of E, Androstenedione:
E-1, static minute water of fermented liquid: the fermented liquid in step D is squeezed into while hot oil phase storage and filled with, standing 4 hours minutes water, squeezes into water storage by water and fills with, and puts into afterwards wastewater disposal basin; Stay in oil phase storage tank for oil phase;
E-2, oil phase heating dewater: the oil phase in oil phase storage tank is squeezed into heating in extractor and dewatered, oil phase in extractor is heated to 85 degree, in heat-processed, regulating frequency transformer to make extractor stirring velocity is 50 revs/min, the static insulation of 85 degree 1 hour, afterwards hot water in coil pipe is changed to service water, makes fermented liquid be cooled to 50 degree.Bleed off lower floor's water in extractor;
E-3, oil phase heating under vacuum dewater: maintaining oil phase temperature is 53 degree, vacuumize as-0.08 MPa, and stirring velocity is 70 revs/min, when temperature rises to 60, stop vacuumizing while spending;
E-4, interpolation methyl alcohol: hot water in coil pipe is changed to service water, makes oil phase be cooled to room temperature; Extraction is stayed 0.7 ton of oil phase in filling with, and remaining oil phase is put into bedroom bubble; The in-line pump of opening methyl alcohol storage tank, measures filling by methyl alcohol, in extraction is filled with, adds 1.05 tons of methyl alcohol;
E-5, extraction: stirring velocity is 110 revs/min, churning time is 30 minutes;
E-6, separation: after extraction, extracting solution pneumatics is entered to buffering and fill with; Open spiral pump, make the extracting solution recirculate mixing in buffering filling; Check whether disk centrifuge cleans; Check after errorless and bring into use disk centrifuge separation and Extraction liquid; The isolated heavy phase of disk centrifuge enters heavy phase buffering and fills with, and gently enters mutually gently buffering mutually and fills with; Heavy phase in simultaneously heavy phase buffering being filled with spiral pump is squeezed into heavy phase storage and is filled with, and the storage mutually that is gently pressed into mutually gently in pneumatics, light buffering mutually being filled with is filled with;
E-7, again extract with separated: the heavy phase in heavy phase tank is squeezed into extractor with spiral pump and according to the method for E-4, E-5 and E-6 step, add methyl alcohol, extraction with separated; By same method, extract again three times;
E-8, concentrated: will gently be pressed into 3 tons of concentration tanks with pneumatics and concentrate; Concentrated condition is: temperature is 60 degree, normal pressure, a upper industrial water condensation of condenser, the refrigerated water condensation of next condenser; Methyl alcohol in 3 tons of concentration tanks mutually surplus with pneumatics, be pressed into while having 1 ton of left and right in the concentration tank of 2 tons, continue concentrated;
The concentrated condition of E-9,2 tons of concentration tanks is: temperature is 60 degree, normal pressure, and a upper industrial water condensation of condenser, the refrigerated water condensation of next condenser is stirred simultaneously, and stirring velocity is 60 revs/min;
E-10, the concentrated solution in 2 tons of concentration tanks are 1 ton hour, stop concentrating; Cooling, making temperature is 40 degree, stirs 2 hours, continues to be cooled to room temperature, then stirs 4 hours;
E-11, filtration: the dense magma in 2 tons of concentration tanks is squeezed into suction filtration in suction filtration cylinder; Mother liquor is squeezed in mother liquor holding tank; Crude product in suction filtration cylinder filter bag continues to drain; Obtain crude product; Weigh.
As a preferred technical solution of the present invention, after step e, also comprise following subsequent technique:
Refining of F, Androstenedione:
F-1, washing: take 200 kilograms of crude products, by weight: volume=1:5 adds ethyl acetate, stir, be heated to 60 degree and dissolve crude product; By crude product: tap water=1:2 adds tap water; 60 degree agitator treatings 30 minutes, static layering, minute falls down a layer water, and be cooled to and do not reflux, suction filtration while hot, mother liquor is squeezed into mother liquor holding tank;
F-2, decolouring: filter cake is squeezed into bleacher, by crude product weight: volume=1:10 adds methyl alcohol, adds gac by 5% of crude product weight; Be heated to 62 degree, stir, reflux 30 minutes;
F-3, filtration: filter activity charcoal in deep bed filter, refluxes and repeatedly filters, until the filtrate clarification of observing from looking cup; Repeatedly filtered a small amount of methanol solution washing of rear use bleacher, by strainer, filtered afterwards; Filtrate is squeezed in crystallizer concentrated;
F-4, concentrated: normal pressure, temperature is 60 degree, is concentrated into a half of volume, stops concentrating;
F-5, crystallization: by change the form of hot water in chuck with service water, cool the temperature to 40 degree, maintain 2 hours, then the water in chuck is all changed to service water, be cooled to room temperature, maintain 2 hours; Afterwards service water in chuck is changed to half and make it cooling, maintain 2 hours, then the water in chuck is all changed to refrigerated water, maintain 4 hours; The simultaneously slow stirred crystallization of whole process; Refrigerated water requires to be back to brine tank;
F-6, filtration: the crystallization in crystallizer and solution are squeezed into suction filtration cylinder, and mother liquor is filtered dry a small amount of methanol wash filter cake of rear use, till making mother liquor colourless; Mother liquor is squeezed into mother liquor holding tank; Get crystallized sample and dry, sample presentation detects; Detected result is carried out to detailed record; If detected result is qualified, directly enter F-8 step; If disqualified upon inspection, enters F-7 step;
F-7, secondary crystal: failed test sample is weighed, by weight: volume=1:5 adds methyl alcohol, be heated to 60 degree it dissolved completely; By F-5 step, carry out crystallization, by F-6 step, filter;
F-8, oven dry: after check product is qualified, product is poured in pallet, pushed in heat-circulation oven, steam regulation, observes thermometer, and making temperature is that 55 degree are dried, and every 1 hour, turns over once; Dry altogether 8 hours;
F-9, packing: after product sieves, pack as requested.
As a preferred technical solution of the present invention, after step D, adopt HPLC method or the right Androstenedione content of HPTLC method to detect.
The beneficial effect that adopts technique scheme to produce is:
The 9-hydroxylase defect bacterial strain that the present invention obtains has the ability of accumulation AD (D), and this bacterial strain also has high concentration substrate tolerance.
The present invention has tested the changing effect of multi-solvents and fermented liquid composition biphasic system, finally determines that aboundresources, cheap soya-bean cake oil coordinate with tensio-active agent, and common and fermented liquid forms bilateral system; Because soya-bean oil has good emulsifying capacity, can promote contacting of sterol and microorganism cells, so changing effect is best, the most applicable large-scale industrial production, microbial transformation rate of the present invention can reach more than 95.5%, more than the concentration that feeds intake reaches 40g/l.
The present invention has also set up the fermentation control technique that a whole set of microbial transformation phytosterin is prepared Androstenedione on the basis of pilot scale, detection method, and extraction process and process for refining, be convenient to the preparation technology of Androstenedione to be amplified in large-scale commercial production.
Preservation explanation
Preservation of bacteria strain ranges the Mycobacterium mycobacterium kind in actinomycetes, and Latin is called Mycobacterium sp, called after Mycobacterium sp.GTF-69, depositary institution is Chinese Typical Representative culture collection center, preservation address: China, Wuhan, Wuhan University; Preservation date on December 11st, 2012, preserving number CCTCCNO:M2012522.
Embodiment
Following examples describe the present invention in detail.Various raw material used in the present invention and items of equipment are conventional commercially available prod, all can be bought directly and be obtained by market.
Embodiment 1. complex mutations obtain 9-hydroxylase Auxotrophie mutant
1.1 starting strains: Mycobacteriumsp.NRRLB-3683
1.2 screening routes: the original strain → separation and purification → bacterium that sets out (Mycobacterium sp.NRRLB-3683) → fresh slant strains → spore suspension → UV irradiation →
60co irradiation → nitrosoguanidine processing → YAG double frequency pulse laser → plate isolation → mono-bacterium colony → primary dcreening operation, multiple sieve → gain mutant variant
The preparation of 1.3 spore suspensions: by the fresh inclined-plane of starting strain (Mycobacterium sp.NRRL B-3683) with under aseptic washing, and transfer in triangular flask sterilizing, that granulated glass sphere is housed, the 30min that vibrates on 28 ℃, the rotary shaker of 200r/min, makes concentration and is about 10
7the bacteria suspension of individual/ml.
1.4UV irradiates: bacteria suspension is put in to (apart from 30cm) under 15W ultraviolet lamp, irradiates respectively 1,2,3,4,5min, keep in Dark Place subsequently.
1.5 gamma-rays (
60co) mutagenic treatment: the suspension 5ml keeping in Dark Place is in diameter 1.3cm, in the transparent glass test tube of long 10cm, carry out gamma-rays (
60co) irradiate.Irradiation dose is respectively 300GY, 600GY, 900GY, 1200GY, 1500kGY.
1.6 nitrosoguanidine mutagenesis: nitrosoguanidine mass concentration is 50 μ g/mL, mutation time is respectively 10,20,30,40min, after mutagenesis finishes, by 1000 times of bacteria suspension dilutions, stops mutagenesis.
1.7YAG double frequency pulse laser: by YAG double frequency pulse laser radiation for bacteria suspension, wavelength 532nm, energy density is 50mJ/ pulse, and pulse-repetition is 1 time/s, and umber of exposures is respectively 200,600,900.
1.8 chromatographicconditions: octadecylsilane bonding reverse-phase chromatographic column (ODS, 4.6mm * 250mm, 5 μ m, Dalian Yi Lite scientific instrument company limited); Mobile phase methanol-water (80: 20); Sample size 5 μ L; Flow velocity 0.8ml/min; 50 ℃ of column temperatures; Detect wavelength 242nm.Fermented liquid 1ml, more than soaking 1h, draws supernatant liquor and measures with HPLC the amount that converted product generates by 4 times of volumes methanol.
Embodiment 2. microorganisms transformation phytosterin in two-phase system is prepared the fermentation control technique of Androstenedione
2.1 first class seed pots (700L) fermentation
2.1.1 medium component
| Composition | Concentration, g/L | Effective volume is the amount of 400L, Kg |
| Corn steep liquor | 50.0 | 20.00 |
| Glucose/brown sugar | 6.0 | 2.40 |
| NaNO 3 | 5.4 | 2.16 |
| (NH 4) 2HPO 4 | 0.6 | 0.24 |
| Bubble enemy | 1.0 | 0.40 |
2.1.2 prepare burden: according to above-mentioned formula, accurately take each composition, and other medium components beyond de-bubble enemy are all dropped into fermentor tank, and constant volume is to 360L.After constant volume, the PH of substratum is greatly between 3.5-4.3, with NaOH(40%) be adjusted between 8.3-8.5, then add bubble enemy, and sterilizing 30 minutes under 124 degree.
2.1.3 culture condition: 1%, 31 ℃ of inoculum size, 180rpm, 0.05MPa, 30-42h
2.2 secondary seed tank (4000L) fermentations
2.2.1 medium component
| Composition | Concentration, g/L | Effective volume is 2.5M3, Kg |
| Corn steep liquor | 50.0 | 125.0 |
| Glucose/brown sugar r | 6.0 | 15.0 |
| NaNO 3 | 5.4 | 13.5 |
| (NH 4) 2HPO 4 | 0.6 | 1.5 |
| Bubble enemy | 1.0 | 2.5 |
2.2.2 prepare burden: according to above-mentioned formula, accurately take each composition, and other medium components beyond de-bubble enemy are all dropped into fermentor tank, and constant volume is to 2000L.After constant volume, the PH of substratum is greatly between 3.5-4.3, with NaOH(40%) be adjusted between 8.3-8.5, then add bubble enemy, and sterilizing 30 minutes under 124 degree.
2.2.3 culture condition: 10%, 31 ℃ of inoculum size, 180rpm, 0.05MPa, 20-30h
2.3 ferment tanks and conversion generate Androstenedione
2.3.1 medium component
| Composition | Concentration, g/L | Effective volume is 21m 3Amount, Kg |
| Corn steep liquor | 50.0 | 1050.0 |
| NaNO 3 | 5.4 | 113.4 |
| (NH 4) 2PO 4 | 0.6 | 12.6 |
| Soya-bean cake oil | 160.0 | 3360.0 |
| Sterol | 40 | 735 |
2.3.2 the configuration of substratum
First accurately take each composition of substratum, oil and sterol are put into fermentor tank and be warming up to 80-90 ℃, and start stirring arm and maintain 20 minutes, and then other compositions are added and use process water constant volume to 16.5m
3.Now medium pH is greatly about 3.5-4.0.Use 40%NaOH to regulate PH to 8.3-8.5. according to following steps sterilizing: first at 100 ℃ of insulation 40min, 125 ℃ of insulation 30min. under real sterilising temp then
2.3.3 the parameters of microbial transformation
Inoculum size 12%; 31 ℃, 0.05MPa, air flow: regulate according to following steps: initial-to28-30
-thh---1 ﹕ 1.2VVM (maximum .) 29-33
-thh-end: air flow is progressively reduced to minimum (1 ﹕ 0.5VVM) according to PH.Rotating speed: regulate initial-28-30 according to following steps
-thh---200rpm (Max.); 29-33
-thh-end: rotating speed progressively need to be adjusted to minimum (150rpm) according to PH.DO: maintain it more than 20%.PH: PH changes in conversion process, but be between 8.2-8.6, to transform the best at PH.
2.3.4 stop transforming
Requiring sterol lower than 0.5%, is weight/volume, TLC immaculate.Now PH8.9-9.0, puts tank.Be warmed up to 90-100 ℃ and maintain 40min. and be then cooled to 40 ℃, stop stirring standing 5 hours.(also can phosphoric acid adjusting PH6.4-6.5 to accelerate layering).
Embodiment 3. microorganisms transformation phytosterin in two-phase system is prepared the detection method of Androstenedione
3.1HPLC detection method:
3.1.1 testing conditions: liquid chromatography pump; Purple place visible detection device; Water: distilled water, then through 0.45 μ m membrane filtration; Methyl alcohol: HPLC reagent.
3.1.2 chromatographic condition: chromatographic column: C18 (4.6*1505um); Moving phase: methyl alcohol: water=65:35; Flow velocity: 1ml/min; Solvent: methyl alcohol; Detect wavelength: 240nm.
3.1.3 step:
1. precision weighing 20mg working standard, is placed in 100ml volumetric flask, with after a small amount of dissolve with methanol, by moving phase, is diluted to scale, shakes up, in contrast product solution.
2. get certain volume fermented liquid oil phase and (claim its weight W
1), add equal-volume trichloromethane and (claim its weight W
2).Mix, centrifugal 15min, claims to obtain its upper water weight W
water, then inhale certain grams subnatant W
subnatant(oil+trichloromethane phase), in beaker, dried 15min and added 2ml ethyl acetate ultrasonic dissolution in 25ml volumetric flask, methanol constant volume, filtration at 65 ℃.
3. under these conditions,, after instrument baseline stability, get respectively each 20ul sample introduction of standard solution and product to be tested solution, and record peak response.
4. the calculating of content:
A
1the peak area of-standard substance; A
2the peak of-sample is long-pending; M
1the weighing of-standard substance; C
1the content % of-reference substance;
3.2HPTLC detection method:
3.2.1 testing conditions: thin layer chromatogram scanner, the electronic point sample instrument of thin layer, uv analyzer.
3.2.2 chromatographic condition: gel GF 254 plate: specification (100 * 100mm) thickness (0.20-0.25mm); Developping agent: ethyl acetate: sherwood oil=3:7; Detect wavelength: 240nm.
3.2.3 step:
1. precision weighing 25mg working standard, is placed in 50ml volumetric flask, with trichloromethane phase dilution, to scale, shakes up, in contrast product solution.
2. get certain volume fermented liquid oil phase and (claim its weight W
1), add equal-volume trichloromethane and (claim its weight W
2).Mix, centrifugal 15min, claims to obtain its upper water weight W
water, then inhale certain volume subnatant W
subnatant(oil+trichloromethane phase) is to 10ml volumetric flask, with trichloromethane dilution, constant volume.
3. under these conditions,, after instrument stabilizer, get respectively contrast solution 2ul and 4ul and product to be tested solution sample introduction, and record peak response.
4. the calculating of content:
N-sample detection concentration
Embodiment 4. microorganisms transformation phytosterin in two-phase system is prepared the extraction process of Androstenedione
Static minute water of 4.1 fermented liquids: fermented liquid is squeezed into while hot oil phase storage and filled with, standing 4 hours minutes water, squeezes into water storage by water and fills with, and puts into afterwards wastewater disposal basin.Stay in oil phase storage tank for oil phase.
4.2 oil phase heating dewater: the oil phase in oil phase storage tank is squeezed into heating in extractor and dewatered.Oil phase in extractor is heated to 85 degree, and in heat-processed, regulating frequency transformer to make extractor stirring velocity is 50 revs/min, the static insulation of 85 degree 1 hour, afterwards hot water in coil pipe is changed to service water, makes fermented liquid be cooled to 50 degree.Bleed off lower floor's water in extractor.
4.3 oil phase heating under vacuum dewater: maintaining oil phase temperature is 53 degree, vacuumize as-0.08 MPa, and stirring velocity is 70 revs/min.When rising to 60, temperature stops vacuumizing while spending.
4.4 add methyl alcohol: hot water in coil pipe is changed to service water, makes oil phase be cooled to room temperature.Extraction is stayed 0.7 ton of oil phase in filling with, and remaining oil phase is put into bedroom bubble.The in-line pump of opening methyl alcohol storage tank, measures filling by methyl alcohol, in extraction is filled with, adds 1.05 tons of methyl alcohol.
4.5 extract: stirring velocity is 110 revs/min, and churning time is 30 minutes.
4.6 separation: after extraction, extracting solution pneumatics is entered to buffering and fill with.Open spiral pump, make the extracting solution recirculate mixing in buffering filling.Check whether disk centrifuge cleans according to the operation of < < disk centrifuge and use rules > >.Check errorless rear beginning according to the operation of < < disk centrifuge and use rules > > to use disk centrifuge separation and Extraction liquid.The isolated heavy phase of disk centrifuge enters heavy phase buffering and fills with, and gently enters mutually gently buffering mutually and fills with.Heavy phase in simultaneously heavy phase buffering being filled with spiral pump is squeezed into heavy phase storage and is filled with, and the storage mutually that is gently pressed into mutually gently in pneumatics, light buffering mutually being filled with is filled with.
4.7 extract again with separated: the heavy phase in heavy phase tank is squeezed into extractor with spiral pump and according to the 4.4th, 4.5, add methyl alcohol, extraction with separated with the method for 4.6 steps.By same method, extract again three times.
4.8 is concentrated: will gently with pneumatics, be pressed into 3 tons of concentration tanks and concentrate.Concentrated condition is: temperature is 60 degree, normal pressure, a upper industrial water condensation of condenser, the refrigerated water condensation of next condenser.Methyl alcohol in 3 tons of concentration tanks mutually surplus with pneumatics, be pressed into while having 1 ton of left and right in the concentration tank of 2 tons, continue concentrated.
The concentrated condition of 4.92 tons of concentration tanks is: temperature is 60 degree, normal pressure, and a upper industrial water condensation of condenser, the refrigerated water condensation of next condenser is stirred simultaneously, and stirring velocity is 60 revs/min.
4.10 concentrated solutions of working as in 2 tons of concentration tanks are 1 ton hour, stop concentrating.Cooling, making temperature is 40 degree, stirs 2 hours, continues to be cooled to room temperature, then stirs 4 hours.
4.2.11 filter: the dense magma in 2 tons of concentration tanks is squeezed into suction filtration in suction filtration cylinder.Mother liquor is squeezed in mother liquor holding tank.Crude product in suction filtration cylinder filter bag continues to drain.Obtain crude product.Weigh.
Embodiment 5. microorganisms transformation phytosterin in two-phase system is prepared the process for refining of Androstenedione
5.1 washings: take 200 kilograms of crude products, by weight: volume=1:5 adds ethyl acetate, stir, be heated to 60 degree and dissolve crude product.By crude product: tap water=1:2 adds tap water.60 degree agitator treatings 30 minutes, static layering, minute falls down a layer water, and be cooled to and do not reflux, suction filtration while hot, mother liquor is squeezed into mother liquor holding tank.
5.2 decolourings: filter cake is squeezed into bleacher, by crude product weight: volume=1:10 adds methyl alcohol, adds gac by 5% of crude product weight.Be heated to 62 degree, stir, reflux 30 minutes.
5.3 filter: filter activity charcoal in deep bed filter, refluxes and repeatedly filters, until the filtrate clarification of observing from looking cup.Repeatedly filtered a small amount of methanol solution washing of rear use bleacher, by strainer, filtered afterwards.Filtrate is squeezed in crystallizer concentrated.
5.4 is concentrated: normal pressure, temperature is 60 degree, is concentrated into a half of volume, stops concentrating.
5.5 crystallizations: cool the temperature to 40 degree by change the form of hot water in chuck with service water, maintain 2 hours, then the water in chuck is all changed to service water, be cooled to room temperature, maintain 2 hours.Afterwards service water in chuck is changed to half and make it cooling, maintain 2 hours, then the water in chuck is all changed to refrigerated water, maintain 4 hours.The simultaneously slow stirred crystallization of whole process.Refrigerated water requires to be back to brine tank.
5.6 filter: the crystallization in crystallizer and solution are squeezed into suction filtration cylinder, and mother liquor is filtered dry a small amount of methanol wash filter cake of rear use, till making mother liquor colourless.Mother liquor is squeezed into mother liquor holding tank.Get crystallized sample and dry, sample presentation detects.Detected result is carried out to detailed record.If detected result is qualified, directly enter the 5.8th step; If disqualified upon inspection, enters the 5.7th step.
5.7 secondary crystals: failed test sample is weighed, by weight: volume=1:5 adds methyl alcohol, are heated to 60 degree it are dissolved completely.By 5.5 steps, carry out crystallization, by 5.6 steps, filter.
5.8 dry: after check product is qualified, product is poured in pallet, pushed in heat-circulation oven, steam regulation, observes thermometer, and making temperature is that 55 degree are dried, and every 1 hour, turns over once.Dry altogether 8 hours.
5.9 packings: pack as requested after product sieves.
Foregoing description only proposes as the enforceable technical scheme of the present invention, not as the Single restriction condition to its technical scheme itself.
Claims (8)
1. for the preparation of the microorganism strains of Androstenedione, its name is called Mycobacterium sp.GTF-69, belongs to mycobacterium Mycobacterium, preservation date on December 11st, 2012, preserving number CCTCC NO:M2012522; Its principal character is as follows: Gram-positive G+, and cell is shaft-like, hydrogen peroxide enzyme positive, V.P. reaction negative, methyl red test is negative, and indole test is negative, and fermenting plant sterol generates 4AD Androstenedione and a small amount of ADD androsadiendione.
2. utilize the bacterial strain of claim 1 to prepare the method for Androstenedione, it is characterized in that: the method comprises the steps:
A, first class seed pot fermentation: according to following table, carry out the preparation of substratum:
The inoculum size of bacterial strain is 0.5-1.5%, and at 27-35 ℃, 160-200rpm, cultivates 30-42h under 0.03-0.07MPa condition;
B, the fermentation of secondary seed tank: according to following table, carry out the preparation of substratum:
The inoculum size of bacterial strain is 8-12%, and at 27-35 ℃, 160-200rpm, cultivates 20-30h under 0.03-0.07MPa condition;
C, ferment tank transform and generate Androstenedione: according to following table, carry out the preparation of substratum:
The inoculum size of bacterial strain is 10-14%; At 27-35 ℃, under 0.03-0.07MPa condition, cultivate;
D, stop transforming: requiring sterol lower than 0.5%, is weight/volume, TLC thin-layer chromatography immaculate, PH8.9-9.0, puts tank, is warmed up to 90-100 ℃ and maintains 30-50min, is then cooled to 30-50 ℃, stops stirring standing 4-6 hour.
3. the method for preparing Androstenedione according to claim 2, it is characterized in that: in steps A, the distribution of substratum is: according to described formula, accurately take each composition, and other medium components beyond de-bubble enemy are all dropped into fermentor tank, and constant volume is to 360L; After constant volume, the PH of substratum is between 3.5-4.3, with NaOH, adjusts between PH to 8.3-8.5, then adds bubble enemy, and sterilizing 30 minutes under 124 degree.
4. the method for preparing Androstenedione according to claim 2, it is characterized in that: in step B, the distribution of substratum is: according to described formula, accurately take each composition, and other medium components beyond de-bubble enemy are all dropped into fermentor tank, and constant volume is to 2000L; After constant volume, the PH of substratum is between 3.5-4.3, with NaOH, adjusts between PH to 8.3-8.5, then adds bubble enemy, and sterilizing 30 minutes under 124 degree.
5. the method for preparing Androstenedione according to claim 2, it is characterized in that: in step C, the distribution of substratum is: first accurately take each composition of substratum, oil and sterol are put into fermentor tank and be warming up to 80-90 ℃, and start stirring arm and maintain 20 minutes, and then other compositions are added and use process water constant volume to 16.5m
3; Now medium pH is between 3.5-4.0, uses NaOH to regulate PH to 8.3-8.5, according to following steps sterilizing: first at 100 ℃ of insulation 40min, be then incubated 30min at 125 ℃ of sterilising temps.
6. the method for preparing Androstenedione according to claim 2, is characterized in that: after step D, also comprise following subsequent technique:
The extraction of E, Androstenedione:
E-1, static minute water of fermented liquid: the fermented liquid in step D is squeezed into oil phase storage tank while hot, standing 4 hours minutes water, squeezes into water storage tank by water, puts into afterwards wastewater disposal basin; Stay in oil phase storage tank for oil phase;
E-2, oil phase heating dewater: the oil phase in oil phase storage tank is squeezed into heating in extractor and dewatered, oil phase in extractor is heated to 85 degree, in heat-processed, regulating frequency transformer to make extractor stirring velocity is 50 revs/min, the static insulation of 85 degree 1 hour, afterwards hot water in coil pipe is changed to service water, make fermented liquid be cooled to 50 degree, bleed off lower floor's water in extractor;
E-3, oil phase heating under vacuum dewater: maintaining oil phase temperature is 53 degree, vacuumize as-0.08 MPa, and stirring velocity is 70 revs/min, when temperature rises to 60, stop vacuumizing while spending;
E-4, interpolation methyl alcohol: hot water in coil pipe is changed to service water, makes oil phase be cooled to room temperature; In extractor, stay 0.7 ton of oil phase; The in-line pump of opening methyl alcohol storage tank by methyl alcohol test tank, adds 1.05 tons of methyl alcohol in extractor;
E-5, extraction: stirring velocity is 110 revs/min, churning time is 30 minutes;
E-6, separation: after extraction, extracting solution pneumatics is entered to surge tank; Open spiral pump, make the extracting solution recirculate mixing in surge tank; Check whether disk centrifuge cleans; Check after errorless and bring into use disk centrifuge separation and Extraction liquid; The isolated heavy phase of disk centrifuge enters heavy phase surge tank, gently enters mutually light phase surge tank; With spiral pump, the heavy phase in heavy phase surge tank is squeezed into heavy phase storage tank simultaneously, with pneumatics, will in light phase surge tank, gently be pressed into mutually light phase storage tank;
E-7, again extract with separated: the heavy phase in heavy phase tank is squeezed into extractor with spiral pump and according to the method for E-4, E-5 and E-6 step, add methyl alcohol, extraction with separated; By same method, extract again three times;
E-8, concentrated: will gently be pressed into 3 tons of concentration tanks with pneumatics and concentrate; Concentrated condition is: temperature is 60 degree, normal pressure, a upper industrial water condensation of condenser, the refrigerated water condensation of next condenser; Methyl alcohol in 3 tons of concentration tanks mutually surplus with pneumatics, be pressed into while having 1 ton of left and right in the concentration tank of 2 tons, continue concentrated;
The concentrated condition of E-9,2 tons of concentration tanks is: temperature is 60 degree, normal pressure, and a upper industrial water condensation of condenser, the refrigerated water condensation of next condenser is stirred simultaneously, and stirring velocity is 60 revs/min;
E-10, the concentrated solution in 2 tons of concentration tanks are 1 ton hour, stop concentrating; Cooling, making temperature is 40 degree, stirs 2 hours, continues to be cooled to room temperature, then stirs 4 hours;
E-11, filtration: the dense magma in 2 tons of concentration tanks is squeezed into suction filtration in suction filtration cylinder; Mother liquor is squeezed in mother liquor holding tank; Crude product in suction filtration cylinder filter bag continues to drain; Obtain crude product; Weigh.
7. the method for preparing Androstenedione according to claim 6, is characterized in that: after step e, also comprise following subsequent technique:
Refining of F, Androstenedione:
F-1, washing: take 200 kilograms of crude products, by weight: volume=1: 5 add ethyl acetate, stir, be heated to 60 degree and dissolve crude product; By crude product: tap water=1: 2 add tap water; 60 degree agitator treatings 30 minutes, static layering, minute falls down a layer water, and be cooled to and do not reflux, suction filtration while hot, mother liquor is squeezed into mother liquor holding tank;
F-2, decolouring: filter cake is squeezed into bleacher, by crude product weight: volume=1: 10 add methyl alcohol, adds gac by 5% of crude product weight; Be heated to 62 degree, stir, reflux 30 minutes;
F-3, filtration: filter activity charcoal in deep bed filter, refluxes and repeatedly filters, until the filtrate clarification of observing from looking cup; Repeatedly filtered a small amount of methanol solution washing of rear use bleacher, by strainer, filtered afterwards; Filtrate is squeezed in crystallizer concentrated;
F-4, concentrated: normal pressure, temperature is 60 degree, is concentrated into a half of volume, stops concentrating;
F-5, crystallization: by change the form of hot water in chuck with service water, cool the temperature to 40 degree, maintain 2 hours, then the water in chuck is all changed to service water, be cooled to room temperature, maintain 2 hours; Afterwards service water in chuck is changed to half and make it cooling, maintain 2 hours, then the water in chuck is all changed to refrigerated water, maintain 4 hours; The simultaneously slow stirred crystallization of whole process; Refrigerated water requires to be back to brine tank;
F-6, filtration: the crystallization in crystallizer and solution are squeezed into suction filtration cylinder, and mother liquor is filtered dry a small amount of methanol wash filter cake of rear use, till making mother liquor colourless; Mother liquor is squeezed into mother liquor holding tank; Get crystallized sample and dry, sample presentation detects; Detected result is carried out to detailed record; If detected result is qualified, directly enter F-8 step; If disqualified upon inspection, enters F-7 step;
F-7, secondary crystal: failed test sample is weighed, by weight: volume=1: 5 add methyl alcohol, be heated to 60 degree it dissolved completely; By F-5 step, carry out crystallization, by F-6 step, filter;
F-8, oven dry: after check product is qualified, product is poured in pallet, pushed in heat-circulation oven, steam regulation, observes thermometer, and making temperature is that 55 degree are dried, and every 1 hour, turns over once; Dry altogether 8 hours;
F-9, packing: after product sieves, pack as requested.
8. the method for preparing Androstenedione according to claim 2, is characterized in that: after step D, adopt HPLC method or HPTLC method to detect Androstenedione content.
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| CN109627275A (en) * | 2018-12-18 | 2019-04-16 | 湖南科瑞生物制药股份有限公司 | A kind of bis- dehydrogenation -17a- hydroxyl progesterone product preparation methods of 1,6- |
| CN109535214A (en) * | 2018-12-18 | 2019-03-29 | 湖南科瑞生物制药股份有限公司 | A method of preparing the bis- dehydrogenation -17a- hydroxyl progesterones of 1,6- |
| CN111454871B (en) * | 2020-03-03 | 2022-06-14 | 天津大学 | Recombinant mycobacterium with high androstenedione yield, construction method and application |
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Denomination of invention: Microbial strains and their applications for the preparation of androstenedione Effective date of registration: 20231127 Granted publication date: 20140827 Pledgee: Hubei Danjiangkou Rural Commercial Bank Co.,Ltd. Pledgor: HUBEI GONGTONG BIOTECHNOLOGY CO.,LTD. Registration number: Y2023980067680 |
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