CN103667247A - Method for improving phytosterol degradation rate of mycobacteria - Google Patents
Method for improving phytosterol degradation rate of mycobacteria Download PDFInfo
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Abstract
The invention relates to a biological pharmaceutical preparation, and particularly relates to a method for improving phytosterol degradation rate to prepare 'androstenedione' by compound mutation of microorganisms. The method comprises the following steps: (1) by taking phytosterol degrading bacteria as original strains, cultivating the bacteria on a bevel for 4 days; (2) preparing a bacterial suspension from the mature bevel, carrying out ion beam irradiation mutagenesis, and then immediately coating the bevel; (3) preparing a seed solution after the bevel is mature, cultivating for 14-16 hours, then coating a flat plate, carrying out ultraviolet mutagenesis, and then primarily screening by the flat plate and secondarily screening by a shake flask, so as to obtain a high-yielding strain; (4) preparing the bevel and the seed solution from a bacterial colony, cultivating in the bottle for 14-16 hours and then coating the flat plate, carrying out ultraviolet and laser compound mutation, and then primarily screening by the flat plate and secondarily screening by the shake flask after mutagenesis, so as to finally obtain the high-yielding strain. The strain obtained by the method has the average titer up to 25-30g/l in a 50L fermentation tank on the basis that substrate sterol is 5% and resin is 6%, and the sterol conversion rate is 86.8%. Thus, the production cost can be greatly reduced.
Description
Technical field
The present invention relates to a kind of biotechnology and prepare the method for Androstenedione, particularly a kind ofly by mycobacterium being carried out to complex mutation, improve its method to plant sterol degradation rate preparation " Androstenedione ".Belong to bioengineering field.
Background technology
4-AD (chemical name: androstane-4-alkene-3,17-diketone, English name 4-Androstenedione, be called for short AD), this product appearance shape is near-white crystal powder, free from extraneous odour, nontoxicity, water insoluble, be that in a kind of world today, purposes is extremely wide, high in technological content biochemical industry intermediate.
Androstenedione is the important intermediate of producing hormones bulk drug, nearly all steroid hormone medicine can be take Androstenedione and produced as starting raw material, as for the production of cortin, sexual hormoue, progestogen and protein anabolic hormone, can be used for again synthetic cortisone, prednisone, Progesterone, estrenol, dexamethasone etc. more than 100 and plant medicine, is also for the production of antiearly pregnancy mifepristone and the requisite raw material of all kinds of birth control medications.
Androstenedione is as the important raw material of synthesizing steroid parahormone medicine and crucial intermediate, and market outlook are wide.Steroid hormone medicine passes through the research and development of decades, has formed a large class steroid drugs of a great variety, that clinical application extensive and needs are vigorous at present.From the industry development of actual steroid hormone medicine, approximately 9.5 tons of external 1980 annual production, sell 1,500,000,000 dollars, account for 4.3% of pharmaceutical prod gross sales (GS); Nineteen ninety, output increased to 105 tons, sold 10,800,000,000 dollars, on average increased progressively 10.4%; 2010 annual sales amounts reach 40,000,000,000 dollars, account for 18% of world's medicine marketing volume, keep fast-developing tendency, and medicine kind reaches 1920 kinds more than.
China's steroid hormone medicine is through 40 years development, and exploitation strength is very powerful, and enterprise development is very fast.Steroid hormone medicine annual value of production surpasses 8,000,000,000 yuan, accounts for 5% of the medicine industry gross output value, becomes the important component part in China's medicine industry system.Refuse statistics, within 1996, China's steroid hormone medicine yearly capacity has reached 220 tons, more than 100 tons of output.Androstenedione customs number is 29372900, and customs's data show, 0.14 ton of steroid hormone in 2005 and derivative import thereof, and 2335 dollars of the value of imports, export 151.891 tons, and export amount of money reaches 2,542 ten thousand dollars.Main exit area is India, Germany, the U.S., Holland, Brazil etc., and import area is mainly from Sweden and Singapore.1-2 month in 2008,26 tons of steroid hormone and derivative imports thereof, 45.3 dollars of the value of imports, export 111.2 tons, 5,655 ten thousand dollars of export amount of moneys, estimate to import and export for 2012 quantity situation and will increase considerably compared with former years, the amount of money also can continue significantly to increase, and is mainly due to our quality product and the reason of class raising.The more active area of domestic export is the provinces such as Beijing, Tianjin, Shanghai, Shaanxi, Zhejiang, Hubei, Yunnan, roughly consistent with its resource and process zone.
At present, 80,000,000,000 dollars of Androstenedione world markets, growth rate of market 20%.The existing throughput of China's Androstenedione is greatly about 1000 tons of left and right, and also there is a big difference with demand.According to world market steroid hormone class medicine material medicine statistics in 2004, Chinese pharmaceutical enterprises is every year to approximately 6000 tons of the demands of Androstenedione, wherein need produce from dioscin for approximately 3000 tons, need produce from plant sterol for approximately 3000 tons in addition, this ratio is along with the requirement of environmental pollution can increase the amount (produce Androstenedione from the separated saponin of Chinese yam (yellow ginger) seriously polluted, many provinces have forbidden producing) that plant sterol transforms production Androstenedione.So produce coming into one's own of Androstenedione with plant sterol or cholesterol, can predict, along with further developing of production technology, the sales volume that on world market, plant sterol is converted into Androstenedione is also by rapid growth.Therefore, very tempting to the market outlook of plant sterol conversion Androstenedione scale operation.
The production technique production cycle of hormone is long, and reaction process is complicated, and it is high that industry enters barrier, and manufacturer both domestic and external is few, rival's comparatively small amt, and very concentrated.Domesticly concentrate on some enterprises such as a day medicine share, celestial jade pendant medicine company, and they mostly adopt chemical synthesis process to produce corticoid at present.Compare chemical synthesis, microbe transformation method has the advantages such as raw materials cost is low, production stage is simplified, the production cycle shortens, the reduction of production Master Cost.From adopted plant sterol since 1998 always, be abroad the production technique of raw material production Androstenedione, the technology of capturing microorganism fermentative production hormone is domestic manufacturers' striving directions for many years always.
My company is through research for many years, develop the new technology of a set of high yield AD, this technology is to adopt to take plant sterol as main, be aided with analysis for soybean powder simultaneously, glucose and other conventional common fermentation Chemicals, integrated use modern biotechnology, a series of technological difficulties that microbial selective degraded side chain obtains the suitability for industrialized production of Androstenedione product have been solved, bacterial classification transformation efficiency is high, compare with chemosynthesis, technical process is short, reaction conditions is gentle, extraction solvent can reclaim and make full use of, there is very high cost advantage, it is a kind of brand new technical that is different from traditional technology, effectively promoted the upgrading of conventional process techniques.
Summary of the invention
At present, improve the ability that plant sterol degradation bacteria is produced, still take selection by mutation as Main Means.For problems of the prior art, the invention provides and a kind ofly by mycobacterium being carried out to complex mutation, improve its method to plant sterol degradation rate preparation " Androstenedione "
1, of the present inventionly a kind ofly by mycobacterium being carried out to complex mutation, improve its method to plant sterol degradation rate preparation " Androstenedione ", it comprises the following steps:
(1) take plant sterol degradation bacteria as starting strain, plant sterol degradation bacteria is obtained to fresh ripe thalline for preculture 3-4 days on slant medium.
(2) the plant sterol degradation bacteria after preculture being prepared into biomass is 1 * 10
6the bacteria suspension of individual/mL carries out ion beam irradiation mutagenesis: mutagenesis temperature is 25 ℃, and mutagenesis dosage is to be coated with immediately medium slant after 50Gy mutagenesis.(3) after inclined-plane maturation, prepare seed liquor, after seed liquor inoculation, cultivate and be coated with flat board after 14-16h and carry out ultraviolet mutagenesis: under the condition that is 20cm with ultraviolet lamp tube and the fixed range of 15W power, carry out split dose irradiation, irradiation dose is 0s, 30s, 60s, 90s, 120s, 150s, 180s, after mutagenesis, through culture medium flat plate primary dcreening operation and shake flask fermentation method, sieve again again, obtain high yield bacterium colony.
(4) this high yield bacterium colony is prepared to inclined-plane, seed liquor again, after seed liquor inoculation, cultivate and be coated with flat board after 14-16h and again carry out ultraviolet and laser Mutation: under the condition that is 20cm with ultraviolet lamp tube and the fixed range of 15W power, irradiate 2 minutes, immediately lucifuge shifts and in laser machining device, uses the laser radiation of YAG double frequency pulse, and umber of exposures is respectively 200,400,600,800,1000,1200.After mutagenesis, through culture medium flat plate primary dcreening operation and shake flask fermentation method, sieve again again, finally obtain high yield bacterium colony.
2, the formula of the foster base in step (1) or step (2) or step (4) inclined-plane is: glucose 10g, and ammonium nitrate 12g, magnesium sulfate 2.5g, potassium primary phosphate 0.25g, yeast soaks powder 4.5g, and agar powder 20g adds water and is settled to 1000ml, and adjust pH is to 7.0-7.2.The formula of the plate culture medium of step (3) or step (4) is: glucose 10g, and ammonium nitrate 12g, magnesium sulfate 0.3g, potassium primary phosphate 0.25g, yeast soaks powder 4.5g, and agar powder 20g adds water and is settled to 1000ml, and adjust pH is to 7.0-7.2.The formula of the seed liquor substratum of step (3) or step (4) is: glucose 2.4g, and ammonium nitrate 2.4g, magnesium sulfate 0.6g, potassium primary phosphate 0.80g, yeast soaks powder 15g, adds water and is settled to 1000ml, adjust pH to 7.0.
3, step (1) or step (2) step (3) or step (4) inclined-plane and dull and stereotyped culture condition are: temperature is 29 ± 1 ℃, and humidity is 30-60%, and dull and stereotyped culture cycle is 6 days, and the slant culture cycle is 4 days.
4, the formula of the seed liquor substratum of step (3) or step (4) is: glucose 2.4g, and ammonium nitrate 2.4g, magnesium sulfate 0.6g, potassium primary phosphate 0.80g, yeast soaks powder 15g, adds water and is settled to 1000ml, adjust pH to 7.0.Its culture condition is 29 ℃, 220 revs/min, and 16 hours.
5, the formula of the shake-flask seed bottle substratum of step (3) or step (4) is: glucose 2.4g, and ammonium nitrate 2.4g, magnesium sulfate 0.6g, potassium primary phosphate 0.80g, yeast soaks powder 15g, adds water and is settled to 1000ml, adjust pH to 7.0.
Medium of shaking flask fermentation formula is: glucose 30g, and yeast soaks powder 24g, magnesium sulfate 2g, dipotassium hydrogen phosphate 2g, ferrous sulfate 0.005g, calcium carbonate 10g, plant sterol 50g, PEG60g, resin 60g, adds water and is settled to 1000ml, adjust pH to 7.0.
6, the shake flask fermentation of step (3) or step (4) is by inoculum size, to be in 10% access fermentation shake flask substratum by cultivating ripe seed liquor in seed culture medium.
7, the shake-flask seed liquid culture condition of step (3) or step (4) is: 29 ℃, and 220 revs/min, shaking culture 32-36h.The shake flask fermentation liquid culture condition of step (3) or step (4) is: 29 ℃, and 220 revs/min, shaking culture 168h.
8, ripe rear its titer detection method of the shake flask fermentation liquid of step (3) or step (4) cultivation is high performance liquid chromatography:
Chromatographic condition: chromatographic column: Kromasil C
185 μ m 200 * 4.6mm, moving phase: CH
3oH:H
2o=80: 20 (v/v), flow velocity: 1.0ml/min, detects wavelength: 242nm.
The mensuration of typical curve:
Prepare the AD standardized solution (50-500 μ g/ml) of a series of different concns, the filtered liquid 20 μ l that get respectively each concentration standard solution inject high performance liquid chromatograph, measure its absorption peak area, then concentration (Y) and absorption peak area (X) are carried out to one-variable linear regression, obtain one-variable linear regression equation.
The method of calculation of tiring:
Testing sample is made to the methanol solution of suitable concn (50-500 μ g/ml), with the filtering with microporous membrane of 0.45 μ m, accurately draw filtrate 20 μ l and inject HPLC, record color atlas, by in the above-mentioned regression equation of AD peak area substitution, calculate AD amount in sample.
9, the shake flask fermentation liquid of step (3) or step (4) is cultivated ripe rear plant sterol transformation efficiency method of calculation: after having fermented, fermented liquid is after methyl alcohol is processed, by liquid chromatography, quantitatively measure again the content of Androstenedione, then calculate the transformation efficiency of plant sterol: the AD content * 100/(sterol content * 0.66*0.95 in oil that feeds intake in transformation efficiency/%=fermented liquid).
10, first class seed pot culture medium prescription: glucose 9g, ammonium nitrate 9g, magnesium sulfate 2.25g, potassium primary phosphate 2.25g, yeast soaks powder 45g, adds water and is settled to 2L, adjust pH to 7.0.
Fermentor cultivation based formulas is: glucose 500g, and yeast soaks powder 375g, ammonium nitrate 125g, magnesium sulfate 50g, dipotassium hydrogen phosphate 30g, ferrous sulfate 0.125g, calcium carbonate 25g, plant sterol 1250g, resin 1500g, adds water and is settled to 20L, adjust pH to 7.0.
11, will cultivate ripe mycobacterium seed liquor according in the inoculum size access 50L fermentor tank of 10% (V/V), 30 ℃, 220 revs/min, aerated culture, about 7 days, obtains Androstenedione, utilizes HPLC to measure and tires.
Advantage of the present invention:
1, the present invention has tempting prospect by its Industrialized processing technique of microorganism fermented extracted " AD "; not only can fundamentally change the weak points such as traditional chemical synthesizing steroid medicine step is many, yield is low, price; also can make full use of and bring into play resources advantage; break away from starting material source because the natural causes such as season, region are to the restriction of producing, to protecting national resource with ecotope, safeguarding that human health plays significant role.
2, the present invention, by three kinds of mutafacient system, four mutagenesis, twice screening, obtains the sudden change mycobacterium higher to plant sterol degradation rate.By present method, obtaining plant sterol degradation bacteria is 5% in substrate sterol concentration, and under the basis that resin concentration is 6%, 50L fermentor tank is put tank and on average tired and can reach 25-30g/ml, to the transformation efficiency of plant sterol, is 86.8%.Will greatly reduce production costs so in process of production.
The present invention realizes by following technical measures:
1 prepares materials and methods
1.1 material
1.1.1 bacterial classification
Bacterial classification source: DY20111816-102(Shandong Dong Yao medicine company limited-liability company)
1.1.2 instrument and analysis condition
HPLC: Agilent-1260, chromatographic condition: chromatographic column---Kromasil C
185 μ m 200 * 4.6mm
Moving phase---CH
3oH:H
2o=80: 20 (v/v), flow velocity---1.0ml/min, detects wavelength---242nm.Reagent: 4-AD (AD) standard substance and methyl alcohol are chromatographically pure, all the other reagent are analytical pure.Content calculates by external standard peak area method.
1.2 implementation method
1.2.1 substratum
Inclined-plane and plate culture medium: glucose 10g, ammonium nitrate 12g, magnesium sulfate 2.5g, potassium primary phosphate 0.25g, yeast soaks powder 4.5g, and agar powder 20g adds water and is settled to 1000ml, and adjust pH is to 7.0-7.2.
Seed bottle substratum:
Glucose 2.4g, ammonium nitrate 2.4g, magnesium sulfate 0.6g, potassium primary phosphate 0.80g, yeast soaks powder 15g, adds water and is settled to 1000ml, adjust pH to 7.0.
Fermentation flask substratum: glucose 30g, yeast soaks powder 24g, magnesium sulfate 2g, dipotassium hydrogen phosphate 2g, ferrous sulfate 0.005g, calcium carbonate 10g, plant sterol 50g, PEG60g, resin 60g, adds water and is settled to 1000ml, adjust pH to 7.0.
First class seed pot culture medium prescription: glucose 9g, ammonium nitrate 9g, magnesium sulfate 2.25g, potassium primary phosphate 2.25g, yeast soaks powder 45g, adds water and is settled to 2L, adjust pH to 7.0.
Fermentor cultivation based formulas is: glucose 500g, and yeast soaks powder 375g, ammonium nitrate 125g, magnesium sulfate 50g, dipotassium hydrogen phosphate 30g, ferrous sulfate 0.125g, calcium carbonate 25g, plant sterol 1250g, resin 1500g, adds water and is settled to 20L, adjust pH to 7.0.
1.2.2 culture condition:
Flat board and inclined-plane: temperature is 29 ± 1 ℃, humidity is 30-60%, and dull and stereotyped culture cycle is 5-7 days, and the slant culture cycle is 4-5 days.
Shake-flask seed: 29 ℃ ± 1,220 rev/min, shaking culture 32-36h.
Shake flask fermentation liquid culture condition is: 29 ± 1 ℃, and 220 revs/min, shaking culture 168h.
1.2.3 technology of preparing--mutagenic treatment
(1) ion beam irradiation mutagenesis: it is 1 * 10 that the plant sterol degradation bacteria after preculture is prepared into biomass
6the bacteria suspension of individual/mL carries out ion beam irradiation mutagenesis: mutagenesis temperature is 25 ℃, and mutagenesis dosage is to be coated with immediately medium slant after 50Gy mutagenesis.
(2) ultraviolet mutagenesis: prepare seed liquor after inclined-plane maturation, after seed liquor inoculation, cultivate and be coated with flat board after 14-16h and carry out ultraviolet mutagenesis: under the condition that is first 20cm with ultraviolet lamp tube and the fixed range of 15W power, carry out split dose irradiation, irradiation dose is 0s, 30s, 60s, 90s, 120s, 150s, 180s, after mutagenesis, through culture medium flat plate primary dcreening operation and shake flask fermentation method, sieve again again, obtain high yield bacterium colony.
(3) Ultra-Violet Laser complex mutation: this high yield bacterium colony is prepared to inclined-plane, seed liquor again, after seed liquor inoculation, cultivate and be coated with flat board after 14-16h and again carry out ultraviolet and laser Mutation: under the condition that is 20cm with ultraviolet lamp tube and the fixed range of 15W power, irradiate 2 minutes, immediately lucifuge shifts and in laser machining device, uses the laser radiation of YAG double frequency pulse, and umber of exposures is respectively 200,400,600,800,1000,1200.After mutagenesis, through culture medium flat plate primary dcreening operation and shake flask fermentation method, sieve again again, finally obtain high yield bacterium colony.
2 test-results and analysis
2.1 ion beam irradiation mutagenesis
It is 1 * 10 that plant sterol degradation bacteria after preculture is prepared into biomass
6the bacteria suspension of individual/mL is 25 ℃ in mutagenesis temperature, carries out mutagenesis under the condition that mutagenesis dosage is 50Gy, after mutagenesis finishes, is coated with immediately medium slant.After inclined-plane maturation, bacterium layer is thick compared with control group, and color is heavier.
2.2 ultraviolet mutagenesis
Random choose some amount is screened through single bacterium colony of various dose UV-irradiation.Found that, although positive mutation rate improves with the increase of mutagenic strain mortality ratio, the bacterial classification that gain mutant amplitude is the highest appears at low dose exposure group (table 1).
Table 1 ultraviolet mutagenesis result
| Irradiation time/s | Mortality ratio/% | Positive mutation rate/% | Transform increase rate/% |
| 0 | 0 | 0 | 0 |
| 30 | 10 | 1.5 | 2.4 |
| 60 | 27 | 2.2 | 5.1 |
| 90 | 59 | 7.3 | 11.6 |
| 120 | 73 | 14.9 | 17.6 |
| 150 | 91 | 6.5 | 9.7 |
| 180 | 100 | 0 | 0 |
The mutagenesis of 2.3 UV-light and laser multiple processing
Random choose some amount is screened through single bacterium colony of UV-light and laser multiple processing mutagenesis, through screening, obtains its transformation efficiency of a strain and with respect to starting strain, improved 23.2% high yield and transform bacterial strain in the experimental group of laser radiation 600 times
The study on the stability of 3 mutant strains
The superior strain continuous passage obtaining is carried out to study on the stability, found that, this bacterial strain conversion capability proterties after 4 times go down to posterity is still stablized (in Table 2).
The study on the stability result of table 2 mutant strain
| Passage number | (g/L) tires |
| 0 | 26.4 |
| 1 | 27.2 |
| 2 | 26.9 |
| 3 | 27.0 |
| 4 | 26.8 |
4 conclusion the present invention, by three kinds of mutafacient system, four mutagenesis, twice screening, obtain the sudden change mycobacterium higher to plant sterol degradation rate.By present method, obtaining plant sterol degradation bacteria is 5% in substrate sterol concentration, and under the basis that resin concentration is 6%, 50L fermentor tank is put tank and on average tired and can reach 25-30g/ml, to the transformation efficiency of plant sterol, is 86.8%.Will greatly reduce production costs so in process of production.
Claims (9)
1. by a kind of mycobacterium being carried out to complex mutation, improve its method to plant sterol degradation rate preparation " Androstenedione ", its feature comprises the following steps successively:
(1) take plant sterol degradation bacteria as starting strain, by plant sterol degradation bacteria preculture 4 days on slant medium;
(2) the plant sterol degradation bacteria after preculture being prepared into biomass is 1 * 10
6the bacteria suspension of individual/mL carries out ion beam irradiation mutagenesis: mutagenesis temperature is 25 ℃, and mutagenesis dosage is to be coated with immediately medium slant after 50Gy mutagenesis; (3) after inclined-plane maturation, prepare seed liquor, after seed liquor inoculation, cultivate and be coated with flat board after 14-16h and carry out ultraviolet mutagenesis: under the condition that is 20cm with ultraviolet lamp tube and the fixed range of 15W power, carry out split dose irradiation, irradiation dose is 0s, 30s, 60s, 90s, 120s, 150s, 180s, after mutagenesis, through culture medium flat plate primary dcreening operation and shake flask fermentation method, sieve again again, obtain high yield bacterium colony;
(4) this high yield bacterium colony is prepared to inclined-plane, seed liquor again, after seed liquor inoculation, cultivate and be coated with flat board after 14-16h and again carry out ultraviolet and laser Mutation: under the condition that is 20cm with ultraviolet lamp tube and the fixed range of 15W power, irradiate 2 minutes, immediately lucifuge is transferred in laser machining device and uses the laser radiation of YAG double frequency pulse, and umber of exposures is respectively 200,400,600,800,1000,1200; After mutagenesis, through culture medium flat plate primary dcreening operation and shake flask fermentation method, sieve again again, finally obtain high yield bacterium colony.
2. according to claim 1ly by a kind of mycobacterium being carried out to complex mutation, improve its method to plant sterol degradation rate preparation " Androstenedione ", it is characterized in that: the formula that base is supported on step (1) or step (2) or step (4) inclined-plane is: glucose 10g, ammonium nitrate 12g, magnesium sulfate 2.5g, potassium primary phosphate 0.25g, yeast soaks powder 4.5g, agar powder 20g, add water and be settled to 1000ml, adjust pH is to 7.0-7.2; The formula of the plate culture medium of step (3) or step (4) is: glucose 10g, and ammonium nitrate 12g, magnesium sulfate 0.3g, potassium primary phosphate 0.25g, yeast soaks powder 4.5g, and agar powder 20g adds water and is settled to 1000ml, and adjust pH is to 7.0-7.2; The formula of the seed liquor substratum of step (3) or step (4) is: glucose 2.4g, and ammonium nitrate 2.4g, magnesium sulfate 0.6g, potassium primary phosphate 0.80g, yeast soaks powder 15g, adds water and is settled to 1000ml, adjust pH to 7.0.
3. according to claim 1ly by a kind of mycobacterium being carried out to complex mutation, improve its method to plant sterol degradation rate preparation " Androstenedione ", it is characterized in that: step (1) or step (2) step (3) or step (4) inclined-plane and dull and stereotyped culture condition are: temperature is 29 ± 1 ℃, humidity is 30-60%, dull and stereotyped culture cycle is 5-7 days, and the slant culture cycle is 4-5 days.
4. according to claim 1ly by a kind of mycobacterium being carried out to complex mutation, improve its method to plant sterol degradation rate preparation " Androstenedione ", it is characterized in that: the formula of the seed liquor substratum of step (3) or step (4) is: glucose 2.4g, ammonium nitrate 2.4g, magnesium sulfate 0.6g, potassium primary phosphate 0.80g, yeast soaks powder 15g, adds water and is settled to 1000ml, adjust pH to 7.0; Its culture condition is 30 ℃, and 220 revs/min, 16 hours, humidity was 30-60%.
5. according to claim 1ly by a kind of mycobacterium being carried out to complex mutation, improve its method to plant sterol degradation rate preparation " Androstenedione ", it is characterized in that: the formula of the shake-flask seed bottle substratum of step (3) or step (4) is: glucose 2.4g, ammonium nitrate 2.4g, magnesium sulfate 0.6g, potassium primary phosphate 0.80g, yeast soaks powder 15g, adds water and is settled to 1000ml, adjust pH to 7.0; The formula of Medium of shaking flask fermentation is: glucose 30g, and yeast soaks powder 24g, magnesium sulfate 2g, dipotassium hydrogen phosphate 2g, ferrous sulfate 0.005g, calcium carbonate 10g, plant sterol 50g, PEG60g, resin 60g, adds water and is settled to 1000ml, adjust pH to 7.0.
6. according to claim 1ly by a kind of mycobacterium being carried out to complex mutation, improve its method to plant sterol degradation rate preparation " Androstenedione ", it is characterized in that: the shake flask fermentation of step (3) or step (4) is by inoculum size, to be in 10% access Medium of shaking flask fermentation by cultivating ripe seed liquor in seed culture medium.
7. according to claim 6ly by a kind of mycobacterium being carried out to complex mutation, improve its method to plant sterol degradation rate preparation " Androstenedione ", it is characterized in that: the shake-flask seed liquid culture condition of step (3) or step (4) is: 30 ℃, 220 revs/min, shaking culture 32-36h; The shake flask fermentation liquid culture condition of step (3) or step (4) is: 30 ℃, and 220 revs/min, shaking culture 168h.
8. according to claim 7ly by a kind of mycobacterium being carried out to complex mutation, improve its method to plant sterol degradation rate preparation " Androstenedione ", it is characterized in that: the shake flask fermentation liquid of step (3) or step (4) cultivate ripe after its titer detection method be high performance liquid chromatography:
Chromatographic condition: chromatographic column: Kromasil C
185 μ m 200 * 4.6mm, moving phase: CH
3oH:H
2o=80: 20 (v/v), flow velocity: 1.0ml/min, detects wavelength: 242nm;
The mensuration of typical curve:
Prepare the AD standardized solution (50-500 μ g/ml) of a series of different concns, get respectively each concentration standard solution 20 μ l and inject high performance liquid chromatograph, measure its absorption peak area, then concentration (Y) and absorption peak area (X) are carried out to one-variable linear regression, obtain one-variable linear regression equation;
The method of calculation of tiring:
Testing sample is made to the methanol solution of suitable concn (50-500 μ g/ml), with the filtering with microporous membrane of 0.45 μ m, accurately draw filtrate 20 μ l and inject HPLC, record color atlas, by in the above-mentioned regression equation of AD peak area substitution, calculate AD amount in sample.
9. according to claim 7ly by a kind of mycobacterium being carried out to complex mutation, improve its method to plant sterol degradation rate preparation " Androstenedione ", it is characterized in that: the shake flask fermentation liquid of step (3) or step (4) is cultivated ripe rear plant sterol transformation efficiency method of calculation: after having fermented, fermented liquid is after methyl alcohol is processed, by liquid chromatography, quantitatively measure again the content of Androstenedione, then calculate the transformation efficiency of plant sterol: the AD content * 100/(sterol content * 0.66*0.95 in oil that feeds intake in transformation efficiency/%=fermented liquid.
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| CN104651261A (en) * | 2014-12-08 | 2015-05-27 | 云南中烟工业有限责任公司 | Microbial strain and applications thereof in degradation of tobacco sterol |
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| CN102586139A (en) * | 2012-01-20 | 2012-07-18 | 广东本科生物工程股份有限公司 | High-yield AD/ADD strain and method for high-efficient production of AD/ADD |
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| CN104651261A (en) * | 2014-12-08 | 2015-05-27 | 云南中烟工业有限责任公司 | Microbial strain and applications thereof in degradation of tobacco sterol |
| CN104651261B (en) * | 2014-12-08 | 2017-09-22 | 云南中烟工业有限责任公司 | A kind of microbial strains and its application in degrading tobacco sterol |
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