CN103667126B - Mycobacterium smegmatis for fermentation production of 9-OH-AD, bactericide, preparation method and application - Google Patents
Mycobacterium smegmatis for fermentation production of 9-OH-AD, bactericide, preparation method and application Download PDFInfo
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Abstract
The invention relates to a strain for producing steroid hormones and a preparation method of the strain, in particular to mycobacterium smegmatis for fermentation production of 9-OH-AD, a bactericide, a preparation method and an application. The mycobacterium smegmatis is collected in China center for type culture collection (CCTCC) and has a collection number of CCTCC NO:M2013544. The 9-OH-AD is prepared by fermenting the strain, so that the dehydrogenation of the positions 1 and 2 of the 9-OH-AD is effectively inhibited, the degradation of the 9-OH-AD is inhibited, and the yield by weight is increased and can reach 50%-55%.
Description
Technical field
The present invention relates to a kind of bacterial strain and the preparation method that produce steroid hormone, specifically M. smegmatics, microbial inoculum, the preparation method and application of a kind of fermentative production 9-OH-AD.
Background technology
Modern age, the achievement in research of steroidal intermediate is prepared by wide coverage and research about using microbial method transformation phytosterin, relevant report can trace back to nineteen thirty-seven the earliest, two sections of patent Ber.70470 and Ber.70 that Mamoli and Vercellone delivers, 2079, in the patent, they are for using yeast by 17-carbonyl reduction for 17-beta-hydroxy is set forth.After this, peterson and Murray has set forth Progesterone 11 α hydroxylations in US Pat.No.2602769.1972, Kraychy etc. set forth at US Pat.No.3684657 and have prepared 4-AD, the method for ADD with Mycobacterium SP.NRRL B-3683 17 aliphatic chains of degrading.1973, Marsheck etc. to set forth with Mycobacterium SP.NRRL B-3805 in patent US Pat.No.3759791, had the steroidal raw material of more than 8 carbon atoms to prepare 4-AD with cholestane etc. 17.
9-OH-AD is the important intermediate preparing cortisone, up to now, a lot of bibliographical information 9-OH-17-carbonyl is had to be correlated with the preparation method of steroidal particularly 9-OH-AD, US patent4397947 reports the method transforming 4-AD to 9-OH-AD with Nocardia bacteria, and EP-A-0027829 reports the method transforming 4-AD to 9-OH-AD with Corynespora cassicola bacterium.Merle G. in 1977 etc. have set forth the process being prepared 9-OH-AD by plant sterol in US Pat.No.40352236.USpatent3759791 reports and uses mycobacterium, take sterol as the method that substrate prepares 9-OH-AD.1988, patent GB2197869 reports by Mycobacterium roseum, take sterol as the method that substrate prepares 9-OH-AD, weight yield 28.5%, 1986, in EP0322081B1, Slijkhuls etc. have set forth the example using bacterial classification Mycobacterium speciesCBS482.86 sterol to be transformed 9-OH-AD, its charging capacity about 1.3% (90% content), transforms 216 hours, HPLC weight yield 42.1%.
Summary of the invention
The technical problem to be solved in the present invention is to provide the M. smegmatics that a kind of fermenting plant sterol produces 9-OH-AD, and microbial inoculum, preparation method and application, the weight yield that the present invention produces 9-OH-AD is high, and fermentation efficiency is high.
Substrate used herein is plant sterol, and bacterial strain is M. smegmatics NK-XHX-118(Mycobacteriumsmegmatis NK-XHX-118), be preserved in China typical culture collection center, deposit number is: CCTCC NO:M2013544.
Above-mentioned M. smegmatics (Mycobacterium smegmatis) CCTCC NO:M2013544 is cultivated by M. smegmatics (Mycobacterium smegmatis) the CD116 mutagenesis collected voluntarily and obtains.
Mutagenic processes is as follows:
1, original strain is M. smegmatics (Mycobacterium smegmatis) CD116, and this bacterial strain can degrading plant sterol, obtains in piling up in the soil near plant sterol place to screen.
By original strain 30 DEG C of cultivations, 180rpm cultivates, substratum: peptone 1%, extractum carnis 0.3%, sodium-chlor 0.5%, yeast extract paste 0.1%, Sodium Propionate 0.05%, and pH value 10% sodium hydroxide solution is adjusted to 7.0,121 DEG C of sterilizings 30 minutes.
When cell density grows to 5 × 108/ml, centrifugal, the aseptic sodium citrate solution of thalline pH=5.6,0.1mol/L rinses, and with lemon sodium solution by mycelium dilution to original volume, add nitrosoguanidine again, nitrosoguanidine concentration: 60 μ g/ml, bacteria suspension 37 DEG C of water-bath half an hour, recentrifuge, the phosphoric acid buffer of the 0.1mol/L of thalline equivalent, pH=7 rinses.Finally, thalline is suspended dispersed in the phosphoric acid buffer of 0.1mol/L, pH=7 again, obtains the bacteria suspension after mutagenesis.
2, bacterial strain screening
Primary dcreening operation substratum: glucose 1%, Sodium phosphate dibasic 0.4%, potassium primary phosphate 0.4%, magnesium sulfate heptahydrate 0.03%, iron vitriol 0.005%, agar 2%, pH=7.5, substratum 121 DEG C of 30min that go out.
Sieve substratum again: 9-OH-AD0.2%, Sodium phosphate dibasic 0.4%, potassium primary phosphate 0.4%, magnesium sulfate heptahydrate 0.03%, iron vitriol 0.005%, agar 2%, pH=7.5.Substratum 121 DEG C of 30min that go out.
Bacteria suspension after mutagenesis is carried out dilution spread, and substratum adopts primary dcreening operation substratum, cultivates 7 days in 30 DEG C.Picking list colony inoculation is in multiple sieve substratum, and every thalline that can not grow in multiple sieve substratum, carries out further culture purified with primary dcreening operation substratum, obtain the bacterial strain after mutagenesis.
3, bacterial classification shaking flask transforms test
Liquid seed culture medium: glucose 1%, yeast extract paste 0.6%, Sodium phosphate dibasic 0.4%, potassium primary phosphate 0.4%, magnesium sulfate heptahydrate 0.03%, iron vitriol 0.005%, pH=7.5, substratum 121 DEG C of 30min that go out.
Bioconversion medium: potassium primary phosphate 0.08%, three water dipotassium hydrogen phosphates 0.42%, Secondary ammonium phosphate 0.3%, glucose 0.5%, beta-cyclodextrin (powder) 2%, plant sterol 0.5%, pH=8.0.
Bacterial strain after mutagenesis is in 30 DEG C, 180rpm cultivates 72 hours, is inoculated in bioconversion medium, inoculum size 20%, conversion condition: 30 DEG C, 220rpm, transforms 72 hours, and Liquid Detection is sent in sampling, good and bad according to liquid phase result determination aimed strain, obtain the dissociant that a strain is outstanding, by it called after M. smegmatics NK-XHX-118(Mycobacterium smegmatisNK-XHX-118), i.e. M. smegmatics (Mycobacterium smegmatis) CCTCC NO:M2013544.
The present invention also provides a kind of liquid bacterial agent containing M. smegmatics, and the cell concentration of described M. smegmatics (Mycobacteriumsmegmatis) CCTCC NO:M2013544 reaches 1,000,000,000/more than ml.
The feature of M. smegmatics of the present invention (Mycobacterium smegmatis) CCTCC NO:M2013544 is: under Different growing environment, thalline presents different shape, but be essentially shaft-like, bacterium colony is canescence or faint yellow, thalline size is generally little compared with other bacteriums, usual length 1.5 ~ 2.5 microns, wide 0.3 ~ 0.4 micron.Its 16S rRNA gene order is SEQ ID No:1.Its sequence is:
TGCAAGTCGAACGGAAAGGCCCTTCGGGGTACTCGAGTGGCGAACGGGTGAGTAACACGTGGGTGATCTGCCCTGCACTTTGGGATAAGCCTGGGAAACTGGGTCTAATACCGAATATGACCACGCGCTTCATGGTGTGTGGTGGAAAGCTTTTGCGGTGTGGGATGGGCCCGCGGCCTATCAGCTTGTTGGTGGGGTAATGGCCTACCAAGGCGACGACGGGTAGCCGGCCTGAGAGGGTGACCGGCCACACTGGGACTGAGATACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCGACGCCGCGTGAGGGATGACGGCCTTCGGGTTGTAAACCTCTTTCAATAGGGACGAAGCGCAAGTGACGGTACCTATAGAAGAAGGACCGGCCAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGTCCGAGCGTTGTCCGGAATTACTGGGCGTAAAGAGCTCGTAGGTGGTTTGTCGCGTTGTTCGTGAAAACTCACAGCTTAACTGTGGGCGTGCGGGCGATACGGGCAGACTAGAGTACTGCAGGGGAGACTGGAATTCCTGGTGTAGCGGTGGAATGCGCAGATATCAGGAGGAACACCGGTGGCGAAGGCGGGTCTCTGGGCAGTAACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGGTGGGTACTAGGTGTGGGTTTCCTTCCTTGGGATCCGTGCCGTAGCTAACGCATTAAGTACCCCGCCTGGGGAGTACGGCCGCAAGGCTAAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGCGGAGCATGTGGATTAATTCGATGCAACGCGAAGAACCTTACCTGGGTTTGACATGCACAGGACGACTGCAGAGATGTGGTTTCCCTTGTGGCCTGTGTGCAGGTGGTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTCTCATGTTGCCAGCACGTTATGGTGGGGACTCGTGAGAGACTGCCGGGGTCAACTCGGAGGAAGGTGGGGATGACGTCAAGTCATCATGCCCCTTATGTCCAGGGCTTCACACATGCTACAATGGCCGGTACAAAGGGCTGCGATGCCGTGAGGTGGAGCGAATCCTTTCAAAGCCGGTCTCAGTTCGGATCGGGGTCTGCAACTCGACCCCGTGAAGTCGGAGTCGCTAGTAATCGCAGATCAGCAACGCTGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACGTCATGAAAGTCGGTAACACCCGAAGCCGGTGGCCTAACCCTTGTGGAGGGAGCCGTCGAA。
Fermentation reaction equation of the present invention can be:
The concrete steps of producing containing the liquid bacterial agent of M. smegmatics are as follows:
(1) inclined-plane seed culture:
Inclined-plane seed culture medium sterilizing, inoculates M. smegmatics (Mycobacterium smegmatis) CCTCC NO:M2013544,28 ~ 32 DEG C after cooling, preferably cultivate 3-5 days for 30 DEG C.Described inclined-plane seed culture based formulas is, described inclined-plane seed culture based formulas is, corn steep liquor 9 ~ 11g/L, ammonium sulfate 1.3 ~ 1.7g/L, potassium primary phosphate 0.45 ~ 0.55g/L, dipotassium hydrogen phosphate 0.5 ~ 0.6g/L, sucrose 0.45 ~ 0.55g/L, magnesium sulfate heptahydrate 0.18 ~ 0.22g/L, iron vitriol 0.004 ~ 0.006g/L, Zinc Sulphate Heptahydrate 0.001 ~ 0.003g/L, agar 18 ~ 22g/L, adjust ph is 7.1 ~ 7.2.Screening formulation is, corn steep liquor 10g/L, ammonium sulfate 1.5g/L, potassium primary phosphate 0.5g/L, dipotassium hydrogen phosphate 0.55g/L, sucrose 0.5g/L, magnesium sulfate heptahydrate 0.2g/L, iron vitriol 0.005g/L, Zinc Sulphate Heptahydrate 0.002g/L, agar 20g/L, adjust ph is 7.1 ~ 7.2.
(2) first order seed is cultivated:
Liquid seed culture medium sterilizing, is cooled to room temperature, and the bacterium colony of above-mentioned inclined-plane seed culture is added in liquid seed culture medium and cultivates, temperature is 28 ~ 32 DEG C, preferably 30 DEG C, and rotating speed is 200rpm, and the time is 48 hours.Liquid seed culture medium formula is, liquid seed culture medium formula is, corn steep liquor 9 ~ 11g/L, ammonium sulfate 1.3 ~ 1.7g/L, potassium primary phosphate 0.45 ~ 0.55g/L, dipotassium hydrogen phosphate 0.5 ~ 0.6g/L, sucrose 9 ~ 11g/L, magnesium sulfate heptahydrate 0.18 ~ 0.22g/L, iron vitriol 0.004 ~ 0.006g/L, Zinc Sulphate Heptahydrate 0.001 ~ 0.003g/L, adjust ph is 7.1 ~ 7.2.Screening formulation is, corn steep liquor 10g/L, ammonium sulfate 1.5g/L, potassium primary phosphate 0.5g/L, dipotassium hydrogen phosphate 0.55g/L, sucrose 10g/L, magnesium sulfate heptahydrate 0.2g/L, iron vitriol 0.005g/L, Zinc Sulphate Heptahydrate 0.002g/L, adjust ph is 7.1-7.2.
(3) secondary seed is cultivated:
Bacterium liquid after being cultivated by first order seed is inoculated in aforesaid liquid seed culture medium, and inoculum size is 9 ~ 11%, and be preferably 10%, temperature is 28 ~ 32 DEG C, and be preferably 30 DEG C, rotating speed is 200rpm, and the time is 48 hours, must containing the liquid bacterial agent of M. smegmatics.
The application of described liquid bacterial agent of the present invention in fermentative production 9-OH-AD, step is as follows,
1, the strain activity after secondary seed cultivation is detected
Detect medium sterilization.Being inoculated into by liquid bacterial agent (liquid bacterial agent after secondary seed cultivation) to be tested detects in substratum, and inoculum size 4.5 ~ 5.5%, 180 ~ 220rpm, cultivates 48 hours for 28 ~ 32 DEG C.Liquid Detection is sent in sampling, if wherein 9-OH-AD concentration is greater than 5g/L, then the liquid bacterial agent after secondary seed cultivation is qualified, is suitable for transforming.Detection substratum is, diammonium hydrogen phosphate 3g/L, potassium primary phosphate 1g/L, dipotassium hydrogen phosphate 4g/L, sucrose 5g/L, magnesium sulfate heptahydrate 0.2g/L, iron vitriol 0.005g/L, Zinc Sulphate Heptahydrate 0.002g/L, beta-cyclodextrin 50g/L, plant sterol 10g/L, pH are 7.1 ~ 7.2.
2, fermentation culture
Above-mentioned qualified liquid bacterial agent is inoculated in the bioconversion medium after sterilizing, and inoculum size is 9-11%, and be preferably 10%, invert point is 28 ~ 32 DEG C, and be preferably 30 DEG C, rotating speed is 400 ~ 600rpm, and transformation time is 120 hours, and air flow quantity is 0.18 ~ 0.22Nm
3/ h, is preferably 0.2Nm
3/ h, tank pressure 0.045 ~ 0.055MPa, be preferably 0.05MPa, described bioconversion medium formulation by weight is, potassium primary phosphate 0.07 ~ 0.09%, three water dipotassium hydrogen phosphates 0.4 ~ 0.5%, Secondary ammonium phosphate 0.2 ~ 0.4%, glucose 0.45 ~ 0.55%, beta-cyclodextrin 6 ~ 7%, plant sterol 1.8 ~ 2.2%, mass concentration is the silicone antifoam agent 0.045 ~ 0.055% of 20-40%, trace nutrient 3 ~ 5%, pH is 7 ~ 9, the composition of described trace nutrient is magnesium sulfate heptahydrate 0.8 ~ 1.2%, green vitriol 0.45 ~ 0.55%, Zinc Sulphate Heptahydrate 0.08 ~ 0.12%, mass concentration is the sulfuric acid 0.05% of 20 ~ 40%, all the other are water.Preferred formulation by weight is, potassium primary phosphate 0.08%, three water dipotassium hydrogen phosphates 0.42%, Secondary ammonium phosphate 0.3%, glucose 0.5%, beta-cyclodextrin 6.65%, plant sterol 2%, and mass concentration is the silicone antifoam agent 0.05% of 30%, and trace nutrient 4%, pH is 8.The composition of described trace nutrient is magnesium sulfate heptahydrate 1%, green vitriol 0.5%, Zinc Sulphate Heptahydrate 0.1%, and mass concentration is the sulfuric acid 0.05% of 30%, and all the other are water.
3, separation and Extraction
Centrifugal, isolate thalline, the fermented liquid chloroform extraction after centrifugal, the chloroform of each use 0.5 times of fermentating liquid volume, extracts three times, combined chloroform layer, be concentrated into small volume, add 500 ml methanol, be concentrated into and about remain 100 ml methanol, be cooled to 4 DEG C, suction filtration, cake layer 20 ml methanol drip washing, obtain white crystal, cake layer 70 DEG C oven dry, weight yield 50 ~ 55%, HPLC external standard content >97%.
4, fermented liquid recycling
Join in the aqueous phase of the fermented liquid after above-mentioned process by recycling substratum, adjusted to ph is 8, concentrated.
The invention has the beneficial effects as follows, strain fermentation of the present invention prepares 9-OH-AD, effectively inhibits 1 of 9-OH-AD, 2 dehydrogenations, and then inhibits it to degrade, and improve yield, weight yield can reach 50 ~ 55%.Relative to prior art, there is obvious progress.
Bacterial strain of the present invention is M. smegmatics NK-XHX-118(Mycobacterium smegmatis NK-XHX-118), be preserved in China typical culture collection center, deposit number is: CCTCC NO:M2013544, preservation address is China. Wuhan. and Wuhan University, preservation date is on November 4th, 2013.
Embodiment
Embodiment 1
Bacterial strain of the present invention is M. smegmatics NK-XHX-118(Mycobacterium smegmatis NK-XHX-118), be preserved in China typical culture collection center, deposit number is: CCTCC NO:M2013544.
Embodiment 2
Preparation method containing the liquid bacterial agent of M. smegmatics is as follows
(1) inclined-plane seed culture:
Inclined-plane seed culture, based on 121 DEG C of sterilizing 30min, inoculates M. smegmatics NK-XHX-118(Mycobacterium smegmatis NK-XHX-118 after cooling), deposit number is: CCTCC NO:M2013544, cultivates 3-5 days for 30 DEG C.Described inclined-plane seed culture based formulas is, corn steep liquor 10g/L, ammonium sulfate 1.5g/L, potassium primary phosphate 0.5g/L, dipotassium hydrogen phosphate 0.55g/L, sucrose 0.5g/L, magnesium sulfate heptahydrate 0.2g/L, iron vitriol 0.005g/L, Zinc Sulphate Heptahydrate 0.002g/L, agar 20g/L, adjust ph is 7.1-7.2.
(2) first order seed is cultivated:
Liquid seed culture medium, in 121 DEG C of 30min that go out, is cooled to room temperature, the bacterium colony of above-mentioned seed culture is added in liquid seed culture medium and cultivates, and temperature is 30 DEG C, and rotating speed is 200rpm, and the time is 48 hours.Liquid seed culture medium formula is, corn steep liquor 10g/L, ammonium sulfate 1.5g/L, potassium primary phosphate 0.5g/L, dipotassium hydrogen phosphate 0.55g/L, sucrose 10g/L, magnesium sulfate heptahydrate 0.2g/L, iron vitriol 0.005g/L, Zinc Sulphate Heptahydrate 0.002g/L, adjust ph is 7.1-7.2.
(3) secondary seed is cultivated:
Bacterium liquid after first order seed cultivation is inoculated in liquid seed culture medium, and inoculum size is 10%, and temperature is 30 DEG C, and rotating speed is 200rpm, and the time is 48 hours, must containing the liquid bacterial agent of M. smegmatics.
Embodiment 3
The application of liquid bacterial agent in fermentative production 9-OH-AD containing M. smegmatics of the present invention, step is as follows,
1, the strain activity after secondary seed cultivation is detected
Detect medium sterilization.Liquid bacterial agent containing M. smegmatics (liquid bacterial agent after secondary seed cultivation prepared by embodiment 2) to be tested is inoculated into and detects in substratum, inoculum size 5%, 200rpm, cultivates 48 hours for 30 DEG C.Liquid Detection is sent in sampling, if wherein 9-OH-AD concentration is greater than 5g/L, then the liquid bacterial agent after secondary seed cultivation is qualified, is suitable for transforming.Detection substratum is, diammonium hydrogen phosphate 3g/L, potassium primary phosphate 1g/L, dipotassium hydrogen phosphate 4g/L, sucrose 5g/L, magnesium sulfate heptahydrate 0.2g/L, iron vitriol 0.005g/L, Zinc Sulphate Heptahydrate 0.002g/L, beta-cyclodextrin 50g/L, plant sterol 10g/L, pH are 7.1-7.2.
2, fermentation culture
Above-mentioned qualified liquid bacterial agent is inoculated in the bioconversion medium after sterilizing, and inoculum size is 10%, and invert point is 30 DEG C, and rotating speed is 400rpm, and transformation time is 120 hours, and air flow quantity is 0.2Nm
3/ h, tank pressure 0.05MPa, bioconversion medium formula is, potassium primary phosphate 0.08%, three water dipotassium hydrogen phosphates 0.42%, Secondary ammonium phosphate 0.3%, glucose 0.5%, beta-cyclodextrin 6.65%, plant sterol 2%, mass concentration is the silicone antifoam agent 0.05% of 30%, and trace nutrient 4%, pH is 8.The composition of described trace nutrient is magnesium sulfate heptahydrate 1%, green vitriol 0.5%, Zinc Sulphate Heptahydrate 0.1%, and mass concentration is the sulfuric acid 0.05% of 30%, and all the other are water.
3, separation and Extraction
Sampling, book layer chromatography (TLC) is put plate and is detected, and transforms completely, centrifugal, isolate thalline, the fermented liquid chloroform extraction after centrifugal, at every turn with the chloroform of 0.5 times of fermentating liquid volume, extract three times, combined chloroform layer, is concentrated into small volume, add 500 ml methanol, be concentrated into and about remain 100 ml methanol, be cooled to 4 DEG C, suction filtration, cake layer 20 ml methanol drip washing, obtain white crystal, cake layer 70 DEG C oven dry, weight yield 55.2%, HPLC external standard content 98.1%.
Embodiment 4
The application of liquid bacterial agent in fermentative production 9-OH-AD containing M. smegmatics of the present invention, step is as follows,
1, the strain activity after secondary seed cultivation is detected
Detect medium sterilization.Liquid bacterial agent (liquid bacterial agent after secondary seed cultivation prepared by embodiment 2) to be tested is inoculated into and detects in substratum, inoculum size 5%, 200rpm, cultivates 48 hours for 30 DEG C.Liquid Detection is sent in sampling, if wherein 9-OH-AD concentration is greater than 5g/L, then the liquid bacterial agent after secondary seed cultivation is qualified, is suitable for transforming.Detection substratum is, diammonium hydrogen phosphate 3g/L, potassium primary phosphate 1g/L, dipotassium hydrogen phosphate 4g/L, sucrose 5g/L, magnesium sulfate heptahydrate 0.2g/L, iron vitriol 0.005g/L, Zinc Sulphate Heptahydrate 0.002g/L, beta-cyclodextrin 50g/L, plant sterol 10g/L, pH are 7.1-7.2.
2, fermentation culture
Above-mentioned qualified liquid bacterial agent is inoculated in the bioconversion medium after sterilizing, and inoculum size is 20%, and invert point is 30 DEG C, and rotating speed is 600rpm, and transformation time is 120 hours, and air flow quantity is 0.22Nm
3/ h, tank pressure 0.045MPa, bioconversion medium formula is, potassium primary phosphate 0.07%, three water dipotassium hydrogen phosphates 0.5%, Secondary ammonium phosphate 0.4%, glucose 0.45%, beta-cyclodextrin 6%, plant sterol 1.8%, mass concentration is the silicone antifoam agent 0.045% of 40%, and trace nutrient 3%, pH is 9.The composition of described trace nutrient is magnesium sulfate heptahydrate 1.2%, green vitriol 0.45%, Zinc Sulphate Heptahydrate 0.08%, and mass concentration is the sulfuric acid 0.05% of 20%, and all the other are water.
3, separation and Extraction
Sampling, book layer chromatography (TLC) is put plate and is detected, and transforms completely, centrifugal, isolate thalline, the fermented liquid chloroform extraction after centrifugal, at every turn with the chloroform of 0.5 times of fermentating liquid volume, extract three times, combined chloroform layer, is concentrated into small volume, add 500 ml methanol, be concentrated into and about remain 100 ml methanol, be cooled to 4 DEG C, suction filtration, cake layer 20 ml methanol drip washing, obtain white crystal, cake layer 70 DEG C oven dry, weight yield 54.4%, HPLC external standard content 97.3%.
Below be only the preferred embodiment of the present invention, protection scope of the present invention be not only confined to above-described embodiment, all technical schemes belonged under thinking of the present invention all belong to protection scope of the present invention.It should be pointed out that for those skilled in the art, some improvements and modifications without departing from the principles of the present invention, should be considered as protection scope of the present invention.
Claims (8)
1. a M. smegmatics of fermentative production 9-OH-AD, is characterized in that, described M. smegmatics (Mycobacteriumsmegmatis) is deposited in China typical culture collection center, and deposit number is CCTCC NO:M2013544.
2., containing a liquid bacterial agent for M. smegmatics described in claim 1, it is characterized in that, in described liquid bacterial agent, the cell concentration of described M. smegmatics (Mycobacterium smegmatis) reaches 1,000,000,000/more than ml.
3. a preparation method for the liquid bacterial agent of M. smegmatics according to claim 2, is characterized in that,
(1) inclined-plane seed culture: M. smegmatics (Mycobacterium smegmatis) CCTCC NO:M2013544 is inoculated in inclined-plane seed culture medium, 28 ~ 32 DEG C of cultivations;
(2) first order seed is cultivated: the bacterium colony of above-mentioned inclined-plane seed culture is added in liquid seed culture medium and cultivates, temperature is 28 ~ 32 DEG C;
(3) secondary seed is cultivated: the bacterium liquid after first order seed cultivation is inoculated in liquid seed culture medium, and inoculum size is 9 ~ 11%, and temperature is 28 ~ 32 DEG C, obtains liquid bacterial agent.
4. the preparation method of the liquid bacterial agent of M. smegmatics according to claim 3, it is characterized in that, described inclined-plane seed culture based formulas is, corn steep liquor 9 ~ 11g/L, ammonium sulfate 1.3 ~ 1.7g/L, potassium primary phosphate 0.45 ~ 0.55g/L, dipotassium hydrogen phosphate 0.5 ~ 0.6g/L, sucrose 0.45 ~ 0.55g/L, magnesium sulfate heptahydrate 0.18 ~ 0.22g/L, iron vitriol 0.004 ~ 0.006g/L, Zinc Sulphate Heptahydrate 0.001 ~ 0.003g/L, agar 18 ~ 22g/L, adjust ph is 7.1 ~ 7.2.
5. the preparation method of the liquid bacterial agent of M. smegmatics according to claim 3, it is characterized in that, liquid seed culture medium formula is, corn steep liquor 9 ~ 11g/L, ammonium sulfate 1.3 ~ 1.7g/L, potassium primary phosphate 0.45 ~ 0.55g/L, dipotassium hydrogen phosphate 0.5 ~ 0.6g/L, sucrose 9 ~ 11g/L, magnesium sulfate heptahydrate 0.18 ~ 0.22g/L, iron vitriol 0.004 ~ 0.006g/L, Zinc Sulphate Heptahydrate 0.001 ~ 0.003g/L, adjust ph is 7.1 ~ 7.2.
6. the application of the liquid bacterial agent of M. smegmatics in fermentative production 9-OH-AD as claimed in claim 2, is characterized in that, is inoculated in bioconversion medium by qualified liquid bacterial agent, inoculum size is 9 ~ 11%, invert point is 28 ~ 32 DEG C, transforms completely, centrifugal, fermented liquid chloroform extraction after centrifugal, concentrated, cooling, suction filtration, cake layer washed with methanol, obtains 9-OH-AD.
7. the application of the liquid bacterial agent of M. smegmatics in fermentative production 9-OH-AD as claimed in claim 6, it is characterized in that, described bioconversion medium formulation by weight is, potassium primary phosphate 0.07 ~ 0.09%, three water dipotassium hydrogen phosphates 0.4 ~ 0.5%, Secondary ammonium phosphate 0.2 ~ 0.4%, glucose 0.45 ~ 0.55%, beta-cyclodextrin 6 ~ 7%, plant sterol 1.8 ~ 2.2%, mass concentration is the silicone antifoam agent 0.045 ~ 0.055% of 20-40%, trace nutrient 3 ~ 5%, pH is 7 ~ 9, the composition of described trace nutrient is magnesium sulfate heptahydrate 0.8 ~ 1.2%, green vitriol 0.45 ~ 0.55%, Zinc Sulphate Heptahydrate 0.08 ~ 0.12%, mass concentration is the sulfuric acid 0.05% of 20 ~ 40%, all the other are water.
8. the application of the liquid bacterial agent of M. smegmatics in fermentative production 9-OH-AD as claimed in claim 6, is characterized in that, the rotating speed of fermentation culture is 400 ~ 600rpm, and air flow quantity is 0.18 ~ 0.22Nm3/h, tank pressure 0.045 ~ 0.055MPa.
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| CN112110970A (en) * | 2020-09-16 | 2020-12-22 | 湖南新合新生物医药有限公司 | Method for recycling 9-OH-AD by-product and method for preparing 9-OH-AD |
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| CN105316385A (en) * | 2015-03-25 | 2016-02-10 | 河北达瑞生物科技股份有限公司 | Culture medium for 9 alpha-hydroxyl androstenedione |
| CN112359087A (en) * | 2020-11-12 | 2021-02-12 | 湖南新合新生物医药有限公司 | Preparation method of 9-hydroxy androstenedione |
| CN112877393B (en) * | 2021-02-03 | 2022-09-02 | 湖南醇康医药科技有限公司 | Method for preparing deoxycholic acid from phytosterol |
| CN115433698A (en) * | 2022-10-18 | 2022-12-06 | 湖北共同药业股份有限公司 | Mycobacterium smegmatis bacterial liquid, preparation method thereof and application of mycobacterium smegmatis bacterial liquid in preparation of steroid drug intermediate |
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| CN112110970A (en) * | 2020-09-16 | 2020-12-22 | 湖南新合新生物医药有限公司 | Method for recycling 9-OH-AD by-product and method for preparing 9-OH-AD |
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