CN103088013A - Rhizopus nigricans as well as microbial conversion method for catalyzing hydroxylation reaction of steroid of rhizopus nigricans - Google Patents
Rhizopus nigricans as well as microbial conversion method for catalyzing hydroxylation reaction of steroid of rhizopus nigricans Download PDFInfo
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- CN103088013A CN103088013A CN 201210468288 CN201210468288A CN103088013A CN 103088013 A CN103088013 A CN 103088013A CN 201210468288 CN201210468288 CN 201210468288 CN 201210468288 A CN201210468288 A CN 201210468288A CN 103088013 A CN103088013 A CN 103088013A
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- epoxyprogesterone
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- 17alpha
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Abstract
The invention relates to rhizopus nigricans as well as a method for catalyzing hydroxylation reaction of steroid of rhizopus nigricans by microorganisms to produce mildew oxides. In the method, the microbial strain Rhizopus nigricans TS-8522 which is induced, screened and acclimatized converts epoxy progesterone into mildew oxide. The method comprises the following steps of: acclimatizing by using a millet culture medium containing epoxy progesterone in concentration gradient in a seed culture stage; fermenting, culturing and converting for 3 days at 28 DEG C by optimizing composition of carbon and nitrogen sources of the culture medium and adding trace elements; extracting by using an acetone method; and performing HPLC (High Performance Liquid Chromatography) detection, wherein the conversion ratio reaches 54%. Compared with chemical synthesis, the method has the advantages of high conversion specifity, extremely low content of by-products, simple production step, high product yield, low cost of extracting method, great cost saving, mild reaction condition and environment friendliness.
Description
Technical field
The present invention relates to a kind of new microbial transformation reaction that is used for and produce bread mould bacterial strain (Rhizopus nigricans) TS-8522 of mold oxide, and utilize this bread mould 16ALPHA,17ALPHA-epoxyprogesterone to be converted into the microbial conversion process of mold oxide.Genus fermentation engineering field.
Background technology
Steroidal drug is that in pharmaceutical industries, output is only second to antibiotic second largest class medicine.In the steroid hormone drug manufacture, a plurality of groups of steroidal parent nucleus need to be modified, and wherein the reaction of C11 'alpha '-hydroxylation is one of most important Steroid Transformation reaction.The hydroxylation of C11 position is introduced high physiologically active group to the steroidal parent nucleus, increases curative effect, reduces side effect significant.
Since adopting first the 1950's biotransformation method to carry out C11 position hydroxylation, bread mould is always as a kind of most widely used C11 'alpha '-hydroxylation microorganism, it passs hydrogen by NADPH, complete the hydroxylating of steroidal substrate, directly 16ALPHA,17ALPHA-epoxyprogesterone is converted into mold oxide, has saved 10 operations of chemosynthesis.1958, Wang Minglong etc. established bread mould steroidal C11 'alpha '-hydroxylation conversion process, after this, domestic this technique that generally adopts, it has the advantages such as stability is high, conversion is single-minded, but because transformation efficiency is not high, the reaction of C11 'alpha '-hydroxylation becomes the conditioning step that steroidal drug is produced always.
Technical scheme
Main purpose of the present invention is to provide and a kind ofly can effectively 16ALPHA,17ALPHA-epoxyprogesterone C11 'alpha '-hydroxylation be converted into the new bacterial strain of mold oxide, and a whole set of comparatively perfect 16ALPHA,17ALPHA-epoxyprogesterone bio-conversion process is provided.
For achieving the above object, the present invention adopts following technical scheme:
The present invention is take the bread mould (Rhizopus nigricans) that obtains from nature screening as starting strain, through femtosecond laser mutagenesis and screening, obtain the new bacterial strain (Rhizopus nigricans TS-8522) that a strain can effectively be converted into 16ALPHA,17ALPHA-epoxyprogesterone C11 'alpha '-hydroxylation mold oxide.
Transform cultivation and fermentation with bacterial strain:
Slant culture: glucose 10g/L, yeast powder 15g/L, sodium-chlor 5g/L, K
2HPO
45g/L, agar 15g/L, 16ALPHA,17ALPHA-epoxyprogesterone 20g/L.Sterilized 20 minutes for 121 ℃.Cooling rear inoculation was cultivated 3 days for 28 ℃.
Seed culture: proportioning is millet: the millet substratum of water=1: 0.8, through packing (15g/50ml triangular flask) after boiling, add 2% 16ALPHA,17ALPHA-epoxyprogesterone (domestication substratum 16ALPHA,17ALPHA-epoxyprogesterone concentration gradient is 0.5%, 1%, 1.5% and 2%), sterilized 20 minutes for 121 ℃, cooling rear inoculation was cultivated 3 days for 28 ℃.Save backup in 4 ℃.
The microbial transformation reaction:
Fermention medium forms: glucose 15g/L, soybean cake powder 15g/L, corn steep liquor 25g/L, dried silkworm chrysalis meal 2g/L, ammonium sulfate 1.5g/L, Iron trichloride hexahydrate 1.5g/L (divide and disappear), sodium hydroxide solution is adjusted pH to 4.5, add 2% 16ALPHA,17ALPHA-epoxyprogesterone, liquid amount is the 4L/5L fermentor tank, 121 ℃ of sterilization 20min.
The fermentation air flow is 4L/min, and mixing speed is 400r/min, and inoculum size is 2% (millet culture), cultivates under 28 ℃.Transform after 24 hours, add 150ml glucose solution (containing 40g glucose), further conversion reaction.
The extraction of product: fermented liquid filters with Büchner funnel, collects filter cake and dries under 70 ℃, grinds, and adopts the acetone of 7.5 times of volumes in 80 ℃ of lower refluxing extraction 20～30min, repeats twice of extraction step.Collect the extracting solution that obtains by filtration, 50 ℃ of lower rotary evaporation in vacuo are dry, and the faint yellow crystallization that obtains adds rare NaOH saponification, and suction filtration is washed to neutrality, and oven dry namely gets crude product.
The HPLC of 16ALPHA,17ALPHA-epoxyprogesterone and mold oxide detects: column type is C18 (Hypersil ODS2,5 μ m, 250 * 4.6mm).Moving phase is: acetonitrile: water=50: 50, flow velocity are 0.7ml/min, and the ultraviolet detection wavelength is 240nm.
Beneficial effect of the present invention:
The present invention adopts biotransformation method to carry out the C11 'alpha '-hydroxylation of 16ALPHA,17ALPHA-epoxyprogesterone, has the following advantages with respect to chemical synthesis: 1. transform specificity high, by-products content is extremely low; 2. production stage is simple, and the product yield is high; 3. the extracting method expense is low, saves in a large number cost; 4. reaction conditions is gentle, environmental friendliness.
The bacterial strain that adopts mutagenesis screening to obtain, compare with traditional conversion process and have the following advantages: 1. mutagenesis screening of the present invention obtain bread mould (Rhizopus nigricans TS-8522) have the ability of stronger opposing 16ALPHA,17ALPHA-epoxyprogesterone pressure; 2. the 16ALPHA,17ALPHA-epoxyprogesterone domestication that all exists at every one-phase of strain growth, so fermenting process do not need the induction period in early stage, greatly shortened fermentation period, improved productive rate; 3. adopt millet culture direct inoculation, not only be conducive to the dispersion of bacterial strain and adhere to, more can make bacterial strain adapt to as early as possible new growing environment, economize on resources; 4. adopt the starch to add the mode that glucose (be long-acting carbon source with utilize fast carbon source) combines, removed the early stage high concentration glucose to the retarding effect of bacterial strain, improved transformation efficiency.
Description of drawings
Fig. 1 is process flow diagram of the present invention.
Specific implementation method
It is following that the present invention will be described by embodiment.Embodiment only limits to that the invention will be further described, does not represent protection scope of the present invention, the nonessential modification of making according to the present invention and adjust and all belong to protection scope of the present invention.
Embodiment 1: the femtosecond laser mutagenesis of bread mould and screening
Get the bread mould inclined-plane of cultivating 3 days, wash and pass through 8 layers of filtered through gauze with ultrapure water, make 10
5The spore suspension of individual spore/ml.Get the 0.2ml spore suspension in clean culture dish, shine.The femtosecond laser radiation condition is: λ=800nm, lasing beam diameter=6mm, repetition rate=76MHz, irradiation distance=6cm, irradiation power=20mW, irradiation time=20s.Draw the spore suspension after irradiation, coat the PDA culture dish after diluting 100 times, be cultured in 28 ℃ of darkrooms and grow single bacterium colony.
With single bacterium colony respectively picking be seeded to new PDA inclined-plane, cultivated 4 days for 28 ℃, spore is washed make spore suspension, be seeded to fermention medium, it consists of: glucose 20g/L, soybean cake powder 15g/L, corn steep liquor 25g/L, dried silkworm chrysalis meal 2g/L, ammonium sulfate 1.5g/L, sodium hydroxide solution is adjusted pH to 4.5, adds 2% 16ALPHA,17ALPHA-epoxyprogesterone, and liquid amount is the 30ml/250ml triangular flask.Under 28 ℃, cultivated 3 days in the 180rpm shaking table, acetone extraction, the HPLC method detects the transformation efficiency of 16ALPHA,17ALPHA-epoxyprogesterone.Wherein, the highest bacterial strain of transformation efficiency has improved approximately 60% than the starting strain transformation efficiency, reaches 26.2%.With this new bacterial strain called after bread mould TS-8522 that obtains.
Embodiment 2: the impact on mutant strain 16ALPHA,17ALPHA-epoxyprogesterone transformation efficiency of multistage domestication and feeding mode
The mutagenic strain bread mould TS-8522 that adopts the present invention to obtain.Be to solve 16ALPHA,17ALPHA-epoxyprogesterone extremely low problem of solubleness in water, the 16ALPHA,17ALPHA-epoxyprogesterone that experiment is added all adds the dehydrated alcohol of 1 times of volume, and 80 ℃ of lower reflux one hour evenly add substratum after cooling again.
Slant culture: substratum 100ml contains glucose 1g, yeast powder 1.5g, sodium-chlor 0.5g, K
2HPO
40.5g 16ALPHA,17ALPHA-epoxyprogesterone 2g (control group does not contain) sterilized 20 minutes for 121 ℃.Cooling rear inoculation was cultivated 3 days under 28 ℃.Grow the mycelium of a large amount of whites on the inclined-plane, high approximately 3mm, there are a large amount of macroscopic black points on its top, is the sporocyst of bread mould.
Seed culture: take the 100g millet, after cleaning, add a small amount of distilled water (millet: water=1: 0.8), boiling 30min waits to dry in the air to tack-free, the packing of 15g/50ml triangular flask, every bottle adds 0.3g 16ALPHA,17ALPHA-epoxyprogesterone (control group does not contain), and fully mixing, sterilized 20 minutes for 121 ℃.After cooling, with sterilized water, the spore on the inclined-plane is rinsed, after 8 layers of filtered through gauze, the 1ml/ bottle graft enters in the millet substratum.Cultivated 3 days for 28 ℃, grow the white hypha of high approximately 1cm on the millet substratum, the upper end black spore that distributes in a large number.Preserve under 4 ℃ and be beneficial to switching.
Fermentation culture and conversion: fermention medium 400ml, contain glucose 8g, soybean cake powder 6g, corn steep liquor 10g, dried silkworm chrysalis meal 0.8g, ammonium sulfate 0.6g, sodium hydroxide adjust pH to 4.5, packing, 50ml/500ml triangular flask, experimental group adds 2% 16ALPHA,17ALPHA-epoxyprogesterone (control group adds 0.2% 16ALPHA,17ALPHA-epoxyprogesterone), sterilizes 20 minutes for 121 ℃.With sterilized water, seed is washed from millet substratum undershoot, 8 layers of filtered through gauze transfer concentration to approximately 10
8Individual/ml is inoculated in fermention medium by 4% inoculum size, and 28 ℃, 180r/min cultivated 3 days.The bacterial strain of experimental group is through the multistage domestication of 0.5%, 1%, 1.5% and 2% 16ALPHA,17ALPHA-epoxyprogesterone, and the cultivation of seed and fermentation stage is all in the substratum that contains 2% 16ALPHA,17ALPHA-epoxyprogesterone.Control group adopts more traditional method, and namely inclined-plane, seed stage all do not contain 16ALPHA,17ALPHA-epoxyprogesterone, adopts the substratum that contains 0.2% 16ALPHA,17ALPHA-epoxyprogesterone first to induce during the fermentation inoculation, ferments to be added into 1.8% 16ALPHA,17ALPHA-epoxyprogesterone after 16 hours again and to transform.Result such as table 1:
Table 1 bread mould Rhizopus nigricans TS-8522 transforms 16ALPHA,17ALPHA-epoxyprogesterone and generates mold oxide
Embodiment 3: the impact of the carbon nitrogen source technique after optimization on the mutant strain transformation efficiency
Adopt 5L self-control type fermentor tank, and adopt millet culture direct inoculation.
Fermention medium 4L, 1. the substratum of control group consists of (g/L): glucose 35, corn steep liquor 40, dried silkworm chrysalis meal 2, ammonium sulfate 1.5; 2. the substratum of experimental group consists of (g/L): glucose 15, and soybean cake powder 15, corn steep liquor 25, dried silkworm chrysalis meal 2, ammonium sulfate 1.5 ferments and adds glucose 10g/L after 24 hours.Sodium hydroxide adjust pH to 4.5 adds 2% 16ALPHA,17ALPHA-epoxyprogesterone of having processed through reflux, sterilizes 20 minutes for 121 ℃.
After sterilization, pass into rapidly water of condensation, and accent is stirred to the dispersion that 400r/min is beneficial to 16ALPHA,17ALPHA-epoxyprogesterone.The fermentation inoculum size is 2%, and air flow is 1vvm.28 ℃ of bottom fermentations were cultivated 72 hours.Conversion results such as table 2.
The fermentation parameter of table 2 bread mould Rhizopus nigricans TS-8522
Embodiment 4: Iron trichloride hexahydrate adds the impact on the mutant strain transformation efficiency
Adopt 5L self-control type fermentor tank, and adopt millet culture direct inoculation.
Fermention medium 4L, substratum consist of (g/L): glucose 15, and soybean cake powder 15, corn steep liquor 25, dried silkworm chrysalis meal 2, ammonium sulfate 1.5, Iron trichloride hexahydrate 1.5 (only add, and control group does not add by experimental group; Adopt the mode that disappears of dividing, be added into fermention medium after dissolving with sterilized water after sterilization), ferment and add glucose 10g/L after 24 hours.Sodium hydroxide adjust pH to 4.5 adds 2% 16ALPHA,17ALPHA-epoxyprogesterone of having processed through reflux, sterilizes 20 minutes for 121 ℃.
After sterilization, pass into rapidly water of condensation, and accent is stirred to the dispersion that 400r/min is beneficial to 16ALPHA,17ALPHA-epoxyprogesterone.The fermentation inoculum size is 2%, and air flow is 1vvm.28 ℃ of bottom fermentations were cultivated 72 hours.
The result demonstration, the transformation efficiency of experimental group bread mould improves 14.4% than control group, reaches 54.2%.
Claims (5)
1. the microbial conversion process of a bread mould and catalysis steroidal hydroxylating thereof, is characterized in that: screening bread mould (Rhizopus nigricans) → mutagenesis, screening → new rhizopus niger (Rhizopus nigricans TS-8522) → slant activation → multistage domestication → fermentation conversion → filtration drying → acetone extraction → evaporation concentration → NaOH saponification → sample.
2. the microbial conversion process of a kind of bread mould according to claim 1 and catalysis steroidal hydroxylating thereof, it is characterized in that: described rhizopus niger is through femtosecond laser mutagenesis and screening, and acquisition can effectively be converted into 16ALPHA,17ALPHA-epoxyprogesterone C11 'alpha '-hydroxylation the new rhizopus niger of mold oxide.
3. the microbial conversion process of 2 described a kind of bread moulds and catalysis steroidal hydroxylating thereof according to claim 1,, it is characterized in that: after the multistage domestication of described new rhizopus niger process 16ALPHA,17ALPHA-epoxyprogesterone, it is carried out seed culture, the domestication substratum is the millet substratum that has added 0.5%, 1%, 1.5% and 2% 16ALPHA,17ALPHA-epoxyprogesterone, and seed culture medium is the millet substratum that contains 2% 16ALPHA,17ALPHA-epoxyprogesterone.
4. the microbial conversion process of a kind of bread mould according to claim 1 and catalysis steroidal hydroxylating thereof is characterized in that: the fermention medium during described fermentation transforms is (g/L): glucose 15, soybean cake powder 15, corn steep liquor 25, dried silkworm chrysalis meal 2, (NH
4)
2SO
41.5, Iron trichloride hexahydrate 1.5, the pH value is 4.5, adds 2% 16ALPHA,17ALPHA-epoxyprogesterone through the alcohol heating reflux processing.
5. the microbial conversion process of 4 described a kind of bread moulds and catalysis steroidal hydroxylating thereof according to claim 1,, it is characterized in that: the culture condition of described fermention medium thalline is: liquid amount 4L-5L fermentor tank, inoculum size is 2%, mixing speed is 400r/min, transforms 3 days under 28 ℃.Wherein, transform disposable 10% glucose of adding in 24 hours backward fermented liquids.
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Cited By (7)
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| CN103396469A (en) * | 2013-07-30 | 2013-11-20 | 安阳九州药业有限责任公司 | Extraction process of 11a-hydroxy-16,17a-epoxyprogesterone |
| CN103627721A (en) * | 2013-10-22 | 2014-03-12 | 浙江工业大学 | Application of G6PDH gene for improving steroid C11 alpha-hydroxylation ability of rhizopus nigricans and strain |
| CN103695455A (en) * | 2013-07-24 | 2014-04-02 | 浙江工业大学 | Method for constructing rhizopus nigericans CRP genetically engineered bacteria by protoplast transformation |
| CN104560724A (en) * | 2014-11-12 | 2015-04-29 | 浙江工业大学 | Cladosporium ZF-35 and application in preparation of hydroxylated epoxy progestin |
| CN104893982A (en) * | 2014-04-08 | 2015-09-09 | 丽江映华生物药业有限公司 | Rhizopus nigricans and application technology thereof |
| CN109852659A (en) * | 2019-02-28 | 2019-06-07 | 武汉大学 | A kind of bioconversion method being hydroxylated for serial steroid 19 |
| CN110863027A (en) * | 2019-11-22 | 2020-03-06 | 盐城工学院 | Method for reducing content of fusidic acid by-product by biotransformation method |
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2012
- 2012-11-06 CN CN 201210468288 patent/CN103088013A/en active Pending
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103695455A (en) * | 2013-07-24 | 2014-04-02 | 浙江工业大学 | Method for constructing rhizopus nigericans CRP genetically engineered bacteria by protoplast transformation |
| CN103396469A (en) * | 2013-07-30 | 2013-11-20 | 安阳九州药业有限责任公司 | Extraction process of 11a-hydroxy-16,17a-epoxyprogesterone |
| CN103396469B (en) * | 2013-07-30 | 2017-06-06 | 安阳九州药业有限责任公司 | A kind of extraction process of mold oxide |
| CN103627721A (en) * | 2013-10-22 | 2014-03-12 | 浙江工业大学 | Application of G6PDH gene for improving steroid C11 alpha-hydroxylation ability of rhizopus nigricans and strain |
| CN103627721B (en) * | 2013-10-22 | 2015-08-05 | 浙江工业大学 | G6PDH gene is improving bread mould to the application in steroidal C11 'alpha '-hydroxylation ability and bacterial strain |
| CN104893982A (en) * | 2014-04-08 | 2015-09-09 | 丽江映华生物药业有限公司 | Rhizopus nigricans and application technology thereof |
| CN104560724A (en) * | 2014-11-12 | 2015-04-29 | 浙江工业大学 | Cladosporium ZF-35 and application in preparation of hydroxylated epoxy progestin |
| CN104560724B (en) * | 2014-11-12 | 2017-06-13 | 浙江工业大学 | The branch mould ZF 35 of spore and the application in hydroxylation epoxy progesterone is prepared |
| CN109852659A (en) * | 2019-02-28 | 2019-06-07 | 武汉大学 | A kind of bioconversion method being hydroxylated for serial steroid 19 |
| CN110863027A (en) * | 2019-11-22 | 2020-03-06 | 盐城工学院 | Method for reducing content of fusidic acid by-product by biotransformation method |
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Application publication date: 20130508 |