CN1322112C - Mycobacterium fortuitum and its use in production of testosterone by conversion of microbe - Google Patents
Mycobacterium fortuitum and its use in production of testosterone by conversion of microbe Download PDFInfo
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- CN1322112C CN1322112C CN 200510024193 CN200510024193A CN1322112C CN 1322112 C CN1322112 C CN 1322112C CN 200510024193 CN200510024193 CN 200510024193 CN 200510024193 A CN200510024193 A CN 200510024193A CN 1322112 C CN1322112 C CN 1322112C
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Abstract
The present invention relates to a mycobacterium fortuitum mutant CGMCC1241 and the application in the production of testosterone through conversion of microbe. The strain can realize the transformation from substrate animal and plant sterol to product testosterone at a high conversion rate, and the present invention has industrial application value.
Description
Technical field
The present invention relates to the microbiological transformation technology field, be specifically related to a kind of mycobacterium fortutitum and the application in production of testosterone by conversion of microbe.
Background technology
Steroidal drug is that a big class has the special treatment effect clinically, and the irreplaceable extremely important medicine of other drug.Wherein, testosterone (17 beta-hydroxy androstane-4-alkene-3-ketone, Testosterone, TS; The following formula I structure of tool)
As a kind of male sex hormone medicine, can promote the growth of buck secondal sexual character and the maturation of sexual organ, in recent years the therapy primary or the secondary hypogonadism that are used to treat the male patient as an alternative.Abroad to this existing big quantity research.In addition, testosterone is the important precursor of protein anabolic hormone, and the main effect of protein anabolic hormone is to improve Proteometabolism, promote protein synthesis and reduce the protein heteroplasia, reduce calcium, phosphorus drainage, alleviate bone marrow depression, recovery and physical strength reinforcing, diuretic antihypertensive etc.Because it has significant anti-ageing and antitumous effect, range of application enlarges day by day.
Producing the testosterone traditional technology is to carry out a series of chemosynthesis with the steroidal saponin from natural plant resource as raw material.Wherein not only relate to the consumption of natural resource, also the participation owing to a large amount of chemical reaction step causes serious environmental to pollute.
The application of microbiological transformation technology makes the Study on Preparation of steroidal drug obtain significant progress.Particularly since the 1980s, microbiological transformation technology has been applied to the progress of the making a breakthrough property of research of steroidal key intermediate preparation.World medicine power, especially the U.S. and Japan, utilize this country agricultural byproducts---plant sterol is that raw material obtains key intermediate through microbial transformation, with this synthesizing steroid medicine, thereby not only solved the limited and raw material bottleneck problem brought of plant resources, and reduced manufacturing cost widely and reduced environmental pollution.
For testosterone, the external also corresponding research of carrying out the microbial conversion process aspect.At present report mainly is to use rotex (androst-4-ene-3,17-dione, 4AD; State formula II structure as follows) carry out a step microbial hydrogenation as substrate and change into testosterone; Perhaps use single sterol such as Sitosterol to carry out microbial transformation and obtain testosterone.
Because involving preparation 4AD substrate itself, the former also need pass through the series reaction step, so the latter is more superior by contrast.It is enough high and be easy to the microbial strains of large scale culturing to obtain conversion capability, and the industrial applications that then can promote this circuit greatly is worth.
Summary of the invention
The objective of the invention is to by the optimization of strain improvement means in conjunction with conversion condition, acquisition can realize the mutant strain that testosterone high conversion, tool commercial application are worth.
It is mycobacterium fortutitum (Mycobacterium fortuitum) mutant strain that the testosterone of high conversion disclosed by the invention is produced bacterial strain, and this bacterial classification microbial preservation number is CGMCCNo.1241.This bacterial strain is the bacterial classification that sets out with mycobacterium fortutitum (Mycobacterium fortuitum) DSMZ2966, through processing such as natural seed selection, chemical physics mutagenesis and obtain.Concrete mutagenesis, screening step comprise:
1, starting strain
Mycobacterium fortutitum (Mycobacterium fortuitum) DSMZ 2966
2, substratum
Inclined-plane/plate culture medium: contain an amount of glucose, yeast powder, peptone and agar.Cultivation, the natural separation of bacterial classification, the bacterial classification routine that this substratum is used for starting strain purpose such as go down to posterity.
Transform substratum: glucose 4.0%, yeast extract powder 1.0%, potassium primary phosphate 0.7% Sitosterol 0.5%, tween 80 0.1%.
3, the analytical procedure-HPLC of converted product
Specimen preparation: quantitatively add acetone 8ml among the conversion fluid 2ml, place behind the vibration mixing and spend the night.Get supernatant liquid 1ml in 15000rpm centrifugal 10 minutes, supernatant liquor detects for HPLC.
Chromatographic column: C
18, ODS, 4.6mm * 250mm, 5 μ m
Detector: Agilent 1100
Detect wavelength: 242nm
Column temperature: 50 ℃
Moving phase: methyl alcohol: water=80: 20 (volume ratio)
Flow velocity: 0.8ml/min
4, natural separation
One on fresh inclined-plane getting bacterial classification adds a small amount of sterilized water, scrapes lawn gently and pours in the triangular flask that sterile glass beads and sterilized water are housed, and shake well 10 minutes filters with the aseptic funnel that is plugged with absorbent cotton, obtains bacteria suspension.Coat on the plate culture medium behind the bacteria suspension gradient dilution, cultivate and grow single bacterium colony.
5, ultraviolet mutagenesis
(1) long wave ultraviolet mutagenesis
By obtaining bacteria suspension with quadrat method in the above-mentioned natural separation, bacterial classification is carried out mutagenic treatment with the radiation wavelength of 380nm.Handle the back gradient dilution and coat plate culture medium, cultivate in 28 ℃ of lucifuges.
(2) shortwave ultraviolet mutagenesis
By obtaining bacteria suspension with quadrat method in the above-mentioned natural separation, bacterial classification is carried out mutagenic treatment with the radiation wavelength of 260nm.Handle the back gradient dilution and coat plate culture medium, cultivate in 28 ℃ of lucifuges.
6, gamma-radiation (
60Co) mutagenesis
By obtaining bacteria suspension with quadrat method in the above-mentioned natural separation, it is the aseptic triangular flask of jumping a queue of 10ml that taking-up 2ml places capacity.Triangular flask is placed
60The Co radiation source irradiates.Handle the back gradient dilution and coat the screening flat board, in 28 ℃ of cultivations.
7, NTG mutagenesis
Add NTG in by the bacteria suspension system of phosphoric acid buffer preparation after cultivating processing on 28 ℃ of shaking tables, sampling gradient dilution coating screening is dull and stereotyped, 28 ℃ of cultivations.
8, shake the bottle screening
Will be after seed selection be handled 28 ℃ on single colony inoculation inclined-plane of obtaining and is cultivated maturation, be inoculated into to contain and transform shaking of substratum and carry out liquid culture in the bottle and transform, HPLC detects converted product.
Finally obtaining a strain transformation efficiency through repeated screening is the mutant strain of starting strain more than 10 times, and this mutant strain is investigated its inheritance stability of proof through going down to posterity, transform the ability that generates testosterone and can remain on peer-level behind rational passage number.The open preserving number of this microbial strains is CGMCC 1241.The strain selection pedigree is seen Fig. 1; Starting strain and sieve mutant strain separately the HPLC collection of illustrative plates of converted product see Fig. 2 and Fig. 3.
CGMCC 1241 bacterial strains that adopt mutagenesis of the present invention, screening to obtain carry out testosterone when preparing, can 4AD, the animals and plants sterol is that substrate directly transforms the generation testosterone; Wherein the animals and plants sterol is selected from cholesterol, Sitosterol, campesterol or their mixture.The interpolation concentration of substrate sterol is 0.1%-3.0%.
Adopt bacterial strain of the present invention to carry out testosterone when preparing, available carbon source is selected from dextrin, Citrate trianion, glycerine, sucrose, maltose, N.F,USP MANNITOL, glucose, Zulkovsky starch in its substratum, and content is 1.0%-6.0%; Nitrogenous source can be selected from yeast powder, aminoacids complex, peptone, corn steep liquor, urea, nitrate, ammonium salt, content 0.2%-5.0%; Can add phosphoric acid salt, magnesium salts in the conversion, receive trace elements such as salt; Also can add tensio-active agents such as tween 80, dissolve better, help transformation efficiency and improve to impel the solid sterol.
The present invention adopts CGMCC 1241 bacterial strains to carry out testosterone when preparing, and preferably transforming substratum is glucose 1.0%-6.0%, yeast powder 0.2%-2.0%, potassium primary phosphate 0.5%-1.5%, animals and plants sterol 0.1%-3.0%, tween 80 0-0.5%; Invert point is 20 ℃-40 ℃, preferred 25 ℃-30 ℃; Conversion pH is 4-9, preferred pH5-7; Transformation time 5-9 days.
Adopt above-mentioned condition optimizing combined preparation testosterone, finally can realize transformation efficiency more than 80% to 10 tons of fermentor tanks shaking bottle from the 250ml triangle.Converted product can obtain purity through steps such as extraction, column chromatographies and be higher than 90% testosterone product.
The mutant strain that the present invention obtains has reached the purpose of high conversion generation testosterone, has commercial application and is worth.
Description of drawings
Fig. 1. mycobacterium fortutitum (Mycobacterium fortuitum) CGMCC 1241 strain improvement pedigrees
Wherein NS represents natural separation, UV
380The UV treatment of expression 380nm wavelength, UV
260The UV treatment of expression 260nm wavelength, NTG represents the nitrosoguanidine processing.
Fig. 2. the HPLC collection of illustrative plates of starting strain converted product
Fig. 3. the HPLC collection of illustrative plates of bacterial strain CGMCC 1241 converted products
Embodiment
Embodiment 1 bacterial classification study on the stability---go down to posterity to the influence that transforms
Slant medium: glucose 1.0%, yeast extract powder 1.0%, polyprotein peptone 0.8% agar powder 1.5%.
Seed culture medium: glucose 1.0%, yeast extract powder 1.0%, potassium primary phosphate 0.2%.
Transform substratum: glucose 4.0%, yeast extract powder 1.0%, potassium primary phosphate 0.7%, Sitosterol 0.5%, tween 80 0.1%; Preceding pH6.5 disappears.
Culture condition: 28 ℃ of slant culture temperature, incubation time 5 days; 28 ℃ of seed liquor culture temperature, incubation time 2 days; Transform 28 ℃ of culture temperature, incubation time 5 days.Shake a bottle rotating speed 220rpm/min, rotary shaking table shaking culture.
| Algebraically goes down to posterity | 1 | 2 | 3 | 4 | 5 |
| The relative growing amount of TS (%) | 100 | 97 | 95 | 94 | 96 |
The comparison of embodiment 2 mutant strains and starting strain transformation efficiency
Substratum and culture condition are with embodiment 1.
The controlled trial result of mutant strain and starting strain is as follows:
| Bacterial classification | Starting strain DSMZ 2966 | Bacterial classification CGMCC 1241 |
| The TS growing amount | 265μg/ml | 4100μg/ml |
| The TS relative quantity | 100％ | 1547％ |
| Improve multiple relatively | —— | 14.5 doubly |
Embodiment 3 shakes a bottle conversion test
Slant medium, seed culture medium and each stage culture condition are with embodiment 1.
Transform substratum: dextrin 5.0%, peptone 1.2%, saltpetre 0.1%, potassium primary phosphate 0.5%, cholesterol 1.0%, tween 80 0.2%; Preceding pH5.8 disappears.
Culture condition is with embodiment 1.
Transformation efficiency is 80.2%.
Embodiment 4 shakes a bottle conversion test
Slant medium, seed culture medium and each stage culture condition are with embodiment 1.
Transform substratum: Zulkovsky starch 3.5%, glucose 1.0%, corn steep liquor 2.0%, potassium primary phosphate 0.8%, mixed phytosterin (Sitosterol: campesterol=2: 1) 2.0%, tween 80 0.1%.
Transform incubation time 7 days, all the other culture condition are with embodiment 1.
Transformation efficiency is 83%.
57 tons of fermentor tank conversion tests of embodiment
Slant medium, seed culture medium, conversion substratum, inclined-plane and seed culture condition are with embodiment 4.The control of 7 tons of fermentor tank conditions is as follows: 25 ℃ of temperature, air flow 1: 1 (vol: vol), mixing speed 300rpm, 7 days cycles.Generating the testosterone transformation efficiency is 81.7%.
Embodiment 6
The conversion fluid that obtains by embodiment 5 soaks through acetone, use ethyl acetate extraction, and extraction liquid concentrates, and (the elutriant hexanaphthene: ethyl acetate=7: 1) obtaining purity is 92% testosterone product to pass through silica gel column chromatography.
Claims (5)
1, a kind of mycobacterium fortutitum (Mycobacterium fortuitum) mutant strain, the microbial strains preserving number is CGMCC No.1241.
2, the application of mycobacterium fortutitum mutant strain CGMCC No.1241 according to claim 1 in the preparation testosterone.
3, a kind of method that adopts claim 1 mycobacterium fortutitum mutant strain CGMCC No.1241 to prepare testosterone, it is characterized in that the conversion culture medium carbon source that this method adopts is selected from dextrin, Citrate trianion, glycerine, sucrose, maltose, N.F,USP MANNITOL, glucose, Zulkovsky starch, content is 1.0%-6.0%;
Nitrogenous source is selected from yeast powder, aminoacids complex, peptone, corn steep liquor, urea, nitrate, ammonium salt, content 0.2%-5.0%;
The substrate sterol is selected from 4AD and animals and plants sterol, and interpolation concentration is 0.1%-3.0%;
Invert point is 20 ℃-40 ℃, and conversion pH is 4-9; Transformation time 5-9 days.
4, mycobacterium fortutitum mutant strain CGMCC No.1241 according to claim 3 prepares the method for testosterone, it is characterized in that wherein said conversion substratum is glucose 1.0%-6.0%, yeast powder 0.2%-2.0%, potassium primary phosphate 0.5%-1.5%, animals and plants sterol 0.1%-3.0%, tween 80 0-0.5%.
5, mycobacterium fortutitum mutant strain CGMCC No.1241 according to claim 3 prepares the method for testosterone, it is characterized in that wherein said invert point is 25 ℃-30 ℃, and conversion pH is 5-7.
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Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103756940B (en) * | 2014-01-22 | 2015-05-13 | 湖南新合新生物医药有限公司 | Mycobacterium fortuitum and application thereof in fermentation production of delta-lactone |
| CN104195209A (en) * | 2014-09-02 | 2014-12-10 | 常州大学 | Method for preparing 4-androstenedione through phytosterin |
| CN106011158A (en) * | 2016-07-20 | 2016-10-12 | 江南大学 | Method using microorganism method to convert androstenedione so as to produce testosterone |
| CN108796021B (en) * | 2017-04-28 | 2021-03-19 | 沈阳博泰生物制药有限公司 | Engineering bacterium and application thereof in preparing testosterone |
| CN112813041B (en) * | 2020-12-31 | 2022-03-18 | 江南大学 | 17 beta-hydroxysteroid dehydrogenase mutant of mycobacterium, engineering bacterium and application of mutant and engineering bacterium |
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