CN106282077A - The bacterial strain of one plant height effect transformation phytosterin and application thereof - Google Patents
The bacterial strain of one plant height effect transformation phytosterin and application thereof Download PDFInfo
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- CN106282077A CN106282077A CN201610978886.6A CN201610978886A CN106282077A CN 106282077 A CN106282077 A CN 106282077A CN 201610978886 A CN201610978886 A CN 201610978886A CN 106282077 A CN106282077 A CN 106282077A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P33/00—Preparation of steroids
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Abstract
The invention belongs to biological technical field, relate to mycobacteria (Mycobacterium sp.) LY 1 and the application thereof of a plant height effect transformation phytosterin, this bacterial strain is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and preserving number is CGMCC No.13031.Mycobacteria (Mycobacterium sp.) LY 1 can transformation phytosterin be 9 α hydroxy-androstane 4 alkene 3,17 diketone, under conditions of substrate inventory is 15g/L, efficiency of pcr product 33%.
Description
Technical field
The invention belongs to biological technical field, be specifically related to a strain mycobacteria (Mycobacterium sp.) LY-1 and
Its transformation phytosterin is 9 Alpha-hydroxy androstane-4-alkene-3s, the method for 17-diketone.
Background technology
9 Alpha-hydroxy 4-ADs (9 α-OH-AD) are the precursors of the important steroid hormone class medicine of a class,
Its 9, by hydroxylated site, through simple halogenation, just can introduce the halogenic substituents such as F or Cl, thus effectively carry
Rise the medicinal drug effect of some corticoid.Owing to conventional chemical synthesis brings serious industrial pollution, and yield is low, cost
Height, significantly limit the development of its synthesis, therefore utilizes microorganism to convert in recent years and be increasingly becoming Main Trends of The Development.Therefore,
One of screening and enrichment bacterial strain emphasis becoming steroid medicine developmental research concern that can effectively accumulate 9 α-OH-AD.
The traditional method synthesizing 9 α-OH-AD is to use same or like structure parent nucleus analog to carry out chemosynthesis, but
It is that 9 α-OH-AD require height to stereo selectivity, it will usually causing efficiency of pcr product low, by-product is many, product later separation purification mistake
The problems such as journey is complicated.In recent years, domestic and international researcher starts to consider that biotransformation method specificity converts 9 α-OH-AD, and this reaction has
Having ready conditions gentleness, safe and low consumption, easily operation, conversion ratio be high and the feature such as environmental friendliness.According to pertinent literature, the most existing red
The bacterial strains such as flat Rhodococcus fascians (Rhodococcus erythropolis) possess conversion 4-AD (AD) be 9 α-
The ability of OH-AD.AD and plant sterol are both at fat-soluble compound, and in aqueous phase, dissolubility is extremely low, causes conversion process
Middle substrate feed concentrations and transformation efficiency are the lowest, and cost is far above plant sterol, and these problems are directly becoming 9 α-OH-
Bottleneck in AD commercial production.Therefore, screen the microorganism that a plant height effect transformation phytosterin is 9 α-OH-AD and have the heaviest
The meaning wanted, and have no for the research of transformation phytosterin about mycobacteria (Mycobacterium sp.) LY-1 at present
Report, its 9 high α-OH-AD convert the big advantage of purity the most industrial one.
Summary of the invention
It is an object of the invention to provide the mycobacteria (Mycobacterium sp.) of a plant height effect transformation phytosterin
LY-1, and the method utilizing this bacterial strain transformation phytosterin to be 9 α-OH-AD, can reach high substrate feed concentrations, high substrate height
Conversion ratio, the requirement of high efficiency of pcr product.
Bolter the present inventor's pedotheque around the chemical plant Factory Building of domestic synthesizing steroid compound
Choosing obtains a strain mycobacteria (Mycobacterium sp.) LY-1, and this bacterium is deposited in JIUYUE in 2016 and is positioned at Beijing on the 22nd
The China Committee for Culture Collection of Microorganisms of Institute of Microorganism, Academia Sinica of North Star West Road, Chaoyang District 1 institute 3 is general
Logical microorganism center, deposit number is CGMCC No.13031, and Classification And Nomenclature is mycobacteria Mycobacterium sp..
The method that application mentioned microorganism transformation phytosterin produces 9 α-OH-AD, its step is as follows:
(1) mycobacteria (Mycobacterium sp.) LY-1 using preserving number to be CGMCC No.13031 is for producing
Strain, conventionally carries out activation culture and obtains seed liquor, this seed liquor be applied in PDA culture medium；
(2) the cellular liquid culture of LY-1 bacterial strain is prepared: the LY-1 bacterium on the PDA solid medium of picking step (1)
Strain one ring, is inoculated in that sterilized cultivation temperature is 20-35 DEG C equipped with in the 500mL conical flask of 70-110mL seed culture medium,
Put and cultivate 60-72h to mid log phase with the rotating speed of 90-150r/min on shaking table, i.e. obtain the cellular liquid of LY-1 bacterial strain
Culture；
(3) fermentation culture: by the cellular liquid culture for preparing in step (2) with the inoculum concentration of 0.5-1.2% (w/w)
Accessing fermentation medium, liquid amount is dress 70-110mL fermentation medium in 500mL conical flask, and cultivation temperature is 20-35 DEG C,
Cultivate 120-168h under the rotating speed of 90-150r/min, obtain fermentation liquid.
(4) product detection: the conversional solution of step (3) equal-volume ethyl acetate is extracted 6 times, revolves in Rotary Evaporators
To there being crystal to occur, acetonitrile redissolves crystal by the organic membrane filter remove impurity of 0.22 μm, and filtrate utilizes high performance liquid chromatography
Analyze the content of 9 α-OH-AD.
Wherein composition and the proportioning of the PDA culture medium described in step (1) is: Rhizoma Solani tuber osi 200-500g/L；Glucose 20-
50g/L；Yeast powder 2-10g/L；Agar powder 10-20g/L；PH is natural, sterilizing 20min under 121 DEG C of high steams；Step (2)
The composition of described seed culture medium and proportioning be: NaNO32-10g/L, yeast powder 5-20g/L, glycerol 1-5g/L, (NH4)2HPO4
0.2-1.5g/L, sterilizing 20min under 121 DEG C of high steams；Composition and the proportioning of step (3) described fermentation medium be: plants
Thing sterol 5-20g/L, NaNO32-10g/L, Semen Maydis pulp 5-25g/L, (NH4)2HPO40.2-1.5g/L, pH 7.5-8.5,121
Sterilizing 20min under DEG C high steam.
Mycobacteria of the present invention (Mycobacterium sp.) LY-1, bacterial strain finds to convert plant steroid first
Alcohol generates 9 α-OH-AD, and can reach high substrate inventory, high substrate conversion efficiency, the requirement of high product purity, is a strain pole
The production bacterial strain that tool developmental research is worth.
Accompanying drawing explanation
Fig. 1 is that mycobacteria (Mycobacterium sp.) LY-1 of the present invention converts plant steroid in the fermentation medium
The process study of alcohol.
Fig. 2 be the present invention mycobacteria (Mycobacterium sp.) LY-1 add Tween 80 fermentation system with
Under normal fermentation system, transformation phytosterin generates the comparison of 9 α-OH-AD content.
Detailed description of the invention
The isolation identification of embodiment 1 mycobacteria (Mycobacterium sp.) LY-1 and the preservation of bacterial strain
(1) mycobacteria (Mycobacterium sp.) separation of LY-1, screening
The present invention obtains soil sample around the chemical plant Factory Building of domestic synthesizing steroid compound, weigh 1.0-2.0g soil sample in
In 250mL triangular flask, suspending with 10-50mL normal saline, be subsequently placed in water-bath, 60 DEG C process 10min, carry out after cooling
Gradient dilution, takes 0.2mL diluent and coats and carry out primary dcreening operation on the solid plate that plant sterol is sole carbon source.Under the conditions of 30 DEG C
After cultivating 120h, choose the preferably single bacterium colony of growing way and carry out fermentation checking.Picking list colony inoculation is to fresh liquid seeds training
Support base, 30 DEG C, 90-150r/min cultivate 72h, then with the inoculum concentration of 1% (w/w) access liquid amount as 100mL/500mL
Ferment culture medium, 120h under same culture conditions, collect conversional solution, after the organic membrane of ethyl acetate extracting and 0.22 μm processes,
Use conversion ratio and the yield of product 9 α-OH-AD of high performance liquid chromatography detection substrate plant sterol.(2) mycobacteria
The qualification of (Mycobacterium sp.) LY-1
16s rDNA strain identification: extract mycobacteria (Mycobacterium sp.) LY-1 by genomic kit
The full-length genome of bacterial strain, uses universal primer 27F and 1492R to transfer the 16s rDNA fragment of this bacterial strain, and sequencing result is in NCBI
On compare.
(3) bacterial strain preserves: picking one ring mycobacteria (Mycobacterium sp.) LY-1 inoculation is in seed liquid
In culture medium, 20-35 DEG C, after 120r/min shaken cultivation 60-72h, the cell culture fluid taking 0.5mL proceeds to equipped with 0.5mL's
In 60% glycerol stocks pipe ,-20 DEG C of freezings.Then in JIUYUE in 2016 preservation on the 22nd to being positioned at the Chaoyang District, Beijing City North Star
In China Committee for Culture Collection of Microorganisms of the Institute of Microorganism, Academia Sinica common micro-organisms of West Road 1 institute 3
The heart, deposit number is CGMCC No.13031, and Classification And Nomenclature is mycobacteria Mycobacterium sp..
Embodiment 2 utilizes mycobacteria CGMCC No.13031 (Mycobacterium sp.) LY-1 transformation phytosterin
(1) the cellular liquid training of mycobacteria CGMCC No.13031 (Mycobacterium sp.) LY-1 bacterial strain is prepared
Support thing
Mycobacteria (Mycobacterium sp.) LY-1 bacterial strain one ring on picking solid PDA medium, is seeded in dress
Have in the 500mL conical flask of 100mL seed culture medium, at 30 DEG C, be placed on shaking table and cultivate 60-with the rotating speed of 120r/min
72h, to logarithmic (log) phase, i.e. prepares the cellular liquid culture of mycobacteria (Mycobacterium sp.) LY-1 bacterial strain.
(2) composition and the proportioning of fermentation medium is: plant sterol 5-20g/L, NaNO32-10g/L, Semen Maydis pulp 5-
25g/L, (NH4)2HPO40.2-1.5g/L, pH 7.5-8.5, sterilizing 20min under 121 DEG C of high steams.
(3) shake flask fermentation: by above-mentioned mycobacteria CGMCC No.13031 (Mycobacterium sp.) LY-1 bacterial strain
Cellular liquid culture is seeded in equipped with sterilized 100mL fermentation medium with the inoculum concentration of 0.5-1.2% (w/w)
In 500mL conical flask, at 20-35 DEG C, the rotating speed bottom fermentation of 90-150r/min cultivates 120-168h, obtains fermentation liquid.
Embodiment 3 under the biphase fermentation system of soybean oil and water, mycobacteria CGMCC No.13031
(Mycobacterium sp.) LY-1 transformation phytosterin
(1) the cellular liquid training of mycobacteria CGMCC No.13031 (Mycobacterium sp.) LY-1 bacterial strain is prepared
Support thing
Mycobacteria (Mycobacterium sp.) LY-1 bacterial strain one ring on picking solid PDA medium, is seeded in dress
Have in the 500mL conical flask of 100mL seed culture medium, at 30 DEG C, be placed on shaking table and cultivate 60-with the rotating speed of 120r/min
72h, to logarithmic (log) phase, i.e. prepares the cellular liquid of mycobacteria CGMCC No.13031 (Mycobacterium sp.) LY-1 bacterial strain
Culture.
(2) add the fermentation system of Tween 80: in the fermentation medium, add the Tween 80 of 0.05%, ultrasonic vibration
30min。
(3) composition and the proportioning of fermentation medium is: plant sterol 5-20g/L, NaNO32-10g/L, Semen Maydis pulp 5-
25g/L, (NH4)2HPO40.2-1.5g/L, pH 7.5-8.5, sterilizing 20min under 121 DEG C of high steams.
(3) shake flask fermentation: by above-mentioned mycobacteria CGMCC No.13031 (Mycobacterium sp.) LY-1 bacterial strain
Cellular liquid culture is seeded in equipped with sterilized 100mL fermentation medium with the inoculum concentration of 0.5-1.2% (w/w)
In 500mL conical flask, at 20-35 DEG C, the rotating speed bottom fermentation of 90-150r/min cultivates 120-168h, obtains fermentation liquid.
Claims (2)
1. the bacterial strain of a plant height effect transformation phytosterin, this bacterial strain is mycobacteria (Mycobacterium sp.) LY-1, this bacterium
The Institute of Microorganism, Academia Sinica being positioned at Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 within 22nd, it is deposited in JIUYUE in 2016
China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number is CGMCC No.13031, Classification And Nomenclature
For mycobacteria Mycobacterium sp..
2. the side utilizing mycobacteria CGMCC No.13031 transformation phytosterin to be 9 Alpha-hydroxy 4-ADs
Method, is characterized by: the fermentation medium of the bottled 70-100mL of 500mL triangle, and inoculum concentration is 0.5-1.2% by volume, cultivates
Temperature 20-35 DEG C, shaking speed 90-150r/min, the fermentation starting stage adds a certain amount of substrate plant sterol, converts 5-
7d；Fermentation medium composition used is as follows: NaNO32-10g/L, Semen Maydis pulp 5-25g/L, (NH4)2HPO40.2-1.5g/L, pH
7.5-8.5, sterilizing 20min under 121 DEG C of high steams.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201610978886.6A CN106282077A (en) | 2016-11-08 | 2016-11-08 | The bacterial strain of one plant height effect transformation phytosterin and application thereof |
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| CN201610978886.6A CN106282077A (en) | 2016-11-08 | 2016-11-08 | The bacterial strain of one plant height effect transformation phytosterin and application thereof |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108056495A (en) * | 2017-12-20 | 2018-05-22 | 河南中烟工业有限责任公司技术开发分公司 | A kind of method of phytosterin compound in legal degrading tobacco of microorganism group |
| CN108913748A (en) * | 2018-06-15 | 2018-11-30 | 浙江大学 | A kind of method that phytosterol bio-conversion prepares androstenedione in quaternary phosphonium salt ionic liquid cosolvent system |
-
2016
- 2016-11-08 CN CN201610978886.6A patent/CN106282077A/en active Pending
Non-Patent Citations (3)
| Title |
|---|
| YANMIN HU,ET AL: "3-Ketosteroid 9a-hydroxylase is an essential factor in the pathogenesis of Mycobacterium tuberculosis", 《MOLECULAR MICROBIOLOGY》 * |
| 王贵娥等: "3-甾酮-9α-羟基化酶基因在分枝杆菌中强化表达", 《药物生物技术》 * |
| 高兴强等: "油水乳化体系中分枝杆菌转化植物甾醇产９α-羟基雄甾烯酮工艺研究", 《华东理工大学学报（自然科学版）》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108056495A (en) * | 2017-12-20 | 2018-05-22 | 河南中烟工业有限责任公司技术开发分公司 | A kind of method of phytosterin compound in legal degrading tobacco of microorganism group |
| CN108913748A (en) * | 2018-06-15 | 2018-11-30 | 浙江大学 | A kind of method that phytosterol bio-conversion prepares androstenedione in quaternary phosphonium salt ionic liquid cosolvent system |
| CN108913748B (en) * | 2018-06-15 | 2021-01-19 | 浙江大学 | Method for preparing androstenedione through phytosterol biotransformation in quaternary phosphonium salt ionic liquid cosolvent system |
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