CN108707553A - It is capable of bacterial strain and its application of Efficient Conversion 4AD specificity synthesis keto lactone clonorchis and ADD - Google Patents
It is capable of bacterial strain and its application of Efficient Conversion 4AD specificity synthesis keto lactone clonorchis and ADD Download PDFInfo
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Abstract
The present invention relates to bacterial strain and its applications that one plant is capable of Efficient Conversion 4AD specificity synthesis keto lactone clonorchis and ADD, the strain isolation is from the endogenetic fungus in Huperzia serrata, Classification And Nomenclature is Plectosphaerella cucumerina, specify entitled IA2, it is stored in China Committee for Culture Collection of Microorganisms's common micro-organisms center at present, deposit number is：CGMCC NO.11605, the deposit date is：On November 24th, 2015.Compared with prior art, bacterial strain provided by the invention being capable of Efficient Conversion substrate 4AD specificity generation keto lactone clonorchis (17a- oxos-D- ring expansions-Androsta-1,4-diene-3,17-dione) and Androsta-1,4-diene-3,17-dione (ADD).The conversion ratio of substrate 4AD is significantly improved with the increase of transformation time, complete to 4AD conversions in the 3rd day.The yield of 3rd day converted product ADD reaches up to 90% or more, continuously decreases later；The production rate of converted product keto lactone clonorchis is proportionate with transformation time, and static balancing has been reached by 10 days, and yield is up to 92% or more after fermented culture.
Description
Technical field
The invention belongs to biotechnologies, being capable of Efficient Conversion 4AD specificity synthesis Testolactone more particularly, to one plant
The bacterial strain and its application of ketone and ADD.
Background technology
Steroidal compounds are also known as sterid, in generally existing animal vegetable tissue, bile acid (cholic acid),
Cortex hormone of aadrenaline (adrenal cortex hormone), steroid saponin, cholesterine (cholestero1), steroidal biology
Alkali etc. belongs to such.Its chemical constitution is made of a five-membered ring and three hexatomic rings, and similar cyclopentanoperhy drophenanthrene is parent nucleus
Compound, as shown in Equation 1, A, B, C ring are hexatomic ring, and D rings are five-membered ring, and steroid compound is in the condensed place of A/B rings and C/D rings
10,13 of condensed place i.e. parent nucleus are usually each, and there are one methyl, A rings and B rings there may be partial double bond, and 17 have length
Different side chains.This structure of steroidal compounds not only has very strong physiological activity, but also due to the double bond on its parent nucleus
Position, substituent group and stereochemical structure freely change combination, form the steroidal drug of distinct, therefore steroid chemical combination
Object has very high medical value.
1 steroidal compounds general structure of formula
Compound with steroid nucleus structure is mostly steroids compound, be widely used in angiocardiopathy, leukemic lymphoblastoid,
The diseases such as bacterial encephalitis, rheumatic arthritis, bronchial asthma, osteoporosis be antitumor.Steroid hormone class drug can be divided into
Five classes：1. corticoid, such as：Cortisone, prednisone, hydrocortisone, dexamethasone, prednisone, fluocinolone acetonide etc.；2. female swash
Element, such as：Ethinylestradiol, Estradiol Valerate etc.；3. androgen, such as：Cortisol, methyl testosterone etc.；4. protein anabolic hormone,
Such as：Propiophenone nandrolone, metandienone etc.；5. contraceptive, such as：Norethindrone, megestrol acetate, acetic acid progesterone etc..Corticoid has
Anti-inflammatory, resisting allergic effect are clinically widely used in treating rheumatoid arthritis, bronchial asthma and eczema etc.
Skin disease；Sex hormone be treat male organs decline and certain gynecological diseases key agents, be oral contraceptive it is main at
Point；Protein anabolic hormone is for improving protein metabolism, recovery and enhancing muscle power and diuretic antihypertensive etc..
Androstenedione (4-AD, Androstenedione), abbreviation 4AD, structure such as 2 institute of formula
Show, molecular formula C19H26O2, white in appearance crystal powder dress, non-toxic, it is organic to be dissolved in acetic acid, ethyl alcohol, ether etc. for free from extraneous odour
Solvent is the indispensable key intermediate of the numerous steroid drugs of synthesis, very important adjustment effect is played to body.Androstane
Diene diketone (Androsta-1,4-diene-3,17-dione), abbreviation ADD, structural formula is as shown in Equation 3, molecular formula C19H24O2, source
The dehydrogenation product of 4AD.
2 4AD of formula (androstane-4-alkene-3,17- diketone) chemical structural formula
Formula 3ADD (androstane -1,4- diene -3,17- diketone) chemical structural formula
Androsterone (Androsterone) is found in 1931, and Butenandt etc. is detached from a male police urine
Go out a kind of compound, and it is named as androsterone.Nineteen thirty-five, Ruzicka and Butenandt etc. are reported from cholesterol biosynthesis
The research of androstenedione.After 1 year, Kochakian has found, androstenedione has male sex hormone and protein anabolic hormone
Characteristic.It is synthesized it may be said that almost all of steroidal drug all can be used as raw material by 4AD, recently as steroid
The demand of drug constantly expands, and prepares steroidal drug using natural material Chinese yam saponin or tigogenin, it is clear that cannot meet
The growing demand of people, faces this problem, and microbiological transformation technology is applied among steroid drugs by domestic and foreign scholars
The preparation research of body androstenedione (androst-4-ene-3,17-dione, 4AD) made breakthrough progress.The U.S. with
Stigmasterol makees raw material, the key intermediate 4AD for preparing steroid drugs is obtained through microorganism conversion, and with this synthesizing steroid medicine
Object, from this not only solve domestic Chinese yam plant it is limited and caused by raw material bottleneck problem, and greatly reduce and be manufactured into
This, and reduce environmental pollution, this is the primary great revolution in steroid drugs field.Steroid drugs obtained in recent years advances by leaps and bounds
Development should be attributed to the fact that the microorganism fungus kind for having screened key, can be by the animals and plants sterol such as sitosterol, stigmasterol and cholesterol
It is converted into key intermediate androstenedione and androstane diene diketone, and has researched and developed and can be used for large-scale industrial production
Transformation technology.Androstenedione and androstane diene diketone are obtained by microorganism conversion, and with this synthesizing steroid drug, not only may be used
To greatly simplify the production technology of steroid drugs, production cost is reduced, improves product yield and quality, and comprehensive profit can be carried out
With reduction " three wastes " pollution.Current advanced country in the world is can be seen that from existing information and data, in the system of steroid drugs
It is standby upper, microorganism conversion is carried out by starting material of animals and plants sterol mostly, after obtaining androstenedione and androstane diene diketone, then
Further prepare a series of related drugs.
Chinese patent CN106282077A discloses the mycobacteria of plant height effect transformation phytosterin
(Mycobacterium sp.) LY-1 and its application, it is common which is deposited in China Committee for Culture Collection of Microorganisms
Microorganism center, preserving number are CGMCC No.13031.Mycobacteria (Mycobacterium sp.) LY-1 can convert plant steroid
Alcohol is 9 Alpha-hydroxy 4-ADs, under conditions of substrate inventory is 15g/L, efficiency of pcr product 33%.
Invention content
The purpose of the present invention is to provide the bacterial strains that one plant is capable of Efficient Conversion 4AD specificity synthesis keto lactone clonorchis and ADD
And its application.Bacterial strain provided by the invention is with steroid drugs key intermediate-androstenedione (4AD) and androstane diene diketone
(ADD) it is substrate, and being capable of high specific bioconversion acquisition keto lactone clonorchis.
The purpose of the present invention can be achieved through the following technical solutions：
The present invention provides the bacterial strain that one plant is capable of Efficient Conversion 4AD specificity synthesis keto lactone clonorchis and ADD, the bacterial strains point
From from the endogenetic fungus in Huperzia serrata, Classification And Nomenclature is Plectosphaerella cucumerina, specifies entitled IA2,
It is stored in China Committee for Culture Collection of Microorganisms's common micro-organisms center at present, deposit number is：CGMCC
NO.11605, the deposit date is：On November 24th, 2015.Preservation place is：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3.
Bacterial strain provided by the invention with steroid drugs key intermediate-androstenedione (4AD) be substrate, being capable of Gao Te
Anisotropic bioconversion obtains keto lactone clonorchis.Specifically,
4AD for substrate, is converted into keto lactone clonorchis (17a- oxygen by the bacterial strain with 4-AD (4AD)
Generation-D- ring expansions-Androsta-1,4-diene-3,17-dione)；And it can efficiently specificity convert, 92% or more yield.Or,
The bacterial strain with steroid drugs key intermediate -4-AD (4AD) be substrate, by androstane -4-
Alkene -3,17- diketone is converted into androstane -1,4- diene -3,17- diketone (ADD)；And it can efficiently specificity convert, yield 90%
More than.Or,
For substrate, 4-AD is converted into 4-AD (4AD) for the bacterial strain
Boldenone.Or,
The bacterial strain with 4-AD (4AD) be substrate, simultaneously by 4-AD (4AD)
It is converted into androstane -1,4- diene -3,17- diketone (ADD), keto lactone clonorchis and boldenone.
One of which bacterial strain provided by the invention converts androstane-4-alkene-3, and the method for 17- diketone (4AD) is as follows：
1) IA2 strains are inoculated in seed culture medium, as primary seed solution, are cultivated, wait for that hypha,hyphae is grown
Afterwards, primary seed solution is transferred in conversion culture medium and is cultivated, add 4-AD substrate, made final
Androstane-4-alkene-3, a concentration of 0.2-0.8g/L (preferably 0.5g/L) of 17- diketone substrates；
2) after adding substrate 5 days, thalline zymotic fluid is taken out, vacuumizes centrifugation, collects filtrate, extraction, vacuum rotary steam extraction
Liquid obtains raw product；
3) separation of converted product：Chloroform is added in raw product, ultrasonic dissolution is admixed silica white, stirred evenly, room temperature
Under the conditions of air-dry, obtain pre-separation object, by pre-separation object be added silicagel column in, it is organic molten with dichloromethane, two kinds of ethyl acetate
Agent carries out gradient elution, fraction collection；It is detected with TLC and collects eluent, each fraction containing same composition is merged, and depressurizes
Concentration obtains each section crude product after silica gel post separation, then is detached through silicagel column and HPLC successively, finally obtains different conversions
Product.
When HPLC is detached, tri- converted products of 5.021min, 11.248min and 16.805min are respectively keto lactone clonorchis, hero
Steroid-Isosorbide-5-Nitrae-diene -3,17- diketone and boldenone.
The present invention also provides a kind of enzyme detached from the bacterial strain, for 4AD, efficiently specificity synthesizes Testolactone to the enzyme
Ketone and ADD, the enzyme are the dehydrogenation reaction and Baeyer- for participating in 4AD and other compounds 1,2 including steroidal
Villiger oxidation reactions and its relevant enzyme.
Mainly efficiently specificity conversion 4AD generates keto lactone clonorchis (17a- oxo-D- ring expansions-to the bacterial strain or enzyme of the present invention
Androsta-1,4-diene-3,17-dione) and Androsta-1,4-diene-3,17-dione (ADD), bacterial strain of the invention or the main needle of enzyme
To generating 1,2 dehydrogenation reactions of the A rings that keto lactone clonorchis occurs and 16,17 Baeyer- occurred of D rings
Villiger oxidation reactions generate corresponding steroid lactone.
Compared with prior art, bacterial strain provided by the invention being capable of Efficient Conversion substrate 4AD specificity generation keto lactone clonorchis
(17a- oxos-D- ring expansions-Androsta-1,4-diene-3,17-dione) and Androsta-1,4-diene-3,17-dione (ADD).Substrate
The conversion ratio of 4AD is significantly improved with the increase of transformation time, complete to 4AD conversions in the 3rd day.3rd day converted product ADD's
Yield reaches up to 90% or more, continuously decreases later；The production rate of converted product keto lactone clonorchis is in positive with transformation time
It closes, static balancing has been reached by 10 days, yield is up to 92% or more after fermented culture；Boldenone is in the 6th day yield highest, conversion
Process product variation of yield is not very big.
Description of the drawings
Fig. 1：Androstane-4-alkene-3, the microorganism conversion primary dcreening operation flow of 17- diketone (4AD)；
Fig. 2：The HPLC collection of illustrative plates of substrate 4AD；
Fig. 3：The HPLC collection of illustrative plates of IA2 bacterial strain converted products；
Fig. 4：IA2 bacterial strain fermentation liquor blank group HPLC collection of illustrative plates；
Fig. 5：Expand culture conversion sample HPLC detection figures；
Fig. 6：The carbon of keto lactone clonorchis is composed；
Fig. 7：The hydrogen of keto lactone clonorchis is composed；
Fig. 8：The carbon of ADD is composed；
Fig. 9：The hydrogen of ADD is composed；
Figure 10：The carbon of boldenone is composed；
Figure 11：The hydrogen of boldenone is composed；
Figure 12：Influence (note of the transformation time to conversion ratio：Conversion ratio：4AD；Production rate：IA2-1 is keto lactone clonorchis, IA2-
2 be ADD, and IA2-3 is boldenone)；
Figure 13：The bioconversion constructive ways of keto lactone clonorchis.
Specific implementation mode
The present invention is described in detail with specific embodiment below in conjunction with the accompanying drawings.
1 androstane-4-alkene-3 of embodiment, 17- diketone (4AD) convert bacterial strain screening
1, the preparation of substrate
300mg 4AD powder is weighed, the absolute ethyl alcohol of 10mL is added, is stirred with glass bar, is placed in ultrasonic wave
The 4AD ethyl alcohol storing solutions of 30g/L, superclean bench ultraviolet lamp is made until 4AD powder is completely dissolved in cleaning device 10min or so
It sterilizes after 30min, places 4 DEG C of refrigerators and preserve.
2, the preparation of PDA plate culture medium
PDA plate culture medium main component is：(in terms of g/L units)
| Potato | 200 | Glucose | 20 | Agar | 18 |
| Vitamin B1 | 0.01 | KH2PO4 | 5 | Anhydrous MgSO4 | 2.94 |
3, the preparation of seed culture medium and conversion culture medium (g/L)
Seed culture medium is identical with conversion medium component, and main component is：(in terms of g/L units)
4, the preparation of sulfuric acid vanillic aldehyde is improved
6% vanillic aldehyde, 10% ethanol solution of sulfuric acid
5, androstane-4-alkene-3,17- diketone (4AD) convert bacterial strain screening
From the bacterial strain to keep in cold storage, a small amount of mycelia of picking is inoculated in PDA plate culture medium with line-of-sight course, is placed in perseverance
28 DEG C of warm incubator is cultivated 4~7 days, then from PDA culture medium picking be about 0.4~0.6mm2Mycelium or spore, are inoculated in
In the seed culture medium of conical flask, it is placed on and shakes fast 120rpm, on the shaking table that temperature is 28 DEG C, wait for that seed mycelia liquid culture is good
Afterwards, it is transferred in conversion culture medium with 10% inoculum concentration, after the same terms culture 7 days, adds the 4AD storing solutions of preparation, make
Convert culture medium in 4AD ultimate density be 0.05g/L, culture 5 days after, take conversion sample, for chromatography detect, screening it is whole
A operating process is shown in Fig. 1.
According to high-efficient liquid phase chromatogram condition (instrument model：Shimadzu LC-20AT, detector models：Shimadzu SPD-20A；Chromatography
Column：WR C18 reversed-phase columns, 15cm × 4.6mm；Mobile phase：Chromatography methanol：Water=53：47；Detection wavelength：242nm；Column temperature：25
℃；Flow velocity：0.8mL/min), the high-efficient liquid phase chromatogram of substrate 4AD such as Fig. 2.4AD retention times are 18.460min.
By the screening of 50 plants of bacterial strains, it is found that IA2 bacterial strains have Efficient Conversion 4AD abilities, secondary screening result is consistent twice, root
According to high-efficient liquid phase chromatogram condition (instrument model：Shimadzu LC-20AT, detector models：Shimadzu SPD-20A；Chromatographic column：WR C18
Reversed-phase column, 15cm × 4.6mm；Mobile phase：Chromatography methanol：Water=53：47；Detection wavelength：242nm；Column temperature：25℃；Flow velocity：
0.8mL/min), the result of IA2 bacterial strains catalysis 4AD is shown in shown in Fig. 3, Fig. 4.Retention time be 5.021min, 5.478min,
The product of 11.248min, 16.805min are the converted product of IA2 bacterial strains.
By the above screening technique, show that IA2 bacterial strains have specific conversion capability to 4AD.The IA2 strain isolations are from feet added to a snake by an ignorant artist
Endogenetic fungus in Shi Shan, it is proposed that Classification And Nomenclature be Plectosphaerella cucumerina, specify entitled IA2,
It is stored in China Committee for Culture Collection of Microorganisms's common micro-organisms center at present, deposit number is：CGMCC
NO.11605, the deposit date is：On November 24th, 2015.
2 bacterial strain IA2 of embodiment expands culture and obtains converted product
1, the preparation of PDA plate culture medium
PDA plate culture medium main component is：(in terms of g/L units)
| Potato | 200 | Glucose | 20 | Agar | 18 |
| Vitamin B1 | 0.01 | KH2PO4 | 5 | Anhydrous MgSO4 | 2.94 |
2, the preparation of seed culture medium and conversion culture medium (g/L)
Seed culture medium is identical with conversion medium component, and main component is：(in terms of g/L units)
3, expand cultural method
IA2 strains are inoculated in seed culture medium in aseptic superclean bench, as primary seed solution, are placed in temperature
For 28 DEG C, shake speed and be on the shaking table of 120rpm, after hypha,hyphae grow it is good after, primary seed solution is transferred to 1L with 10% amount and is contained
In the conical flask for having conversion culture medium 200mL, 38 bottles in total, after the same terms culture 7 days, 4AD substrates is added, final substrate is made
A concentration of 0.5g/L.
After adding substrate 5 days, thalline zymotic fluid is taken out, vacuumizes centrifugation, filtrate is collected, with dichloromethane equal-volume extraction
4 times, it is sample A to merge all extract liquors.By mycelium acetone soak 2 times, centrifugation stays acetone solution, then extracted with dichloromethane
It takes 4 times, is sample B after merging.Merge sample A and B, on a rotary evaporator vacuum rotary steam extract liquor, rotary evaporation water-bath temperature
Degree is 45 DEG C, finally obtains raw product, is detected by HPLC, and testing conditions are same as above (instrument model：Shimadzu LC-20AT, detection
Type number：Shimadzu SPD-20A；Chromatographic column：WR C18 reversed-phase columns, 15cm × 4.6mm；Mobile phase：Chromatography methanol：Water=53：47；
Detection wavelength：242nm；Column temperature：25℃；Flow velocity：0.8mL/min), it obtains expanding culture converted product, as shown in Figure 5.
Fig. 5 show amplification cultured products and Fig. 3 preliminary experiment converted products retention time (5.021min, 5.478min,
11.248min and 16.805min) it is completely the same, and the converted product of retention time 5.021min obtains special sexual clorminance conversion,
This result also obtains reflecting card in substrate transformation time is to the result of the influence of conversion ratio.
4, the separation of converted product
Chloroform is added in raw product, ultrasonic dissolution makes it fully dissolve, and admixes silica white (silica white and raw product
Weight ratio be 1:1), glass bar stirs evenly, and is air-dried under normal temperature condition, referred to as pre-separation object.Pre-separation object is slowly added to
(ratio of height to diameter 10 in silicagel column:1, dichloromethane wet method dress post), with two kinds of dichloromethane, ethyl acetate organic solvents by centainly matching
Than gradient elution, (when gradient elution, it is 1 to match:0,10:0.2,10:0.5,10:1,10:2,10:4,10:6,0:1), branch receives
Collection；It is detected with TLC and collects eluent, each fraction containing same composition is merged, is concentrated under reduced pressure with Rotary Evaporators, successively
Each section crude product after to silica gel post separation, then through silicagel column and HPLC preparative separations, finally obtain different converted products.Through
Nuclear-magnetism identifies that the structure of tri- converted products of 5.021min, 11.248min and 16.805min is respectively keto lactone clonorchis (abbreviation
4Adc1), Androsta-1,4-diene-3,17-dione (ADD) and boldenone (17 β-hydroxyandrosta-1,4-dien-3-
One, abbreviation 4Ad3).The structure of keto lactone clonorchis and boldenone is respectively as shown in formula 4,5.
Carbon spectrum, the hydrogen spectrum of keto lactone clonorchis are as shown in Figure 6,7 respectively.
Carbon spectrum, the hydrogen spectrum of ADD is as shown in Figure 8,9 respectively.
The carbon spectrum of boldenone, hydrogen are composed respectively as shown in Figure 10,11.
4AD and its converted product keto lactone clonorchis, the structure of ADD and boldenone are as follows：
4AD and its converted product keto lactone clonorchis, ADD nuclear magnetic data related to boldenone are shown in Table 1, table 2.
1 4AD of table and its converted product carbon-13 nmr spectra data (13C NMR:100MHz, solvent：CDCl3)
2 4AD of table and its converted product hydrogen nuclear magnetic resonance modal data (1H NMR:400MHz, solvent：CDCl3)
5, influence of the substrate transformation time to conversion ratio
Bacterium solution culture puts into 30g/L 4AD ethyl alcohol storing solutions, control substrate 4AD a concentration of 0.8 (g/ in bacterium solution after 7 days
L), sampling daily, continues 11 days, investigates the length of transformation time and the relationship of conversion ratio, sample is detected through HPLC, passes through IA2
After conversion, the production quantity of converted product and the residual quantity concentration of 4AD are indicated with peak area, obtain conversion of the transformation system to 4AD
Rate is as shown in figure 12 with the variation dynamic relationship of transformation time.
As seen from Figure 12, the conversion ratio of substrate 4AD was significantly improved with the increase of transformation time, to the 3rd day 4AD
Conversion is complete.The yield of 3rd day converted product ADD reaches highest, continuously decreases later；The production rate of converted product keto lactone clonorchis
It is proportionate with transformation time, has reached static balancing by 10 days；Boldenone is obtained in the 6th day yield highest, conversion process product
Rate variation is not very greatly.There are the correlated process of a dynamic change between ADD and keto lactone clonorchis, due to ADD yield with when
Between increase and significantly reduce, the yield of keto lactone clonorchis increases as time increases and significantly, therefore has reason to show that 4AD is arrived
ADD, ultimately produces the conversion process of keto lactone clonorchis, and the bioconversion constructive ways of keto lactone clonorchis are as shown in figure 13.
The above description of the embodiments is intended to facilitate ordinary skill in the art to understand and use the invention.
Person skilled in the art obviously easily can make various modifications to these embodiments, and described herein general
Principle is applied in other embodiment without having to go through creative labor.Therefore, the present invention is not limited to the above embodiments, ability
Field technique personnel announcement according to the present invention, improvement and modification made without departing from the scope of the present invention all should be the present invention's
Within protection domain.
Claims (10)
1. one plant of bacterial strain for capableing of Efficient Conversion 4AD specificity synthesis keto lactone clonorchis and ADD, which is characterized in that the strain isolation
From the endogenetic fungus in Huperzia serrata, Classification And Nomenclature is Plectosphaerella cucumerina, specifies entitled IA2, mesh
Before be stored in China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number is：CGMCC NO.11605,
The deposit date is：On November 24th, 2015.
2. bacterial strain application as described in claim 1, which is characterized in that the bacterial strain using 4-AD as substrate,
4-AD is converted into 17a- oxos-D- ring expansions-Androsta-1,4-diene-3,17-dione.
3. bacterial strain application according to claim 2, which is characterized in that bacterial strain using 4-AD as substrate,
4-AD is converted into 17a- oxos-D- ring expansions-Androsta-1,4-diene-3,17-dione, it can be efficiently special
Opposite sex conversion, 92% or more yield.
4. bacterial strain application as described in claim 1, which is characterized in that the bacterial strain using 4-AD as substrate,
By androstane-4-alkene-3,17- diketone is converted into androstane -1,4- diene -3,17- diketone.
5. bacterial strain application according to claim 4, which is characterized in that bacterial strain using 4-AD as substrate,
4-AD is converted into Androsta-1,4-diene-3,17-dione, can efficiently specificity be converted, yield
90% or more.
6. bacterial strain application as described in claim 1, which is characterized in that the bacterial strain using 4-AD as substrate,
By androstane-4-alkene-3,17- diketone is converted into boldenone.
7. bacterial strain application as described in claim 1, which is characterized in that the bacterial strain using 4-AD as substrate,
By androstane-4-alkene-3,17- diketone is converted into keto lactone clonorchis, androstane -1,4- diene -3,17- diketone and boldenone.
8. bacterial strain application according to claim 7, which is characterized in that the bacterial strain converts androstane-4-alkene-3,17- bis-
The method of ketone is as follows：
1) IA2 strains are inoculated in seed culture medium, as primary seed solution, are cultivated, it, will after hypha,hyphae is grown well
Primary seed solution is transferred in conversion culture medium and is cultivated, and adds 4-AD substrate, makes final androstane-
A concentration of 0.2-0.8g/L of 4- alkene -3,17- diketone substrates；
2) after adding substrate 5 days, thalline zymotic fluid is taken out, vacuumizes centrifugation, collects filtrate, extraction, vacuum rotary steam extract liquor obtains
To raw product；
3) separation of converted product：
Chloroform is added in raw product, ultrasonic dissolution is admixed silica white, stirred evenly, and is air-dried under normal temperature condition, obtains pre-separation
Pre-separation object is added in silicagel column object, carries out gradient elution with two kinds of dichloromethane, ethyl acetate organic solvents, branch receives
Collection；It is detected with TLC and collects eluent, each fraction containing same composition is merged, and is concentrated under reduced pressure, obtain silicagel column point successively
Each section crude product from after, then detached through silicagel column and HPLC, finally obtain different converted products.
9. bacterial strain application according to claim 8, which is characterized in that HPLC detach when, 5.021min, 11.248min and
Tri- converted products of 16.805min are respectively keto lactone clonorchis, Androsta-1,4-diene-3,17-dione and boldenone.
10. a kind of enzyme detached from bacterial strain described in claim 1, which is characterized in that the enzyme is for 4AD efficiently specificity synthesis
Keto lactone clonorchis and ADD, the enzyme are the dehydrogenation reaction and Baeyer- for participating in 4AD and other compounds 1,2 including steroidal
Villiger oxidation reactions and its relevant enzyme.
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