CN103740799B - A kind of bioconversion phytosterol prepares the method for Steroid medicine intermediates - Google Patents
A kind of bioconversion phytosterol prepares the method for Steroid medicine intermediates Download PDFInfo
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Abstract
The invention provides a kind of method that bioconversion phytosterol prepares Steroid medicine intermediates, comprise the following steps: the suspension liquid that 1) plant sterol is provided; 2) a kind of resting cell plant sterol being converted into the microbial strains of Steroid medicine intermediates is provided; 3) by the suspension liquid of described plant sterol and the mixing of described resting cell, and the beta-cyclodextrin added after beta-cyclodextrin or chemically modified fully mixes, and has cultivated conversion; 4) by centrifugal for step 3) gained conversion fluid, cleer and peaceful thalline in separation, is separated through solvent extraction and obtains described Steroid medicine intermediates from supernatant.Method provided by the invention is improve sterol feed concentrations, is increasing object product amount, while the shortening conversion time length, also achieve the simple and easy separation of thalline, solubility promoter and product simultaneously, and achieve repeatedly using of thalline and solubility promoter, greatly save production cost, be applicable to suitability for industrialized production.
Description
Technical field
The present invention relates to sterol technical field of biotransformation, relate more specifically to a kind of method that bioconversion phytosterol prepares Steroid medicine intermediates.
Background technology
The compound of steroidal compounds to be a class with perhydrocyclopentanophenanthrene be parent nucleus, structural similitude, is extensively present in animal vegetable tissue and certain micro-organisms cell, compares and common are plant sterol, cholesterol, androsterone and diosgenin etc.Steroidal compounds is the important source material of producing steroid drugs.Steroid drugs plays very important regulating effect to body, is to be only second to antibiotic second largest class medicine.Pharmaceutically occupying the main component of hormone medicine of critical role as hydrocortisone, testosterone, androsterone, progesterone and estradione etc. are all the medicines with steroidal structure.AD (AD), ADD (ADD) and 9-hydroxyl-AD (9-OH-AD) are the irreplaceable intermediates of steroid drugs.Steroid medicine intermediates, steroidal compounds all contain the mother nucleus structure of identical perhydrocyclopentanophenanthrene with various steroid drugs.Have been found that a variety of microorganism can utilize plant sterol to change into this several Steroid medicine intermediates.These microorganisms comprise joint bacterium, bacillus, mycobacterium, Nocardia bacteria and tyrothricin etc.
For a long time, AD and ADD is used as the precursor substance synthesizing male hormone and assimilation class medicine always.And 9-OH-AD is the important precursor compound of a class in synthesis preparation is as medicines such as reflunomides.The exploitation of 9-OH-AD have become study hotspot in recent years, because this intermediate can be used for synthetic glucocorticoid, antiphlogiston and the efficient cortin containing halogen elements such as F, Cl, because of the existence of 9-OH in its chemical structure, chemosynthesis means by routine form C9,11-double bond systems, thus introduce a halogen atom in C9-position smoothly, C11 β-position forms requisite function hydroxy.This just can solve the bottleneck problem of domestic existing diosgenin operational path---and mould transforms the hydroxylated inefficiencies problem of C11-of epoxy Progesterone, can produce domestic existing 9-halogen (F, Cl) cortin, as dexamethasone, Betamethasone Valerate and C11-ketone structure, as cortisone, prednisone synthesis technique are integrated and carried out cortin production generation tremendous influence.Utilize the mycobacterium fortutitum mutant strain Sitosterol that ferments to break side chain, effectively accumulation useful intermediates 9-OH-AD as far back as Pu Qiang drugmaker of the U.S. in 1979 report, with this intermediate through 7 step chemosynthesis, obtained acetic acid hydrocortisone, total recovery reaches 36.7%.
But plant sterol is a class hydrophobic compound, the solubleness in water is very low, limits its bioavailability; And plant sterol (substrate), AD, ADD and 9-OH-AD (product) may suppress and poison microorganism, limit the accumulation volume of substrate charging capacity and product.The low-solubility of sterol can cause mass transfer obstacle, and sterol has certain toxicity to be the bottleneck problem that plant sterol transforms, so sterol transformation efficiency is often very low to thalline.Investigators taked a lot of measure to promote that sterol transforms.In aqueous phase reforming system, increase sterol transformation efficiency by adding the solubility promoters such as tensio-active agent, cyclodextrin and organic solvent.In addition, two-phase system and emulsification system are equally also used for improving the biotransformation efficiency of plant sterol.In prior art, also reporting some by adding the single-phase fermentation system of the organic solvent such as ethanol, glycerine, the solvability of sterols material can be improved, improve the biological transformation ratio of microorganism.WO9949075A1 discloses the microbial conversion process of a plant sterols to AD and ADD, and specifically discloses following technical characteristic: utilize a kind of substratum to breed the mycobacterium MB3683 bacterial classification of Mycobacterium; Utilize one or more solubilizing agent that phytosterol compositions is dissolved into solution; Mycobacterium MB3683 culture and phytosterol compositions solution are placed in bio-reactor, and keep time enough, phytosterol compositions is converted into AD, ADD.
In addition a kind of utilization with the inconsistent organic solvent of water as octanol etc. is also had, inconsistent Bi-liquid phase system is formed with fermented liquid, the advantage of this system is that the solubleness of sterol material and biological transformation ratio have obvious raising, and AD and ADD almost all concentrates on organic phase, segregation ratio is easier to, but shortcoming is solvent to the toxic action of microorganism and the safety problem that produces due to solvent evaporates, is all unfavorable for commercial introduction.
Present industrial process is oil-containing fermentation mostly, and be that grown cell cultivates method for transformation, namely be before microorganism cells is cultivated or after cultivating for some time, be directly added to by substrate in microbial transformation substratum, microorganism carries out bio-transformation to substrate while self-reproduction growth.This is a kind of simple and the most the most frequently used method of biotransformation method.The advantage of this method is that microbial transformation bio-transformation is easy, and shortcoming is that the fermentation time of product is long, easily produces multi-products, process separation and purification difficulty.As illustrated in CN03804973, during feed concentrations according to 1%, plant sterol is converted into the time of AD/ADD at 87-91h.If reduce the feed concentrations of substrate, the reaction times can shorten, but corresponding industrial cost can improve greatly.AD and ADD is produced in bio-transformation through about 120-168h, then with suitable organic solvent AD with ADD carried out extract, be separated, crystallization refining, AD and ADD of the crystalline powder of white can be obtained.Chinese patent CN101525651A discloses biodegradation of phytosterol in a kind of Bi-liquid phase system and prepares the method for Androstenedione, this patent is by adding Trisun Oil R 80 as organic phase, profit two phase culture system is formed with aqueous phase fermented liquid, and utilize mycobacterium Mycobacteriumsp-UV-8 to breed in such a system and effective transformation phytosterin production AD, transformation in planta rate is about 80%, but in this system, Trisun Oil R 80 is excellent at least reaches 20%V/V, and charging capacity only has 0.6%, although its transformation efficiency can reach 80%, but single turnout is still very low, far be not by far up to the mark.
Mention in the Chinese patent of " a kind of resting cell that adopts carries out bio-transformation in cloud point system " (CN200310108967) by name, mycobacterium MycobacteriumNRRLB-3683 resting cell is utilized to obtain hero-1 in cloud point system fermentation cholesterol, 4-diene-3,17-diketone (ADD).But because this cloud point system needs to use nonionogenic tenside tritonX100 or 114, this kind of tensio-active agent is generally harmful to thalline, and from ecological point, they contact underground water, water channel or sink drainage, even if a small amount of product permeates the ground, water will work the mischief to water body, if anarchy license, material is not entered surrounding environment, realize industrialization difficulty.This patent inventor mentions in " Chinese engineering science " that " about the report of large-scale surface active agent recovery technique is few, in research product and cloud point system, the separating technology of tensio-active agent has practical significance to improving the application of cloud point system in bioprocess further for 2005 in 7 volume 5 phase 73-78 pages " application of two-phase distributing bioreactor---cloud point system in bio-transformation " simultaneously." illustrate that the recovery problem of tensio-active agent in cloud point system not yet solves.
Chinese patent CN101760494A discloses a kind of biofermentation method adopting the Androstenedione of resting cell, fermentation process described in this patent obtains the resting cell of the microbial strains of AD and ADD by having degrading plant sterol, join in plant sterol conversion fluid and carry out first time conversion of resting cells reaction, after making in conversion fluid plant sterol transform to obtain AD and ADD to terminate, remove somatic cells, and conversion fluid is obtained AD and ADD through solvent extraction.The essence of the method is two-steps tissue culture method, first obtains thalline and then carries out bio-transformation.In this patents state, cell concentration is limited, and sterol feeds intake lower, and it is limited that thalline reuses number of times, is not suitable for suitability for industrialized production.And interpolation thalline Protective substances not yet in effect (or against sterols toxicity or anti-living contaminants), bacterial classification is easily degenerated and is had microbiological contamination or even infect the risk of wet phage.
In sum, the defect of prior art mainly concentrate on following some:
1) routine growth cell fermentation method fermentation time is long, and cell concentration is limited, and sterol charging capacity is limited; If reduce the feed concentrations of substrate, although the reaction times can shorten, corresponding industrial cost can improve greatly.
2) solubility promoter used in prior art has nonionogenic tenside tritonX100 or 114, and this kind of tensio-active agent generally reclaims problem and not yet solves, and can not effectively reuse, and can make separation and purification of products difficulty.And general tensio-active agent one is harmful to water body, belongs to the unfriendly material of environment; Two is be harmful to thalline, if lacking of adding is not enough to play hydrotropy effect, if dosage conference restriction sterol substrates charging capacity.
3) tensio-active agent as solubility promoter use can used in prior art or organic solvent, wherein, tensio-active agent causes fermenting process bubble-related issues serious, and microbiological contamination has a big risk; Organic solvent, to the toxic action of microorganism and the safety problem that produces due to solvent evaporates, is all unfavorable for commercial introduction.
4) common conversion of resting cells method, thalline is separated with steroidal compounds difficulty, and thalline is easily degenerated, and thalline repeat usage is low, and there is microbiological contamination risk.
Summary of the invention
The object of this invention is to provide a kind of method that bioconversion phytosterol prepares Steroid medicine intermediates, thus it is low to overcome substrate charging capacity in prior art, cell concentration is limited, long reaction time, solubility promoter reclaims difficulty, and the defect to thalline and bad environmental.
In order to solve the problems of the technologies described above, the present invention by the following technical solutions:
There is provided a kind of bioconversion phytosterol to prepare the method for Steroid medicine intermediates, comprise the following steps: the suspension liquid that 1) plant sterol is provided; 2) a kind of resting cell plant sterol being converted into the microbial strains of Steroid medicine intermediates is provided; 3) by the suspension liquid of described plant sterol and the mixing of described resting cell, and the beta-cyclodextrin added after beta-cyclodextrin or chemically modified fully mixes, and has cultivated conversion; 4) by centrifugal for step 3) gained conversion fluid, cleer and peaceful thalline in separation, is separated through solvent extraction and obtains described Steroid medicine intermediates from supernatant.
Use as solubility promoter by the beta-cyclodextrin after beta-cyclodextrin or chemically modified is added reaction soln, make the resting cell of thalline present good dispersion state, be not only conducive to the carrying out of conversion reaction, and be conducive to the separation of thalline; Secondly, self high-dissolvability due to the beta-cyclodextrin after beta-cyclodextrin or chemically modified and the high hydrotropy effect to plant sterol, in the process of cleer and peaceful thalline in separation, this solubility promoter overwhelming majority is in aqueous phase, and in conversion process, the nontoxic character of this solubility promoter makes the activity of thalline be able to good maintenance.
The beta-cyclodextrin in the aqueous phase that the thalline of acquisition and/or supernatant obtain after solvent extraction is separated or the beta-cyclodextrin after chemically modified is directly reused in described step 4).
Described microbial strains is the mycobacterium having lacked 1,2-desaturase (KstD), or enhances the mycobacterium of 1,2-desaturase (KstD), or has first lacked 1,2-desaturase (KstD), after enhance the mycobacterium of 9-hydroxylase (KsH).
Beta-cyclodextrin after described chemically modified is selected from: methyl-B-cyclodextrin, hydroxy-beta-cyclodextrin, sulphonyl group-beta-cyclodextrin or hydroxypropyl-beta-cyclodextrin, is preferably hydroxypropyl-beta-cyclodextrin or methyl-B-cyclodextrin.Wherein, hydroxypropyl-beta-cyclodextrin solubleness in water, up to 600-800g/L, and is insoluble to organic solvent, by the extraction of organic solvent, facilitates its being separated with product.
Described plant sterol is selected from: Sitosterol, Stigmasterol, campesterol, at least one in brassicasterol or any mixture.
Described Steroid medicine intermediates comprises: AD (AD), ADD (ADD) or 9-hydroxyl-AD (9-OH-AD).
Described resting cell obtains by adopting two-stage seed culture method to cultivate described microbial strains.The energy for growth of thalline is enhanced by two-stage seed enlarged culturing.
Described microbial strains, after two-stage seed culture method is cultivated, also comprises proceeding in growth medium and continues to cultivate, thus be more conducive to transforming.
The formula of described growth medium is as follows: glucose 20g/L, citric acid 2g/L, (NH
4)
2hPO
43.5g/L, ferric ammonium citrate 0.05g/L, K
2hPO
43H
2o0.5g/L, MgSO
47H
2o0.5g/L, Zulkovsky starch 2g/L, corn steep liquor 1g/L.The formula of this substratum is that contriver gropes to optimize by experiment, proves that thalline energy for growth after the cultivation of this growth medium is farthest improved through experiment.
In described step 3), the mol ratio of beta-cyclodextrin and plant sterol is between 2:1 to 1:2, is preferably 1:1.Being best as the mol ratio 1:1 of beta-cyclodextrin and plant sterol, both because making system toughness increase adding of too much cyclodextrin, also can not can not can not playing hydrotropy Inclusion property not because cyclodextrin amount.
The solvent used in described step 4) is preferably ethyl acetate.
Method of the present invention is also included in conversion of resting cells process adds microbiotic, and object is the growth preventing miscellaneous bacteria, reduces microbiological contamination risk.
Conversion of resting cells is compared with grown cell, and transformation time shortens, and microbiological contamination risk reduces; Biomass can regulate, and can increase substrate feed concentrations simultaneously, shortens the conversion reaction time; The cyclodextrin that conversion of resting cells needs and thalline can multiplely utilize, and and convenient separation between product and cyclodextrin.
The invention provides a kind of method that efficient bioconversion phytosterol prepares Steroid medicine intermediates, improve sterol feed concentrations, increase object product amount, while the shortening conversion time length, also achieve the simple and easy separation of thalline, solubility promoter and product simultaneously, and achieve repeatedly using of thalline and solubility promoter.The transformation efficiency of the plant sterol that method provided by the present invention obtains is close to 100%, and the output of Steroid medicine intermediates is high, and thalline and solubility promoter even can be reused up to 8 times, and production cost reduces greatly, is applicable to suitability for industrialized production.
Accompanying drawing explanation
Fig. 1 is the schematic flow sheet according to multiple preferred embodiment of the present invention.
Fig. 2 is the metabolism train of thought figure of plant sterol.
Embodiment
Below in conjunction with specific embodiment, the present invention will be further described.Should be appreciated that following examples only for illustration of the present invention but not for limiting the scope of the invention.
1. materials and methods
1.1 material
1.1.1 bacterial classification
Embodiments of the invention mainly have employed three bacterial strains, are mycobacterium strain MycobacteriumneoaurumNwIB-02, MycobacteriumneoaurumNwIB-04, the MycobacteriumneoaurumNwIB-V built by molecular modification respectively.
Wherein, MycobacteriumneoaurumNwIB-02 is that (preserving number is CCTCCM209094 to starting strain with MycobacteriumneoaurumNwIB01, see document 1 [WeiWei, ShuyueFan, FengqingWang, DongzhiWei (2010) ANewSteroid-TransformingStrainofMycobacteriumneoaurumand Cloningof3-Ketosteroid9 α-HydroxylaseinNwIB-01.ApplBiochemBiotechnol.162:1446 – 1456]), lack 1, the mycobacterium of 2-desaturase (KstD) is (see document 2 [WeiWei, Feng-qingWang, Shu-yueFan, Dong-zhiWei (2010) InactivationandAugmentationofthePrimary3-Ketosteroid-Δ 1-DehydrogenaseinMycobacteriumneoaurumNwIB-01:Biotransfo rmationofSoybeanPhytosterolsto4-Androstene-3, 17-Dioneor1, 4-Androstadiene-3, 17-Dione.Appl.Environ.Microbiol.76 (13): 134578-4582].
MycobacteriumneoaurumNwIB-04 is starting strain with MycobacteriumneoaurumNwIB01, enhances the mycobacterium (see also document 2) of 1,2-desaturase (KstD).
MycobacteriumneoaurumNwIB-V take MycobacteriumneoaurumATCC25795 as starting strain, first lacked 1,2-desaturase (KstD), after enhance the mycobacterium of 9-hydroxylase (KsH).According to common practise and the above-mentioned information of this area, the construction process of this bacterial strain is ordinary skill in the art means, simultaneously can with reference to the structure of above-mentioned MycobacteriumneoaurumNwIB-02 and MycobacteriumneoaurumNwIB-04, specifically see above-mentioned document 1 and document 2.
Wherein, as shown in Figure 2, plant sterol (Sitosterol, Stigmasterol, campesterol and brassicasterol) can be converted into AD (AD) under the effect of mycobacterium, hero-4-alkene-3,17-diketone (AD) then can 1, ADD (ADD) is generated under 2-desaturase (KstD) effect, or under 9-hydroxylase (KsH) effect, generate 9-hydroxyl-AD (9-OH-AD).
1.1.2 instrument and analysis condition
Shaking flask conversion, seed culture, yeast culture carry out on conventional rotational oscillation formula shaking table.
Fermentor tank transforms, thalline prepares: protect emerging fermentor tank 5L.
TLC analyzes: the sample taken out at regular intervals, and with the ethyl acetate continuous extraction 3 times of 4 times of volumes, the ethyl acetate merging extracted 3 times, gets 10 μ L point samples in tlc silica gel plate HSGF254.Developing agent is sherwood oil (60-90 DEG C)/ethyl acetate (6:4, v/v), after exhibition layer terminates, 20% vitriol oil is sprayed on silica-gel plate, and juxtaposition 110 DEG C baking 10-15min colour developing, qualitative observation sterol transforms situation.
HPLC:AD, ADD, 9-OH-AD can be detected by HPLC.Agilent 1100 liquid chromatographic system, chromatographic column is: HypersilODS2, filler granularity: 5 μm, size: 4.6mm × 250mm, determined wavelength 254nm, detected temperatures 40 DEG C, and moving phase is methyl alcohol: water (70:30, v/v).
1.2 implementation method
1.2.1 substratum
Seed culture medium MYC/01 (g/L): glycerine 20, citric acid 2, NH
4nO
32, ferric ammonium citrate 0.05, K
2hPO
43H
2o0.5, MgSO
47H
2o0.5.Initial pH8,121 DEG C, 20min, sterilizing is for subsequent use.
Growth medium MYC/03 (g/L): glucose 20, citric acid 2, (NH
4)
2hPO
43.5, ferric ammonium citrate 0.05, K
2hPO
43H
2o0.5, MgSO
47H
2o0.5, Zulkovsky starch 2, corn steep liquor 1.Initial pH8,115 DEG C, 20min, sterilizing is for subsequent use.
1.2.2 culture condition:
Seed culture: 100mL seed culture medium MYC/01(1000mL shaking flask), 30 DEG C, 200rpm, culture cycle 2-3d.
Yeast culture: shake-flask culture, 100mL growth medium MYC/03 (1000mL shaking flask), 30 DEG C, 200rpm, culture cycle 2-3d.
Sterol transforms: shaking flask transforms 100mL conversion of resting cells system (1000mL shaking flask), 30 DEG C, 200rpm, culture cycle 3d.Fermentor tank transforms 3L conversion of resting cells system (5L fermentor tank), 30 DEG C, air flow 0.5vvm.
1.2.3 seed culture: seed culture takes two-stage seed culture.Mycobacterium frozen for glycerine is inoculated in primary-seed medium MYC/01, to be generated when reaching mid-log phase, be inoculated in secondary seed medium with 5% inoculum size and be cultured to mid-log phase.Wherein, secondary seed medium and primary-seed medium are the same, and first order seed is as activated strains use, and secondary seed strengthens thalli growth ability as enlarged culturing and uses.
1.2.4 yeast culture process: be inoculated in growth medium MYC/03 with 10% inoculum size by ready secondary seed, cultivates 3d, collects thalline.
1.2.5 microorganism collection treating processes: by cultured thalline 6000rpm, 10min is centrifugal, washes twice with deionized water (pH8), centrifugal thalline of weighing.
1.2.6 plant sterol mother liquor: because plant sterol is slightly water-soluble compound, in advance it will be made into uniform suspension liquid.The process (every L) of plant sterol suspension liquid configuration: precise 200g plant sterol powder, add a small amount of water, be placed in and electric furnace stir limit and be heated to sterol and be in the state of being suspended, continuing heated and stirred for some time to sterol is in comparatively after uniform state, move in magnetic stirring apparatus and continue stirring until room temperature, for subsequent use.
1.2.7 plant sterol conversion process: in transformation system, by the thalline that the beta-cyclodextrin after beta-cyclodextrin or chemically modified and the plant sterol for preparing are suspended mother liquor and get ready, short mix is even, is settled to system nominal volume.The regular replenishment water yield is to nominal volume.Whole system is deionized water (pH8).
1.2.8 the reusing of thalline: the adding of beta-cyclodextrin after beta-cyclodextrin or chemically modified makes thalline and product be easy to be separated.In addition, due to adding of the beta-cyclodextrin after beta-cyclodextrin or chemically modified, thalline also presents extraordinary dispersion state, is easy to the separation of carrying out and the thalline reacted.High-dissolvability due to the beta-cyclodextrin after beta-cyclodextrin or chemically modified and the high hydrotropy effect to sterid, when centrifugation thalline, beta-cyclodextrin after beta-cyclodextrin or chemically modified and the steroid overwhelming majority in system are in aqueous phase, and in conversion process, the nontoxic character of the beta-cyclodextrin after beta-cyclodextrin or chemically modified makes the activity of thalline keep preferably.Thalline after centrifugal can be re-used in second time and transform after water or buffer solution, and so repeat, thalline can be reused repeatedly.
1.2.9 the reusing of beta-cyclodextrin: although this material of beta-cyclodextrin after beta-cyclodextrin or chemically modified is not advantageous in price, the present invention is by increasing the access times of the beta-cyclodextrin after beta-cyclodextrin or chemically modified and then reducing the input of cost.And the beta-cyclodextrin itself after beta-cyclodextrin or chemically modified belongs to soluble in water, be insoluble to organic solvent.The beta-cyclodextrin after product and beta-cyclodextrin or chemically modified can be made to be separated when product extraction, after removing organic phase, the product of the few part be dissolved with in aqueous phase, do not hinder the use next time of the beta-cyclodextrin after beta-cyclodextrin or chemically modified, repetition like this, the beta-cyclodextrin after beta-cyclodextrin or chemically modified can be reused repeatedly.
1.2.10 the separation of product: each conversion group institute sample thief, with extraction into ethyl acetate 3 times, merge 3 extraction liquids, after first TLC preliminary observation conversion situation, measure concrete content with HPLC.
As shown in Figure 1, the schematic flow sheet according to multiple preferred embodiment of the present invention is.Wherein, plant sterol is not limited to Sitosterol listed in figure, Stigmasterol, campesterol, and brassicasterol can also be the mixture of other plant sterol, and the kind of final product is then only relevant with bacterial strain, has nothing to do with concrete sterol kind.
Embodiment 1:
1. seed preparation and yeast culture: mycobacterium MycobacteriumneoaurumNwIB-V frozen for glycerine is inoculated in primary-seed medium MYC/01, to be generated when reaching mid-log phase (3d), be inoculated in secondary seed medium with 5% inoculum size and be cultured to mid-log phase (2d), be inoculated into growth medium MYC/03 with 10% inoculum size again, when cultivating 3d, collect thalline.
2. microorganism collection treating processes: collect thalline during 3d, 6000rpm, 10min are centrifugal, wash twice with deionized water (pH8), and it is 35g that centrifugal thalline of weighing obtains wet thallus weight.
3. conversion of resting cells process: set specified conversion liquid amount 100mL in 1L shaking flask, accurately measure plant sterol mother liquor 50mL, weighing hydroxypropyl-beta-cyclodextrin 400g(and plant sterol mol ratio are 1:1), weigh the heavy 10-15g of wet bacterium, three fully mixes at once, be settled to 100mL, regulate initial pH to be 8, cultivate 4-5d.
4. transform after terminating, by cleer and peaceful thalline in conversion fluid 10000rpm centrifugation, thalline is washed one time with the deionization that pH is 8, cleer and peaceful water in centrifugation.Twice supernatant merged, with the ethyl acetate continuous extraction 3 times of 4 times of volumes, 3 gained ethyl acetate merged, TLC, HPLC detect.
5. thalline repeating step 3, does thalline and reuses number of times experiment; The ethyl acetate extracting phase obtained in step 4, after fully vaporing away residual ethyl acetate, repeating step 3 replaces the hydroxypropyl cyclodextrin weighed, and does cyclodextrin and reuses experiment.
6. first time conversion of resting cells experiment, record the transformation efficiency of plant sterol through HPLC close to 100%, 9-OH-AD output 50g/L, molar yield can reach 72%.
7. repeatedly test 8 times through step 5, thalline and cyclodextrin can reuse 8 times, and the 8th time, 9-OH-AD output can have 45g/L.
Embodiment 2: bacterial strain and cultural method, with embodiment 1, are not all and do conversion of resting cells experiment on 5L fermentor tank.Experiment proves, the transformation experiment that 5L fermentor tank carries out obtains the experiment effect similar to shaking flask substantially.
Embodiment 3,4: with embodiment 1,2, difference is that bacterial strain is mycobacterium MycobacteriumneoaurumNwIB-02.No matter be 1000mL shaking flask or 5L fermentor tank, record the transformation efficiency of plant sterol through HPLC close to 100%, AD output 37g/L.Thalline and cyclodextrin can reuse 8 times.
Embodiment 5,6: with embodiment 1,2, difference is that bacterial strain is mycobacterium MycobacteriumneoaurumNwIB-04.No matter be 1000mL shaking flask or 5L fermentor tank, record the transformation efficiency of plant sterol through HPLC close to 100%, ADD output 38g/L.Thalline and cyclodextrin can reuse 8 times.
Embodiment 7,8: with embodiment 1,2, difference is only that solubility promoter is methyl-B-cyclodextrin.No matter be 1000mL shaking flask or 5L fermentor tank, record the transformation efficiency of plant sterol through HPLC close to 100%, 9-OH-AD output 55g/L.Thalline and cyclodextrin can reuse 8 times.
According to above-described embodiment, method provided by the present invention is not only applicable to small-scale laboratory requirement, and is applicable to large-scale industrial production.
Above-described, be only preferred embodiment of the present invention, and be not used to limit scope of the present invention, the above embodiment of the present invention can also make a variety of changes.Namely every claims according to the present patent application and description are done simple, equivalence change and modify, and all fall into the claims of patent of the present invention.The not detailed description of the present invention be routine techniques content.
Claims (8)
1. bioconversion phytosterol prepares a method for Steroid medicine intermediates, it is characterized in that, comprises the following steps:
1) concentration is provided to be that the suspension liquid 50mL of the plant sterol of 200g/L is in every 1L shaking flask;
2) a kind of wet thallus plant sterol being converted into the resting cell of the heavy 10-15g of the microbial strains of Steroid medicine intermediates is provided;
3) by the mixing of the wet thallus of the suspension liquid of described plant sterol and described resting cell, and add 400g hydroxypropyl-beta-cyclodextrin and fully mix, be settled to 100mL, regulate initial pH to be 8, cultivate 4-5d and complete conversion;
4) by step 3) gained conversion fluid is centrifugal, and cleer and peaceful thalline in separation, is separated through solvent extraction and obtains described Steroid medicine intermediates from supernatant.
2. method according to claim 1, is characterized in that, described step 4) in the hydroxypropyl-beta-cyclodextrin be separated in the thalline and/or the aqueous phase that obtains after solvent extraction of supernatant obtained directly reuse.
3. method according to claim 1, is characterized in that, described microbial strains has lacked 1, the mycobacterium of 2-desaturase, or the mycobacterium enhancing 1,2-desaturase, or first lacked 1,2-desaturase, after enhance the mycobacterium of 9-hydroxylase.
4. method according to claim 1, is characterized in that, described plant sterol is selected from: Sitosterol, Stigmasterol, campesterol, at least one in brassicasterol or any mixture.
5. method according to claim 1, is characterized in that, described Steroid medicine intermediates comprises: AD, ADD or 9-hydroxyl-AD.
6. method according to claim 1, is characterized in that, described resting cell obtains by adopting two-stage seed culture method to cultivate described microbial strains.
7. method according to claim 6, is characterized in that, described microbial strains, after two-stage seed culture method is cultivated, also comprises proceeding in growth medium and continues to cultivate.
8. method according to claim 7, is characterized in that, the formula of described growth medium is as follows: glucose 20g/L, citric acid 2g/L, (NH
4)
2hPO
43.5g/L, ferric ammonium citrate 0.05g/L, K
2hPO
43H
2o0.5g/L, MgSO
47H
2o0.5g/L, Zulkovsky starch 2g/L, corn steep liquor 1g/L.
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| CN111662858A (en) * | 2020-07-17 | 2020-09-15 | 湖北工业大学 | Application of mutant of HGMS2 strain in preparation of 4-androstene-3, 17-dione (4-AD) |
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| CN101020919A (en) * | 2007-03-19 | 2007-08-22 | 天津科技大学 | Biological sterol transforming process with cyclodextrin and its application |
| CN101760494A (en) * | 2008-11-06 | 2010-06-30 | 天津金耀集团有限公司 | Biofermentation method for androstendione by using resting cell |
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| CN101760494A (en) * | 2008-11-06 | 2010-06-30 | 天津金耀集团有限公司 | Biofermentation method for androstendione by using resting cell |
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