CN102586358B - Biosynthesis method for improving yield of epothilone B - Google Patents
Biosynthesis method for improving yield of epothilone B Download PDFInfo
- Publication number
- CN102586358B CN102586358B CN 201210006937 CN201210006937A CN102586358B CN 102586358 B CN102586358 B CN 102586358B CN 201210006937 CN201210006937 CN 201210006937 CN 201210006937 A CN201210006937 A CN 201210006937A CN 102586358 B CN102586358 B CN 102586358B
- Authority
- CN
- China
- Prior art keywords
- epothilone
- bottled
- shake
- liquid
- culture
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- QXRSDHAAWVKZLJ-PVYNADRNSA-N epothilone B Chemical compound C/C([C@@H]1C[C@@H]2O[C@]2(C)CCC[C@@H]([C@@H]([C@@H](C)C(=O)C(C)(C)[C@@H](O)CC(=O)O1)O)C)=C\C1=CSC(C)=N1 QXRSDHAAWVKZLJ-PVYNADRNSA-N 0.000 title claims abstract description 52
- QXRSDHAAWVKZLJ-OXZHEXMSSA-N Epothilone B Natural products O=C1[C@H](C)[C@H](O)[C@@H](C)CCC[C@@]2(C)O[C@H]2C[C@@H](/C(=C\c2nc(C)sc2)/C)OC(=O)C[C@H](O)C1(C)C QXRSDHAAWVKZLJ-OXZHEXMSSA-N 0.000 title claims abstract description 49
- HESCAJZNRMSMJG-HGYUPSKWSA-N epothilone A Natural products O=C1[C@H](C)[C@H](O)[C@H](C)CCC[C@H]2O[C@H]2C[C@@H](/C(=C\c2nc(C)sc2)/C)OC(=O)C[C@H](O)C1(C)C HESCAJZNRMSMJG-HGYUPSKWSA-N 0.000 title claims abstract description 49
- 238000000034 method Methods 0.000 title claims abstract description 31
- 230000015572 biosynthetic process Effects 0.000 title abstract description 4
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims abstract description 30
- 239000007788 liquid Substances 0.000 claims abstract description 30
- 238000000855 fermentation Methods 0.000 claims abstract description 17
- 230000004151 fermentation Effects 0.000 claims abstract description 17
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims abstract description 15
- 239000011347 resin Substances 0.000 claims abstract description 13
- 229920005989 resin Polymers 0.000 claims abstract description 13
- 239000002243 precursor Substances 0.000 claims abstract description 11
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 claims abstract description 10
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 claims abstract description 10
- 238000001291 vacuum drying Methods 0.000 claims abstract description 8
- 238000000605 extraction Methods 0.000 claims abstract description 5
- 230000001954 sterilising effect Effects 0.000 claims description 21
- 239000002609 medium Substances 0.000 claims description 19
- 241000894006 Bacteria Species 0.000 claims description 16
- 238000004519 manufacturing process Methods 0.000 claims description 16
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 11
- 230000001580 bacterial effect Effects 0.000 claims description 11
- 239000000203 mixture Substances 0.000 claims description 11
- 239000013543 active substance Substances 0.000 claims description 10
- 230000001851 biosynthetic effect Effects 0.000 claims description 10
- 239000007791 liquid phase Substances 0.000 claims description 9
- 239000000843 powder Substances 0.000 claims description 9
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 8
- 229920002472 Starch Polymers 0.000 claims description 8
- 238000002425 crystallisation Methods 0.000 claims description 8
- 230000008025 crystallization Effects 0.000 claims description 8
- 239000008103 glucose Substances 0.000 claims description 8
- 235000019698 starch Nutrition 0.000 claims description 8
- 239000008107 starch Substances 0.000 claims description 8
- 238000012546 transfer Methods 0.000 claims description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 7
- 239000012074 organic phase Substances 0.000 claims description 6
- 239000007787 solid Substances 0.000 claims description 6
- 238000012549 training Methods 0.000 claims description 5
- 229930191978 Gibberellin Natural products 0.000 claims description 4
- 229940041514 candida albicans extract Drugs 0.000 claims description 4
- 239000003448 gibberellin Substances 0.000 claims description 4
- IXORZMNAPKEEDV-OBDJNFEBSA-N gibberellin A3 Chemical class C([C@@]1(O)C(=C)C[C@@]2(C1)[C@H]1C(O)=O)C[C@H]2[C@]2(C=C[C@@H]3O)[C@H]1[C@]3(C)C(=O)O2 IXORZMNAPKEEDV-OBDJNFEBSA-N 0.000 claims description 4
- 239000001963 growth medium Substances 0.000 claims description 4
- 239000003375 plant hormone Substances 0.000 claims description 4
- 238000011218 seed culture Methods 0.000 claims description 4
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 claims description 4
- 239000012138 yeast extract Substances 0.000 claims description 4
- 229920001817 Agar Polymers 0.000 claims description 3
- 239000001888 Peptone Substances 0.000 claims description 3
- 108010080698 Peptones Proteins 0.000 claims description 3
- DPDMMXDBJGCCQC-UHFFFAOYSA-N [Na].[Cl] Chemical compound [Na].[Cl] DPDMMXDBJGCCQC-UHFFFAOYSA-N 0.000 claims description 3
- 239000008272 agar Substances 0.000 claims description 3
- 238000001035 drying Methods 0.000 claims description 3
- 239000012535 impurity Substances 0.000 claims description 3
- 238000002955 isolation Methods 0.000 claims description 3
- 230000002503 metabolic effect Effects 0.000 claims description 3
- 239000003960 organic solvent Substances 0.000 claims description 3
- 238000012856 packing Methods 0.000 claims description 3
- 235000019319 peptone Nutrition 0.000 claims description 3
- 238000005406 washing Methods 0.000 claims description 3
- 238000005119 centrifugation Methods 0.000 claims description 2
- 238000001816 cooling Methods 0.000 claims description 2
- 150000003839 salts Chemical class 0.000 claims description 2
- 238000004659 sterilization and disinfection Methods 0.000 claims 4
- 239000000411 inducer Substances 0.000 claims 2
- 240000008005 Crotalaria incana Species 0.000 claims 1
- 241000863434 Myxococcales Species 0.000 claims 1
- 235000019764 Soybean Meal Nutrition 0.000 claims 1
- 238000013019 agitation Methods 0.000 claims 1
- 230000035622 drinking Effects 0.000 claims 1
- 229910000403 monosodium phosphate Inorganic materials 0.000 claims 1
- 235000019799 monosodium phosphate Nutrition 0.000 claims 1
- 239000004455 soybean meal Substances 0.000 claims 1
- 238000012258 culturing Methods 0.000 abstract description 3
- 238000000926 separation method Methods 0.000 abstract description 2
- 238000003306 harvesting Methods 0.000 abstract 1
- 238000009776 industrial production Methods 0.000 abstract 1
- 238000002360 preparation method Methods 0.000 abstract 1
- 238000009738 saturating Methods 0.000 abstract 1
- 239000000047 product Substances 0.000 description 12
- 239000000243 solution Substances 0.000 description 12
- 239000000523 sample Substances 0.000 description 10
- 238000012807 shake-flask culturing Methods 0.000 description 8
- 239000013558 reference substance Substances 0.000 description 6
- 239000000126 substance Substances 0.000 description 6
- 244000068988 Glycine max Species 0.000 description 4
- 235000010469 Glycine max Nutrition 0.000 description 4
- 241001052560 Thallis Species 0.000 description 4
- 238000010521 absorption reaction Methods 0.000 description 4
- 239000000306 component Substances 0.000 description 4
- 235000011187 glycerol Nutrition 0.000 description 4
- 230000014759 maintenance of location Effects 0.000 description 4
- 238000003756 stirring Methods 0.000 description 4
- 238000001514 detection method Methods 0.000 description 3
- 238000004128 high performance liquid chromatography Methods 0.000 description 3
- 239000002054 inoculum Substances 0.000 description 3
- 230000004060 metabolic process Effects 0.000 description 3
- 229940045641 monobasic sodium phosphate Drugs 0.000 description 3
- 235000015097 nutrients Nutrition 0.000 description 3
- 239000003463 adsorbent Substances 0.000 description 2
- 239000000428 dust Substances 0.000 description 2
- 239000003120 macrolide antibiotic agent Substances 0.000 description 2
- 239000012533 medium component Substances 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- 238000004321 preservation Methods 0.000 description 2
- 239000012488 sample solution Substances 0.000 description 2
- 238000003892 spreading Methods 0.000 description 2
- 239000008399 tap water Substances 0.000 description 2
- 235000020679 tap water Nutrition 0.000 description 2
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- FBHLXXMDOGRQOQ-FCIKGTDHSA-N Epothilone B10 Natural products O=C1[C@H](C)[C@H](O)[C@H](C)CCC[C@@]2(C)O[C@H]2C[C@@H](/C(=C\c2nc(CC)sc2)/C)OC(=O)C[C@H](O)C1(C)C FBHLXXMDOGRQOQ-FCIKGTDHSA-N 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 206010061217 Infestation Diseases 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 238000000862 absorption spectrum Methods 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000008485 antagonism Effects 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- 230000000975 bioactive effect Effects 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 239000002775 capsule Substances 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 239000002131 composite material Substances 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000013016 damping Methods 0.000 description 1
- 230000007850 degeneration Effects 0.000 description 1
- 238000003795 desorption Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 229930013356 epothilone Natural products 0.000 description 1
- 150000003883 epothilone derivatives Chemical class 0.000 description 1
- 238000010812 external standard method Methods 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- 239000012467 final product Substances 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 235000001727 glucose Nutrition 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 229910017053 inorganic salt Inorganic materials 0.000 description 1
- 238000011031 large-scale manufacturing process Methods 0.000 description 1
- 238000004811 liquid chromatography Methods 0.000 description 1
- 229940041033 macrolides Drugs 0.000 description 1
- 239000002075 main ingredient Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 239000013580 millipore water Substances 0.000 description 1
- QJGQUHMNIGDVPM-UHFFFAOYSA-N nitrogen group Chemical group [N] QJGQUHMNIGDVPM-UHFFFAOYSA-N 0.000 description 1
- 230000000050 nutritive effect Effects 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 239000010453 quartz Substances 0.000 description 1
- 239000000700 radioactive tracer Substances 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- 230000024053 secondary metabolic process Effects 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N silicon dioxide Inorganic materials O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 235000013619 trace mineral Nutrition 0.000 description 1
- 239000011573 trace mineral Substances 0.000 description 1
- 238000000870 ultraviolet spectroscopy Methods 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention relates to a biosynthesis method for improving yield of epothilone B. The biosynthesis method comprises the following steps of: selecting robust strains for producing the epothilone B by using a centrifugal process, inoculating the strains into a sterilized seed medium, performing shaking culture at 30DEG C on a shaker, and fermenting; adding a precursor inductor phenylalanine into a fermentation medium; harvesting a culture solution after culturing at 30DEG C for 10 to 11 days; and collecting, adsorbing and saturating by using macroporous resin, eluting by using ethyl acetate, separating and purifying, performing vacuum concentration, extracting, crystalizing at low temperature, and performing vacuum drying. The purity of the epothilone B can be effectively improved by a liquid preparation column, and is over 99.5 percent; and methanol is adopted for extraction and separation, so that the epothilone B is crystallized in the methanol, and the finished product reaches the pharmaceutical grade. By the method, the yield of the epothilone B is greatly improved and is 30-45 percent, and the method has a good industrial production prospect.
Description
Technical field
The present invention relates to a kind of biosynthetic means of ebormycine, specifically a kind of suitability for industrialized production improves the biosynthetic means of epothilone B productive rate, belongs to antibiotic microorganism fermentation culture field.
Background technology
Epothilone B belongs to the macrolide antibiotic newcomer, and ebormycine is that a class has unique texture and unique bioactive microbiotic.Its molecular structure is as follows:
Ebomycin A
Epothilone B
Wherein epothilone B is the macrolides compound produced by slime bacteria (fiber is to the capsule bacterium), epothilone B not only has antitumor action, tetter for fungi infestation also has good antagonism, therapeutic action, epothilone B toxicity is low, side effect is little, have a good application prospect, but it is high at present by fermentation, to prepare the method production cost of epothilone B, in the active substance that cultivation produces, the epothilone B productive rate is low, fluctuation is large, the highest accounts for below 22%, and what have even only has tracer level.
Summary of the invention
The objective of the invention is in order to solve the problem of the high and low-yield of epothilone B production cost in prior art, and biosynthetic means a kind of high yield, that can adapt to suitability for industrialized production the ebormycine that can effectively reduce production costs is provided, adopt present method to spread cultivation, ferment the productive rate of epothilone B in the active result produced up to 30～45%, there is good suitability for industrialized production prospect.
In order to realize above-mentioned purpose, the technical solution used in the present invention is: a kind of biosynthetic means that improves the epothilone B productive rate is provided, comprises the steps:
(1), produce the bacterial classification that sets out, Soce90-G15: the slime bacteria of choosing robust growth by the centrifugal shaking flask bacterium of 3000rpm liquid is as the production bacterial classification that sets out;
(2), the solid inclined-plane spreads cultivation: the component of substratum is by mass%, glucose 0.5～1%, peptone 0.3～0.5%, starch 0.5～1%, sodium-chlor 0.1～0.5%, Plant hormones regulators,gibberellins 0.002% and agar powder 2%, surplus is water; Substratum is arranged to inclined-plane after sterilizing in 120 ℃, 30 minutes is cooling, after 24 hours, the production bacterial classification that sets out is coated on inclined-plane, at 30 ± 2 ℃, 55%, 7～9 days plentiful, flavous lawns of visible growth of relative humidity, refrigerates stand-by;
(3), shaking flask spreads cultivation: the shake-flask culture base is starch 5～8g/L, yeast extract 2～4g/L, glucose 0.5～1.5g/L, MgSO
41g/L, CaCl
21g/L, glycerine 0.5～1g/L, SODIUM PHOSPHATE, MONOBASIC 0.05g/L, soybean cake powder 2～4g/L, 1 liter of tap water, to adjust PH be 7.20, sterilizing in 120 ℃, 30 minutes; The lawn on solid inclined-plane is scraped to access shake-flask culture base gently, under 29 ℃ ± 1 ℃, rotating speed 150rpm, grow 5～6 days; Choosing the shaking flask strain liquid of thalli growth stalwartness transfers into first class seed pot;
(4), first order seed is cultivated: the composition of first order seed substratum is identical with the shake-flask culture base, also sterilizing in 120 ℃, 30 minutes; The shaking flask strain liquid of having grown is accessed in first class seed pot and spreads cultivation by pressure differential method, 29 ± 2 ℃ of culture temperature, rotating speed 120rpm, air flow quantity than 1 to 0.4, tank pressure 0.04kg/cm
2, cultivate 5～6 days, eugonic strain liquid is transferred into the secondary seed tank;
(5), the secondary seed tank is cultivated: the composition of secondary seed medium is identical with the shake-flask culture base, also sterilizing in 120 ℃, 30 minutes; The first class inoculum liquid of having grown is accessed in the secondary seed tank and spreads cultivation by pressure differential method, 29 ± 2 ℃ of culture temperature, rotating speed 120rpm, air flow quantity than 1 to 0.5, tank pressure 0.04kg/cm
2, cultivate 2～3 days, eugonic strain liquid is transferred into fermentor tank;
(6), fermentor cultivation: the composition of fermention medium is identical with the shake-flask culture base, but additional precursor inductor 0.2～0.5g/L, macroporous adsorbent resin 20g/L; PH is adjusted to sterilizing in 7.30,120 ℃, 30 minutes; The second class inoculum liquid of having grown is accessed in fermentor tank and spreads cultivation by pressure differential method, 29 ± 2 ℃ of the temperature of cultured product, rotating speed 150rpm, air flow quantity than 1 to 0.8, tank pressure 0.04kg/cm
2, cultivate 10～11 days;
(7), washing, wash-out: ferment to residual sugar lower than 0.2%, stop fermentation when the high point of metabolic active substance output, by fermented liquid resin isolation out eluriate in the resin bed of totally packing into; With 1 times of post bed amount of 50% methanol wash, flow velocity 25ml/min, decolouring, removal of impurities; Use 6 times of post bed amount ethyl acetate, flow velocity 15ml/min wash-out ebomycin A, epothilone B active substance, collect elutriant again;
(8), vacuum concentration: the elutriant vacuum concentration containing active substance of collecting, 40～45 ℃ of temperature, vacuum tightness-0.085～-0.095MPa, be concentrated into below 1/15 of initial body accumulated amount;
(9), the liquid phase preparative column separates: concentrated solution is passed through to liquid phase preparative column separated and collected epothilone B part parting liquid;
(10), extraction: contain epothilone B parting liquid vacuum concentration to what collect to remaining water, add organic solvent to carry out extracting and separating and collect organic phase;
(11), crystallization: the organic phase vacuum concentration of collecting, 40～45 ℃ of temperature, vacuum tightness-0.085～-0.09MPa, be concentrated into below 1/10 of initial body accumulated amount; Concentrated solution is put into to clean crystallizer-20 ℃ low temperature crystallization;
(12), vacuum-drying; Collect xln and put into clean vacuum drying oven, 45～50 ℃ of temperature, vacuum tightness-0.085～-0.09MPa; Drying obtains the active result epothilone B finished product of content more than 99.5%.
Of the present invention spreading cultivation adopts the shaking flask shaking culture or stirs the sinking training method, the first-selected sinking mode that stirs of suitability for industrialized production.
Precursor inductor additional in fermention medium of the present invention is phenylalanine, or its organic salt, and its method that adds substratum is for once dropping into or intermittent flow adds.
The present invention should add the carbon source that can be utilized as starch, glucose, glycerine etc. for the substratum of culturing micro-organisms, and nitrogenous source is as soybean cake powder, fishbone powder, yeast powder etc., inorganic salt and various trace elements and the precursor substance that can be utilized.These compositions can disposablely add in substratum or the successional high yield that fills into to realize epothilone B.The fermentation culture based component that the present invention is used and the composition of seed culture medium are basic identical, just will add precursor substance.The precursor substance that adds epothilone B in fermention medium is phenylalanine, and its add-on is 0.2～0.5g/L; Specifically, the component of fermention medium is starch 5～8g/L, yeast extract 2～4g/L, glucose 0.5～1.5g/L, MgSO
41g/L, CaCl
21g/L, glycerine 0.5～1g/L, SODIUM PHOSPHATE, MONOBASIC 0.05g/L, soybean cake powder 2～4g/L, phenylalanine 0.2～0.5g/L macroporous adsorbent resin 20g/L; PH is adjusted to sterilizing in 7.30,120 ℃, 30 minutes.
Used medium of the present invention all needs sterilising treatment, generally in 120 ℃ of sterilizings, within 30 minutes, gets final product.Described spreading cultivation can adopt the shaking flask shaking culture or stir the sinking training method, the first-selected sinking mode that stirs of suitability for industrialized production.Culture condition is made respective change with the metabolism situation, medium component is changed different.That metabolism biological activity in fermented liquid reaches is the highest, residual sugar 0.2% stops cultivating when following.Prepare separation and purification gained active result epothilone B through liquid phase, gained epothilone B content ratio can reach 30～50%.
In brief, a kind of biosynthetic means that improves the epothilone B productive rate of the present invention, be to produce the slime bacteria of epothilone B, is inoculated in the seed culture medium that the configuration sterilizing is good, is used further to fermentation after the shaking table shaking culture of 30 ℃; To adding the precursor inductor phenylalanine of epothilone B in the fermention medium of slime bacteria, make its growth metabolism fermentation culture during fermentation culture; Gather in the crops nutrient solution 30 ℃ of cultivations after 10～11 days, and reclaim epothilone B from substratum; Again through collecting the saturated macroporous resin of absorption; With ethyl acetate to solution separating purify, vacuum concentration, extraction, low temperature crystallization, vacuum-drying, obtain content and reach 30～50% epothilone Bs.
Method of the present invention compared with prior art has following advantage:
1, in method of the present invention, adopt centrifugation to choose the shaking flask bacterium liquid of robust growth, can effectively isolate nutritive medium, more be conducive to the preservation of bacterial classification, prevent variation in spawn degeneration and preservation.
2, add this class tethelin of Plant hormones regulators,gibberellins in method of the present invention in bacteria culture medium, the growth of raising and balanced slime bacteria, be conducive to shorten the production cycle, improves the output capacity of secondary metabolism.At substratum, add phenylalanine as the precursor inductor, improved the output capacity of purpose meta-bolites ebormycine.
3, in method of the present invention, the dynamic desorption method is used for to the suitability for industrialized production of epothilone B, makes this product large-scale production become possibility.By can effectively the purify purity of epothilone B of liquid phase preparative column, finished product purity 99.5% adopts methyl alcohol to carry out extracting and separating simultaneously, makes epothilone B crystallization in methyl alcohol, xln has good crystalline form and color base, and finished product can reach the pharmaceutical grade level.
Embodiment
Below in conjunction with embodiment, the inventive method is described in further detail.
Embodiment 1: a kind of biosynthetic means that improves the epothilone B productive rate of the present invention, and synthesis step comprises:
(1), produce the bacterial classification that sets out, choose the slime bacteria bacterial classification that sets out of robust growth for producing bacterial classification by the centrifugal shaking flask bacterium of 3000rpm liquid;
(2), the solid inclined-plane spreads cultivation: its slant medium component is by mass%, glucose 0.5%, peptone 0.5%, starch 1%, sodium-chlor 0.5%, Plant hormones regulators,gibberellins 0.002% and agar powder 2%, surplus is water; In 120 ℃ of sterilizings 30 minutes, the bacterial classification that after 24 hours, well-grown production set out was coated on the solid slant culture base, at 30 ℃ ± 2 ℃, relative humidity 55%, cultivated 8 days;
(3), well-grown bacterial classification is inoculated in through 120 ℃ of sterilizings shaking flask of 30 minutes and spreads cultivation in base, the spread cultivation component of base of shaking flask is starch 5g/L, yeast extract 2g/L, glucose 1g/L, MgSO
41g/L, CaCl
21g/L, glycerine 1g/L, SODIUM PHOSPHATE, MONOBASIC 0.05g/L, soybean cake powder 2g/L, 1 liter of tap water, PH is adjusted to 7.20,29 ℃ ± 1 ℃ rotating and culturing after 120 hours; Choosing the shaking flask strain liquid of thalli growth stalwartness transfers into first class seed pot;
(4), first order seed is cultivated: the composition of first order seed substratum is identical with the shake-flask culture base, also sterilizing in 120 ℃, 30 minutes; Inoculation after sterilizing, 29 ± 2 ℃ of culture temperature, rotating speed 120rpm, air flow quantity than 1 to 0.4, tank pressure 0.04kg/cm
2, cultivate 5 days, when thalli growth is vigorous, transfer into the secondary seed tank;
(5), the secondary seed tank is cultivated: the composition of secondary seed medium is identical with the shake-flask culture base, also sterilizing in 120 ℃, 30 minutes; The first class inoculum liquid of having grown by pressure differential method access in the secondary seed tank 29 ± 2 ℃ of the culture temperature that spread cultivation, rotating speed 120rpm, air flow quantity than 1 to 0.4, tank pressure 0.04kg/cm
2, cultivate 3 days, when thalli growth is vigorous, transfer into fermentor tank;
(6), fermentor cultivation: the composition of fermention medium is identical with the shake-flask culture base, just separately adds phenylalanine 0.2g/L, macroporous resin 20g/L, adjusts PH7.30, equally in 120 ℃ of sterilizings, sterilizing 30 minutes; By 29 ℃ ± 2 ℃, rotating speed 120rpm, air flow quantity in cultured secondary seed nutrient solution access fermention medium than 1 to 0.8, tank pressure 0.04kg/cm
2, cultivate after 10 days and gather in the crops nutrient solution; Adopt macroporous resin to collect absorption composite reactive product;
(7), washing, wash-out: ferment to residual sugar lower than 0.2%, metabolic active substance output vertex stops fermentation, by fermented liquid resin isolation out eluriate in the resin bed of totally packing into; With 1 times of post bed amount of 50% methanol wash, flow velocity 25ml/min, decolouring, removal of impurities; Use 6 times of post bed amount ethyl acetate, flow velocity 15ml/min wash-out ebomycin A, epothilone B active substance, collect elutriant again;
(8), vacuum concentration: the elutriant vacuum concentration containing active substance of collecting, temperature 45 C, vacuum tightness-0.095MPa, be concentrated into below 1/15 of initial body accumulated amount;
(9), the liquid phase preparative column separates: concentrated solution is passed through to liquid phase preparative column separated and collected epothilone B part parting liquid;
(10), extraction: add the epothilone B parting liquid of collecting organic solvent to carry out extracting and separating and collect organic phase;
(11), crystallization: the organic phase vacuum concentration temperature 45 C, the vacuum tightness-0.09MPa that collect, be concentrated into below 1/10 of initial body accumulated amount; Concentrated solution is put into to clean crystallizer-20 ℃ low temperature crystallization;
(12), vacuum-drying; Collect xln and put into clean vacuum drying oven, temperature 50 C, vacuum tightness-0.085MPa; Drying obtains the active result epothilone B finished product of high-content.
Adopt the inventive method, the gained main ingredient is epothilone B, through productive rate shared ratio in activated complex of Liquid Detection epothilone B, can reach 45%.
Through HPLC, check its product retention time identical with the epothilone B standard substance, ultraviolet maximum absorption degree is identical with the epothilone B standard substance.Liquid phase is consistent with epothilone B standard substance retention time.
Attached detection method:
1. key instrument and material
1.11 chromatographic instrument: Shimadzu 10A-Tvp HPLC.
1.12 chromatographic column: Shim-pack MRC-ODS (250mm * 4.6mm, 4.60 μ m) analytical column or Kromasil C18 (250mm * 4.6mm) Dalian Yi Lite scientific instrument company limited.
1.13EpoB reference substance: Sigma E2656, (-)-Epothilone B 10 μ g.
1.2 chromatographic condition
With 65% methyl alcohol (HPLC level, Merck Co.) and 35% damping fluid (0.2%A.P.acetate acid/18MR Millipore Water), be moving phase, flow velocity is 1.0ml/min, and sample size is 10 μ l.The detection wavelength is 249nm, 28 ℃ of column temperatures.The peak sequence of main chromatographic peak is followed successively by ebomycin A (relative retention time is 0.8) and epothilone B (relative retention time is 1.0).
1.3 working method
Get the inventive method products therefrom and make in right amount sample, accurately weighed, add dissolve with methanol and quantitatively dilute and make the solution that approximately contains 0.5mg in every 1ml, as test sample solution.Separately get epothilone B reference substance 10 μ g, add methyl alcohol 20 μ l constant volumes, obtain reference substance solution (0.5mg/ml).
Get respectively each 10 μ l of sample solution of the present invention and reference substance solution, the injection liquid chromatography, record color atlas.By external standard method, with calculated by peak area, obtain.
1.4 the expression of calculating and result
In formula: A
sample: the peak area of sample of the present invention; C
sample: the concentration of sample of the present invention, mg/ml;
A
right: the peak area of reference substance; C
right: the concentration of reference substance, mg/ml.
1.5 criterion
Sample of the present invention is pressed dry product and is calculated, containing C
27h
41nO
6s must not be less than 96.0%.
Ultra-violet absorption spectrum
2.1 instrument apparatus
The UV-7502PCS ultraviolet-visible divides spectrophotometer, AL104 ten thousand/electronic balance, volumetric flask (10ml, 100ml), weighing bottle, transfer pipet (1ml), quartz colorimetric utensil.
2.2 working method
Get sample 10mg of the present invention, be placed in the 100ml volumetric flask, be dissolved in water and be diluted to scale, get in 1ml to 10ml volumetric flask, be diluted with water to scale, obtain sample liquid of the present invention.Measure according to " ultraviolet visible spectrophotometry ", in 200nm～300nm wavelength region interscan, record collection of illustrative plates.
2.3 criterion
There is maximum absorption at wavelength place at 211nm and 249nm.
Invention sample: add phenylalanine to reach 6 grams with the slime bacteria fermentation 200L fermentation output product that produces the rich B of dust.
Comparative Examples: under the same conditions, do not add phenylalanine.With the slime bacteria fermentation of producing the rich B of dust.200L fermentation output product is only 3.3 gram left and right.Productive rate is low 45%.
Claims (3)
1. a biosynthetic means that improves the epothilone B productive rate, is characterized in that: comprise the steps:
⑴, production starting strain, Soce90-G15: Shake bottled select robust growth of bacteria by centrifugation at 3000rpm myxobacteria starting as a production strain;
, the solid inclined-plane spreads cultivation: the component of substratum is by mass%, glucose 0.5～1%, peptone 0.3～0.5%, starch 0.5～1%, sodium-chlor 0.1～0.5%, Plant hormones regulators,gibberellins 0.002% and agar powder 2%, surplus is water; Substratum is arranged to inclined-plane after sterilizing in 120 ℃, 30 minutes is cooling, after 24 hours, the production bacterial classification that sets out is coated on inclined-plane, at 30 ± 2 ℃, 55%, 7～9 days plentiful, flavous lawns of visible growth of relative humidity, refrigerates stand-by;
⑶, bottled spread shake culture: medium was shake bottled starch 5 ~ 8g / L, yeast extract 2 ~ 4g / L, glucose 0.5 ~ 1.5g / L, MgSO <sub TranNum="166"> 4 </ sub? > 1g / L, CaCl <sub TranNum="167"> 2 </ sub>? 1g / L, glycerol 0.5 ~ 1g / L, sodium dihydrogen phosphate 0.05g / L, soybean meal 2 ~ 4g / L, drinking 1 liter, adjust PH to 7.20 at 120 ℃, 30 minute sterilization; put solid beveled lawn shake gently scraped access bottled medium at 29 ℃ ± 1 ℃, speed 150rpm, the growth of 5 to 6 days ; selected bacterial strains grown strong shaking bottled liquid transfer access to a seed tank;
⑷, primary seed culture: composition and shake bottled same level of seed culture medium, but also 120 ℃, 30 minute sterilization; the growth of good bacteria shake bottled liquid tank through an access level seed differential pressure method the spread cultivation, culture temperature 29 ± 2 ℃, speed 120rpm, air flow than a ratio of 0.4, the tank pressure 0.04 kg / cm <sup TranNum="169"> 2 </ sup>, cultured for 5 to 6 days, the vigorous growth strain liquid transfer access two seed tank;
⑸, second seed pot culture: the composition of the secondary seed medium and shake bottled same medium, but also 120 ℃, 30 minute sterilization; the growth of good bacteria liquid level access secondary seed by differential pressure method Pei expansion tank, temperature 29 ± 2 ℃, speed 120rpm, air flow than a ratio of 0.5, the tank pressure 0.04 kg / cm <sup TranNum="171"> 2 </ sup>, cultured for 2 to 3 days, the growth strain liquid transfer access exuberant fermenter;
⑹, fermenter culture: the composition of the fermentation medium and shake bottled same medium, but additional precursor inducer 0.2 ~ 0.5g / L, macroporous resin 20g / L, a plus in the fermentation medium precursor inducer phenylalanine, or its organic salts; PH adjusted to 7.30,120 ℃, 30 minute sterilization; put a good two strains grown by liquid fermentation tank pressure method to expand access to training, training product temperature 29 ± 2 ℃, speed 150rpm, air flow than a ratio of 0.8, the tank pressure 0.04 kg / cm <sup TranNum="173"> 2 </ sup>, cultured for 10 to 11 days;
, washing, wash-out: ferment to residual sugar lower than 0.2%, stop fermentation when the high point of metabolic active substance output, by fermented liquid resin isolation out eluriate in the resin bed of totally packing into; With 1 times of post bed amount of 50% methanol wash, flow velocity 25ml/min, decolouring, removal of impurities; Use 6 times of post bed amount ethyl acetate, flow velocity 15ml/min wash-out ebomycin A, epothilone B active substance, collect elutriant again;
(8), vacuum concentration: the elutriant vacuum concentration containing active substance of collecting, 40～45 ℃ of temperature, vacuum tightness ﹣ 0.085～﹣ 0.095 MPa, be concentrated into below 1/15 of initial body accumulated amount;
(9), the liquid phase preparative column separates: concentrated solution is passed through to liquid phase preparative column separated and collected epothilone B part parting liquid;
(10), extraction: contain epothilone B parting liquid vacuum concentration to what collect to remaining water, add organic solvent to carry out extracting and separating and collect organic phase;
(11), crystallization: the organic phase vacuum concentration of collecting, 40～45 ℃ of temperature, vacuum tightness ﹣ 0.085～﹣ 0.09MPa, be concentrated into below 1/10 of initial body accumulated amount; Concentrated solution is put into to 20 ℃ of low temperature crystallizations of clean crystallizer ﹣;
(12), vacuum-drying; Collect xln and put into clean vacuum drying oven, 45～50 ℃ of temperature, vacuum tightness ﹣ 0.085～﹣ 0.09MPa; Drying obtains the active result epothilone B finished product of content more than 99.5%.
2 according to claim 1, wherein a biosynthetic method for improving the yield of epothilone B, wherein: said step of ⑶, ⑷, ⑸ ⑹ and expanded in culture using shake shake bottled bottled shaking or agitation sank training methods.
3. a kind of biosynthetic means that improves the epothilone B productive rate according to claim 1 is characterized in that: the described method that additional precursor inductor adds substratum in fermention medium is for once dropping into or intermittent flow adds.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201210006937 CN102586358B (en) | 2012-01-11 | 2012-01-11 | Biosynthesis method for improving yield of epothilone B |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201210006937 CN102586358B (en) | 2012-01-11 | 2012-01-11 | Biosynthesis method for improving yield of epothilone B |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102586358A CN102586358A (en) | 2012-07-18 |
| CN102586358B true CN102586358B (en) | 2013-06-12 |
Family
ID=46475582
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 201210006937 Active CN102586358B (en) | 2012-01-11 | 2012-01-11 | Biosynthesis method for improving yield of epothilone B |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102586358B (en) |
Families Citing this family (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103014092B (en) * | 2012-12-15 | 2014-10-01 | 湖北宏中药业有限公司 | Preparation method for improving productivity of mitomycin C |
| CN103243134B (en) * | 2013-04-15 | 2015-04-22 | 陕西科技大学 | Fermentation production method based on epothilone B metabolic pathways |
| CN104593444B (en) * | 2013-10-31 | 2019-07-09 | 重庆乾泰生物医药有限公司 | A kind of method that fermentation prepares epothilone B |
| CN103667387B (en) * | 2013-11-27 | 2015-09-30 | 陕西科技大学 | A kind of method of separation and Extraction epothilone B from sorangium cellulosum fermented liquid |
| CN104694601A (en) * | 2013-12-30 | 2015-06-10 | 中国科学院成都生物研究所 | High-efficiency preparation method of Iturin A and homologue of Iturin A |
| CN103772407B (en) * | 2014-01-23 | 2016-05-18 | 陕西科技大学 | A kind of epothilone B separating and extracting process based on membrane filtration technique |
| CN110964029B (en) * | 2019-12-19 | 2020-11-03 | 鲁南制药集团股份有限公司 | Pretreatment method of epothilone B fermentation liquor |
| CN112630369A (en) * | 2020-12-18 | 2021-04-09 | 卓和药业集团有限公司 | High performance liquid chromatography detection and analysis method for content of epothilone B |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1769468A (en) * | 2005-10-19 | 2006-05-10 | 华南理工大学 | Method for highly-effective producing epothilone using myxobacteria sorangium cellulosum |
-
2012
- 2012-01-11 CN CN 201210006937 patent/CN102586358B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1769468A (en) * | 2005-10-19 | 2006-05-10 | 华南理工大学 | Method for highly-effective producing epothilone using myxobacteria sorangium cellulosum |
Non-Patent Citations (2)
| Title |
|---|
| 纤维堆囊菌发酵产生埃博霉素条件的优化;罗立新等;《华南理工大学学报(自然科学版)》;20060531;第34卷(第5期);48-51 * |
| 罗立新等.纤维堆囊菌发酵产生埃博霉素条件的优化.《华南理工大学学报(自然科学版)》.2006,第34卷(第5期),48-51. |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102586358A (en) | 2012-07-18 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102586358B (en) | Biosynthesis method for improving yield of epothilone B | |
| CN105524840B (en) | One plant of new rattan storehouse sickle-like bacteria and its fermentation production of gibberellin A4Method | |
| CN106222098B (en) | One plant of monascus strain and its application | |
| CN104342390B (en) | A kind of Sinorhizobium meliloti strain and combinations thereof and application | |
| CN103898004B (en) | The method of Selective medium and fermentative production U-32070E thereof | |
| CN102433364B (en) | Process for preparing rapamycin by using microbial fermentation method | |
| CN101555503A (en) | Method for separating and extracting L-arabinose from waste wood sugar mother liquid from wood sugar production | |
| CN102399837A (en) | Method for synthesizing acarbose through microbial fermentation | |
| CN103882080A (en) | Effective method for preparing avermectin | |
| CN103740606A (en) | Streptomyces phytohabitans, method for producing new antibiotics Novonestmycin from Streptomyces phytohabitans, and application of Novonestmycin | |
| CN110791462B (en) | Bacillus subtilis and application thereof in fermentation production of adenosine | |
| CN102965288A (en) | Strain for biosynthesis of 3beta, 7alpha, 15alpha-trihydroxyandrost-5-ene-17-one and application thereof | |
| CN108823110B (en) | Strain for producing griseofulvin and application thereof | |
| CN104774888A (en) | Cordycepin fermentation solid medium and preparation method and application thereof | |
| CN108841889B (en) | Method for producing griseofulvin serving as major component of tranexamycin by microbial fermentation | |
| CN101720772A (en) | Macrolide composition for preventing and controlling fungal disease of crop and preparation process thereof | |
| CN103146595B (en) | Bacillus subtilis and method for fermentation production of D- ribose | |
| CN103014092B (en) | Preparation method for improving productivity of mitomycin C | |
| CN101319199B (en) | Method for cell abduction generation of taxone with Chinese yew | |
| CN103146594B (en) | Sorangiumcellulosum strain and application thereof to synthesis of epothilone | |
| CN114875093A (en) | Method for improving fermentation yield of PF1022A | |
| CN108794502B (en) | Trichothecene compound and preparation method and application thereof | |
| CN103173398B (en) | Short bacillus and method for preparing trehalose by virtue of fermentation | |
| CN103276028A (en) | Method for preparing epothilone B by fermentation method | |
| CN102757443A (en) | Sulfur-substituted podophyllum derivative and bioconversion, separation and purification method thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CP01 | Change in the name or title of a patent holder |
Address after: 435300 Li Shizhen Pharmaceutical Industrial Park, Qichun, Huanggang, Hubei Patentee after: HUBEI HONCH PHARMACEUTICAL Co.,Ltd. Address before: 435300 Li Shizhen Pharmaceutical Industrial Park, Qichun, Huanggang, Hubei Patentee before: HUBEI HONCH PHARMACEUTICAL Co.,Ltd. |
|
| CP01 | Change in the name or title of a patent holder | ||
| PE01 | Entry into force of the registration of the contract for pledge of patent right | ||
| PE01 | Entry into force of the registration of the contract for pledge of patent right |
Denomination of invention: A Biosynthetic Method to Improve the Yield of Epsomycin B Effective date of registration: 20230105 Granted publication date: 20130612 Pledgee: Bank of China Limited Huanggang branch Pledgor: HUBEI HONCH PHARMACEUTICAL Co.,Ltd. Registration number: Y2023420000006 |
|
| PC01 | Cancellation of the registration of the contract for pledge of patent right | ||
| PC01 | Cancellation of the registration of the contract for pledge of patent right |
Date of cancellation: 20230823 Granted publication date: 20130612 Pledgee: Bank of China Limited Huanggang branch Pledgor: HUBEI HONCH PHARMACEUTICAL Co.,Ltd. Registration number: Y2023420000006 |
|
| PE01 | Entry into force of the registration of the contract for pledge of patent right | ||
| PE01 | Entry into force of the registration of the contract for pledge of patent right |
Denomination of invention: A Biosynthetic Method for Improving the Yield of Ebomycin B Effective date of registration: 20230825 Granted publication date: 20130612 Pledgee: Bank of China Limited Huanggang branch Pledgor: HUBEI HONCH PHARMACEUTICAL Co.,Ltd. Registration number: Y2023980053856 |