CN1511943A - Engineering strain and its preparing method, and method for preparing taxol thereof - Google Patents
Engineering strain and its preparing method, and method for preparing taxol thereof Download PDFInfo
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- CN1511943A CN1511943A CNA021588775A CN02158877A CN1511943A CN 1511943 A CN1511943 A CN 1511943A CN A021588775 A CNA021588775 A CN A021588775A CN 02158877 A CN02158877 A CN 02158877A CN 1511943 A CN1511943 A CN 1511943A
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Abstract
The present invention relates to a kind of microbial strain for raising yield of taxol. The engineering strain is named as Nodulisporium sylviforme and preserved in China typical culture preserving center with the preservation number of CCTCC No. M202049. The strain is endogenous fungus autonomously separated from Northeast-China taxad and is bred via composite conventional bioengineerying measures. The engineering strain is transferred onto solid PDA culture medium for activation and culture, activated fusant is transferred into liquid PDA culture medium, the seed culture medium is transferred into universal mechanical stirring tank for fermentation in modified S-7 culture liquid, and the fermented liquid is extracted and purified chemically to obtain taxol.
Description
Technical field: the present invention relates to a kind of microorganism strains.
Background technology: at present, existing to be used to produce its taxol output of taxol bacterial strain all lower, is difficult to carry out large-scale industrial production.
Summary of the invention: the purpose of this invention is to provide the high engineering strain of a kind of taxol output, and a kind of preparation method of engineering strain is provided, and the method for preparing taxol with engineering strain.Technical scheme: this engineering strain, the classification tree-shaped more piece spore of called after (Nodulisporium sylviforme) is preserved in Chinese typical culture collection center, and preserving number is CCTCC NO.M202049.
A kind of method for preparing aforementioned engineering strain, with original strain through ultraviolet mutagenesis, ethylmethane sulfonate mutation,
60Obtain high productive mutant behind cobalt mutagenesis, the nitrosoguanidine mutagenesis, high productive mutant is prepared into mycelium, the enzymolysis mycelium prepares protoplastis, and wherein enzyme liquid obtains the protoplastis suspension with the prozyme system of 3% lywallzyme, 2% helicase, 1% N,O-Diacetylmuramidase composition after protoplastis is purified; After the protoplastis suspension is purified, adopt two kinds of method mutagenesis, a kind of is ultraviolet mutagenesis, a kind of is that ultraviolet ray adds lithium chloride mutagenesis, the mutant strain suspension that ultraviolet mutagenesis is obtained carries out ultraviolet inactivation, and 30cm, 30W uv irradiating shone 85 seconds, ultraviolet ray is added the mutant strain suspension that lithium chloride mutagenesis obtains carry out hot deactivation, suspension places 54 degree thermostatic water-circulator bath devices insulations 5 minutes; The two kinds of deactivation protoplastis suspensions fusions, the screening of regeneration back that obtain are previously obtained engineering strain.
A kind of method for preparing taxol with engineering strain, it is characterized in that: engineering strain is transferred to activation culture on the solid PDA substratum, fusant after the activation is transferred in the liquid PDA substratum, changing seed culture fluid over to the general form mechanical agitator tank ferments in the s-7 nutrient solution of improvement, wherein Gai Liang S-7 substratum is meant and adds phenylalanine 1.0mg/L, tyrosine 1.5mg/L on the basis of S-7 substratum, linolic acid 1.5mg/L obtains taxol with fermented liquid behind the chemical extraction purifying.
Beneficial effect: can reach more than the 450 μ g/L through the content of taxol that the present invention extracts, content of taxol is compared with the content of taxol that existing bacterial strain obtains and is greatly improved, and engineering strain good stability of the present invention, after the engineering strain continuous passage 10 times, carry out 3 batches of fermentation tests, this bacterial strain taxol produces ability still can keep stable, and mean yield reaches more than the 450 μ g/L.Breeding method involved in the present invention is to adopt the serial compound selection-breeding method of physics and chemically composited mutagenesis and cell engineering, in order to the high-yielding engineering bacterial strain that selects taxol at a high speed.
Engineering strain HDF-68 involved in the present invention has been preserved in Chinese typical culture collection center on December 26th, 2002, the classification tree-shaped more piece spore of called after (Nodulisporium sylviforme), and preserving number is CCTCC NO.M202049.Cultivate and storage conditions: use the PDA substratum, 25-28 ℃ of cultivation 2-3 days 4 ℃ of preservations or make cold dry strain-20 ℃ of refrigerations, or made 4 ℃ of preservations of spore sandy soil pipe.
Embodiment: engineering strain of the present invention is prepared by following method.
1, the acquisition of original strain, original strain can be directly by foreign procurement, and the present invention utilizes taxus chinensis in northeast to separate and obtains original strain.The taxus chinensis in northeast sample picks up from the veteran of 48 age of trees in ground such as forest zone, Dongning County, Heilongjiang Province and forest zone, Muleng more than a century, presses bark, phloem and the sprig etc. of the trunk of different ecological zone, different azimuth, different sexes, different sites and different thicknesses and takes a sample respectively.The sample for preparing is planted respectively on solid medium plates such as Ma Dingshi, Cha Shi, PDA through surface sterilization, 25 ℃ of constant temperature culture, and separation and purification obtains single bacterium colony.Adopt the S-7 liquid nutrient medium shaking culture of improvement after the single bacterium colony enlarged culturing that obtains, the S-7 substratum of improvement is meant and adds phenylalanine 1.0mg/L, tyrosine 1.5mg/L on the basis of S-7 substratum, composition among the linolic acid 1.5mg/L, 14 days post analysis tunnings.Through thin-layer chromatography (TLC) and the analysis of high performance liquid chromatography (HPLC) method, prove that wherein 3 strain endogenetic fungus (HQD33, HQD48 and HQD54) can be paclitaxel produced.After the optimization of fermentation conditions, the content of taxol reaches 51.06-125.70 μ g/L in the fermented liquid.Bacterial strain HQD33 grows on solid plate comparatively fast, and especially bacterium colony densification on the PDA substratum is fine and soft shape, full edge, and the initial stage bacteria colony white, the later stage is dark-grey brown, back side black.Give birth in the mycelia part, the part hypergene has every, multiple-limb, and the mycelia Vandyke brown shoals brown near colourless gradually to the top, and diameter is 2.58～6.45 μ m.The end is seen sclerotium and is produced.Conidiophore Dan Sheng, majority is arborization, and brown is thin out to the top, and wart is obviously arranged, long 35～157 μ m.The conidiogenous cell column, single giving birth to or the colyliform arrangement, 6.25～22.50 μ m * 2.65um～3.75 μ m, the sympodium formula is produced spore, and the top is expanded slightly or is not expanded, and has little tooth and dashes forward.Conidium Dan Sheng, ellipse or pseudovum shape, the level and smooth or little wart of tool, no barrier film, 5.16～7.74 μ m * 2.58～3.35 μ m, conidium is head and arranges in the product spore point position of conidiogenous cell.Bacterial strain HQD33 finds no the sexual stage after cultivating on the various substratum.Being accredited as tree-shaped more piece spore, is the new record Pseudomonas of China, is to find first that in the world this bacterial strain can be paclitaxel produced.Bacterial strain HQD33 selects for use and is original strain.
2, the complex mutation breeding of superior strain
Original strain passes through following mutagenesis successively, obtains high productive mutant, and substratum is the PDA substratum.
The ultraviolet mutagenesis spore concentration is 10
6Individual/ml, the intensity of ultraviolet lamp is 15W, and irradiation distance is 25cm, irradiation time 5min.
The ethylmethane sulfonate mutation ethylmethane sulfonate dissolves with phosphoric acid buffer, and pH is 7.0-7.4, and ethylmethane sulfonate treatment solution ultimate density reaches 0.1mol/L, and be 5min action time.
60 cobalt irradiation mutagenesis mutagenesis dosage are 800Gy, and mutation time is 24h.
The nitrosoguanidine mutagenesis nitrosoguanidine is prepared with the phosphoric acid buffer of pH6.0, and treatment solution concentration is 1mg/ml, and the treatment time is 20min.
Mutagenic treatment through as above orderly physics, chemical supramutagen (comprising nuclear power irradiation), and after primary dcreening operations such as growth potential, bacterium colony, mycelia, spore shape, eventually sieve again by sieve, HPLC and UV spectroscopic analysis for the TLC of extract in the fermented liquid, the high yield mutagenic fungi that screening at last obtains, its taxol output reaches 314.07 μ g/L, and the output of more former bacterial strain has improved 178.7%.
3, the preparation of protoplastis
A, substratum preparation
(1) malt extract medium; (2) Cha Shi substratum; (3) yeast extract medium; (4) PDA cultivates
Base; (5) CM culture medium prescription and compound method are referring to " microbiology experiment " (Shen Ping etc., 1999).
(6) regeneration solid medium: add corresponding homeo-osmosis agent in the substratum.
(7) regeneration semisolid medium: the agar concentration in the solid medium of will regenerating is reduced to 5-6g/L and is got final product.
The preparation of b, enzyme liquid
Helicase (Snailase); N,O-Diacetylmuramidase (Lysozyme); Lywallzyme (Lywallzyme).
The prozyme system that 3% lywallzyme+2% helicase+1% N,O-Diacetylmuramidase is formed, the amount that adds 1ml enzyme liquid with every 300mg wet thallus accurately takes by weighing required enzyme, and after homeo-osmosis agent dissolving, 3000r/min low-temperature centrifugation 15min uses after getting the supernatant liquid filtering degerming.
C, homeo-osmosis agent 0.7mol/L NaCl
D, preparation mycelium
It is the 50ml liquid nutrient medium that bacterial classification on the inclined-plane is transferred to loading amount, and volume is in the Erlenmeyer flask of 250ml, adopts static cultivation, temperature 23-25 ℃.By 2% inoculum size, being forwarded to loading amount is the 200ml liquid nutrient medium after 3 days, and volume is in the Erlenmeyer flask of 500ml, static cultivation 3 days.3000r/min, centrifugal 10min collects mycelium.The mycelium of collecting is broken up, and washed with the homeo-osmosis agent, weighing is standby.
E, enzymolysis mycelium prepare protoplastis
Use the mercaptoethanol (the phosphate buffer solution preparation of 0.1mol/L pH6.0) of 0.2%0.5% to handle mycelium 30min enzymolysis then before the enzymolysis respectively.The prozyme system that forms with 3% lywallzyme+2% helicase+1% N,O-Diacetylmuramidase carries out enzymolysis, 30 ℃ of constant temperature enzymolysis 7h.Be equipped with protoplastis with this legal system, the 300mg wet thallus can obtain 2.00 * 10
7-5.80 * 10
7Individual protoplastis.
The purifying of f, protoplastis
Enzymolysis solution is filtered with aseptic 3 layers of lens paper, and filtrate is centrifugal 10min under the 3000r/min condition, removes supernatant liquor, with homeo-osmosis agent washing 2 times, suspends in the homeo-osmosis agent then, obtains the protoplastis suspension of purifying.
G, protoplasts regenerated
With the liquid regeneration culture medium protoplastis suspension is carried out 10 times of serial dilutions, adopt the double-layer plate culture method: draw 0.5ml protoplastis serial dilutions mixing in the test tube that the 4.5ml height oozes semisolid medium is housed, be poured into corresponding solid height then and ooze on the regenerated plate, cultivated 3-5 days for 23-25 ℃.
In addition, get protoplastis behind the purifying and be suspended in the liquid regeneration culture medium that does not add the homeo-osmosis agent and handle 30min, coating is cultivated in the solid medium that does not add the homeo-osmosis agent then, in contrast, calculates the protoplasts regenerated frequency.
Our result of study shows, cultivates with the double-layer plate method with the PDA regeneration culture medium that contains 0.7mol/L NaCl, and the protoplast regeneration frequency can reach 72%.
4, protoplastis selection by mutation
Prepare the protoplastis suspension as stated above, carry out purifying after, adopt two kinds of method mutagenesis: (1) ultraviolet mutagenesis: ultraviolet lamp power is 30W, irradiation distance 25cm, irradiation time 50s; (2) ultraviolet ray adds lithium chloride mutagenesis: uv irradiating 4 0s add 0.6%LiCl in the substratum then.Protoplastis after the mutagenesis oozes regeneration culture medium with the PDA height and carries out the cultivation of double-layer plate lucifuge.Method (1) obtains high productive mutant UV40-19, and method (2) obtains high productive mutant UL50-6.Its taxol output is increased to more than the 380-390 μ g/L.Be significantly improved than the starting strain before the mutagenesis.
5, protoplast fusion breeding
The protoplastis deactivation
1 hot deactivation: place 54 ℃ of thermostatic water-circulator bath devices to be incubated 5min the UL50-6 protoplastis suspension behind the purifying.
2 ultraviolet inactivations: the UV40-19 protoplastis suspension behind the purifying with 30cm, 30W uv irradiating, is shone 85s.
The method that protoplastis merges
Getting concentration is 10
7Each 0.7ml of parents' deactivation protoplastis suspension of individual/ml mixes, and the centrifugal 10min of 3000r/min collects protoplastis, this protoplastis is suspended in the 0.7mol/L sodium-chlor of 0.2ml, 35% the PEG6000 that adds 30 ℃ of preheatings of 1.8ml, simultaneously, Ca in the fusion system
2+Maintain 0.01mol/L and 0.05mol/L respectively with the concentration of Gly, place 30 ℃ of thermostatic water-circulator bath insulations to merge to add 6ml 0.7mol/L NaCl solution behind the 20min to stop merging.After 0.7mol/L sodium-chlor washing, suspension is done suitable dilution, be poured into height and ooze in semi-solid and the solid double-layer culture medium flat plate and just putting cultivation, 28 ℃ of cultivations 120 hours.Fusion rate can reach 6.92%.
After the protoplastis deactivation regeneration with the protoplastis suspension after the deactivation be poured into height ooze semi-solid and the solid double-layer culture medium flat plate in carry out bilayer cultivation, 28 ℃ of cultivations 72-120 hour.
6, the screening of engineering strain
A large amount of pickings merge the single bacterium colony (hundreds of strain fusant) that grows on the regenerated plate, the bacterial strain that obtains behind the primary dcreening operation is through fermentation culture, its extract sieves again by thin-layer chromatography, efficient liquid phase chromatographic analysis on the bacterial strain extract that multiple sieve back obtains, the result shows, the taxol output that merges strain HDF-68 is significantly improved again than parents' strain, on average reaches 451.20 μ g/L.
Engineering strain prepares the method for taxol
Substratum PDA substratum, improvement S-7 substratum.
The activation culture of bacterial strain is transferred to engineering strain on the solid PDA substratum, cultivates 3 days for 28 ℃.Activate 2-3 time.
The fusant of seed culture after with activation culture is transferred in the liquid PDA substratum, 28 ℃ of shaking culture 3 days.
Fermentation is transferred seed culture fluid in the improvement S-7 substratum with 3% shaking flask inoculation kind amount, 28 ℃ shaking culture 16-18 days, the qualitative and semiquantitative determination content of taxol every 3 days.
Fermentor tank adopts the general form mechanical agitating fermentation tank, and this kind fermentor tank meets the biological characteristics of engineering strain, can effectively improve taxol output, and buys easily in market.
The extracting method filtering fermentation liquor of taxol, filter residue oven dry (being lower than 50 ℃), filtrate weighing volume.An amount of ethyl acetate extraction of filter residue, filtrate be with the ethyl acetate extraction (30min) of 1/2 filtrate volume, lower aqueous layer more once with ethyl acetate extraction, combined ethyl acetate, underpressure distillation.Solid with an amount of methanol wash underpressure distillation obtains washs 15min with normal hexane again, collects lower layer methanol, add ethyl acetate and water (v: v=1: 1), extract (30min), the ethyl acetate of collecting is carried out underpressure distillation, wash solid with a certain amount of acetonitrile, 0 ℃ of sealing is preserved.
The taxol quantivative approach is qualitative and semiquantitative determination content of taxol with thin-layer chromatography, with high performance liquid chromatography quantitative assay content of taxol.
Thin layer chromatography analysis (TLC): the extracting solution of each mutagenic fungi is put on the silica gel column chromatography plate of 10-40 μ m granularity, carried out chromatography.The use chloroform-methanol (7: 1, v/v) as developing agent.Immediately with the concentrated sulfuric acid solution spraying that contains 1% Vanillin, observe taxol characteristic Bluepoint color and computation migration rate behind the chromatography, the taxol standard substance made from Sigma company in contrast.
Efficient liquid phase chromatographic analysis: the Waters highly effective liquid phase chromatographic system, diode-array detector, the chromatogram strain is the C18 post, 200mm * 4.6mm, column temperature is a room temperature, moving phase be methanol-water mixtures (60: 40, v/v), wavelength 228nm, flow velocity 1.0ml/min, volume injected 10 μ l.
Fermented liquid is used the qualitative and semiquantitative determination content of taxol of thin-layer chromatography after thick the extraction, the result shows that the tree-shaped more piece spore of endogenetic fungus fusant can adopt the general form mechanical agitating fermentation tank paclitaxel produced next life.Thin-layer chromatography is the result show, fermentation 16d-18d taxol output reaches maximum value substantially.Fermented liquid is through extracting and separating, the high pressure liquid chromatography quantitative assay, and the taxol concentration stabilize is more than 400 μ g/L.
Claims (4)
1, a kind of engineering strain, the classification tree-shaped more piece spore of called after (Nodulisporium sylviforme) is preserved in Chinese typical culture collection center, and preserving number is CCTCC NO.M202049.
2, a kind of method for preparing claim 1 engineering strain, with original strain through ultraviolet mutagenesis, ethylmethane sulfonate mutation,
60Obtain high productive mutant behind cobalt mutagenesis, the nitrosoguanidine mutagenesis, high productive mutant is prepared into mycelium, the enzymolysis mycelium prepares protoplastis, and wherein enzyme liquid obtains the protoplastis suspension with the prozyme system of 3% lywallzyme, 2% helicase, 1% N,O-Diacetylmuramidase composition after protoplastis is purified; After the protoplastis suspension is purified, adopt two kinds of method mutagenesis, a kind of is ultraviolet mutagenesis, a kind of is that ultraviolet ray adds lithium chloride mutagenesis, the mutant strain suspension that ultraviolet mutagenesis is obtained carries out ultraviolet inactivation, and 30cm, 30W uv irradiating shone 85 seconds, ultraviolet ray is added the mutant strain suspension that lithium chloride mutagenesis obtains carry out hot deactivation, suspension places 54 ℃ of thermostatic water-circulator bath devices insulations 5 minutes; The two kinds of deactivation protoplastis suspensions fusions, the screening of regeneration back that obtain are previously obtained engineering strain.
3, a kind of method for preparing taxol with claim 1 engineering strain, it is characterized in that: engineering strain is transferred to activation culture on the solid PDA substratum, fusant after the activation is transferred in the liquid PDA substratum, changing seed culture fluid over to the general form mechanical agitator tank ferments in the s-7 nutrient solution of improvement, wherein Gai Liang S-7 substratum is meant and adds phenylalanine 1.0mg/L, tyrosine 1.5mg/L on the basis of S-7 substratum, linolic acid 1.5mg/L obtains taxol with fermented liquid behind the chemical extraction purifying.
4, a kind of approach of producing taxol with the strain of deriving of claim engineering strain and seed selection thereof.
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1304561C (en) * | 2004-11-13 | 2007-03-14 | 西南师范大学 | Process for raising producing rate of taxad alcohol of Taxus chinensis endogeny fungus fermenting material |
| CN101113414B (en) * | 2006-05-19 | 2010-04-21 | 建德市大洋化工有限公司 | Yew trichoderma strain and its application in preparation of trichoderma ester prime |
| CN101280281B (en) * | 2008-02-29 | 2010-07-14 | 黑龙江大学 | Paclitaxel genome rearrangement bacterial strain HDFS4-26 |
| CN101386874B (en) * | 2008-10-28 | 2011-05-11 | 南开大学 | Two-liquid phase fermentation method for improving paclitaxel yield from fungi |
| CN102796800A (en) * | 2012-07-19 | 2012-11-28 | 绍兴文理学院 | Method for screening paclitaxel strains and semi-quantitative analysis of paclitaxel strains |
| CN103756917A (en) * | 2014-01-14 | 2014-04-30 | 上海交通大学 | Preparation method of protoplasts of catalpa bungei endophytic fungi |
| CN107674890A (en) * | 2016-08-02 | 2018-02-09 | 武汉臻智生物科技有限公司 | The method for preparing taxol |
| CN108904548A (en) * | 2018-09-14 | 2018-11-30 | 江苏安惠生物科技有限公司 | The method of room temperature extraction edible and medical fungi effective component |
-
2002
- 2002-12-30 CN CNA021588775A patent/CN1511943A/en active Pending
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1304561C (en) * | 2004-11-13 | 2007-03-14 | 西南师范大学 | Process for raising producing rate of taxad alcohol of Taxus chinensis endogeny fungus fermenting material |
| CN101113414B (en) * | 2006-05-19 | 2010-04-21 | 建德市大洋化工有限公司 | Yew trichoderma strain and its application in preparation of trichoderma ester prime |
| CN101280281B (en) * | 2008-02-29 | 2010-07-14 | 黑龙江大学 | Paclitaxel genome rearrangement bacterial strain HDFS4-26 |
| CN101386874B (en) * | 2008-10-28 | 2011-05-11 | 南开大学 | Two-liquid phase fermentation method for improving paclitaxel yield from fungi |
| CN102796800A (en) * | 2012-07-19 | 2012-11-28 | 绍兴文理学院 | Method for screening paclitaxel strains and semi-quantitative analysis of paclitaxel strains |
| CN103756917A (en) * | 2014-01-14 | 2014-04-30 | 上海交通大学 | Preparation method of protoplasts of catalpa bungei endophytic fungi |
| CN107674890A (en) * | 2016-08-02 | 2018-02-09 | 武汉臻智生物科技有限公司 | The method for preparing taxol |
| CN108904548A (en) * | 2018-09-14 | 2018-11-30 | 江苏安惠生物科技有限公司 | The method of room temperature extraction edible and medical fungi effective component |
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