CN105524954B - A method of producing the deacetylated baccatin IIIs of 10- using microbial fermentation - Google Patents
A method of producing the deacetylated baccatin IIIs of 10- using microbial fermentation Download PDFInfo
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Abstract
The present invention relates to a kind of methods producing 10 deacetylated baccatin IIIs using microbial fermentation; use Tolumonas osonensis for fermenting microbe; through first order seed culture, second order fermentation culture; substrate baccatin III is put into obtained thalline zymotic fluid; carry out C10 deacetylation conversions; 10 DAB are generated, bacterial strain is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, number CGMCCNo.7562.The present invention uses one step C10 deacetylations biosynthesis of microorganism, 10 DAB using baccatin III as substrate; have many advantages, such as that easy to operate, reaction specificity is good, at low cost, reaction condition is mild, product is easily isolated purifying, overcomes that existing method complex steps, yield are low, by-product is more, isolate and purify difficulty, the shortcomings of environment is unfriendly.
Description
Technical field
The invention belongs to microbiological pharmacy and pharmaceutical engineering fields, and in particular to utilize Tolumonas to a kind of
The method that osonensis produces the deacetylated baccatin IIIs of 10-, this method is with Tolumonas osonensis CGMCC
No.7562 is strain, and using baccatin III as substrate, it is deacetylated to convert production 10- using Microbial biomass C 10- deacetylations
The method of baccatin III.
Background technology
The deacetylated baccatin IIIs of 10- (10-DAB) are Typical Representative -- the Docetaxels of taxane anti-tumor medicament
Precursor substance.It is distributed widely in the leaf of Chinese yew, root, skin, limb, not kindred plant content difference compared with
Greatly, but overall content is relatively low, generally in 0.00513%-0.175% or so.Its traditional preparation methods is carried from plant
It takes, but this process step is tediously long, needs by repeatedly chromatographing, could obtain highly finished product after gradient elution, repeated recrystallize,
The a large amount of losses for not only causing 10-DAB reduce yield, and also largely organic using acetonitrile and petroleum ether etc. in the process
Solvent causes harmful effect to environment.
Microorganism conversion refers to the mistake that a kind of substance (substrate) is transformed into another substance (product) by a certain microorganism
Journey, essence are to carry out enzymic catalytic reaction to xenobiontics using enzyme caused by organism, it has reaction selectivity strong
(regioselectivity, stereoselectivity), reaction condition is mild, by-product is few and does not cause the advantages that environmental pollution.Tolumonas
Osonensis is the novel species for belonging to toluene zygosaccharomyces that Matthew in 2011 et al. has found, is turned currently with the bacterial strain
The research for changing the baccatin III production deacetylated baccatin IIIs of 10- has not been reported.
Invention content
It is an object of the invention to overcome the deficiencies of the prior art and provide it is a kind of it is simple for process, by-product is few, economic reality
The method of mild with, reaction condition and environment amenable production 10-DAB.
The technical solution adopted by the present invention to solve the technical problems is：
A method of the deacetylated baccatin IIIs of 10- being produced using microbial fermentation, using Tolumonas
Osonensis is fermenting microbe, and through first order seed culture, second order fermentation culture, substrate is put into obtained thalline zymotic fluid
Baccatin III carries out C10- deacetylation conversions, generates 10-DAB, the Tolumonas osonensis are by China
Microbiological Culture Collection administration committee common micro-organisms center preservation, deposit number are CGMCC No.7562.
Moreover, the mode for carrying out C10- deacetylations in manufactured thalline zymotic fluid input baccatin III
It is：With organic solvent by baccatin III hydrotropy, the solution being configured to is added in thalline zymotic fluid, the end of baccatin III input
A concentration of 0.04-1g/L, the volume ratio that organic solvent is added are 2-25%.
Moreover, the organic solvent is methanol, ethyl alcohol, acetonitrile, acetone, dichloromethane, chloroform, ethyl acetate
One of which.
Moreover, the conversion condition of the C10- deacetylations is：28-37 DEG C, 150-300r/min conversion cultures
60-144h。
Moreover, the mode of the first order seed culture of the Tolumonas osonensis is：By Tolumonas
Osonensis CGMCC No.7562 are cultivated on inclined-plane, at 22 DEG C -30 DEG C after constant temperature incubation 2-5d, take a full ring to seed
In culture medium, 22 DEG C -30 DEG C, 120-280r/min shaken cultivation 18-30h obtain the seed culture fluid suitable for inoculation.
Moreover, the mode of the second order fermentation culture of the Tolumonas osonensis is：By seed culture fluid with
The inoculum concentration of 5%-15% accesses fermentation medium, 22 DEG C -30 DEG C, 120-280r/min shaken cultivations 20-32h progress two level hairs
Ferment culture obtains thalline zymotic fluid.
A method of the deacetylated baccatin IIIs of 10- being produced using microbial fermentation, using Tolumonas
Osonensis is fermenting microbe, through first order seed culture, second order fermentation culture, obtained thalline, by bacterial cell disruption, broken
Substrate baccatin III is put into thalline bacteria suspension, carries out C10- deacetylation conversions, generates 10-DAB, it is described
Tolumonas osonensis are compiled by China Committee for Culture Collection of Microorganisms's common micro-organisms center's preservation, preservation
Number be CGMCC No.7562.
Moreover, the breaking method is chemically fragmenting method, physical disruption methods or dry broken.
Moreover, the breaking method is that toluene is broken, acetone is broken, isopropanol is broken, ultrasonication, multigelation are broken
Broken, hot and cold alternation is crushed or drying is broken.
The beneficial effects of the invention are as follows：
The preparation method of 10-DAB provided by the invention, using Tolumonas osonensis as strain, baccatin III is made
10-DAB is prepared using one step C10- deacetylations of microorganism for substrate.This method has easy to operate, reaction specificity
The advantages that good, at low cost, reaction condition is mild, product is easily isolated purifying, overcome existing method complex steps, yield it is low,
By-product is more, isolates and purifies the shortcomings of difficult, environment is unfriendly.
Microbial conversion process of the present invention is instead of traditional directly extraction preparation 10-DAB from taxodiaceae plant
Method, have many advantages, such as that easy to operate, reaction specificity is good, at low cost, reaction condition is mild, product is easily isolated purifying.
It has very important significance to preparing natural drug with microbial conversion process.
Description of the drawings
Fig. 1 is the reaction equation that baccatin III is converted into 10-DAB in the present invention；
Fig. 2 is that bacterial strain Tolumonas osonensis CGMCC No.7562 of the present invention convert baccatin III generation 10-
The TLC results of DAB.1：Phage control, 2：Conversion fluid sample, 3：Baccatin III standard items (0.3mg/mL), 4：10-DAB is marked
Quasi- product (0.1mg/mL)；
Fig. 3 is that bacterial strain Tolumonas osonensis CGMCC No.7562 of the present invention convert baccatin III generation 10-
The HPLC results of DAB.Fig. 3-1：The hybrid standard product of baccatin III (0.3mg/mL) and 10-DAB (0.1mg/mL)；Fig. 3-2：
Conversion fluid sample；
Fig. 4 is that bacterial strain Tolumonas osonensis CGMCC No.7562 of the present invention convert turning for baccatin III reaction
Change the LC-MS figures of product, Fig. 4-1：TOF MS anion scan patterns, Fig. 4-2：TOF MS cation scan patterns.
Specific implementation mode
The invention will be further described below in conjunction with the accompanying drawings and by specific embodiment, and following embodiment is descriptive
, it is not restrictive, protection scope of the present invention cannot be limited with this.
Bacterial strain Tolumonas Osonensis P2-A-1 (hereinafter referred to as T.Osonensis P2- according to the present invention
A-1), it has been preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number CGMCC
No.7562, involved open source literature include CN103509735A.
Used culture medium is with buffer solution in following embodiment：
Used medium：
Pancreas peptone soybean broth culture medium 1. (TSB) (L-1)：Tryptone 17.0g, soy peptone 3.0g, NaCl
5.0g、K2HPO42.5g, glucose 2.5g, pH=7.3,115 DEG C of sterilizing 20min.
2. LB (Luria-Bertani) fluid nutrient mediums (L-1)：10 g of tryptone 10g, yeast extract 5g, NaCl,
Adjust pH=7.2.121 DEG C of sterilizing 20min.
Buffer solution used：
50mmol/L phosphate buffers (pH 7.2)：50mmol/L dipotassium hydrogen phosphate solution 300mL are prepared, are used
PH is adjusted to 7.2 by 50mmol/L potassium dihydrogen phosphates.
Specifically it is discussed below：
One, it is as follows to convert baccatin III experiment key step by bacterial strain T.Osonensis P2-A-1：
(1) first order seed culture
A full ring is connect from TSB slant mediums to 50mL LB liquid using T.Osonensis P2-A-1 as production bacterial strain
In the 250mL triangular flasks of body culture medium, 22 DEG C of 200r/min shaken cultivation 1d obtain the seed culture fluid suitable for inoculation.
(2) second order fermentation culture
Seed culture fluid is inoculated into 5% inoculum concentration in the 250mL triangular flasks equipped with 50mL LB liquid mediums,
22 DEG C of 200r/min shaken cultivations 1d.
(3) transformation experiment
Into second order fermentation culture solution be added 1.5mg/mL baccatin III methanol solution 2mL, 37 DEG C of 200r/min after
Persistent oscillation culture 3d.In experiment, not add the culture solution of substrate solution as blank control.
50mL chloroforms are added after conversion, then 37 DEG C of 200r/min oscillation extraction 1h are centrifuged through 5000r/min
5min obtains chloroform extract liquor.Three times with isometric chloroform extraction, combining extraction liquid is rotated to volatilize organic solvent.It is evaporated
The 1mL methanol of sample afterwards redissolves, and is filtered with 0.22 μm of filter membrane, filtrate is testing sample solution, uses high liquid chromatography
Method calculates conversion ratio and product production rate.
Converted product is analyzed
1. thin layer chromatography analysis (TLC)
Solvent uses acetone：Petroleum ether=2：3 (v/v), color developing agent use sulfuric acid：Ethyl alcohol=1：9 (v/v), 105 DEG C
Lower baking colour developing 1-3min, observes the color of spot and calculates Rf value.
2. efficient liquid phase chromatographic analysis (HPLC)
Chromatographic condition is：Phenomenex C18 (5 μm, 250mm × 4.6mm)；Mobile phase：Methanol：Acetonitrile：Water (10：
33：57, v/v/v)；Detection wavelength：227nm；Flow velocity：0.8mL/min；Column temperature：30℃；Sample size：10μL.
3. high performance liquid chromatography mass spectrometry analyzes (LC-MS)
Converted product is detached using preparative silica gel plate, solvent is acetone：Petroleum ether=2：3 (v/v), colour developing
Agent is sulfuric acid：Ethyl alcohol=1：9 (v/v) observe chromatoplate under 254nm ultraviolet lamps, use at 105 DEG C after baking colour developing 1-3min
Pencil marks range in converted product spot position, carefully scraped with blade and collect the silica gel within the scope of this in
In 2.0mL Eppendorf pipes, product is eluted from silica gel repeatedly with 1mL methanol, carries out LC-MS analyses.
Chromatographic condition is：Waters C18 chromatographic columns (100mm × 2.1mm)；Mobile phase：5-95% acetonitriles (0.1%FA-
Acetonitrile), 0-10min；Flow velocity：0.4mL/min；Column temperature：40 DEG C, 10 μ L of sample size.Mass Spectrometry Conditions：Ionization source：Electron spray ionisation
(ESI)；Detection pattern：Multiple-reaction monitoring；Capillary voltage：3kV；Desolvation temperature：400℃；Desolventizing gas flow：
800L/h；Taper hole throughput：30L/h；Ion source temperature： 350℃；Collision gas flow velocity 0.12mL/min.
(4) according to said method tested, as a result, it has been found that the conversion fluid sample of bacterial strain T.Osonensis P2-A-1 on the tlc plate
Occur three apparent spots in product, wherein according to the spot A and B arrived apart from the sequential observation of point sample position from the near to the remote
Rf value RfRespectively with the R of 10-DAB and baccatin III standard itemsfBe worth it is identical, respectively 0.12 and 0.25.Conversion fluid sample
HPLC figures occur apparent detached peaks near 10-DAB retention times, can tentatively judge to contain 10- in conversion fluid sample
DAB.And observe that molecular weight is 544.2256 detached peaks in the TOF MS anion spectrograms of converted product, just can be compared to substrate
Baccatin III lacks an acetyl group, and is C with molecular formula29H36O1010-DAB molecular weight it is consistent.
The result of comprehensive TLC, HPLC, LC-MS analysis illustrates bacterial strain it is found that the product that conversion obtains is really 10-DAB
Baccatin III can be converted into 10-DAB by T.Osonensis P2-A-1.
Two, bacterial strain T.Osonensis P2-A-1 convert the ability of baccatin III after the processing of different crumbling methods
The modes such as chemical method, Physical, seasoning are respectively adopted to be crushed somatic cells, then carry out substrate conversion in fact
It tests, using HPLC methods, using the production rate of substrate conversion efficiency and 10-DAB as index, more different bacterial cell disruption modes are to conversion
The influence of reaction efficiency.
(1) bacterial strain T.Osonensis P2-A-1 are inoculated into the 250mL triangular flasks equipped with 50mL LB liquid mediums,
22 DEG C of 200r/min cultivate 2d.Medium centrifugal (7000r/min, 4 DEG C, 10min) removes supernatant, collects and precipitates wet bacterium mud,
And bacterium mud quality is measured, for use.
(2) the preparation of transformed cells
1. intact cell
Wet bacterium mud 6g is collected, thallus suspension liquid 5mL is made with 50mmol/L phosphate buffers (pH 7.2), for follow-up
Experiment.
2. the broken cell of different modes
I. chemical method
(1) toluene handles crush method
Wet bacterium mud 6g is collected, according to addition 0.04g NaHCO in the wet bacterium muds of every 2g3With the ratio of 8mL toluene, 37 DEG C,
200r/min vibrates 2.5h, centrifuges (5000r/min, 4 DEG C, 10min), removes toluene, lower sediment is collected, with 50mmol/L phosphorus
Thallus suspension liquid 5mL is made in phthalate buffer (pH 7.2), is used for subsequent experimental.
(2) acetone treatment crush method
Wet bacterium mud 6g is collected, the acetone that 200mL is cooled to 4 DEG C in advance is added, mixing is placed in 30min in 4 DEG C of refrigerators, centrifuges
(5000r/min, 4 DEG C, 10min), discards supernatant liquid, and dry removal residual acetone collects lower sediment, with 50mmol/L phosphoric acid
Thallus suspension liquid 5mL is made in salt buffer (pH 7.2), is used for subsequent experimental.
(3) isopropanol handles crush method
Wet bacterium mud 6g is collected, impregnates wet bacterium mud (isopropanol with isopropanol：Wet bacterium mud=9：1 (v/w)) 120min, goes different
Propyl alcohol collects lower sediment, 5 mL of thallus suspension liquid is made with 50mmol/L phosphate buffers (pH 7.2), for follow-up real
It tests.
II. Physical
(1) ultrasonic fragmentation
Wet bacterium mud 12g is collected, thallus suspension liquid 10mL is made with 50mmol/L phosphate buffers (pH 7.2), it will be made
Suspension is divided into two groups, every group of 5mL.Ultrasonic cell disruptor amplitude transformer is stretched into 1cm under the liquid level of suspension, power is
90W, frequency are 20k Hz.Two groups of operating conditions are taken respectively.A groups：Work 15s, rest 30s, 10 cycles；B groups：Work
60s, rest 60s, 8 cycles.Above-mentioned bacterial cell disruption liquid is centrifuged into (5000r/min, 4 DEG C, 10min), supernatant is collected, is used for
Subsequent experimental.
(2) multigelation crush method
Wet bacterium mud 6g is collected, thallus suspension liquid 5mL is made with 50mmol/L phosphate buffers (pH 7.2), is placed in -20
Freeze in DEG C refrigerator, then slowly melt at room temperature, 6 times repeatedly, obtained cellular lysate liquid is used for subsequent experimental.
(3) hot and cold alternation crush method
Wet bacterium mud 6g is collected, thallus suspension liquid 5mL is made with 50mmol/L phosphate buffers (pH 7.2), is put into 90 DEG C
30min is maintained in water-bath, is taken out at once and is placed in ice bath, stands 15min, be allowed to rapid cooling[28], obtained cellular lysate liquid uses
In subsequent experimental.
III. crush method is dried
Wet bacterium mud 6g is collected, is placed in 30 DEG C or so stream of hot air and dries up.With 50mmol/L phosphate buffers (pH
7.2) suspension 5mL is made in bacterium mud suspension, is used for subsequent experimental.
(3) transformation experiment
The above-mentioned baccatin III first through the broken bacteria suspension 4.5mL and 1.5mg/mL of various methods is added into test tube
Alcoholic solution 0.5mL (the final concentration of 0.15mg/mL of baccatin III at this time), vortex mixer 1800r/min oscillations 5min are mixed
It is even.Test tube is placed in 37 DEG C of shaking tables, 200r/min carries out conversion reaction, reacts 72h.Reaction is terminated by the way that 2.5mL chloroforms are added,
And extracted after persistent oscillation (37 DEG C, 200r/min) 3h, finally chloroform organic phase is moved in vial, is volatilized at room temperature
Solvent, and 2.5mL methanol sample dissolutions are added into vial, with 0.45 μm of membrane filtration, filter after supersonic oscillations mixing
Liquid is testing sample solution.In experiment, as a contrast with the conversion fluid of the intact cell of phase homogenous quantities.Sample is measured using HPLC
The content of baccatin III and 10-DAB in product.
(4) conversion results
(1) according to said method tested, as a result, it has been found that when intact cell converts, the conversion ratio of substrate baccatin III is
45%, 10-DAB production rate are 2.1%.
(2) use toluene to be crushed, isopropanol is broken, multigelation is broken, acetone is broken, the processing of hot and cold alternation breaking method
When thalline afterwards is converted, the conversion ratio of substrate baccatin III more complete cell (45.0%) when increase, respectively
77.0%, 74.4%, 56.7%, 53.3%, 52.4%, wherein thalline carries the conversion ratio of substrate pair after toluene break process
It is high significantly.
(3) use toluene to be crushed, acetone is broken, multigelation is broken, ultrasonication B groups, dry broken, ultrasonication A groups
Treated when thalline converted, and the production rate of product 10-DAB increases than intact cell, respectively 12.0%,
4.0%, 3.9%, 3.8%, 3.2%, 3.1%, wherein thalline is converted after toluene break process, and product production rate carries
It is high significantly.Thus it can be inferred that, after toluene break process, thalline shows highest transformation efficiency.
10-DAB is distributed widely in the leaf of Chinese yew, root, skin, limb.Preparation method is mainly from Taxodiaceae
It is extracted in plant, but this method the shortcomings of that there are yields is relatively low, environment unfriendly property.
Bacterial strain provided by the invention can direct fermentation produce 10-DAB, with microbiological transformation technology by baccatin III turn
Metaplasia then has specificity high at the process of 10-DAB, and by-product generates few, and reaction condition is mild, advantages of environment protection.
Claims (9)
1. a kind of method producing the deacetylated baccatin IIIs of 10- using microbial fermentation, it is characterised in that：Using Tolumonas
Osonensis is fermenting microbe, and through first order seed culture, second order fermentation culture, substrate is put into obtained thalline zymotic fluid
Baccatin III carries out C10- deacetylation conversions, generates the deacetylated baccatin IIIs of 10-, the Tolumonas
Osonensis is by China Committee for Culture Collection of Microorganisms's common micro-organisms center's preservation, deposit number CGMCC
No.7562。
2. the method according to claim 1 for producing the deacetylated baccatin IIIs of 10- using microbial fermentation, feature exist
In：The described mode that C10- deacetylations are carried out in obtained thalline zymotic fluid input baccatin III is：With organic molten
By baccatin III hydrotropy, the solution being configured to is added in thalline zymotic fluid for agent, the final concentration of 0.04- of baccatin III input
1g/L, the volume ratio that organic solvent is added are 2-25%.
3. the method according to claim 2 for producing the deacetylated baccatin IIIs of 10- using microbial fermentation, feature exist
In：The organic solvent is the one of which of methanol, ethyl alcohol, acetonitrile, acetone, dichloromethane, chloroform, ethyl acetate.
4. the method according to claim 1 for producing the deacetylated baccatin IIIs of 10- using microbial fermentation, feature exist
In：The conversion condition of the C10- deacetylations is：28-37 DEG C, 150-300r/min conversion cultures 60-144h.
5. the method according to claim 1 for producing the deacetylated baccatin IIIs of 10- using microbial fermentation, feature exist
In：The mode of the first order seed culture of the Tolumonas osonensis is：By Tolumonas osonensis
CGMCC No.7562 are cultivated on inclined-plane, at 22 DEG C -30 DEG C after constant temperature incubation 2-5d, are taken in a full ring to seed culture medium, 22
DEG C -30 DEG C, 120-280r/min shaken cultivation 18-30h obtain the seed culture fluid suitable for inoculation.
6. the method according to claim 1 for producing the deacetylated baccatin IIIs of 10- using microbial fermentation, feature exist
In：The mode of the second order fermentation culture of the Tolumonas osonensis is：By seed culture fluid connecing with 5%-15%
Kind amount access fermentation medium, 22 DEG C -30 DEG C, the 20-32h progress second order fermentation cultures of 120-280r/min shaken cultivations, obtains
Thalline zymotic fluid.
7. a kind of method producing the deacetylated baccatin IIIs of 10- using microbial fermentation, it is characterised in that：Using Tolumonas
Osonensis is fermenting microbe, through first order seed culture, second order fermentation culture, obtains thalline, by bacterial cell disruption, in broken bacterium
Substrate baccatin III is put into body bacteria suspension, carries out C10- deacetylation conversions, generates the deacetylated baccatin IIIs of 10-
, the Tolumonas osonensis by China Committee for Culture Collection of Microorganisms's common micro-organisms center's preservation,
Deposit number is CGMCC No.7562.
8. the method according to claim 7 for producing the deacetylated baccatin IIIs of 10- using microbial fermentation, feature exist
In：The breaking method is chemically fragmenting method, physical disruption methods or dry broken.
9. the method according to claim 7 for producing the deacetylated baccatin IIIs of 10- using microbial fermentation, feature exist
In：The breaking method is that toluene is broken, acetone is broken, isopropanol is broken, ultrasonication, multigelation are broken, hot and cold alternation
It is broken or dry broken.
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