CN101684451B - Microorganism for hydrolyzing 7-xylose group and 13-side chain of taxane - Google Patents
Microorganism for hydrolyzing 7-xylose group and 13-side chain of taxane Download PDFInfo
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Abstract
The invention relates to enterobacter cloacae which are found in soil and have the collection number of CGMCC No.2487 or resting cell with equivalent function, variant and mutant as well as enzyme produced thereby. The invention also provides a preparation method of taxane, namely comprising the following steps: the enterobacter cloacae are used for hydrolyzing 7-xylose group in 7-xylose taxane molecule in wheat bran culture medium, and 13-side chain in taxane molecule with 13-side chain can be hydrolyzed in peptone beef extract culture medium; and the produced taxane can be used for preparing antitumor drugs, such as intermediates of taxane drugs, docetaxel drugs or taxane drugs.
Description
Technical field:
the present invention relates to a kind of culture presevation and number be the enterobacter cloacae (Enterobactercloacae) of CGMCC No.2487 or the resting cell of its functional equivalent, the enzyme of varient and mutant and production thereof and the preparation method of Taxan, specifically, relate to and a kind ofly both can be hydrolyzed the 7-xylosyl of 7-wood sugar Taxan in the wheat bran substratum, can be hydrolyzed again the enterobacter cloacae (Enterobacter cloacae) with the 13-side chain in 13-side chain Taxan molecule in peptone extractum carnis substratum, a kind of and method of utilizing enterobacter cloacae (Enterobacter cloacae) to prepare Taxan.
Background technology:
The taxol isolated diterpene alkaloid with unique antitumour activity that is Wani in 1971 etc. from red bean section Chinese yew genus plants yewtree bark.Its chemical structural formula is:
Taxol has been got permission listing in more than 40 countries since being used for the treatment of ovarian cancer and metastatic breast cancer from 1992 by the FDA approval.Find also that in addition they also have good curative effect to nonsmall-cell lung cancer, prostate cancer etc., demand is increasing, and its scarcity of resources problem is still the focus that whole world medical research and industry member are paid close attention to.Because taxol content in Ramulus et folium taxi cuspidatae is lower, and Ramulus et folium taxi cuspidatae is poky gymnosperm, thus at present taxol supply falls short of demand in clinical application, resource is most deficient.
At present, the means of acquisition taxol mainly contain:
(1) directly extract from bark of Ramulus et folium taxi cuspidatae: the Chinese yew genus plants poor growth, the content of taxol in bark only accounts for 0.01%～0.02%, extracts the very big destruction that taxol has caused taxus resource from bark.But so far, this remains the main source approach of clinical taxol.
(2) chemistry is complete synthesis: the research group that the countries such as the U.S., Japan, France carry out full chemosynthesis taxol has more than 30 individual, finally succeeds through the effort of two more than ten years.1993, as initiator, the complex chemical reaction through 30 step left and right obtained taxol to some scientists of the U.S. with cheap camphor.But because its complete synthesis route is long, yield is low, cost is expensive, and theoretical meaning is only arranged at present, there is no actual application value.
(3) molecular design: the paclitaxel analogs (removing the acetyl baccatin III as baccatin III and 10-) of extraction itself and non-activity is as intermediate, by the chemical process taxol biosynthesis from the plant leaf that can regenerate.This is also one of approach of producing at present taxol.
(4) cell culture method is produced taxol: utilize the yew cell of isolated culture to produce taxol and synthetic precursor thereof, but due to yew cell throughput is unstable still can not suitability for industrialized production.
(5) endogenetic fungus fermentation: the vertical organic chemist Stierle of university of Montana, United States in 1993 etc. are separated to a strain endogenetic fungus (An Delie Japanese yew bacterium), every liter of taxol that contains several nanograms of the fermented liquid in these three weeks of bacterial strain from the yewtree stem.They were separated to from the Xizang Taxus chinensis withe again that a strain endogenetic fungus---little spore pestalotia bacteria, its fermentation level are slightly higher than the former again afterwards.Because content is extremely low, still can not practical application.
In sum, seeking the novel method do not destroy resource and can produce taxol is one of major issue of taxol research field, is also one of focus of this area research.
Numerous studies show that, in the extraction and sepn process of taxol, paclitaxel analogs Chang Zuowei byproduct and discarding, and serve difficulty to the separation and purification band, cause the great wasting of resources and environmental pollution.These analogues comprise: 10-DAXT, and 7-wood sugar taxol, 7-wood sugar-10-removes the acetyl baccatin III, XB III, 7-wood sugar-10-removes the acetyl Cephalomannine, 7-wood sugar Cephalomannine, 10-DAXT C, 7-wood sugar taxol C; And the C-13 position is connected with bearing taxanes such as the 10-deacetyl taxol of side chain, and 10-removes the acetyl Cephalomannine, Cephalomannine, 10-deacetyl taxol C, taxol C.Have been reported thereby utilize chemical process to obtain taxol to the structure of modification of these paclitaxel analogs, but exist selectivity low, easily generate isomer, by product is many and reactions steps is long, productive rate is low, cause the problem such as environmental pollution.And by the biocatalysis means, these " refuses " are used, for the production of the taxone important intermediate and carry out molecular design, this method not only can take full advantage of resource, and can welding.
Especially, numerous documents show, the content that is grown in the 10-DAXT in the renewable branches and leaves of the Ramulus et folium taxi cuspidatae of China such as Chinese Ramulus et folium taxi cuspidatae, taxusyunnanensis, Taxus x media is high especially, be about 10 times of content of taxol, how this compound being used is a problem that is significant.The present invention has adopted the method for microorganism and enzyme bio-transformation thereof to carry out directed bio-transformation to this compounds, as be hydrolyzed the 7-xylose group of 7-wood sugar taxanes, obtain to produce the intermediate of antitumor drug taxol, Docetaxel etc., not only can solve the deficient problem in medicine source of this type of medicine, and, method therefor also has the advantages such as environmental protection, has larger society and economic benefit.
In the analysis to existing document, the report of relevant this type of research of publishing is arranged at present, but the microorganism strains that relates to only can a kind of reaction of catalysis, i.e. C-7 position wood sugar hydrolysis or C-13 position side chain hydrolysis.And yet there are no the bacterial strain with dual-use function, and cause preparation process complexity, the cost of Taxan high, the productive rate of Taxan is lower.
Summary of the invention:
In order to overcome the deficiencies in the prior art, the object of the present invention is to provide a kind of microorganism and enzyme thereof with dual-use function;
Another goal of the invention of the present invention is to provide the purposes of this microorganism;
Another goal of the invention of the present invention is to provide a kind of preparation method of Taxan.
Enterobacter cloacae (Enterobacter cloacae) and resting cell, varient and the mutant of functional equivalent and the enzyme of producing thereof that a kind of culture presevation is numbered CGMCC No.2487 have been the present invention relates to; Described enterobacter cloacae is preserved by China Committee for Culture Collection of Microorganisms's common micro-organisms center on May 9th, 2008, and deposit number is: CGMCC No.2487.China Committee for Culture Collection of Microorganisms's common micro-organisms is centered close to the Datun Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica.This enterobacter cloacae is that enterobacter cloacae belongs to enterobacter cloacae, for biology pure.Enterobacter cloacae can be deposited on wheat bran or peptone extractum carnis culture medium slant, and the preservation temperature is 4 ℃ of left and right, also can be deposited in the liquid nutrient medium that is added with glycerine, and the preservation temperature is-20～-80 ℃.
Enterobacter cloacae is to obtain by the screening of more than the 200 strain microorganisms that derive from the pedotheque below taxus chinensis in northeast arboreal growth peripheral ground 5cm being carried out 10-DAXT xylose group hydrolysis ability.
When utilizing enterobacter cloacae, can use complete wet cell or stem cell, the cell that exists as the cells form of freeze-drying, spray-dired or heat drying; The cell that the perhaps cell material to process, as the cell that breaks or cell extract form exist; Also comprise varient, the mutant cells that obtains by method mutagenesis such as physics, chemistry; And the cell that uses the engineering strain of genetic engineering acquisition, host cell can be any cell, as the cells such as colon bacillus, saccharomyces cerevisiae or Pichia yeast that one or more are used for expressing one or more enzyme genes that can carry out catalyzed reaction of the present invention that contain through improvement.
The invention still further relates to the purposes of the enzyme of resting cell, varient and the mutant of enterobacter cloacae or its functional equivalent and production thereof, the 7-xylose group of the 7-wood sugar Taxan in the wheat bran substratum in the hydrolysis substrate bearing taxanes, in peptone extractum carnis substratum in the hydrolysis substrate bearing taxanes with the 13-side chain of 13-side chain Taxan.
The enzymatic reaction condition of the enzymic hydrolysis substrate bearing taxanes of the resting cell of enterobacter cloacae of the present invention or its functional equivalent, varient and mutant and production thereof is: concentration is Tris-HCl damping fluid or the phosphoric acid buffer of 0.01～1.0mol/L; Preferred buffer is phosphoric acid buffer, and the preferred concentration of phosphoric acid buffer is 0.5mol/L; PH is 4.5～8.0, is preferably 6.5; Temperature is 15～50 ℃, preferred 37 ℃.
The invention still further relates to the enzyme that enterobacter cloacae produces, by several different methods extract, the enzyme of purifying, comprise thick enzyme and single enzyme.
The invention still further relates to a kind of preparation method of Taxan, utilize enterobacter cloacae or with the 7-xylose group of resting cell, varient and the mutant 7-wood sugar Taxan in the hydrolysis substrate bearing taxanes in the wheat bran substratum of its functional equivalent.
In the preparation method of Taxan provided by the invention, enterobacter cloacae or with resting cell, varient and the mutant temperature of reaction in the wheat bran substratum of its functional equivalent be 15～50 ℃, be preferably 20～35 ℃, most preferably 28 ℃.
Wherein, contain wheat bran 10～100g/L in the wheat bran liquid nutrient medium, (NH
4)
2 HPO
31～5g/L, K
2HPO
40.05～1.0g/L, MgSO
47H
2O0.01～0.5g/L; Be preferably: wheat bran 20～60g/L, (NH
4)
2HPO
31～3g/L, K
2HPO
40.08～0.3g/L, MgSO
47H
2O0.08～0.3g/L; Most preferably be: wheat bran 40g/L, (NH
4)
2HPO
32g/L, K
2HPO
40.1g/L, MgSO
47H
2O0.1g/L.
Wherein, contain wheat bran 10～100g/L in the wheat bran solid medium, (NH
4)
2 HPO
31～5g/L, K
2HPO
40.05～1.0g/L, MgSO
47H
2O0.01～0.5g/L, agar 10～20g/L; Be preferably: wheat bran 20～60g/L, (NH
4)
2 HPO
31～3g/L, K
2HPO
40.08～0.3g/L, MgSO
47H
2O0.08～0.3g/L, agar 12～18g/L; Most preferably be: wheat bran 40g/L, (NH
4)
2HPO
32g/L, K
2HPO
40.1g/L, MgSO
47H
2O0.1g/L, agar 15g/L.
The another preferred version of wheat bran substratum is: can also add weight percent in substratum is 0.01%～1% xylan; Preferred 0.03～0.1%; Most preferably 0.06%.Described xylan is selected from oat xylan, birch xylan.After having added xylan, the transformation efficiency of substrate can improve 10～40%.
A preferred version again of wheat bran substratum is: can also add weight percent in the wheat bran substratum and be 0.01～1.0% ammonium sulfate; Preferred 0.2～0.5%; Most preferably 0.4%.Ammonium sulfate has promoter action for the hydrolysis reaction to substrate of enterobacter cloacae, and after adding ammonium sulfate, the transformation efficiency of substrate improves 20～40%.
A preferred version again of wheat bran substratum is: also comprise macroporous resin 5～40g/L in liquid wheat bran substratum; Be preferably 10～30g/L, most preferably be 20g/L.
Wherein, the wheat bran Medium's PH Value is 5.0～8.0; Be preferably 6.0～7.0; Most preferably be 6.5.
Described bearing taxanes is selected from from Ramulus et folium taxi cuspidatae and extracts monomer bearing taxanes or its crude extract that obtains, monomer bearing taxanes or its crude extract that passes through Ramulus et folium taxi cuspidatae histocyte cultivation acquisition.Wherein: 7-wood sugar Taxan is selected from: 10-DAXT, 7-wood sugar taxol, 7-wood sugar-10-removes the acetyl baccatin III, XB III, 7-wood sugar-10-removes the acetyl Cephalomannine, 7-wood sugar Cephalomannine, 10-DAXT C, 7-wood sugar taxol C.
The invention still further relates to the preparation method of another kind of Taxan, utilize enterobacter cloacae or with resting cell, varient and the mutant of its functional equivalent in peptone extractum carnis substratum in the hydrolysis substrate bearing taxanes with the 13-side chain of 13-side chain Taxan.
In the preparation method of Taxan provided by the invention, enterobacter cloacae or with resting cell, varient and the mutant temperature of reaction in peptone extractum carnis substratum of its functional equivalent be 15～50 ℃, preferred 25 ℃.
Wherein, contain peptone 2～20g/L in peptone extractum carnis liquid nutrient medium, beef extract 1～5g/L, NaCl1～10g/L; Be preferably: peptone 5～15g/L, beef extract 1～3g/L, NaCl3～6g/L; Most preferably be: peptone 10g/L, beef extract 2g/L, NaCl5g/L.
Wherein, contain peptone 2～20g/L in peptone extractum carnis solid medium, beef extract 1～5g/L, NaCl1～10g/L, agar 10～20g/L; Be preferably: peptone 5～15g/L, beef extract 1～3g/L, NaCl3～6g/L, agar 12～18g/L; Most preferably be: peptone 10g/L, beef extract 2g/L, NaCl5g/L, agar 15g/L.
The preferred version of peptone extractum carnis substratum is: also comprise macroporous resin 5～40g/L in liquid protein peptone extractum carnis substratum; Be preferably 10～30g/L, most preferably be 20g/L.
Wherein, the pH value of peptone extractum carnis substratum is 5.0～8.0, is preferably 6.5.
Substrate bearing taxanes in the present invention is selected from from Ramulus et folium taxi cuspidatae and extracts monomer bearing taxanes or its crude extract that obtains, monomer bearing taxanes or its crude extract that passes through Ramulus et folium taxi cuspidatae histocyte cultivation acquisition.Wherein: the Taxan that the C-13 position is connected with side chain is selected from: 10-deacetyl taxol, taxol, 10-remove the acetyl Cephalomannine, Cephalomannine, 10-deacetyl taxol C, taxol C.
10-DAXT in substrate and the 10-deacetyl taxol in product all have and suppress very doughtily the cell fission effect.In order to strengthen the input amount of substrate, add a small amount of macroporous resin can improve productive rate in substratum.Macroporous resin can adsorb substrate and product, plays the effect of the slow controlled release of compound in conversion process.Majority of compounds is not entered in the middle of reaction system by resin absorption, after substrate reactions, substrate content in reaction system reduces, at this moment the part substrate can the system of entering be proceeded enzyme catalysis, and the product that generates can be broken away from reaction system by macroporous resin adsorption, thereby reduces product to the effect of microorganism.
The substrate bearing taxanes can be cyclodextrin encapsulated, polyvinylpyrrolidone hydrotropy, tween hydrotropy and make the modes such as liposome and add substratum.
Resting cell, varient and mutant or engineering strain with the enterobacter cloacae functional equivalent, and the enzyme of producing, can react in substratum, damping fluid, also can add immobilized cell or the reactions of being fixed of enzyme material such as resin, sodium alginate, wilkinite.Add a small amount of fixing reagent can improve productive rate in substratum.
The resting cell of enterobacter cloacae or its functional equivalent, varient and mutant or engineering strain, and the enzyme of producing can liquid-liquid diphase such as the form of water/ethyl acetate etc. carry out the two-phase bioconversion reaction.
The taxane compounds of the present invention's preparation can be used for treating cancer and preparation antitumor drug: the intermediate of taxol, Docetaxel or other taxones.
The useful technique effect that the present invention has is:
1. enterobacter cloacae of the present invention has dual function, namely can be hydrolyzed the 7-xylosyl in 7-wood sugar bearing taxanes molecule in the wheat bran substratum, can be hydrolyzed with the 13-side chain in 13-side chain Taxan molecule in peptone extractum carnis substratum again.Thereby simplified the preparation technology of Taxan.
2. the biological activity of enterobacter cloacae of the present invention is strong, and is low with the fostering requirement condition for going down to posterity, and is suitable for large-scale industrial production.
3. the preparation method of Taxan of the present invention, utilized fully and extracted the waste of taxol in the prior art, turns waste into wealth, and produces huge economic benefit, protected ecotope.
4. the preparation method of Taxan of the present invention carries out directed hydrolysis for different substrate bearing taxanes, can directly obtain the intermediate of taxol or taxol biosynthesis and Docetaxel.
Abbreviation and term
XDT:7-wood sugar-10-deacetyl taxol
DT:10-removes the acetyl Japanese yew
PDA: potato dextrose agar
DMF:N, dinethylformamide
TLC: thin-layer chromatography
NMR: nuclear magnetic resonance spectroscopy(NMR spectroscopy)
HPLC: high performance liquid chromatography
MS: mass spectroscopy
Description of drawings:
The liquid chromatogram of Fig. 1 standard substance (upper figure) and enterobacter cloacae reaction solution (figure below)
The one-level mass spectrum of chromatographic peak P and second order ms figure in Fig. 2 enterobacter cloacae reaction solution
Fig. 3 ethyl acetate residue silica gel column chromatography gradient elution schema
Figure 41 0-removes the acetyl taxol
1The H-NMR collection of illustrative plates
Figure 51 0-removes the acetyl taxol
13The C-NMR collection of illustrative plates
(the NH of Fig. 6 different concns
4)
2SO
4Impact on the 10-DAXT conversion
The impact that Fig. 7 xylan transforms 10-DAXT
The impact that Fig. 8 temperature transforms 10-DAXT
Fig. 9 enterobacter cloacae transforms the performance graph that 10-DAXT generates 10-deacetyl taxol
Figure 107-wood sugar-10-deacetyl taxol liquid-solid two-phase bio-transformation
Embodiment:
In order further to understand the present invention, the following examples only are used for further illustrating the present invention, but and do not mean that any limitation of the invention.
Embodiment 1: the screening of enterobacter cloacae and evaluation
More than the 200 strain microorganisms that derive from the pedotheque below taxus chinensis in northeast arboreal growth peripheral ground 5cm have been carried out the screening of 10-DAXT xylose group hydrolysis ability, result shows that the bacterial strain enterobacter cloacae has 10-DAXT xylose group hydrolysis ability.
Isolated individual plant microorganism is stored on potato dextrose agar (PDA) inclined-plane.When carrying out the microorganism primary dcreening operation with each strain strain culturing in the triangular flask of 2 splendid attire wheat bran substratum, cultivate after 2 days, one bottle only adds DMF (DMF) solvent as blank, and another bottle adds the DMF solution of substrate 10-DAXT.Continue to cultivate after 7 days the nutrient solution ethyl acetate extraction after filtration.Analyze technology for detection by thin-layer chromatography, high performance liquid chromatography, liquid phase mass spectrometry etc. after the extraction liquid concentrating under reduced pressure and confirm product.Subsequently, be prepared bio-transformation, obtain product by column chromatography repeatedly, and confirm by the NMR (Nuclear Magnetic Resonance) spectrum technology.
1. the hydrolysis of enterobacter cloacae to 10-DAXT
The bacterium colony of the enterobacter cloacae that on picking, step obtains is in the wheat bran liquid nutrient medium, and culture condition is: 110 rev/mins, incubated at room temperature two days is as the seed of microbial.Wheat bran substratum food preparation is: wheat bran 50g, (NH
4)
2HPO
32g, K
2HPO
42g, MgSO
47H
2The O0.1 gram is supplied 1000ml with distilled water, and the pH value is 6.5.
(1), experimental group: draw 1ml (2% inoculum size) seed with aseptic straw, join in the 250ml triangular flask that fills 50ml wheat bran substratum, continue to cultivate 48 hours.The solution 0.5ml that adds the 10-DAXT of 30mg/ml in the growth logarithmic phase of bacterium.Continue to cultivate 7 days.
(2), control group: draw 1ml (2% inoculum size) seed with aseptic straw, join in the 250ml triangular flask that fills 50ml wheat bran substratum, continue to cultivate 48 hours.Growth logarithmic phase bacterium adds DMF0.5ml.Continue to cultivate 7 days.
2. the TLC (thin-layer chromatography) of product 10-deacetyl taxol detected
After a large amount of screenings, find in the reaction solution of enterobacter cloacae except the brown spot (Rf value=0.3) of 10-DAXT, also there is another brown spot at polarity smaller part (Rf value=0.5), and the position of this spot and reference substance 10-deacetyl taxol position, solid colour.
3. the HPLC (high performance liquid chromatography) of product 10-deacetyl taxol detected
With the reaction solution extract of enterobacter cloacae with the dissolve with methanol constant volume to 1ml, with after 0.22 μ m membrane filtration as test liquid detection use to be analyzed.
Analysis condition: with Apollo C
18(5 μ m, 250mm * 4.6mm i.d.) as analytical column, acetonitrile/water=1/1 (V/V) detects wavelength and is made as 230nm as moving phase, and flow velocity is 1ml/min, and sampling volume is 10 μ l; The HPLC chromatographic instrument is Agilent1100 type HPLC chromatographic instrument.10-deacetyl taxol is that standard substance are preserved in the laboratory.Can find out that the reaction solution of enterobacter cloacae is at the retention time (t identical with standard substance in the liquid chromatogram of standard substance and enterobacter cloacae reaction solution
R15.5min) time have absorption peak P to occur, the ultraviolet full wavelength scanner shows that also they have identical ultra-violet absorption spectrum.See accompanying drawing 1.
4. the liquid chromatography mass coupling (LC/MS/MS) of product 10-deacetyl taxol detected
The HPLC condition: as moving phase, sampling volume is 0.1 μ l with acetonitrile/water/formic acid=50/50/0.1 (V/V/V), test sample, analyze chromatographic column, detect wavelength, flow velocity is the same.
MS condition: with N
2As carrier gas, flow velocity 6v/min; Pressure 30.0psi, cracking temperature are made as 325 ℃;
Positive ion scanning, sweep limit is located at m/z750-1050.
Chromatographic peak P in reaction solution HPLC collection of illustrative plates is carried out the LC-MS analyzing and testing, see accompanying drawing 2.M/z812[M+H obviously detected in the one-level mass spectrum]
+, m/z834[M+Na]
+, m/z859[M+K]
+Quasi-molecular ion peak, the molecular weight that shows this compound is 811, the molecular weight of this and 10-deacetyl taxol coincide.Further carry out second order ms and detect, can find m/z268,286,527,794,751 equimolecular fragment peaks.And these also all match with the cleavage of mass spectrum feature of 10-deacetyl taxol.
5.10-the preparation of removing the acetyl taxol with separate
Main raw and method are with step 1.500mg7-wood sugar-10-deacetyl taxol is dissolved in 17mlDMF, makes the DMF solution of substrate.Add substrate DMF solution 0.5ml in 35 1000ml triangular flasks that 350ml wheat bran substratum is housed; Transform under room temperature condition and cultivate after 7 days, filter.Ethyl acetate extraction 4 times of bacterium liquid, the pressure reducing and steaming ethyl acetate gets residue 4.78g.Silica gel column chromatography, gradient elution.Detailed process is seen ethyl acetate residue silica gel column chromatography gradient elution schema, sees accompanying drawing 3.
Through after column chromatography repeatedly, obtain 10-deacetyl taxol 92.4mg, yield is 18.8%.The substrate that does not react is total to 162.5mg after reclaiming, account for 32.5% of input amount.The structure warp of purpose Product Taxol
1H-NMR and
13C-NMR confirmed, its
1H-NMR and
13The C-NMR spectrogram is seen respectively accompanying drawing 4 and accompanying drawing 5.
6), the nuclear magnetic resonance spectrum data of converted product 10-deacetyl taxol
White powder.
1H-NMR(400MHz，CDCl
3)δ：8.11(2H，d，J＝7.6Hz，o-H?of?OBz)，7.75(2H，d，J＝7.6Hz，o-H?of?NH-Bz)，7.61(1H，m，p-H?of?OBz)，7.50(2H，m，m-H?of?OBz)，7.48(1H，m，p-H?of?NH-Bz)，7.45(2H，m，o-H?of3′-C
6H
5)，7.40(2H，m，m-H?of3′-C
6H
5)，7.37(2H，m，m-H?ofNH-Bz)，7.33(1H，m，p-H?of3′-C
6H
5)，7.17(1H，d，J＝8.8Hz，N
H-CO)，6.18(1H，t，J＝8.8Hz，H-13)，5.77(1H，dd，J＝9.2，2.0Hz，H-3′)，5.66(1H，d，J＝7.2Hz，H-2)，5.18(1H，s，H-10)，4.92(1H，d，J＝8.8Hz，H-5)，4.77(1H，d，J＝2.0Hz，H-2′)，4.30(1H，d，J＝8.8Hz，H-20a)，4.20(1H，d，J＝8.8Hz，H-20b，overlapped)，4.20(1H，m，H-7，overlapped)，3.88(1H，d，J＝6.8Hz，H-3)，2.55(1H，ddd，J＝14.8，9.7，6.7Hz，H-6a)，2.37(3H，s，H-4OCOC
H 3)，2.29(2H，m，H-14)，1.85(1H，m，H-6b)，1.75(3H，s，H-18)，1.74(3H，s，H-19)，1.19(3H，s，H-16)，1.10(3H，s，1.10，H-17)。
13C-NMR(100MHz，CDCl
3)δ207.1(C-9)，172.5(C-1′)，170.5(4-
COCH
3)，167.1(NH-
CO)，167.0(2-
COC
6H
5)，138.1(C-12)，137.9(quaternary?C?of3-CO
C 6H
5)，136.1(C-11)，133.7(p-C?of2-CO
C 6H
5)，133.6(quaternaryCof3′-
C 6H
5)，131.9(p-Cof3′-
C 6H
5)，130.2(o-C?of2-CO
C 6H
5)，129.17(quaternary?C?of2-CO
C 6H
5)，128.96(m-C?of?NH-CO
C 6H
5)，128.71(m-C?of2-
COC
6H
5)，128.68(m-C?of3′-
C 6H
5)，128.3(p-C?of?NH-CO
C 6H
5)，127.05(o-C?of3′-
C 6H
5)，127.02(o-C?of?NH-CO
C 6H
5)，84.1(C-5)，81.1(C-4)，78.7(C-1)，76.7(C-20)，74.8(C-10)，74.5(C-2)，73.2(C-2′)，72.4(C-13)，72.0(C-7)，57.7(C-8)，55.0(C-3′)，46.4(C-3)，43.0(C-15)，36.9(C-14)，35.9(C-6)，26.5(C-17)，22.5(4-CO
CH
3)，20.6(C-16)，14.3(C-18)，9.8(C-19)。
Embodiment 2: the impact that different culture media transforms 10-DAXT
Use following 4 kinds of substratum as the microbial transformation system:
Culture medium A: wheat bran 50g, K
2HPO
42g, (NH
4)
2 HPO
32 grams, MgSO
47H
2O, 0.1 gram, distilled water is settled to 1000ml;
Substratum B: extractum carnis extract 10g, NaCl5g, peptone 10g, water 1000ml;
Culture medium C: glucose (or in alpha-lactose, semi-lactosi, sucrose, N.F,USP MANNITOL, fructose, glycerine, inositol, sorbyl alcohol, D-wood sugar a kind of) 20g, peptone 2g, yeast extract 2g, K
2HPO
41g, KH
2PO
41g, MgSO
47H
2O0.2g, FeSO
47H
2O0.01g, NaCl0.01g, MnSO
44H
2O0.01g;
Substratum D: potato 200g (filtering after boiling 20min), glucose 20g, water 1000ml;
Prepare above-mentioned 4 kinds of each 50ml of substratum and pack in the 250ml triangular flask, regulate pH value to 6.0.Under aseptic condition, add 1ml to cultivate 2 days enterobacter cloacae suspensions to each bottle.Culture condition is: 28 ℃, and 200rpm.Continue to cultivate 2 days, add the DMF solution of 250 μ l substrate 10-DAXTs.Continue to cultivate conversion and use isopyknic each bottle of ethyl acetate extraction reaction solution 3 times after 7 days, merge organic phase, carry out TLC and HPLC after concentrating under reduced pressure and analyze.Each be treated to 2 parallel.
Result shows: bacterial strain only could be hydrolyzed the xylose group of 10-DAXT after fermentation in culture medium A, and the side chain hydrolysis reaction mainly occurs in substratum B, and substrate is had an effect hardly in culture medium C.Find when adding different carbon source (as fructose and semi-lactosi etc.) in culture medium A, the productive rate of 10-deacetyl taxol does not but almost improve.See Table 1.
Table 1: enterobacter cloacae in 4 kinds of substratum to the conversion of 10-DAXT
Embodiment 3: the impact of the ammonium sulfate of different concns on the hydrolysis of 10-DAXT xylose group
Preparation wheat bran substratum: wheat bran 50g, K
2HPO
42g, (NH
4)
2 HPO
32 grams, MgSO
47H
2O, 0.1 gram, distilled water is settled to 1000ml; Add ammonium sulfate in the wheat bran substratum, adopt 5 (NH
4)
2SO
4Concentration is respectively 0,0.4%, 1%, 2%, 3%, and the corresponding N that is numbered
0, N
1, N
2, N
3, N
4With the each (NH for preparing
4)
2SO
4The substratum of concentration, the 50ml wheat bran substratum of packing in every 250ml triangular flask.Working method is with embodiment 2.The content of converted product detects by HPLC.With the residue that obtains after concentrated with dissolve with methanol and be settled to 10ml, after filtering as the HPLC test liquid.Each concentration gradient do 3 times parallel.
1), standardized solution preparation: precision takes 10-DAXT 6.1mg respectively, 10-deacetyl taxol 9.7mg, but 7-wood sugar-10-removes acetyl bar booth III1.6mg, with dissolve with methanol and constant volume to 5ml.Accurate absorption 3 kinds of solution 10 μ l, 20 μ l, 50 μ l, 100 μ l, 150 μ l and constant volumes are to the standardized solution of 2ml as different concns respectively.
2), the liquid phase analysis condition: analytical column is BDS HYDERSIL C18, and column temperature is 30 ℃, and the detection wavelength is 230nm, and flow velocity is 1.0ml/min, sample size 5 μ l, water and acetonitrile are as the eluent gradient wash-out.Gradient condition table 2:
The eluent gradient table of table 2 water and acetonitrile
3), regression equation and the linearity range of above-mentioned 3 kinds of materials under this condition sees Table 3:
Table 3
4), content detection result:
(NH4) in the wheat bran substratum
2SO
4Productive rate to product has promoter action.When content was 0.4%, the output of converted product was up to 9.4mg/L, and transformation efficiency also reaches 52.8%.See accompanying drawing 6.Embodiment 4: the impact of xylan on the hydrolysis of 10-DAXT xylose group
Preparation wheat bran substratum: wheat bran 50g, K
2HPO
42g, (NH
4)
2 HPO
32 grams, (NH
4)
2SO
44 grams, MgSO
47H
2The O0.1 gram, distilled water is settled to 1000ml; The xylan that adds again different concns makes the concentration that adds be respectively 0,0.04%, 0.06%, 0.08%, 0.16%, and reference numeral is A-E.Heating makes xylan dissolve moist heat sterilization afterwards fully.The extraction and analysis process of cultivation, substrate and product that transforms is with embodiment 1.Each process to do 3 times parallel.The content of DT and XDT is analyzed by HPLC and is got.
Experimental result: in the finite concentration scope, xylan can strengthen the ability of selective hydrolysis 10-DAXT C-7 xylose group.When the concentration of xylan was 0.06%, the output of DT had reached 12.69mg/L, and transformation efficiency is up to 73.7%.Continue to increase xylan, the output of DT begins to descend.See accompanying drawing 7.Embodiment 5: the impact of temperature on the hydrolysis of 10-DAXT xylose group
Preparation wheat bran substratum: wheat bran 50g, K
2HPO
42g, (NH
4)
2 HPO
32 grams, (NH
4)
2SO
44 grams, MgSO
47H
2The O0.1 gram, xylan 0.6 gram, distilled water is settled to 1000ml.Sterilizing adds 1ml to cultivate the seed of 2 days in backward every triangular flask, adds the substrate 10-DAXT after continuing to cultivate 2 days under 24 ℃.Transform under different temperature condition.Temperature setting is set to 24 ℃, and 28 ℃, 37 ℃.The extraction and analysis process of method for transformation, substrate and product is with embodiment 1.Each is processed parallel 3 times.10-goes the output of acetyl Japanese yew (DT) and the residual content of 10-DAXT (XDT) to detect by HPLC.The result demonstration, in tested temperature range, temperature is higher, and hydrolysis reaction carries out also more thoroughly.Transform 7 days under 37 ℃ of conditions, transformation efficiency is 18.1%, sees accompanying drawing 8.
Embodiment 6:7-wood sugar-10-deacetyl taxol xylose group hydrolysis generates the performance graph of 10-deacetyl taxol
Preparation wheat bran substratum: wheat bran 50g, K
2HPO
42g, (NH
4)
2 HPO
32 grams, (NH
4)
2SO
44 grams, MgSO
47H
2The O0.1 gram, xylan 0.6 gram, distilled water is settled to 1000ml.In each 250ml triangular flask, Intake Quantity is 50ml.Cultivation, conversion operation method are with embodiment 1.Change each triangular flask over to 37 ℃ after substrate adds and transform cultivation.The residual content of the content of converted product 10-deacetyl taxol (DT) and substrate 10-DAXT (XDT) detects by high performance liquid chromatography.With each bottle of equivalent ethyl acetate extraction fermented liquid 3 times, concentrating under reduced pressure is removed organic solvent.With the residue that obtains after concentrated with dissolve with methanol and be settled to 10ml, solution after 0.45 μ m membrane filtration as the HPLC test liquid.Primary treatment is only done in this experiment.
After having determined to cultivate the primary condition that transforms, we transform the optimum reacting time of 10-DAXT for determining enterobacter cloacae, sampling in every 24 hours in conversion process, with the consumption of high-efficient liquid phase chromatogram technique analysis substrate XDT and the generation of primary product DT, with acetonitrile-water system gradient elution.
With enterobacter cloacae, 10-DAXT is carried out in the process of bio-transformation, two reactions having occured altogether: the hydrolysis of C-7 wood sugar and the hydrolysis of C-13 side chain, investigated the dynamic change of principal reaction C-7 wood sugar hydrolysis in this experiment.It is 12.82mg/L that the residual content of XDT reached Schwellenwert at the 7th day, and this moment, DT also was in higher level, and output reaches 3.78mg/L, and transformation efficiency reaches 25.53%.See accompanying drawing 9.After reaction 7 days, the trend of rising is in gently.
Embodiment 7:7-wood sugar-10-deacetyl taxol liquid-solid two-phase bio-transformation
Preparation wheat bran substratum: wheat bran 50g, K
2HPO
42g, (NH
4)
2 HPO
32 grams, MgSO
47H
2O, 0.1 gram, (NH
4)
2SO
44 grams, xylan 0.6 gram, distilled water is settled to 1000ml; In each 250ml triangular flask, Intake Quantity is 50ml.Put into the cloth bag that the 1g macroporous resin is housed again in each triangular flask.Cultivate the conversion operation method with the step 1 of embodiment 1.5 processing are used in this experiment, and it is 50mg, 100mg, 200mg, 500mg, 1000mg and difference reference numeral A-E that substrate adds concentration.Converted product 10-goes the content of acetyl Japanese yew (DT) and the residual content of substrate 10-DAXT (XDT) to detect by high performance liquid chromatography.Take out in pocket macroporous resin and repeatedly rinse with ethanol after transforming 7 days, fermented liquid is with ethyl acetate extraction 3 times, merging 2 part organic solvents.With the residue that obtains after concentrated with dissolve with methanol and be settled to 10ml, solution after 0.45 μ m membrane filtration as the HPLC test liquid.
10-DAXT and 10-deacetyl taxol all have and suppress very doughtily the cell fission effect.In order to strengthen the input amount of substrate, we have studied liquid-solid two-phase bio-transformation situation.Add a small amount of macroporous resin can adsorb substrate and product in substratum, play the effect of the slow controlled release of compound in conversion process.Majority of compounds is not entered in the middle of reaction system by resin absorption, after substrate reactions, substrate content in reaction system reduces, at this moment the part substrate can the system of entering be proceeded enzyme catalysis, and the product that generates can be broken away from reaction system by macroporous resin adsorption, thereby reduces product to the effect of microorganism.
In this experiment, add the substrate of different concns there is no considerable influence to transformation efficiency, but it is low to compare former experimental study transformation efficiency.But when dropping into substrate 500mg in every liter of fermented liquid, DT output has obtained raising, reaches 43.63mg/L, and the fermentation of prompting two-phase can improve the output of purpose product to a certain extent.
In E processed, the residual content of substrate XDT and the output of DT and D were roughly suitable in processing, but the input amount of XDT is but 2 times that D processes.Reason may be that the XDT add-on is excessive in E processes, and macroporous resin adsorption not exclusively makes substrate exist in a large number in fermented liquid.Little because of substrate solubleness in ethyl acetate again, cause extraction not exclusively.See accompanying drawing 10.
Embodiment 8:
Use resting cell thalline and cell free system as the system that transforms, testing sequence is as follows:
1) collect the growth cloaca intestines rod suspension 1L of 2 days in the wheat bran substratum;
2) with bacteria suspension centrifugal 20min under 20,000 * g condition.Pour out supernatant (extracellular fluid), precipitation part (living cell body) Eddy diffusion is in 50mmol, and pH is in 6.0 potassium phosphate buffer;
3) all add substrate DMF solution in upper cleer and peaceful precipitation part.At 37 ℃, the 24h that vibrates under the 200rpm condition, TLC detects enzyme and lives;
4) rinse the centrifugal living cell body that obtains several all over residual to guarantee acellular outer liquid with distilled water.With the cell Eddy diffusion in distilled water, lyophilize;
5) collect stem cell, with the potassium phosphate buffer Eddy diffusion of 50mmol pH6.0, ultrasonication.Condition: 220V, ultrasonic 2s, interval 8s processes ice bath 80 times.In its process, discontinuity is rocked.
6) with cell centrifugal 20min under 20,000 * g condition of fragmentation, inclining supernatant (tenuigenin), precipitation part (large cell membrane fragments and not broken cell);
7) all add substrate DMF solution in upper cleer and peaceful precipitation part.At 37 ℃, the 24h that vibrates under the 200rpm condition, TLC detects enzyme and lives;
Detected the various piece xylosidase activity by TLC, result only shows that 2 precipitation partial displays after centrifugal have xylosidase activity.This explanation xylosidase is an intracellular enzyme, and is not free in tenuigenin.
Claims (19)
1. a culture presevation number is CGMCC No.2487 enterobacter cloacae (Enterobacter cloacae) or its resting cell.
2. the purposes of enterobacter cloacae as claimed in claim 1 (Enterobacter cloacae) or its resting cell, it is characterized in that, the 7-xylose group of the 7-wood sugar Taxan in the wheat bran substratum in the hydrolysis substrate bearing taxanes, in peptone extractum carnis substratum in the hydrolysis substrate bearing taxanes with the 13-side chain of 13-side chain Taxan.
3. the preparation method of a Taxan, it is characterized in that, utilize the 7-xylose group of enterobacter cloacae as claimed in claim 1 (Enterobacter cloacae) or its resting cell 7-wood sugar Taxan in the hydrolysis substrate bearing taxanes in the wheat bran substratum; Described wheat bran substratum comprises liquid wheat bran substratum and solid wheat bran substratum.
4. the preparation method of Taxan as claimed in claim 3, is characterized in that, enterobacter cloacae (Enterobacter cloacae) or the temperature of reaction that is hydrolyzed 7-wood sugar Taxan with its resting cell in the wheat bran substratum are 15～50 ℃.
5. the preparation method of Taxan as claimed in claim 3, is characterized in that, contains wheat bran 10～100g/L, (NH in liquid wheat bran substratum
4)
2HPO
31～5g/L, K
2HPO
40.05～1.0g/L, MgSO
47H
2O0.01～0.5g/L, the pH value is 5.0～8.0.
6. the preparation method of Taxan as claimed in claim 3, is characterized in that, also comprises macroporous resin 5～40g/L in liquid wheat bran substratum, and the pH value is 5.0～8.0.
7. the preparation method of Taxan as claimed in claim 3, is characterized in that, contains wheat bran 10～100g/L, (NH in solid wheat bran substratum
4)
2HPO
31～5g/L, K
2HPO
40.05～1.0g/L, MgSO
47H
2O0.01～0.5g/L, agar 10～20g/L, the pH value is 5.0～8.0.
8. the preparation method of the arbitrary Taxan described in claim 5,6,7, is characterized in that, comprises also in described wheat bran substratum that weight percent is 0.01%～1.0% xylan.
9. the preparation method of Taxan as claimed in claim 8, is characterized in that, described xylan is selected from oat xylan, birch xylan.
10. the preparation method of arbitrary Taxan described in claim 5,6,7, is characterized in that, comprises also in described wheat bran substratum that weight percent is 0.01～1.0% ammonium sulfate.
11. the preparation method of Taxan as claimed in claim 3, it is characterized in that, substrate 7-wood sugar Taxan is selected from 10-DAXT, 7-wood sugar taxol, 7-wood sugar-10-and goes acetyl baccatin III, XB III, 7-wood sugar-10-to remove acetyl Cephalomannine, 7-wood sugar Cephalomannine, 10-DAXT C or 7-wood sugar taxol C.
12. the preparation method of a Taxan, it is characterized in that, utilize enterobacter cloacae as claimed in claim 1 (Enterobacter cloacae) or its resting cell in peptone extractum carnis substratum in the hydrolysis substrate bearing taxanes with the 13-side chain of 13-side chain Taxan; Described peptone extractum carnis substratum comprises it being liquid protein peptone extractum carnis substratum and solid protein peptone extractum carnis substratum.
13. the preparation method of Taxan as claimed in claim 12, it is characterized in that, the temperature of reaction that enterobacter cloacae (Enterobacter cloacae) or its resting cell are hydrolyzed with 13-side chain Taxan in peptone extractum carnis substratum is 15～50 ℃.
14. the preparation method of Taxan as claimed in claim 12 is characterized in that, contains peptone 2～20g/L in liquid protein peptone extractum carnis substratum, beef extract 1～5g/L, and NaCl 1～10g/L, the pH value is 5.0～8.0.
15. the preparation method of Taxan as claimed in claim 12 is characterized in that, also comprises macroporous resin 5～40g/L in liquid protein peptone extractum carnis substratum, the pH value is 5.0～8.0.
16. the preparation method of Taxan as claimed in claim 12 is characterized in that, contains peptone 2～20g/L in solid protein peptone extractum carnis substratum, beef extract 1～5g/L, and NaCl 1～10g/L, agar 10～20g/L, the pH value is 5.0～8.0.
17. the preparation method of Taxan as claimed in claim 12, it is characterized in that, the bearing taxanes that the C-13 position is connected with side chain is selected from 10-deacetyl taxol, taxol, 10-and removes acetyl Cephalomannine, Cephalomannine, 10-deacetyl taxol C or taxol C.
18. the preparation method of a Taxan, it is characterized in that, utilize enterobacter cloacae as claimed in claim 1 (Enterobacter cloacae) or its resting cell, 7-xylose group in the hydrolysis substrate bearing taxanes and the enzymatic reaction condition of 13-side chain are: concentration is Tris-HCl damping fluid or the phosphoric acid buffer of 0.01mol/L～1.0mol/L, pH is 4.5～8.0, and temperature is 15～50 ℃.
19. the preparation method as claim 3 or 12 described Taxans, it is characterized in that, described substrate bearing taxanes is selected from from Ramulus et folium taxi cuspidatae and extracts monomer bearing taxanes or its crude extract that obtains, monomer bearing taxanes or its crude extract that passes through Ramulus et folium taxi cuspidatae histocyte cultivation acquisition.
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| 曾宇成等.海枣曲霉β-木糖苷酶的提纯和性质.《微生物学报》.1993,第33卷(第6期),摘要. * |
| 郑莉等.β-木糖苷酶的研究进展.《浙江林业科技》.2005,第25卷(第1期),第60页倒数第6行. * |
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