CN101684451A - Microorganism for hydrolyzing 7-xylose group and 13-side chain of taxane - Google Patents

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 preparingantitumor drugs, such as intermediates of taxane drugs, docetaxel drugs or taxane drugs.

Description

A kind of 7-xylose group of hydrolysis taxanes and the microorganism of 13-side chain

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 a kind of both can be in the wheat bran substratum 7-xylosyl of hydrolysis 7-wood sugar Taxan, again can be in peptone extractum carnis substratum the enterobacter cloacae (Enterobacter cloacae) of the 13-side chain in the hydrolysis band 13-side chain Taxan molecule, and a kind of method of utilizing enterobacter cloacae (Enterobacter cloacae) to prepare Taxan.

Background technology:

Taxol is isolated diterpene alkaloids with unique antitumour activity from red bean section Chinese yew genus plants yewtree bark such as Wani in 1971.Its chemical structural formula is:

Taxol has been got permission listing in more than 40 countries since 1992 are used for the treatment of ovarian cancer and metastatic breast cancer by 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 still is 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, so supply falls short of demand in clinical application for present taxol, 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 countries such as the U.S., Japan, France carry out full chemosynthesis taxol has more than 30, succeeds finally through the effort of two more than ten years.1993, as initiator, the complex chemical reaction through about 30 steps 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, does not still have actual application value.

(3) chemistry is semi-synthetic: extraction itself there is no active paclitaxel analogs (removing the acetyl baccatin III as baccatin III and 10-) as intermediate, by the chemical process taxol biosynthesis from can the regenerated plant leaf.This also is 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 because yew cell throughput instability still can not suitability for industrialized production.

(5) endogenetic fungus fermentation: the upright 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 omits height 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, also is 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 bring some difficulties to separation and purification, cause the great wasting of resources and environmental pollution.These analogues comprise: 7-wood sugar-10-removes the acetyl taxol, 7-wood sugar taxol, and 7-wood sugar-10-removes the acetyl baccatin III, 7-xylose baccatin III, 7-wood sugar-10-removes the acetyl Cephalomannine, 7-wood sugar Cephalomannine, 7-wood sugar-10-removes acetyl taxol C, 7-wood sugar taxol C; And bearing taxanes such as 10-that the C-13 position is connected with side chain remove the acetyl taxol, and 10-removes the acetyl Cephalomannine, and Cephalomannine, 10-remove acetyl taxol C, taxol C.Utilize chemical process that thereby the structure of modification of these paclitaxel analogs is obtained the existing report of taxol, but exist selectivity low, easily generate isomer, by product is many and reactions steps is long, productive rate is low, cause problem such as environmental pollution.And by the biocatalysis means these " refuses " are used, be used to produce the taxone important intermediate and carry out chemistry semi-synthetic, this method not only can make full use of resource, and can not destroy environment.

Especially, numerous documents show, 7-wood sugar-the 10-that is grown 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 goes the content of acetyl taxol 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 that this compounds is carried out directed bio-transformation, 7-xylose group as hydrolysis 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 advantages such as environmental protection, has bigger society and economic benefit.

In analysis, 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 the side chain hydrolysis of C-13 position to existing document.And yet there are no bacterial strain with dual-use function, and causing preparation process complexity, the cost height of Taxan, 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 provides the purposes of this microorganism;

Another goal of the invention of the present invention provides 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 being produced 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 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 the peptone extractum carnis culture medium slant, and the preservation temperature is about 4 ℃, also can be deposited in the liquid nutrient medium that is added with glycerine, and the preservation temperature is-20～-80 ℃.

Enterobacter cloacae be by to derive from pedotheque below the taxus chinensis in northeast arboreal growth peripheral ground 5cm 200 surplus the strain microorganism carry out 7-wood sugar-10-and go the screening of acetyl taxol xylose group hydrolysis ability to obtain.

When utilizing enterobacter cloacae, can use complete wet cell or stem cell, the cell that exists as the cells form of freeze dried, spray-dired or heat drying; Perhaps cell material to handle, the cell that exists as disruptive cell or cell extract form; 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 to express 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, the 13-side chain of band 13-side chain Taxan in the hydrolysis substrate bearing taxanes in peptone extractum carnis substratum.

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 a 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, the enzyme by several different methods extraction, purifying comprises 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 ₂O 0.01～0.5g/L; Be preferably: wheat bran 20～60g/L, (NH ₄) ₂HPO ₃1～3g/L, K ₂HPO ₄0.08～0.3g/L, MgSO ₄7H ₂O 0.08～0.3g/L; Most preferably be: wheat bran 40g/L, (NH ₄) ₂HPO ₃2g/L, K ₂HPO ₄0.1g/L, MgSO ₄7H ₂O 0.1g/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 ₂O 0.01～0.5g/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 ₂O 0.1g/L, agar 15g/L.

The another preferred version of wheat bran substratum is: can also add weight percent in the 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 behind the interpolation 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 the liquid wheat bran substratum; Be preferably 10～30g/L, most preferably be 20g/L.

Wherein, wheat bran medium 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: 7-wood sugar-10-removes the acetyl taxol, 7-wood sugar taxol, 7-wood sugar-10-removes the acetyl baccatin III, 7-xylose baccatin III, 7-wood sugar-10-removes the acetyl Cephalomannine, 7-wood sugar Cephalomannine, 7-wood sugar-10-remove acetyl taxol 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 13-side chain of band 13-side chain Taxan in the hydrolysis substrate bearing taxanes in peptone extractum carnis substratum.

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 the peptone extractum carnis liquid nutrient medium, beef extract 1～5g/L, NaCl 1～10g/L; Be preferably: peptone 5～15g/L, beef extract 1～3g/L, NaCl 3～6g/L; Most preferably be: peptone 10g/L, beef extract 2g/L, NaCl 5g/L.

Wherein, contain peptone 2～20g/L in the peptone extractum carnis solid medium, beef extract 1～5g/L, NaCl 1～10g/L, agar 10～20g/L; Be preferably: peptone 5～15g/L, beef extract 1～3g/L, NaCl 3～6g/L, agar 12～18g/L; Most preferably be: peptone 10g/L, beef extract 2g/L, NaCl 5g/L, agar 15g/L.

The preferred version of peptone extractum carnis substratum is: also comprise macroporous resin 5～40g/L in the 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 among 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-removes the acetyl taxol, and taxol, 10-remove the acetyl Cephalomannine, and Cephalomannine, 10-remove acetyl taxol C, taxol C.

7-wood sugar-10-in the substrate goes the 10-in acetyl taxol and the product to go the acetyl taxol all to have to suppress the cell fission effect very doughtily.In order to strengthen the input amount of substrate, in substratum, add a small amount of macroporous resin and can improve productive rate.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 the reaction system by resin absorption, behind substrate reactions, substrate content in the 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 action of microorganisms.

The substrate bearing taxanes can be cyclodextrin encapsulated, polyvinylpyrrolidone hydrotropy, tween hydrotropy and make 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 being produced, can in substratum, damping fluid, react, also can add immobilized cells such as resin, sodium alginate, wilkinite or enzyme material and carry out immobilized reactant.In substratum, add a small amount of fixed reagent and can improve productive rate.

The resting cell of enterobacter cloacae or its functional equivalent, varient and mutant or engineering strain, and the enzyme of being produced can liquid liquid two-phase 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 beneficial technical effects that the present invention has is:

1. enterobacter cloacae of the present invention has dual function, 7-xylosyl in promptly can hydrolysis 7-wood sugar bearing taxanes molecule in the wheat bran substratum, again can be in peptone extractum carnis substratum the 13-side chain in the hydrolysis band 13-side chain Taxan molecule.Thereby simplified the preparation technology of Taxan.

2. the biological activity of enterobacter cloacae of the present invention is strong, for going down to posterity and to cultivate requirement condition low, is suitable for large-scale industrial production.

3. the preparation method of Taxan of the present invention has utilized the waste that extracts taxol in the prior art fully, turns waste into wealth, and produces huge economic benefit, has protected ecotope.

4. the preparation method of Taxan of the present invention carries out directed hydrolysis at different substrate bearing taxanes, can directly obtain the intermediate of taxol or taxol biosynthesis and Docetaxel.

Abbreviation and term

XDT:7-wood sugar-10-removes the acetyl 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 (last 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 ₄7-wood sugar-10-is gone the influence of acetyl taxol conversion

The influence that Fig. 7 xylan goes the acetyl taxol to transform to 7-wood sugar-10-

The influence that Fig. 8 temperature goes the acetyl taxol to transform to 7-wood sugar-10-

Fig. 9 enterobacter cloacae transforms 7-wood sugar-10-and goes the acetyl taxol to generate the performance graph that 10-removes the acetyl taxol

Figure 10 7-wood sugar-10-goes the bio-transformation of acetyl taxol liquid-solid two-phase

Embodiment:

In order further to understand the present invention, the following examples only are used for further specifying the present invention, but and do not mean that any limitation of the invention.

Embodiment 1: screening of enterobacter cloacae and evaluation

To derive from pedotheque below the taxus chinensis in northeast arboreal growth peripheral ground 5cm 200 surplus the strain microorganism carried out the screening that 7-wood sugar-10-removes acetyl taxol xylose group hydrolysis ability, the result shows that the bacterial strain enterobacter cloacae has 7-wood sugar-10-and removes acetyl taxol 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 (N, dinethylformamide) solvent as blank, and another bottle adds the DMF solution that substrate 7-wood sugar-10-removes the acetyl taxol.Continue to cultivate after 7 days the nutrient solution ethyl acetate extraction after the filtration.Analyze technology for detection by thin-layer chromatography, high performance liquid chromatography, liquid phase mass spectrometry etc. behind the extraction liquid concentrating under reduced pressure and confirm product.Subsequently, be prepared bio-transformation, obtain product, and confirm by the NMR (Nuclear Magnetic Resonance) spectrum technology by column chromatography repeatedly.

1. enterobacter cloacae goes the hydrolysis of acetyl taxol to 7-wood sugar-10-

The bacterium colony of the enterobacter cloacae that step obtains on the picking 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 that transforms microorganism.Wheat bran substratum food preparation is: wheat bran 50g, (NH ₄) ₂HPO ₃2g, K ₂HPO ₄2g, MgSO ₄7H ₂O 0.1 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.7-wood sugar-the 10-that adds 30mg/ml in the growth logarithmic phase of bacterium removes the solution 0.5ml of acetyl taxol.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. go the TLC (thin-layer chromatography) of acetyl taxol to detect to product 10-

After a large amount of screenings, find in the reaction solution of enterobacter cloacae except 7-wood sugar-10-removes the brown spot (Rf value=0.3) of acetyl taxol, also have another brown spot at polarity smaller part (Rf value=0.5), and the position of this spot and reference substance 10-remove acetyl taxol position, solid colour.

3. go the HPLC (high performance liquid chromatography) of acetyl taxol to detect to product 10-

With the reaction solution extract of enterobacter cloacae with the dissolve with methanol constant volume to 1ml, with behind the 0.22 μ m membrane filtration as test liquid detection usefulness 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 Agilent 1100 type HPLC chromatographic instruments.It is that standard substance are preserved in the laboratory that 10-removes the acetyl taxol.In the liquid chromatogram of standard substance and enterobacter cloacae reaction solution as can be seen the reaction solution of enterobacter cloacae at the retention time (t identical with standard substance _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. go the liquid chromatography mass coupling (LC/MS/MS) of acetyl taxol to detect to product 10-

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), for test agent, 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/z 750-1050.

Chromatographic peak P in the reaction solution HPLC collection of illustrative plates is carried out the LC-MS analyzing and testing, see accompanying drawing 2.In the one-level mass spectrum, obviously detect m/z 812[M+H] ⁺, m/z 834[M+Na] ⁺, m/z 859[M+K] ⁺Quasi-molecular ion peak, the molecular weight that shows this compound is 811, this and 10-go the molecular weight of acetyl taxol to coincide.Further carry out second order ms and detect, can find m/z 268,286,527,794,751 equimolecular fragment peaks.And these also all go the cleavage of mass spectrum feature of acetyl taxol to match with 10-.

5.10-the preparation of removing the acetyl taxol with separate

Main raw and method are with step 1.Go the acetyl taxol to be dissolved among the 17mlDMF 500mg 7-wood sugar-10-, make the DMF solution of substrate.In 35 1000ml triangular flasks that 350ml wheat bran substratum is housed, add substrate DMF solution 0.5ml; Room temperature condition transforms down to be cultivated after 7 days, filtered.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 behind the column chromatography repeatedly, obtain 10-and remove acetyl 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 accompanying drawing 4 and accompanying drawing 5 respectively.

6), converted product 10-goes the nuclear magnetic resonance spectrum data of acetyl 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?of?3′-C ₆H ₅)，7.40(2H，m，m-H?of3′-C ₆H ₅)，7.37(2H，m，m-H?of?NH-Bz)，7.33(1H，m，p-H?of?3′-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?of?3-CO C ₆H ₅)，136.1(C-11)，133.7(p-C?of?2-CO C ₆H ₅)，133.6(quaternary?C?of?3′- C ₆H ₅)，131.9(p-Cof?3′- C ₆H ₅)，130.2(o-C?of?2-CO C ₆H ₅)，129.17(quaternary?C?of?2-CO C ₆H ₅)，128.96(m-C?of?NH-CO C ₆H ₅)，128.71(m-C?of?2- COC ₆H ₅)，128.68(m-C?of?3′- C ₆H ₅)，128.3(p-C?of?NH-CO C ₆H ₅)，127.05(o-C?of?3′- 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 influence that different substratum go the acetyl taxol to transform to 7-wood sugar-10-

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, NaCl 5g, peptone 10g, water 1000ml;

Culture medium C: glucose (or in the 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 ₂O 0.2g, FeSO ₄7H ₂O 0.01g, NaCl 0.01g, MnSO ₄4H ₂O 0.01g;

Substratum D: potato 200g (boiling the 20min after-filtration), 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 the aseptic condition, add 1ml to each bottle and cultivated 2 days enterobacter cloacae suspensions.Culture condition is: 28 ℃, and 200rpm.Continue to cultivate 2 days, add the DMF solution that 250 μ l substrate 7-wood sugar-10-remove the acetyl taxol.With each bottle of equal volume of ethyl acetate reaction solution 3 times, merge organic phase after continue to cultivate transforming 7 days, carry out TLC and HPLC behind the concentrating under reduced pressure and analyze.Each be treated to 2 parallel.

The result shows: bacterial strain only could remove the xylose group of acetyl taxol by hydrolysis 7-wood sugar-10-after fermentation in culture medium A, and the side chain hydrolysis reaction mainly takes place in substratum B, and substrate is had an effect hardly in culture medium C.Find when in culture medium A, adding different carbon sources (as fructose and semi-lactosi etc.) that 10-goes the productive rate of acetyl taxol but almost not improve.See Table 1.

Table 1: enterobacter cloacae removes the acetyl taxol to 7-wood sugar-10-in 4 kinds of substratum conversion

Embodiment 3: the ammonium sulfate of different concns goes the influence of acetyl taxol xylose group hydrolysis to 7-wood sugar-10-

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; In the wheat bran substratum, add ammonium sulfate, 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.The residue that obtains after concentrating with dissolve with methanol and be settled to 10ml, is filtered the back as the HPLC test liquid.Each concentration gradient do 3 times parallel.

1), standard solution preparation: precision takes by weighing 7-wood sugar-10-and removes acetyl taxol 6.1mg respectively, and 10-removes acetyl taxol 9.7mg, but 7-wood sugar-10-removes acetyl crust booth III 1.6mg, with dissolve with methanol and constant volume to 5ml.Accurate respectively 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.

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:

(NH in the wheat bran substratum ₄) ₂SO ₄Productive rate to product has promoter action.Content is 0.4% o'clock, and the output of converted product is up to 9.4mg/L, and transformation efficiency also reaches 52.8%.See accompanying drawing 6.

Embodiment 4: xylan goes the influence of acetyl taxol xylose group hydrolysis to 7-wood sugar-10-

Preparation wheat bran substratum: wheat bran 50g, K ₂HPO ₄2g, (NH ₄) ₂ HPO ₃2 grams, (NH ₄) ₂SO ₄4 grams, MgSO ₄7H ₂O 0.1 gram, distilled water is settled to 1000ml; The xylan that adds different concns again makes the concentration of adding 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 that transforms cultivation, substrate and product is with embodiment 1.Each handle 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 that selective hydrolysis 7-wood sugar-10-removes acetyl taxol 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: temperature is gone the influence of acetyl taxol xylose group hydrolysis to 7-wood sugar-10-

Preparation wheat bran substratum: wheat bran 50g, K ₂HPO ₄2g, (NH ₄) ₂ HPO ₃2 grams, (NH ₄) ₂SO ₄4 grams, MgSO ₄7H ₂O 0.1 gram, xylan 0.6 gram, distilled water is settled to 1000ml.The sterilization back adds 1ml and has cultivated 2 days seed in every triangular flask, add substrate 7-wood sugar-10-and remove the acetyl taxol after 24 ℃ continuation is cultivated 2 days down.Under different temperature condition, transform.Temperature 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 handled parallel 3 times.10-goes the output of acetyl Japanese yew (DT) and 7-wood sugar-10-to go the residual content of acetyl taxol (XDT) to detect by HPLC.The result shows that in the temperature range of being tested, temperature is high more, and hydrolysis reaction carries out also thoroughly more.Transform 7 days under 37 ℃ of conditions, transformation efficiency is 18.1%, sees accompanying drawing 8.

Embodiment 6:7-wood sugar-10-goes the hydrolysis of acetyl taxol xylose group to generate the performance graph that 10-removes the acetyl taxol

Preparation wheat bran substratum: wheat bran 50g, K ₂HPO ₄2g, (NH ₄) ₂ HPO ₃2 grams, (NH ₄) ₂SO ₄4 grams, MgSO ₄7H ₂O 0.1 gram, xylan 0.6 gram, distilled water is settled to 1000ml.Intake Quantity is 50ml in each 250ml triangular flask.Cultivation, conversion operation method are with embodiment 1.Change each triangular flask over to 37 ℃ after substrate adds and transform cultivation.Converted product 10-goes the content of acetyl taxol (DT) and substrate 7-wood sugar-10-to go the residual content of acetyl taxol (XDT) to detect 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 concentrating with dissolve with methanol and be settled to 10ml, solution behind 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 that 7-wood sugar-10-removes the acetyl taxol for determining enterobacter cloacae, sampling in per 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.

Go the acetyl taxol to carry out in the process of bio-transformation with enterobacter cloacae to 7-wood sugar-10-, two reactions have taken place altogether: 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 than 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-goes the bio-transformation of acetyl taxol liquid-solid two-phase

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; Intake Quantity is 50ml in each 250ml triangular flask.In each triangular flask, put into the cloth bag that the 1g macroporous resin is housed again.Cultivate the step 1 of conversion operation method with 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 substrate 7-wood sugar-10-to go the residual content of acetyl taxol (XDT) to detect by high performance liquid chromatography.Take out in the pocket macroporous resin and wash repeatedly with ethanol after transforming 7 days, fermented liquid usefulness ethyl acetate extraction 3 times merges 2 part organic solvents.With the residue that obtains after concentrating with dissolve with methanol and be settled to 10ml, solution behind 0.45 μ m membrane filtration as the HPLC test liquid.

7-wood sugar-10-goes acetyl taxol and 10-to go the acetyl taxol all to have to suppress the cell fission effect very doughtily.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 in the substratum and can adsorb substrate and product, in conversion process, play the effect of the slow controlled release of compound.Majority of compounds is not entered in the middle of the reaction system by resin absorption, behind substrate reactions, substrate content in the 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 action of microorganisms.

In this experiment, the substrate that adds different concns does not have 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 handled, the residual content of substrate XDT and the output of DT and D were roughly suitable in handling, but the input amount of XDT but is 2 times that D handles.Reason may be that the XDT add-on is excessive in E handles, 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) collects 2 days cloaca intestines rod suspension 1L of growth in the wheat bran substratum;

2) with bacteria suspension centrifugal 20min under 20,000 * g condition.Topple over and supernatant (extracellular fluid), precipitation part (living cell body) is suspended in 50mmol again, and pH is in 6.0 the potassium phosphate buffer;

3) all add substrate DMF solution in last cleer and peaceful precipitation part.At 37 ℃, the 24h that vibrates under the 200rpm condition, TLC detects enzyme and lives;

4) the centrifugal living cell body that obtains is several all over residual to guarantee acellular outer liquid with the distilled water flushing.Cell is suspended in the distilled water again lyophilize;

5) collect stem cell, suspend ultrasonication again with the potassium phosphate buffer of 50mmol pH 6.0.Condition: 220V, ultrasonic 2s, 8s handles ice bath 80 times at interval.Discontinuity is rocked in its process.

6) with cell centrifugal 20min under 20,000 * g condition of fragmentation, inclining supernatant (tenuigenin), precipitation part (big cell membrane fragments and not broken cell);

7) all add substrate DMF solution in last 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, the result only shows that 2 precipitations after centrifugal partly show xylosidase activity.This explanation xylosidase is an intracellular enzyme, and is not free in the tenuigenin.

Claims (19)

1. a culture presevation number is CGMCC No.2487 enterobacter cloacae (Enterobacter cloacae) or resting cell, varient and the mutant of its functional equivalent and the enzyme of production thereof.

2. the purposes of resting cell, varient and the mutant of enterobacter cloacae as claimed in claim 1 (Enterobacter cloacae) or its functional equivalent and the enzyme of production thereof, it is characterized in that, at the 7-xylose group of wheat bran substratum at wheat bran substratum 7-wood sugar Taxan in the hydrolysis substrate bearing taxanes in the wheat bran substratum, the 13-side chain of band 13-side chain Taxan in the hydrolysis substrate bearing taxanes in peptone extractum carnis substratum.

3. the preparation method of a Taxan, it is characterized in that, utilize enterobacter cloacae as claimed in claim 1 (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.

4. the preparation method of Taxan as claimed in claim 3, it is characterized in that, enterobacter cloacae (Enterobacter cloacae) or with the temperature of reaction of resting cell, varient and the mutant hydrolysis 7-wood sugar Taxan in the wheat bran substratum of its functional equivalent be 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 the 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 the 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 the 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 the described substratum that weight percent is 0.01%～1.0% xylan.

9. as the preparation method of Taxan as described in the claim 8, it 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 the 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 7-wood sugar-10-and goes acetyl taxol, 7-wood sugar taxol, 7-wood sugar-10-to go acetyl baccatin III, 7-xylose baccatin III, 7-wood sugar-10-to go acetyl Cephalomannine, 7-wood sugar Cephalomannine, 7-wood sugar-10-to remove acetyl taxol 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 with resting cell, varient and the mutant of its functional equivalent 13-side chain of band 13-side chain Taxan in the hydrolysis substrate bearing taxanes in peptone extractum carnis substratum.

13. the preparation method of Taxan as claimed in claim 12, it is characterized in that the temperature of reaction of resting cell, varient and the mutant of enterobacter cloacae (Enterobacter cloacae) or its functional equivalent hydrolysis band 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 the 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 the 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 the 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 selects white 10-to go acetyl taxol, taxol, 10-to go acetyl Cephalomannine, Cephalomannine, 10-to remove acetyl 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 with resting cell, varient and the mutant of its functional equivalent and the enzyme of production thereof, the 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. 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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