WO2009030098A1

WO2009030098A1 - A cellulosimicrobium cellulans, its hydrolase and in the use of transformation of taxanes

Info

Publication number: WO2009030098A1
Application number: PCT/CN2008/000618
Authority: WO
Inventors: Ling Yang; Hongwei Luan; Xingbao Liu
Original assignee: Dalian Institute Of Chemical Physics, Chinese Academy Of Sciences
Priority date: 2007-09-04
Filing date: 2008-03-28
Publication date: 2009-03-12
Also published as: CN101381708A

Abstract

A cellulosimicrobium cellulans and its hydrolase, which effectively transforms taxane xylosides into paclitaxel or its analogs. A method of high effectively preparing paclitaxel and its analogs by biotransformation reactions using the hydrolase.

Description

Pseudomonas aeruginosa, hydrolase and its use in taxane conversion

The invention belongs to the technical field of biochemistry, and in particular relates to a method for preparing a microorganism for hydrolyzing taxane xylosides and using the same to prepare paclitaxel and the like. Background technique

Taxus is a class of diterpenoids, of which paclitaxel (formula) and docetaxel are widely used as a first-line clinical treatment for a variety of tumors. At present, the main routes for the production of paclitaxel are: (1) direct extraction of paclitaxel from yew plants; (2) semi-synthesis method - extraction and separation of compounds with paclitaxel core structure from yew plants, followed by chemistry Methods Semi-synthetic paclitaxel, docetaxel and the like. Such compounds having a paclitaxel core structure mainly include baccatin III and 10-deacetylbaccatin (abbreviated 10-DABin).

Wherein Ph is a phenyl group, Ac is an acetyl group, and Bz is a benzoyl group. In addition to the above-mentioned Baccatin III and 10DAB, another compound having a paclitaxel core structure, that is, a C-7 xylosyl taxane such as 7-xylose, is widely present in the genus Taxus. Paclitaxel (XT), 10-deacetyl-7-xylose-taxol (10-DAXT), 10-deacetyl-7-xylose cephalosporin (10-DAXC:), 10-deacetyl-7-wood Taxol C ( 10-DAXTC), a compound that can be removed by biological or/and chemical means to produce a taxol compound of paclitaxel or a C-7 hydroxyl group, which can be used as a preparation for the anticancer activity of docetaxel An important intermediate to achieve efficient use of yew resources. Therefore, the removal of C-7 xylosyl has become an effective use of such compounds. The key factor. At present, there are mainly chemical methods and biological methods for the removal of C-7 taxane xylosidose glycosyl groups. The chemical method is to first oxidize xylose to hemiacetal by periodate, and then hydrolyze it under acidic conditions to obtain a C-7 hydroxyl taxane (US5412116, US6028206, US5856532, US6437154) _. The method requires two steps. The reaction removes the C-7 xylosyl group, resulting in a generally low yield (<50%). In addition, due to the use of highly toxic substances such as hydrazine hydrate in chemical methods, such as industrialization, it will cause serious pollution to the environment.

Because of its environmental friendliness, biological law has attracted more and more people's attention. European Patent No. 95300135.1 discloses a method for hydrolyzing taxol glycosidic glycosyl groups using Moraxdla sp., Bacillus circulans, Bacillus circulans and Micrococcus sp. . Among them, the transformation ability of Moraxella bacillus is strong, 62.5mg of Moraxella (from 20ml fermentation broth) can completely convert 0.5mg 7-xylose-10-deacetylpaclitaxel into 10_deacetylpaclitaxel in 7 hours (Biotechnol Appl Biochem 1997, 26, 153- 158). Other than that, no other relevant reports have been reported.

The microbial enzyme production in the existing technology is generally low, and both are intracellular enzymes, resulting in a decrease in hydrolysis efficiency, and the cells are easily adsorbed to substrates and products, and thus are not suitable for industrial production. Disclosure of invention

The object of the present invention is to provide a fibroblastic cellulase and a hydrolase thereof which can efficiently convert a taxane xyloside to paclitaxel or an analogue thereof, and provide a novel preparation of paclitaxel and the like by biotransformation reaction. An effective method.

The present invention provides a hydrolase produced by the metabolism of Cellulosi microbium cellulans.

The hydrolase provided by the present invention is used for transforming a C-7 taxane xyloside into paclitaxel or an analog thereof. The invention also provides a biotransformation preparation method of paclitaxel and the like: hydrolyzing the hydrolase produced by releasing the hydrolyzed enzyme produced by the cellulosic cellulosum Cdlulosi microbium cellulans with the C-7-position taxane xyloside as a raw material; The xylosyl group at the C-7 position is removed to obtain paclitaxel or an analog thereof.

In the biotransformation preparation method of the above-mentioned paclitaxel and the like, the hydrolysis reaction conditions are as follows: the raw material is added to the hydrolase at a ratio of 0.1 to 2 g/L, and the temperature is 20-40 degrees. 6.0-8.0, reaction time 3-48h.

The present invention provides a starting material of the C-7 taxane xylosidide compound having the formula I:

(Formula I)

Wherein 11 or an acetyl group; R ₂ and the same or different, a T or an OT; wherein T is H, an aryl group, an aralkyl group, a linear, branched or cyclic hydrocarbon group of C 1 to C 20 , and the above Substituents for three hydrocarbyl groups, including hydroxy, C 1 to C 8 alkoxy, acetal, ketal, halogen, nitro or amino.

The present invention provides a raw material of a taxane lignin compound, which is a natural product derived from a monomer having a xylosyl-containing docetaxel compound at the C-7 position or a mixture thereof, or via biosynthesis, chemical semi-synthesis or A xylan-containing taxane obtained at the above position obtained by a total synthesis means.

In the method for biotransformation preparation of paclitaxel and the like, the hydrolase is used in a free state, or is immobilized on a support by physical adsorption or trapping.

The present invention provides a new species of Cellulosi microbium celIulans XZ-5 CCTCC No. M 207130.

In order to achieve the object of the present invention, the inventors screened soil microbial transformation activities in various regions, and found that Pseudomonas aeruginosa can produce secreted xylosidase, hydrolyze taxane xylosides, and obtain paclitaxel and the like. Things. However, the new strain of C. fibrosis (Ce// a? z'crob M cellulans ) XZ-5 CCTCC No. M 207130 obtained from the root soil of Taxus chinensis in Zhejiang Yiwu has the strongest biotransformation activity. The secreted xylosidase can be produced at a high level, and the taxane xyloside is hydrolyzed to obtain paclitaxel and the like.

The cellulosic fibrosis according to the present invention, such as CCTCC M207130, AS 1.2019, AS 1 .1938, AS 1.1920, ACCC0101, is characterized in that: the mycelium is produced in the early stage of the growth cycle, and is broken in the late stage; The coryneform bacteria are transformed into short rods or even spherical cells; on the peptone yeast extract-glucose agar plate, the colonies are round, 0.9~5.0 mm in diameter, yellow-white protrusions, reflective, and neat edges. The following substances can be used as the sole carbon source: glucose, mannose, maltose, sucrose, D-xylose, glycerol, cellobiose, lactose, L-lactate, acetate, pyruvate, propionate, pentane Acid salt, proline, asparagine, aspartic acid, histidine. The raffinose, DL-malic acid and D-lactate cannot be used as the sole carbon source; the catalase is positive; the peptidoglycan contains L-lysine, and the endopeptide bridge is composed of D-Ser-D-Asp, type A4ot Composition; the main sugar component of the cell wall is rhamnose, while fucoid. Sugar and galactose are secondary components; the main menaquinone is MK-9 (H ₄ ), while MK-9 (H ₂ ), MK-8 (H ₄ ) and MK-7 (H ₄ ) are secondary components; the GC content of genomic DNA is 74 mol%. The cellulosimicrobium cellulans XZ-5 of the present invention has been deposited at the China Microbial Collection Center of Wuhan in August 2007 under the accession number CCTCC No. M 207130.

The biologically pure microbial fibrosis, is a newly discovered β-xylosidase-producing microbe. Mutants of such microorganisms, such as mutant strains engineered for chemical hydrolysis (e.g., ultraviolet radiation) or biological methods (e.g., molecular biology techniques) for hydrolysis reactions, are also within the scope of the present invention.

The enzymes used in the present invention are hydrolyzed hydrazines, especially xylosidase. The microorganisms provided by the present invention produce the above enzymes, which can be isolated by extraction and purification. The genetically modified form of the microorganism provided by the present invention is also contemplated by the present invention. The host cell may be any cell such as Escherichia coli, and a gene or a group of genes of the microorganism provided by the present invention is transferred into a host to express an enzyme or a plurality of enzymes required for catalyzing a hydrolysis reaction.

The raw material to which the present invention relates is a taxane glucoside compound having the structural formula of Formula I -'

Wherein 11 or acetyl; R ₂ and R _{3 are the} same or different and are a T or an OT; wherein T is H, aryl, aryl fluorenyl, a C1 to C20 linear, branched or cyclic hydrocarbon group, and Substituents for the above three hydrocarbon groups, the substituent includes a hydroxyl group, a C1 to C8 decyloxy group, an acetal, a ketal, a halogen, a nitro group or an amino group.

In particular, the taxane xylosidide compound can be either a natural product or a biosynthesis, or chemical semi-synthesis or total synthesis, which can be obtained by a xylan-containing taxane at the C-7 position, such as 7 - Xylose Paclitaxel, 7-Xylose-10-Deacetyl Paclitaxel, 7-Xylose Baccarat III, 7-Xylose- 10-Deacetylbaccatin III, 7-Xoseose Cephalosporin, 7 - Xylose - 10-deacetyl cephalosporin, 7-xylocheolol C or 7-xylose-10-deacetylpacepol C and 7-xylose-10-deacetylbaccatin and the like. The hydrolysis method provided by the present invention has taken into account all stereo configurations of the chiral centers of the compounds having the molecular structure I, which are either hydrolyzed alone or mixed with other stereoisomers to be hydrolyzed.

The method provided by the present invention preferentially retains the stereo configuration of the C-7 group of the hydrolyzed taxane in the product. The absolute stereo configuration of the C-7 substituent is identical to the absolute stereo configuration of the paclitaxel C-7 hydroxyl group.

The hydrolysis method provided by the present invention can be carried out after microbial fermentation (two-stage fermentation and hydrolysis), or simultaneously with fermentation (single-stage in situ fermentation and hydrolysis), or immobilization of the enzyme onto a solid support (such as an anion exchanger) ), the hydrolysis reaction is carried out.

With the method provided by the present invention, the yield is over 90%, and the C-7 position can be specifically selected for hydrolysis. Depending on the characteristics of the hydrolyzate, it may be purified by using it singly or in combination as follows: an organic solvent extraction method, an adsorption-desorption method, a precipitation method, a recrystallization method, and the like. For example, the hydrolyzate is extracted from the reaction liquid using ethyl acetate, and the obtained organic layer is concentrated under reduced pressure, and the obtained concentrate is adsorbed on a silica gel column, and then chloroform-methanol, hexane-ethyl acetate Chromatography with a mixed solvent system of hexane-acetone. Further, if necessary, the fraction containing the hydrolyzate may be crystallized from an organic solvent such as ethyl acetate or methanol.

The method of the invention can prepare paclitaxel and its analogues efficiently, inexpensively and environmentally, and provides an effective way to adapt to industrial production for making full use of the taxol resources. The best way to implement the invention

The following examples are intended to further illustrate the invention and are not intended to limit the scope thereof.

Example 1: Cellulomonas fibrosus CCTCC No. M 207130 Hydrolyze 7-xylose paclitaxel A medium containing 1% glycerol, 0.2% peptone, 0.2% yeast extract, 0.2% dipotassium hydrogen phosphate (before sterilization) pH 7.0) as a seed medium.

20 ml of the above seed culture medium was dispensed into a 100 ml Erlenmeyer flask, sterilized at 120 degrees for 15 minutes, and a 1 inoculum of the agarose slant culture of the P. fibrosus XZ-5 strain was inoculated therein, and at 30 degrees. The cells were cultured under shaking for 2 days as a seed liquid.

Place 500ml Erlenmeyer flask in 100ml containing 1°/. Starch, 0.2% yeast extract, 0.2% ammonium chloride, 0.1% NaH ₂ PO ₄ and 0.1% Na ₂ HPO ₄ medium, pH 7, autoclaved at 121 ° 30 min. 1 ml of the seed solution was inoculated to the medium, and cultured at 150 rpm for 5 days in a shake flask at 30 °C. Centrifugation The supernatant is collected, which is the crude enzyme solution.

To 0.9 ml of the crude enzyme solution, 1 ml of 50 mM potassium phosphate buffer (pH 7) was added, and 0.1 ml of a 7-xylose-paclitaxel methanol solution (concentration: 5 mg/ml) was further added. Mix lOOrpm at 30 ° C for 5 hours. The reaction was stopped by adding 2 ml of methanol, and the supernatant was taken for 1 minute at 15,000 rpm for HPLC analysis. There was no residue of 7-xylose-paclitaxel in the reaction solution, and 0.42 mg of paclitaxel was produced (yield 94%). HPLC method: Column: Kromasil ODS (4.6x200mm, 5μηι)

Mobile phase: Methanol: Water (60: 40)

Flow rate: lml/min

Column temperature: room temperature

Detection wavelength: 227 nm

Example 2: Cellulomonas fibrosus CCTCC No. M 207130 hydrolysis 7-xylose-10-deacetylisolol and purification of the product

10 ml of 7-xylose-10-deacetyl paclitaxel in methanol (concentration: 5 mg/m) was added to 90 ml of the crude enzyme solution described in Example 1, reacted at 50 rpm, 3 CTC for 20 hours, and then added with 20 ml of acetic acid. The ester was extracted and co-extracted 3 times. The organic layer was combined and evaporated to dryness to give a solid substance (105 mg), which was applied to a silica gel column (20 g) and eluted with chloroform:methanol (98:2) to give 40 mg of 10-deacetyl-taxol ( Yield 91%).

Example 3: Cellulomonas fibrosus CCTCC No. M 207130 hydrolyzed and mixed taxane and glycoside and purification of the product

500ml of 1% birchwood xylan, 0.5% urea, in a 2000ml Erlenmeyer flask

0.2% KH ₂ P0 ₄ and 0.1% K ₂ HP0 ₄ medium, pH 7, autoclaved for 30 min. 10 ml of the seed solution described in Example 1 was inoculated into this medium, and cultured at 200 rpm, shake flask for 2 days at 30 °C. 'The supernatant was collected by centrifugation and stored at -20 ° for use. 200 ml of a mixture containing 7-xylose-10-deacetylpaclitaxel, 7-xylose-10-deacetyl cephalosporin, 7-xylose-10-deacetylpaclitaxel C (from natural yew extract The methanol solution (5 mg/ml) separated in the amounts of 65%, 9.9%, and 3.3%, respectively, was added to 2000 ml of the crude enzyme solution as described above, and reacted at 35 rpm for 8 hours at 35 °C. The reaction solution was extracted with ethyl acetate three times, 200 ml each time, and the combined organic phases were evaporated to dryness in 100 ml of methanol, and quantitatively detected by the above HPLC method, and it was found that there were no residues of three kinds of substrates with xylose groups. The content of 10-deacetylpaclitaxel, 10-deacetyl cephalosporin, 10-deacetylpaclitaxel C was 503 mg, 77 mg, 26 mg, respectively, and the recovery rate was 90%.

Example 4: Cellulomonas fibrosus CCTCC No. M 207130 immobilized enzyme hydrolyzed mixed taxane lignin

1 L of the crude enzyme solution as described in Example 1 was added to 100 μm of the pretreated DE52 anion exchange packing, and after thorough mixing, the supernatant was centrifuged, and the filler was washed with 50 mM potassium phosphate buffer (pH 7). After centrifugation again, the pellet was resuspended in 200 ml of the above phosphate buffer, which was an immobilized enzyme. 20 ml of a mixture of the taxane xylosides as described in Example 3 was added thereto, and reacted at 12 rpm for 2 hours at 28 °C. The supernatant was taken for 5 minutes at 5000 rpm, and extracted with ethyl acetate three times, 20 ml each time. The combined organic phases were evaporated to dryness in 10 ml of methanol, and quantitatively detected by the above HPLC method, and it was found that there were no three kinds of xylose. The base substrate remained, and the contents of 10-deacetylpaclitaxel, 10-deacetyl cephalosporin, 10-deacetylpaclitaxel C were 51.5 mg, 7.9 mg, and 2.6 mg, respectively, and the recovery rate was 92%.

Example 5: Cellulomonas fibrosus AS1.1920 hydrolysis 7-xylose-10-deacetylpacepol Inoculation of 1 agarose slant culture of the inoculated ring of Fibrocystis cellulosic AS1.1920

20 ml of the seed medium described in Example 1 was shaken and cultured at 30 °C for 2 days as a seed liquid.

A 500 ml Erlenmeyer flask was placed in 100 ml of medium containing 1% bran, 0.5% potassium nitrate, 0.1% NaH ₂ P0 ₄ and 0.1% Na ₂ HPO ₄ , and autoclaved at pH 121 for 30 min. 1 ml of the seed solution was inoculated to the medium, and cultured at 150 rpm in a 30-inch shake flask for 5 days. The supernatant was collected by centrifugation, which was a crude enzyme solution.

10 ml of 50 mM potassium phosphate buffer (pH 7) was added to 9 ml of the supernatant, and then 1 ml of a 7-xylose-10-deacetylpaclitaxel solution in methanol (concentration: 5 mg/ml) was added. lOOrpm, mix at 30 °C 8 hours. The reaction was stopped by adding 20 ml of methanol, and the supernatant was taken for 1 minute at 15,000 rpm for HPLC analysis. There was no residue of 7-xylose-10-deacetylpacillin in the reaction mixture, and 4.2 mg of l0-deacetylisol (yield 98%) was produced. · Example 6: Cellulomonas fibrosus AS1.1938 Hydrolyzed mixed taxane lignin

An agar slant culture of 1 inoculated loop of Pseudomonas aeruginosa AS1.1938 was inoculated into 20 ml of the seed medium described in Example 1, and shake cultured at 30 °C for 2 days as a seed liquid. 500 ml Erlenmeyer flask was filled with 100 ml of medium containing 1% birchwood xylan, 0.2% yeast extract, 0.2% ammonium chloride, 0.1% NaH ₂ PO ₄ and 0.1% Na ₂ HPO ₄ , pH 6 The bacteria were 121 degrees for 30 minutes. 1 ml of the seed solution was inoculated to the medium, and cultured at 150 rpm for 5 days in a shake flask at 30 °C. The supernatant was collected by centrifugation, which was a crude enzyme solution.

10 ml containing 7-xylose-10-deacetylpaclitaxel, 7-xylose-10-deacetyl cephalosporin,

A methanol solution (5 mg/ml) of a mixture of 7-xylose-10-deacetylpacaconol C was added to 100 ml of the crude enzyme solution as described above, and reacted at 35 rpm for 8 hours at 35 °C. 100ml methanol was added to the reaction solution, an HPLC and found no three kinds of substrates with xylosyl residues, respectively, to produce 25mg 10- deacetyl taxol, 38m _g 10- deacetyl cephalomannine base, 13mg l0- Deacetylpacillin C, the recovery rate reached 90%.

Example 7: Cellulomonas fibrosus AS 1.2019 Hydrolysis 7-xylose 10- 10-deacetylbaccatin III A 1 inoculum of agarose culture of Pseudomonas aeruginosa AS 1.2019 was inoculated into 20 ml of Example 1 The seed medium was shaken and cultured at 30 degrees for 2 days as a seed liquid.

A 500 ml Erlenmeyer flask was placed in 100 ml of medium containing 1% bran, 0.4% yeast extract, 0.1% Na3⁄4P0 ₄ and 0.1% Na ₂ HPO ₄ , pH 8, autoclaved at 121 degrees for 30 min. 1 ml of the seed solution was inoculated to the medium, and cultured at 150 rpm for 30 days in a shake flask at 30 °C. The supernatant was collected by centrifugation, which was a crude enzyme solution.

0.05 ml of a solution containing 0.5 mg of 7-xylose-10-deacetylbaccatin III was added to 0.9 m of the crude enzyme solution and 1 ml of 50 mM potassium phosphate buffer (pH 7) at 80 rpm and reacted at 30 ° C. hour. The reaction was stopped by adding 2 ml of methanol, and the supernatant was centrifuged for HPLC analysis. 10-Deacetylbaccatin III 0.39 mg (yield 98%) was detected.

Example 8: Cellulomonas fibrinos ACCC01019 hydrolysis 7-xylose paclitaxel

An agar slant culture of 1 inoculated loop of Pseudomonas aeruginosa ACCC01019 was inoculated into 20 ml of the seed medium described in Example 1, and shake cultured at 30 °C for 2 days as a seed liquid.

500ml Erlenmeyer flask containing 100ml of 1% birchwood xylan, 0.2% yeast extract,

0.2% ammonium chloride, 0.1% NaH ₂ PO ^ P 0.1% Na ₂ HP0 ₄ medium, pH 7, autoclaved 121 degrees 30 min. 1 ml of the seed solution was inoculated to the medium, and cultured at 150 rpm for 30 days in a shake flask at 30 °C. The supernatant was collected by centrifugation, which was a crude enzyme solution. 1 ml of 50 mM potassium phosphate buffer (pH 7) was added to 0.9 ml of the supernatant, and 0.1 ml of a 7-xylose-paclitaxel methanol solution (concentration of 5 mg/ml) was further added. Mix lOO rpm at 30 ° C for 7 hours. The reaction was stopped by adding 2 ml of methanol, and the supernatant was taken for 1 minute at 15,000 rpm for HPLC analysis. There was no 7-xylose-paclitaxel residue in the reaction solution, and 0.42 mg of paclitaxel was produced (yield 94%). Industrial applicability

The present invention provides a fibrosis cellulase and a hydrolase thereof which efficiently convert a taxane xyloside to paclitaxel or an analogue thereof, and provides a novel effective method for preparing paclitaxel and the like by biotransformation. This method can prepare paclitaxel and its analogues efficiently, inexpensively and environmentally, and provides an effective way to adapt to industrial production in order to make full use of taxane resources.

Claims

Rights request

1. A hydrolase produced by the metabolism of Cellulosimicrobium cellular^.

2. A hydrolase according to claim 1 for use in transforming C-7 docetaxel glycosides into paclitaxel or an analogue thereof.

3. A method for biotransformation of paclitaxel and the like, which comprises: hydrolyzing a hydrolyzing enzyme produced by the release of Cellulosi microbium cellulans using a C-7-bit taxane xyloside as a raw material; Hydrolysis, removal of the xylosyl group at the C-7 position, to obtain paclitaxel or an analogue thereof.

A method for biotransformation of paclitaxel or the like according to claim 3, characterized in that the hydrolysis reaction conditions are as follows: the raw material is added to the hydrolase in an amount of from 0.1 to 2 g/L, and the temperature is from 20 to 40 degrees, pH 6.0-8.0, reaction time 3-48 h.

A method for biotransformation of paclitaxel and an analogue thereof according to claim 3, wherein: said C-7 taxane xyloside has a structural formula of formula I:

(Formula I)

Wherein 1 is 11 or an acetyl group; and the same or different, a T or an OT; wherein T is H, an aryl group, an aralkyl group, a C1 to C20 linear, branched or cyclic hydrocarbon group, and the above three A substituent of a hydrocarbon group, the substituent including a hydroxyl group, a C1 to C8 decyloxy group, an acetal, a ketal, a halogen, a nitro group or an amino group.

A method for biotransformation of paclitaxel or the like according to claim 3, wherein: the taxol glycoside compound raw material is a natural product derived taxane having a xylosyl group at the C-7 position. A monomer or a mixture thereof, or a xylan-containing taxane obtained at the above position, obtained by biosynthesis, chemical semi-synthesis or total synthesis.

A method for biotransformation of paclitaxel or an analogue thereof according to claim 3, wherein the hydrolase is used in a free state, or is immobilized on a support by physical adsorption or trapping.

8. A cellulomonas <i Cellulosimicrobium cellulans) ~ XZ-5 CCTCC No. M 207130.