CN113736835A

CN113736835A - Industrial preparation process for producing taxol by fermenting taxus chinensis endophytic fungi mutant

Info

Publication number: CN113736835A
Application number: CN202111061717.3A
Authority: CN
Inventors: 陈开云; 柏凤静
Original assignee: Individual
Current assignee: Individual
Priority date: 2021-09-10
Filing date: 2021-09-10
Publication date: 2021-12-03

Abstract

The inventionAn industrial process for preparing taxol from the endophytic fungus of yew includes such steps as separating the HQ-24 strain from the fruit of yew, mutagenesis, slant culture in test tube, seed culture, fermentation culture, membrane filtering, membrane concentration, extraction separation, macroporous resin purification, and recrystallizing₂Combining a fluid drying technology and a full-automatic control technology, and carrying out fermentation of the taxus chinensis endophytic fungi under the synergistic effect of the fluid drying technology and the full-automatic control technology to produce the taxol compound, thereby realizing non-chemical synthesis of the taxol compound and non-biological synthesis of taxus chinensis extraction; the paclitaxel compound produced by the technology has the advantages of mild reaction conditions, relatively simple operation, low product cost, high yield, high purity, low residual solvent, low energy consumption, high productivity and small environmental pollution, is suitable for industrial mass production, and has good economic benefit and application prospect.

Description

Industrial preparation process for producing taxol by fermenting taxus chinensis endophytic fungi mutant

Technical Field

The invention belongs to the technical field of biological metabolism synthesis and purification, and particularly relates to an industrial preparation process for producing taxol by fermenting a yew endophytic fungus mutant.

Background

Paclitaxel (registered as Taxol) is a diterpenoid derivative, a natural product with taxane ring structure with unique anti-leukemia and anti-cancer mechanism, and was first isolated from taxus plants. The paclitaxel is white crystalline powder, is insoluble in water, is easily soluble in organic solvents such as methanol, ethanol, acetone, ethyl acetate, diethyl ether, etc., and is tricyclic diterpene compound composed of 113 atoms, with molecular formula of C47H51NO14, relative molecular weight of 853.92, melting point of 213-.

Paclitaxel is an important anticancer drug in clinical application, taxane is a metabolic precursor or a branch metabolite of paclitaxel, and some taxanes have special physiological activity. The low yield of paclitaxel and taxane is always the main bottleneck restricting the application, and how to expand the drug source is always a difficult problem. Some techniques for producing paclitaxel and taxane have been developed, and among them, plant cell engineering methods are generally regarded as important because they do not occupy cultivated land, are environmentally friendly, and are controllable, and thus meet the strategy of sustainable development.

1963, Wall [1 ]]The extract of the bark of Taxus brevifolia (Taxus brevifolia) was found to have antitumor activity. Paclitaxel (paclitaxel) was isolated in 1967 by Wall and Wani, and its structure was resolved in 1971 by X-ray diffraction and nuclear magnetic techniques. In 1979, Horwitz disclosed the mechanism of paclitaxel anti-tumor: it can stabilize microtubule aggregation and prevent normal physiological microtubule depolymerization, and can "freeze" tumor cells with extremely high proliferation rate"mitotic spindle, stopping tumor cell division at G2 and M until death. Paclitaxel was the first drug discovered to promote microtubule polymerization. First and second phase clinical trials of paclitaxel were performed in 1984 and 1985, respectively. In 1989 and 1991, significant activity of paclitaxel in inhibiting ovarian cancer and breast cancer, respectively, was discovered. Paclitaxel was approved by the food and drug administration (FDI) in 1992 and 1994 as a clinical drug for the treatment of ovarian and breast cancer. 1993, Baishi noble corporation (Bristol-Myers Squibb company) to

Paclitaxel is marketed under the trademark paclitaxel. In 2000, sales have reached $ 16 billion. Paclitaxel and its semisynthetic derivative docetaxel (Taxotere) have now become the first-line drug for clinical treatment of ovarian cancer, breast cancer, non-small cell lung cancer. In addition, paclitaxel and docetaxel are also used for treating lung cancer, Kaposi's sarcoma, colon cancer, melanoma and lymphoma. Paclitaxel is a major secondary metabolite of Taxus genus plant of Taxaceae family, and is mainly present in bark and needle. The chemical structure is divided into 2 parts: the basic skeleton is a taxane (taxane) tricyclic diterpene, the side chain comprises 3 aromatic rings (1 benzene ring and 2 benzoyl rings) and 1 propylene oxide ring (D ring), the two rings are combined to form 7 rings, and the core is tetracyclic diterpene.

The paclitaxel is extracted from the bark of the short-leaf taxus chinensis only in ten-thousandth, and 2000-3000 barks or more than 10 tons of branches and leaves are needed for 1kg of paclitaxel. The problem of paclitaxel supply has attracted extensive attention, and insufficient resources have become a major factor restricting the clinical application of paclitaxel. Although the Holton and Nicolaou research groups independently and completely synthesize paclitaxel in 1994 respectively, and then have a plurality of different approaches to successfully synthesize paclitaxel, the procedures are complicated, and the yield is low, so that the industrialization cannot be realized. The National Cancer Institute (NCI) authorizes the hundred-hour American noble band company to solve the problem of medicine sources, and the developed medicine sources comprise: extracting reproducible branches and leaves of yew; semi-synthesis of paclitaxel from the extracted taxanes; obtained by plant tissue culture. A major breakthrough was found by Potier to contain 10-deacetylbaccatin III10-deacetylbaccatin III, 10-DAB in the needles of Taxus baccata (Taxus baccata) and baccatin III (baccatin III). The Holton research group started with 10-deacetylbaccatin II (I is contained in various yew plants and extracted from reproducible branches and leaves) and synthesized paclitaxel through 4-step reactions, thereby supplying a large amount of paclitaxel to the clinic. The sales of paclitaxel increased from $ 4 billion in 1994 to $ 16 billion in 2000. Currently, commercially available paclitaxel is classified into 2 types according to the source of raw materials: one is directly extracted from taxus chinensis plants; the other is semi-synthetic paclitaxel, namely 10-deacetylbaccatin III is extracted from renewable resources such as tender branches or leaves of taxus chinensis, or by-products in the process of extracting paclitaxel, such as 7-xylose, 10-deacetyl paclitaxel, cephalomannine and the like, are used as raw materials to produce paclitaxel or docetaxel through chemical semi-synthesis. However, the semisynthetic method has the problem of environmental pollution, and the extraction and processing from the original plants are mainly a physical and chemical method, which destroys resources and is not beneficial to the sustainable development of the environment. Although Stierle et al reports in 1993 that taxol is found in the fungus Taxomyces andreanae and opens up a new way for supplying taxol, the yield of taxol in the fungus is too low (25-50 ng/L), and the commercialization is possible only by greatly increasing the yield by improving the strain. In contrast, the production of paclitaxel and taxanes using plant cell and tissue culture techniques has unparalleled advantages.

Endophytic fungi refer to microorganisms that live inside healthy plant tissues or organs at some or all stages of their life history, but do not cause significant disease symptoms to the plant. Endophytes invade into the body of a host plant, live in plant cells or in a special environment in the intercellular spaces for a long time, and co-evolve with the host plant to form a stable ecological relationship of mutual benefit and symbiosis. In 1993, a Strobel group firstly isolated an endophytic fungus for synthesizing paclitaxel from Taxus brevifolia Nutt, and proves that the endophytic fungus has the function of synthesizing the same or similar active ingredients with a host plant. This finding has stirred the research heat of isolating endophytes from medicinal plants, and then researchers have isolated various endophytes from yew plants in sequence, and found out the yew endophytes with the same or similar active ingredient taxol to the host plants in the culture after artificial culture.

Patent numbers: 2014102694407A method for producing paclitaxel by using Chinese yew seed endophyte comprises sterilizing Chinese yew seed as explant, inoculating, sterilizing twice, inoculating, inducing to generate Chinese yew seed endophyte, culturing Chinese yew seed endophyte to generate paclitaxel, wherein the average content is 0.55mg/L, and the yield of paclitaxel is far lower than that of patent number: 2017103468093, the production cost is still higher than that of the plant yew, therefore, the patent number technology still has no condition for the industrial production of taxol.

Patent numbers: 2017103468093A paclitaxel-producing Penicillium BP6T3 and its application, the invention discloses a paclitaxel-producing endophyte separated and screened from tissue of torreya grandis of Taxaceae, which is classified, identified and named as Penicillium (Penicillium sp) BP6T3 with a preservation number of CCTCC NO: M2016320. The strain has the activity of resisting staphylococcus aureus and escherichia coli, the content of a fat-soluble extract in a fermentation broth can reach 105mg/L, wherein the relative percentage content of paclitaxel is 15.8%, the yield is 16.59mg/L, and the yield is higher than that disclosed in the prior art; the yield of the paclitaxel can be increased by 425.17 percent by taking the L-phenylalanine as a precursor. However, the yield of paclitaxel is only 16.59mg/L, and the production cost is much higher than that of paclitaxel extracted from plant Taxus chinensis, therefore, the patent number technology still has no condition for industrial production of paclitaxel.

The two patent technologies utilize the fermentation of the endophytic fungi of the taxus chinensis to produce the taxol, and although the taxol is produced by utilizing the fermentation of the endophytic fungi of the taxus chinensis plants, the reaction conditions are mild, the pollution is small, and the operation is relatively simple, the yield of the taxol compounds is very low, so the taxol compounds still have no industrial application prospect.

Disclosure of Invention

The invention aims to provide an industrial preparation process for producing taxol by fermenting the yew endophytic fungi mutant, aiming at the defects of the prior art.

In order to meet the requirements, the technical scheme adopted by the invention is as follows: the industrial preparation process for producing the taxol by fermenting the taxus chinensis endophytic fungi mutant comprises the following steps:

s1: cutting fresh Taxus chinensis fruit in Yunnan into 0.5-1 cm by conventional tissue separation method²And (5) small blocks. Soaking the fruits in 75% alcohol for about 5min, washing with sterile water for 3-4 times, and soaking with 2.5% sodium hypochlorite for 3 min; planting the cutting part on a contact culture medium, and checking whether the surface is disinfected completely by adopting a tissue blotting method as a control;

s2: the culture medium was incubated in a constant temperature incubator at 28 ℃. Observing at regular time, sequentially transferring the newly grown hyphae to a new solid culture medium by using an inoculating needle, purifying each strain for 2-3 times, and transferring to a test tube inclined plane to preserve two strains;

s3: preparation of bacterial suspension: taking several inclined planes of the dominant strains cultured for one week, respectively adding physiological saline containing synergistic isonicotin, scraping off thalli on the inclined planes by using an inoculating loop, uniformly shaking, pouring the mixed solution into a sterile triangular flask, placing the sterile triangular flask on a constant temperature shaking table to resist oscillation, and filtering the mixture by using a funnel with a layer of sterile mirror wiping paper after the oscillation is finished to obtain a monospore suspension of the strains. Counting spores with a hemocytometer (hemocytometer) to adjust the spore concentration to about 10⁶Per ml;

s4: ultraviolet mutagenesis: 5ml of the prepared monospore suspension was taken out into a sterile plate with a diameter of 9 mm. The 15W UV lamp was turned on to preheat and stabilize the light. The dish containing the bacterial suspension is placed on a magnetic stirrer, is away from a lamp tube by a certain distance, the dish cover is opened, and the cell is stirred and irradiated at the same time, so that the cell can uniformly absorb ultraviolet light waves. The top was operated in a red background, avoiding photorepair. The thallus after ultraviolet mutagenesis is immediately transferred into a sterile test tube and immersed in ice water for 30min to inhibit enzyme activity and repair. According to the principle of delay phenomenon, adding the irradiated bacterial suspension subjected to ice bath into a PDA culture medium for post-culture so as to improve mutation rate;

s5: and (3) performing compound mutagenesis by combining ultraviolet rays with nitrous acid: prepared according to the method described above to about 10⁶Performing ultraviolet mutagenesis on spore suspension per mL, sucking 1mL of mutagenized spore suspension, placing the spore suspension into a large test tube, adding 2mL of acetic acid buffer solution with pH4.5 and 1mL of sodium nitrite solution with 0.1mol/L (the final treatment concentration is 0.025mol/L), preserving the temperature at 25-26 ℃ for 10-25 min, adding 20mL of disodium hydrogen phosphate solution with 0.07mol/L, PH 8.6.6 to reduce the pH to about 6.8 so as to stop the reaction, properly diluting, coating a flat plate, selecting a bacterial colony, performing shaking table fermentation, using the ultraviolet mutagenesis firstly and then evaluating;

s6: and (3) taking test tubes with the same size, respectively filling quantitative culture media, and preparing basically same inclined planes for later use. The activated strains were picked and streaked onto a medium slant. Placing the inclined planes in a thermostat at 28 ℃ for culturing for 9 days;

s7: the liquid shake flask strain culture medium is PDA, PH 7. The initial pH was 5.0. Preparing 250mL of the mixture, subpackaging the mixture into 250mL of triangular bottles, sterilizing the mixture for 30 minutes at 121 ℃ in each bottle, and cooling the mixture to room temperature for later use;

s8: picking and transferring the cultured test tube slant strains into a 250ml triangular flask filled with a shake flask culture medium, and carrying out shake culture at 28 ℃ for 24 hours at 120 r/min;

s9: preparing a seed culture medium according to a formula standard, respectively pumping the seed culture medium into No. 1-8 500L first-stage seed culture tanks, and sterilizing by using a tank steam sterilization method, wherein the sterilization temperature is more than or equal to 121 ℃, and the sterilization time is more than or equal to 30 min;

s10: after the first-stage seed culture tank is sterilized, the temperature of a culture medium in the tank is reduced to 28 ℃, then the triangular flask fermentation liquor is respectively inoculated into No. 1-8 500L first-stage seed fermentation tanks through an aseptic inoculation system, seed culture is carried out by using the seed culture medium, the inoculation amount is 1%, the fermentation temperature is 26 ℃, the tank pressure is 0.10Mpa, the fermentation culture is carried out for 18h under the condition that the pH is 7.2, the rotation speed of the seed tank is 120r/min for 0-9 h, and the ventilation volume is 0.036m³Min, after 9h, the rotation speed of the seeding tank is 180r/min, and the ventilation volume is 0.043m³Min, collecting seed liquid after fermentation culture;

s11: preparing a seed culture medium according to a formula standard, respectively filling the seed culture medium into No. 1-8 1000L secondary seed fermentation tanks, and sterilizing by using a retort steam sterilization method, wherein the sterilization temperature is more than or equal to 121 ℃, and the sterilization time is more than or equal to 30 min;

s12: after the secondary seed culture tank is sterilized, when the temperature is reduced to 28 ℃, respectively inoculating the primary seed fermentation liquor fermented for 18 hours into a No. 1-8 1000L secondary seed fermentation tank through an aseptic inoculation system, and performing seed culture by using a seed culture medium, wherein the inoculation amount is 1-2%, the fermentation temperature is 28 ℃, the tank pressure is 0.10Mpa, the fermentation culture is 18 hours under the conditions that the pH is 7.4, the rotation speed of the seed tank is 120r/min for 0-9 hours, and the ventilation volume is 1.2m³Min, after 9h, the rotation speed of the seeding tank is 150r/min, and the ventilation volume is 1.5m³Min, collecting seed liquid after fermentation culture;

s13: accurately weighing the culture medium materials according to the formula in batches, putting the culture medium materials into a 40000L multiplied by 3 culture medium preparation tank, pumping purified water according to the formula, and stirring for 30 min;

s14: pumping fermentation medium into No. 1-10 100000L fermentation single tank or No. 1-3 medium liquid supplementing tank in batches, filling the tank with coefficient of 0.7, sterilizing by using a real tank steam sterilization method, wherein the sterilization temperature is more than or equal to 121 ℃, and the sterilization time is more than or equal to 30 min;

s15: after single-tank sterilization is finished, cooling culture medium in the tank to 28 ℃ by using chilled water, respectively inoculating the secondary seed fermentation liquor fermented for 18 hours from No. 1 to No. 8 to a No. 100000L fermentation tank through a sterile inoculation system for fermentation culture, wherein the inoculation amount is 1%, the fermentation temperature is 26 ℃, the tank pressure is 0.03MPa, the pH is 7, the fermentation culture is carried out for 240 hours, the stirring rotation speed is 130r/min, the ventilation amount is 0.5-0.8 vvm/min, and after 16 hours, the fermentation tank rotation speed is 150r/min, and the ventilation amount is 1 vvm/min;

s16: supplementing the culture medium in real time according to the standard of the culture medium amount in the fermentation period; after the culture medium is complemented and the fermentation time reaches the standard, finishing the fermentation to prepare a mixed solution of endophytic fungi, mycelia and paclitaxel;

s17: pumping the prepared mixed solution of the endophytic fungi, the mycelia and the paclitaxel into a No. 1-2 40000L temporary storage tank of fermentation liquor;

s18: pumping the obtained fermentation liquid into coarse filtration equipment, filtering, performing microfiltration with membrane microfiltration unit, and filtering to remove endophytic fungi and mycelium to obtain taxol mixed crude liquid;

s19: centrifuging the filtered endophytic fungi and mycelium with an automatic scraper centrifuge for desolventizing to obtain the wastes such as the endophytic fungi and the mycelium;

s20: pumping the obtained paclitaxel mixed crude solution into a membrane ultrafiltration concentration unit for concentration to obtain paclitaxel mixed concentrated solution and wastewater;

s21: pumping the prepared taxol mixed concentrated solution into an automatic scraper centrifuge for centrifugation and desolventizing to prepare a taxol crude product filter cake A;

s22: sending the prepared taxol crude product filter cake B into a No. 1 extraction kettle, extracting and decoloring for 1-3 times by using a lipophilic solvent with the weight 3 times that of the extract and a decoloring agent with the weight 0.1-0.3 times that of the extract, and filtering the extract to obtain taxol filtrate;

s23, pumping the paclitaxel filtrate into a concentration unit for concentration, and recovering the solvent to obtain a paclitaxel crude product concentrated solution;

s24, pumping the paclitaxel concentrated solution into an automatic scraper centrifuge for centrifugal desolventizing and solvent recovery to obtain a paclitaxel crude product filter cake B;

s25: feeding the obtained crude extract of paclitaxel into No. 2 extraction kettle, extracting with lipophilic solvent 3 times of the extract for 3 times, filtering the extractive solutions, and mixing the extractive solutions to obtain paclitaxel extractive solution;

s26: pumping the paclitaxel extract into a concentration unit for concentration, and recovering the solvent to obtain a paclitaxel crude product concentrated solution;

s27: pumping the taxol crude product concentrated solution into an automatic scraper centrifuge for centrifugal desolventizing and recovering the solvent to prepare a taxol crude product filter cake C;

s28: mixing the taxol crude product filter cake C with the crystallized mother liquor extract, loading into a column feed preparation tank, and dissolving with ethanol to prepare column feed;

s29: feeding the obtained upper column liquid into a macroporous resin column for adsorption; eluting with deionized water and solvent, and collecting eluate; taking the eluate as the upper column liquid for secondary macroporous resin adsorption, eluting with ethanol, and collecting the upper column adsorption effluent liquid and the eluate;

s30: respectively pumping the effluent liquid and the gradient eluent into a concentration unit for concentration and solvent recovery to prepare a taxol crude product concentrated solution;

s31: pumping the obtained paclitaxel crude product concentrated solution into an automatic scraper centrifuge for centrifugal desolventizing, and recovering ethanol solvent to obtain paclitaxel crude product filter cake;

s32: feeding the obtained paclitaxel crude product filter cake into a freezing crystallization kettle, dissolving with a solvent, crystallizing at low temperature, filtering crystals, concentrating mother liquor by a concentration unit, recovering the solvent, centrifuging and desolventizing to obtain paclitaxel crude crystals and mother liquor extract;

s33: feeding the obtained paclitaxel coarse crystal into 8000L crystallization kettle No. 1 or No. 2, dissolving with solvent, recrystallizing at low temperature, filtering crystal, concentrating mother liquor with concentration unit, recovering solvent, centrifuging, and removing solvent to obtain paclitaxel wet crystal and mother liquor extract;

s34: feeding the mother liquor extract into step S28, and mixing with the paclitaxel crude product filter cake to prepare upper column liquid;

s35: delivering the obtained paclitaxel wet crystal into supercritical CO2 drying device for desolventizing, drying, and sterilizing to obtain paclitaxel crystal powder with content of 98% or more;

s36: and (3) sending the prepared taxol crystal powder with the concentration of more than or equal to 98% into a batch V-shaped mixer for batch mixing to prepare a taxol crystal powder product with the concentration of more than or equal to 98%.

In conclusion, the industrial preparation method for producing taxol by fermenting the taxus chinensis endophytic fungi has the following advantages:

1) the paclitaxel produced by the invention adopts a non-taxus chinensis extraction process and a non-chemical synthesis process, and opens up a brand new process route for the industrial production of paclitaxel.

2) The process for producing the paclitaxel saves a large amount of resources because the paclitaxel is not extracted from the barks, branches and leaves of the taxus chinensis, and solves the problem of insufficient raw materials for extracting the paclitaxel; the problem of protection of the taxus chinensis is also solved.

3) The process for producing taxol of the invention has the production cost less than half of the unit cost of extracting and purifying taxol from barks, branches and leaves of taxus chinensis, so the product cost is low.

4) The invention designs 8 sets of full-automatic control primary seed fermentation tanks, 8 sets of full-automatic control secondary seed fermentation tanks, 3 sets of full-automatic control culture medium preparation tanks, 2 sets of full-automatic control culture medium supplement tanks and 6 sets of full-automatic control process fermentation tanks to meet the requirement of circular production, wherein a single industrial fermentation tank corresponds to a corresponding single secondary seed fermentation tank, a single secondary seed fermentation tank corresponds to a corresponding single primary seed fermentation tank, and 8 sets of fermentation systems perform continuous circular fermentation. Therefore, the method for producing the taxol has the advantages of high capacity and continuous production.

5) In the method for preparing the taxol, the modern full-automatic control technology is adopted to prepare the culture medium, control the filling amount of the culture medium, control the temperature, control the ventilation quantity, control the fermentation time, automatically clean each tank, automatically control sterilization and the like, so that the product quality is ensured, the product yield is improved, the energy consumption is optimized and saved, and a large amount of labor force is saved.

6) In the process for preparing the taxol fermentation liquor, the trichoderma HQ-24 strain is subjected to mutagenesis by utilizing an ultraviolet irradiation strain, an elicitor is added into a suspension culture medium, precursor substances such as L-phenylalanine, arachidonic acid and the like are added into the culture medium, the taxol produced by the taxus endophytic fungi with higher yield is obtained, and the taxol yield in the fermentation liquor reaches more than 134 mg/L. Is 7-230 times of that of taxol produced by endophytic fungi of taxus chinensis in the similar patent technology.

7) The invention adopts the membrane separation and membrane concentration device, and the sewage discharged by the device can be used for plant irrigation besides generating part of solid waste, thereby avoiding the discharge of a large amount of polluted water and reducing the environmental pollution.

8) The invention adopts an MVR evaporator or a full-electric evaporator for vacuum concentration or membrane concentration, desolventizing by an automatic scraper centrifuge and recovering the solvent, improves the recovery rate of the solvent, achieves the recovery rate of the solvent above 90 percent, and greatly reduces the energy consumption of the product and the pollution of air.

9) The invention adopts centralized linkage control power, steam, cooling water and sterile air supply, and utilizes an automatic control technology to automatically control and distribute the use time and supply quantity of the power, the steam, the cooling water and the sterile air according to the time difference of the circulating fermentation of 8 fermentation production systems, thereby improving the utilization rate of equipment, reducing the purchase cost of the equipment and reducing the energy consumption of the equipment.

10) The invention feeds the crystallized mother liquor extract generated in the step of crystallizing the paclitaxel into a column liquor preparation tank to be combined with a paclitaxel crude product filter cake to prepare column liquor, and the macroporous resin is used for adsorbing, eluting and recrystallizing higher paclitaxel in the crystallized mother liquor extract, thereby increasing the yield of the paclitaxel by more than 98 percent.

11) The invention prepares the taxol compound by adopting supercritical CO₂The fluid device is used for simultaneously drying, removing residual solvent and sterilizing, the product has low solvent residue and uniform color, and the sanitation index meets the relevant standard.

12) The invention separates a trichoderma HQ-24 strain from taxus chinensis fruits, uses the strain as an original strain, and carries out the techniques of mutagenesis, test tube slant culture, seed culture, fermentation culture, membrane filtration, membrane concentration, extraction decoloration, extraction separation, macroporous resin purification, recrystallization and the like, and supercritical CO₂Combining a fluid drying technology and a full-automatic control technology, and carrying out fermentation on the taxus chinensis endophytic fungi under the synergistic effect of the fluid drying technology and the full-automatic control technology to produce the taxol compound, thereby realizing non-chemical synthesis of the taxol compound and biosynthesis of non-taxus chinensis plant extraction; the paclitaxel compound produced by the technology has the advantages of mild reaction conditions, relatively simple operation, high yield, high purity, low cost, low residual solvent, low energy consumption, high productivity and small environmental pollution, is particularly suitable for industrial mass production, and has good economic benefit and application prospect.

Detailed Description

In order to make the objects, technical solutions and advantages of the present application more apparent, the present application is further described in detail with reference to specific embodiments below.

In the following description, references to "one embodiment," "an embodiment," "one example," "an example," etc., indicate that the embodiment or example so described may include a particular feature, structure, characteristic, property, element, or limitation, but every embodiment or example does not necessarily include the particular feature, structure, characteristic, property, element, or limitation. Moreover, repeated use of the phrase "in accordance with an embodiment of the present application" although it may possibly refer to the same embodiment, does not necessarily refer to the same embodiment.

Certain features that are well known to those skilled in the art have been omitted from the following description for the sake of simplicity.

According to an embodiment of the present application, an industrial preparation process for paclitaxel produced by fermentation of a yew endophytic fungus mutant is provided, which comprises the following steps:

s1: cutting fresh Taxus chinensis fruit in Yunnan into 0.5-1 cm by conventional tissue separation method²Soaking the small pieces of fruits in 75% alcohol for 5min, washing with sterile water for 3-4 times, and soaking with 2.5% sodium hypochlorite for 3 min; planting the cutting part on a contact culture medium, and checking whether the surface is disinfected completely by adopting a tissue blotting method as a control;

s2: culturing the culture medium in a constant-temperature incubator at 28 ℃, observing regularly, sequentially transferring newly grown hyphae to a new solid culture medium by using an inoculation needle, purifying each strain for 2-3 times, and transferring to a test tube inclined plane to preserve two strains;

s3: preparing bacterial suspension, taking the slope of the dominant bacterial strain cultured for one week, respectively adding physiological saline containing synergistic isonicotin, scraping the thallus on the slope by using an inoculating loop, uniformly shaking, pouring the mixed solution into a sterile triangular flask, placing the sterile triangular flask on a constant temperature shaking table for resisting oscillation, filtering the mixed solution by using a funnel with a layer of sterile mirror paper after oscillation to obtain monospore suspension of the bacterial strain, counting spores by using a hemocytometer, and adjusting the concentration of the spores to be about 10⁶Per ml;

s4: ultraviolet mutagenesis, namely taking 5ml of prepared monospore suspension into a sterile plate with the diameter of 9mm, turning on a 15W ultraviolet lamp for preheating, putting the plate containing the bacterial suspension on a magnetic stirrer to stabilize light waves, putting the plate on the magnetic stirrer at a certain distance from a lamp tube, opening a dish cover, stirring and irradiating simultaneously, trying to enable cells to uniformly absorb the ultraviolet light waves, operating under a red light background to avoid photorepair, immediately transferring the thalli subjected to ultraviolet mutagenesis into the sterile test tube, immersing the thalli in ice water for 30min to inhibit enzyme activity and repair, and adding the bacterial suspension subjected to ice bath after irradiation into a PDA culture medium for post-culture according to the principle of delay phenomenon to improve the mutation rate;

s5: complex mutagenesis with UV-combined nitrous acid, prepared as described above for about 10⁶Performing ultraviolet mutagenesis on spore suspension per mL, sucking 1mL of the mutagenized spore suspension, placing the spore suspension into a large test tube, adding 2mL of acetic acid buffer solution with pH4.5 and 1mL of sodium nitrite solution with 0.1mol/L, keeping the final treatment concentration at 0.025mol/L, preserving the temperature at 25-26 ℃ for 10-25 min, adding 20mL of disodium hydrogen phosphate solution with 0.07mol/L, PH 8.6.6 to reduce the pH to 6.8 so as to stop the reaction, diluting, coating a flat plate, picking a bacterial colony, performing shaking table fermentation, using the ultraviolet mutagenesis firstly and then evaluating;

s6: respectively loading test tubes with the same size into a quantitative culture medium, preparing a uniform inclined plane for later use, selecting an activated strain, streaking and inoculating the strain onto the inclined plane of the culture medium, and placing the inclined plane in a constant temperature box at 28 ℃ for culturing for 9 days;

s7: the liquid shake flask strain culture medium is PDA with a pH of 7, the initial pH value is 5.0, 250mL is prepared, the mixture is subpackaged into 250mL triangular flasks with each flask containing 30mL, the mixture is sterilized at 121 ℃ for 30 minutes and then cooled to room temperature for later use;

s15: after single-tank sterilization is finished, cooling culture medium in the tank to 28 ℃ by using chilled water, respectively inoculating the secondary seed fermentation liquor fermented for 18 hours from No. 1 to No. 6 to a No. 1 100000L fermentation tank through a sterile inoculation system for fermentation culture, wherein the inoculation amount is 1%, the fermentation temperature is 26 ℃, the tank pressure is 0.03MPa, the pH is 7, the fermentation culture is carried out for 240 hours, the stirring rotation speed is 130r/min, the ventilation amount is 0.5-0.8 vvm/min, and after 16 hours, the fermentation tank rotation speed is 150r/min, and the ventilation amount is 1 vvm/min;

s19: centrifuging and desolventizing the filtered endophytic fungi and mycelium by using an automatic scraper centrifuge to prepare endophytic fungi and mycelium waste;

s22: feeding the prepared taxol crude product filter cake A into a No. 1 extraction kettle, extracting and decoloring for 1-3 times by using a solvent with the weight 3 times that of the extract and a decoloring agent with the weight 0.1-0.3 times that of the extract, and filtering the extract to obtain taxol filtrate;

The strain name is Trichoderma HQ-24 strain; the preservation place is as follows: the preservation number of the China general microbiological culture Collection center (address: No. 3 Xilu No. 1 Beijing of Chaoyang district, Beijing) is CGMCC No. 9251.

The preparation and industrial fermentation method of the strain comprises the following steps:

i. the liquid shake flask strain culture medium is PDA with a pH of 7, the initial pH value is 5.0, 250mL is prepared, the mixture is subpackaged into 250mL triangular flasks with each flask containing 30mL, the mixture is sterilized at 121 ℃ for 30 minutes and then cooled to room temperature for later use;

ii. Picking and transferring the cultured test tube slant strains into a 250ml triangular flask filled with a shake flask culture medium, and carrying out shake culture at 28 ℃ for 24 hours at 120 r/min;

iii, preparing a seed culture medium according to a formula, wherein the formula of the seed culture medium comprises 40g of sucrose, 10g of peptone, 10g of yeast extract powder, 15g of agar, 1000mL of distilled water and pH 7; respectively pumping the seed culture medium into No. 1-8 500L first-stage seed culture tanks, and sterilizing with steam sterilization method at sterilization temperature of more than or equal to 121 deg.C for more than or equal to 30 min;

iv, after the first-stage seed culture tank is sterilized, reducing the temperature of a culture medium in the tank to 28 ℃, respectively inoculating the triangular flask fermentation liquor into No. 1-8 500L first-stage seed fermentation tanks through an aseptic inoculation system, and performing seed culture by using the seed culture medium, wherein the inoculation amount is 1%, the fermentation temperature is 28 ℃, the tank pressure is 0.10Mpa, the fermentation culture is performed for 18 hours under the conditions that the pH is 7.2, the rotation speed of the seed tank is 120r/min for 0-9 hours, and the ventilation volume is 0.036m³Min, after 9h, the rotation speed of the seeding tank is 180r/min, and the ventilation volume is 0.043m³Min, collecting seed liquid after fermentation culture;

v, preparing a seed culture medium according to the standard of the step iii, respectively filling the seed culture medium into No. 1-8 1000L secondary seed fermentation tanks, and sterilizing by using a real tank steam sterilization method, wherein the sterilization temperature is more than or equal to 121 ℃, and the sterilization time is more than or equal to 30 min;

vi, after the secondary seed culture tank is sterilized, when the temperature is reduced to 28 ℃, respectively inoculating the primary seed fermentation liquor fermented for 18 hours into a No. 1-8 1000L secondary seed fermentation tank through an aseptic inoculation system, and performing seed culture by using a seed culture medium, wherein the inoculation amount is 1-2%, the fermentation temperature is 28 ℃, the tank pressure is 0.10Mpa, the fermentation culture is performed for 18 hours under the conditions that the pH is 7.4, the rotation speed of the seed tank is 120r/min for 0-9 hours, and the ventilation volume is 1.2m³Min, after 9h, the rotation speed of the seeding tank is 150r/min, and the ventilation volume is 1.5m³Min, collecting seed liquid after fermentation culture;

vii, accurately weighing the culture medium materials in batches according to the formula, then putting the culture medium materials into a 40000L multiplied by 3 culture medium preparation tank, pumping purified water according to the formula, and stirring for 30 min; the formula of the culture medium comprises 40g of cane sugar, 10g of peptone, 10g of yeast extract powder, 0.3mg of L-phenylalanine, 0.05mg of sodium acetate, 0.02mg of sodium benzoate, 1000mL of distilled water and pH 7;

viii, pumping the fermentation medium into a No. 1-10 100000L fermentation single tank or a No. 1-3 medium liquid supplementing tank in batches, filling the tank with the coefficient of 0.7, and sterilizing by using a real tank steam sterilization method, wherein the sterilization temperature is more than or equal to 121 ℃, and the sterilization time is more than or equal to 30 min;

ix, after single-tank sterilization, cooling a culture medium in the tank to 28 ℃ by using chilled water, respectively inoculating the secondary seed fermentation liquor fermented for 18 hours from No. 1 to No. 10 to a No. 100000L fermentation tank through a sterile inoculation system for fermentation culture, wherein the inoculation amount is 1%, the fermentation temperature is 26 ℃, the tank pressure is 0.03MPa, the fermentation culture is carried out for 240 hours under the conditions of pH 7, the stirring rotation speed is 130r/min, the ventilation volume is 0.5-0.8 vvm/min, and after 16 hours, the fermentation tank rotation speed is 150r/min, and the ventilation volume is 1 vvm/min;

x, supplementing the culture medium in real time according to the standard of the culture medium amount in the fermentation period; and after the culture medium is replenished and the fermentation time reaches the standard, ending the fermentation to prepare the mixed solution of the endophytic fungi, the mycelium and the paclitaxel.

In the step i, the number of days of culture in the incubator is 3-7 days; the formula of the seed culture medium is as follows: 200g of potato, 20g of glucose, 20g of agar, 0.1mg of arachidonic acid, 0.05mg of cinnamic acid, 1000mL of distilled water and pH 7;

in the step ii, the shaking table culture temperature is 28 ℃, and the rotating speed is 120 r/min;

in the step iv, the inoculation amount is 1-2%, the fermentation temperature is 28 ℃, the tank pressure is 0.10Mpa, the pH is 7.2, the fermentation culture is carried out for 18h, the rotating speed of the seeding tank is 120r/min for 0-9 h, the ventilation rate is 0.6-1.2 vvm/min, and after 9h, the rotating speed of the seeding tank is 180r/min, and the ventilation rate is 0.6-1.2 vvm/min;

in the step vi, the inoculation amount is 1-2%, the fermentation temperature is 28 ℃, the tank pressure is 0.10Mpa, the pH is 7.4, the fermentation culture is carried out for 18h, the rotating speed of the seeding tank is 120r/min for 0-9 h, the ventilation rate is 0.6-1.2 vvm/min, and after 9h, the rotating speed of the seeding tank is 150r/min, and the ventilation rate is 0.6-1.2 vvm/min;

in step vii, the optimized medium formula is as follows: 40g of sucrose, 10g of peptone, 10g of yeast extract powder, 0.3mg of L-phenylalanine, 0.05mg of sodium acetate, 0.02mg of sodium benzoate, 1000mL of distilled water and pH 7;

in the step vi, the inoculation amount is 1-2%, the fermentation temperature is 26 ℃, the tank pressure is 0.03Mpa, the pH is 7.4, the fermentation culture is carried out, the stirring rotation speed is 130r/min, the ventilation rate is 0.6-1.2 vvm/min, and after 16 hours, the fermentation tank rotation speed is 150r/min, and the ventilation rate is 0.8-1.2 vvm/min;

in the step ix, the fermentation time is 240 hours;

in steps i, iii, v and viii, the sterilization temperature is more than or equal to 121 ℃, and the sterilization time is more than or equal to 30 min.

The paclitaxel purification method is as follows:

1. pumping the prepared mixed solution of the endophytic fungi spores, the mycelia and the paclitaxel into a No. 1-2 40000L temporary storage tank of fermentation liquor;

2. pumping the prepared fermentation liquor into a coarse filtration device to filter the filtrate, pumping the filtrate into a full-automatic membrane micro-filtration unit to carry out micro-filtration, and filtering endophytic fungi spores and mycelia to obtain a crude mixed ginkgolide solution;

3. centrifuging the filtered endophytic fungi spores and mycelia by using an automatic scraper centrifuge for exsolution to prepare waste materials such as taxus chinensis endophytic fungi spores and mycelia;

4. pumping the prepared taxol mixed crude solution into a full-automatic control membrane ultrafiltration concentration unit for concentration to prepare taxol mixed concentrated solution and wastewater;

5. pumping the prepared taxol mixed concentrated solution into an automatic scraper centrifuge for centrifugation and desolventization to prepare a taxol crude product filter cake A;

6. delivering the taxol crude product filter cake A into a No. 1 3000L extraction kettle, adding a lipophilic solvent with the weight 3 times that of the extract into a decolorizing agent with the weight 0.1-0.3 times that of the extract for extraction and decolorization, filtering to remove the decolorizing agent, and combining the filtrates to obtain taxol filtrate;

7. pumping the paclitaxel filtrate into a 2000L/h concentration unit for concentration, and recovering the solvent to obtain a paclitaxel crude product concentrated solution;

8. pumping the taxol crude product concentrated solution into an automatic scraper centrifuge for centrifugal desolventizing and recovering the solvent to prepare a taxol crude product filter cake B;

9. feeding the obtained paclitaxel crude product filter cake B into a No. 2 3000L extraction kettle, extracting with lipophilic solvent with weight 3 times of that of the filter cake B, filtering, and mixing extractive solutions to obtain extractive solution;

10. pumping the paclitaxel extract into a 2000L/h concentration unit for concentration, and recovering the solvent to obtain a paclitaxel crude product concentrated solution;

11. pumping the taxol crude product concentrated solution into an automatic scraper centrifuge for centrifugal desolventizing and recovering the solvent to prepare a taxol crude product filter cake C;

12. mixing the taxol crude product filter cake C and the crystallized mother liquor extract, filling into a column feed preparation tank of a full-automatic control chromatography system, and dissolving with ethanol to prepare column feed;

13. feeding the prepared upper column liquid into a full-automatic control HZ-818 macroporous resin column for adsorption; eluting with deionized water and ethanol, and collecting eluate and eluate; taking the eluate as upper column liquid for next macroporous resin adsorption, eluting with deionized water and ethanol, and collecting the upper column adsorption effluent liquid and eluate;

14. loading the second macroporous resin on the column to adsorb the effluent and the eluate, respectively pumping into a concentration unit for concentration, and recovering the solvent to obtain a concentrated solution of crude paclitaxel;

15. pumping the prepared taxol crude product concentrated solution into an automatic scraper centrifuge for centrifugal desolventizing and recovering an ethanol solvent to prepare a taxol crude product filter cake D;

16. feeding the obtained paclitaxel crude product filter cake D into a full-automatic controlled freezing crystallization kettle, dissolving with solvent, crystallizing at-20 deg.C, filtering crystals, concentrating mother liquor with a concentration unit, recovering solvent, centrifuging, and removing solvent to obtain paclitaxel crude crystal and mother liquor extract;

17. feeding the obtained paclitaxel coarse crystal into No. 1 or No. 2 full-automatic controlled crystallization kettle, recrystallizing with solvent at-5 deg.C, filtering crystal, concentrating mother liquor with concentration unit, recovering solvent, centrifuging, and desolventizing to obtain paclitaxel wet crystal and mother liquor extract;

18. feeding the obtained paclitaxel wet crystal into full-automatic control supercritical CO2 drying device for desolventizing, drying and sterilizing to obtain paclitaxel crystal powder with concentration of 98% or more;

19. feeding the crystallization mother liquor into the step 12, and combining the crystallization mother liquor with the taxol crude product filter cake C to prepare upper column liquor;

20. and (3) feeding the taxol crystal powder which is prepared by three batches of fermentation and is more than or equal to 98% into a batch V-shaped mixer for mixing to prepare the taxol crystal powder product which is more than or equal to 98%.

In the step 2, the rough filtering equipment is an automatic scraper centrifuge or a bag type filter or a plate and frame type filter press; in the step 2, the capacity of the full-automatic control microfiltration unit is 4000L/h;

in the step 3, the model of the automatic scraper centrifuge is PGZ-1000;

in the step 4, the capacity of the full-automatic control ultrafiltration concentration unit is 4000L/h;

in step 5, the model of the automatic scraper centrifuge is PGZ-1000;

in step 8, the model of the automatic scraper centrifuge is PGZ-1000;

in step 15, the model of the automatic scraper centrifuge is PGZ-1000;

in step 14, the concentration unit is a fully-automatically controlled 2000L-4000L/h MVR evaporator or a full-electric evaporator or a membrane concentrator;

in the step 20, the batch V-shaped mixer is V-1000; the batch mixing time is 30-60 min.

In the step 6, the solvent is ethanol with volume fraction of 90-95%; the solvent extraction temperature is 40-50 ℃;

in steps 7, 10, 14, 16 and 17, the concentration unit is an MVR evaporator or a full-electric evaporator or a membrane concentration unit; the concentration temperature of the MVR evaporator or the full-electric evaporator is 55-60 ℃; the vacuum degree is 0.07-0.085 Mpa;

in step 9, the extraction lipophilic solvent is ethyl acetate or dichloromethane; the extraction temperature is 40-50 ℃; the extraction times are 3 times;

in the step 9, the solvent is ethanol or methanol with the volume fraction of 70-85% and the PH of 4-6; the solute concentration of the upper column liquid is 5 g-12 g/L;

in step 12, the macroporous resin of the full-automatic control chromatographic column system is used by combining HZ-818 and XAD-2 or HZ-818 and XAD-4 or HZ-818 and XAD-7 or HZ-818 and XAD-8, namely, the HZ-818 macroporous resin is firstly used for adsorption, the eluent is eluted by using a solvent, and then the eluent is used for adsorption and then elution by using XAD-2 or XAD-4 or XAD-7 or XAD-8.

In step 6, the decolorant is activated clay or activated carbon;

in step 19, the mother liquor extract is a crystallized mother liquor extract with the paclitaxel content of more than or equal to 30-60%.

In step 16, the crystallization solvent is absolute ethyl alcohol, methanol, isopropanol or acetone; stirring for 10-30 min each time; the crystallization temperature is 40-50 ℃→ -20 ℃; the crystallization time is 12-24 h each time; the freezing crystallization kettle is a full-automatic control 8000L crystallization kettle;

in the step 17, the No. 1 and No. 2 crystallization kettles are all automatically controlled 8000L crystallization kettles; the crystallization solvent is ethanol or methanol or acetone with volume fraction of 75-95% for recrystallization; stirring for 10-30 min each time; the crystallization temperature is 40-50 ℃→ 5 ℃; the crystallization time is 12-24 h each time.

In the step 13, the adsorption flow rate is 3-4 BV/h; respectively using deionized water, PH 4-6 and 85-95% ethanol or methanol in volume fraction as eluent; the elution flow rate of the deionized water is 2-3 BV/h; the gradient elution flow rate of the ethanol or the methanol is 3-5 BV/h;

in step 18, the supercritical drying device is a 200L supercritical drying device with double-extraction double-separation electric heating full-automatic control; supercritical drying and residual solvent removal are carried out at the pressure of 20-30 Mpa, the temperature of 40-60 ℃ and CO₂The flow rate is 1500-4500L/h, and the drying time is 360-480 min.

Example 1

The method for separating, mutating and preserving the Trichoderma HQ-24 strain comprises the following steps:

a. cutting fresh Taxus chinensis fruit in Yunnan into 0.5-1 cm by conventional tissue separation method²And (5) small blocks. Soaking the fruits in 75% alcohol for about 5min, washing with sterile water for 3-4 times, and soaking with 2.5% sodium hypochlorite for 3 min; planting the cutting part on a contact culture medium, and checking whether the surface is disinfected completely by adopting a tissue blotting method as a control;

b. the culture medium was incubated in a constant temperature incubator at 28 ℃. Observing at regular time, sequentially transferring the newly grown hyphae to a new solid culture medium by using an inoculating needle, purifying each strain for 2-3 times, and transferring to a test tube inclined plane to preserve two strains;

c. preparation of bacterial suspension: taking several inclined planes of the dominant strains cultured for one week, respectively adding physiological saline containing synergistic isonicotin, scraping off thalli on the inclined planes by using an inoculating loop, uniformly shaking, pouring the mixed solution into a sterile triangular flask, placing the sterile triangular flask on a constant temperature shaking table to resist oscillation, and filtering the mixture by using a funnel with a layer of sterile mirror wiping paper after the oscillation is finished to obtain a monospore suspension of the strains. Counting spores with a hemocytometer (hemocytometer) to adjust the spore concentration to about 10⁶Per ml;

d. ultraviolet mutagenesis: 5ml of the prepared monospore suspension was taken out into a sterile plate with a diameter of 9 mm. The 15W UV lamp was turned on to preheat and stabilize the light. The dish containing the bacterial suspension is placed on a magnetic stirrer, is away from a lamp tube by a certain distance, the dish cover is opened, and the cell is stirred and irradiated at the same time, so that the cell can uniformly absorb ultraviolet light waves. The top was operated in a red background, avoiding photorepair. The thallus after ultraviolet mutagenesis is immediately transferred into a sterile test tube and immersed in ice water for 30min to inhibit enzyme activity and repair. According to the principle of delay phenomenon, adding the irradiated bacterial suspension subjected to ice bath into a PDA culture medium for post-culture so as to improve mutation rate;

e. and (3) performing compound mutagenesis by combining ultraviolet rays with nitrous acid: prepared according to the method described above to about 10⁶Performing ultraviolet mutagenesis on spore suspension per mL, sucking 1mL of mutagenized spore suspension, placing the spore suspension into a large test tube, adding 2mL of acetic acid buffer solution with pH4.5 and 1mL of sodium nitrite solution with 0.1mol/L (the final treatment concentration is 0.025mol/L), preserving the temperature at 25-26 ℃ for 10-25 min, adding 20mL of disodium hydrogen phosphate solution with 0.07mol/L, PH 8.6.6 to reduce the pH to about 6.8 so as to stop the reaction, properly diluting, coating a flat plate, selecting a bacterial colony, performing shaking table fermentation, using the ultraviolet mutagenesis firstly and then evaluating;

i. the liquid shake flask strain culture medium is PDA, PH 7. The initial pH was 5.0. Preparing 250mL of the mixture, subpackaging the mixture into 250mL of triangular bottles, sterilizing the mixture for 30 minutes at 121 ℃ in each bottle, and cooling the mixture to room temperature for later use;

iii, preparing a seed culture medium according to a formula standard, pumping the seed culture medium into a No. 1 500L first-stage seed culture tank, and sterilizing by using a solid tank steam sterilization method, wherein the sterilization temperature is more than or equal to 121 ℃, and the sterilization time is more than or equal to 30 min;

iv, after the first-stage seed culture tank is sterilized, reducing the temperature of a culture medium in the tank to 28 ℃, inoculating the triangular flask fermentation liquor into a No. 1 500L first-stage seed fermentation tank through an aseptic inoculation system, and performing seed culture by using the seed culture medium, wherein the inoculation amount is 1%, the fermentation temperature is 28 ℃, the tank pressure is 0.10Mpa, the fermentation culture is performed for 18 hours under the condition that the pH is 7.2, the rotation speed of the seed tank is 120r/min and the ventilation volume is 0.036m within 0-9 hours³Min, after 9h, the rotation speed of the seeding tank is 180r/min, and the ventilation volume is 0.043m³Min, collecting seed liquid after fermentation culture;

v, preparing a seed culture medium according to a formula standard, filling the seed culture medium into a No. 1 1000L secondary seed fermentation tank, and sterilizing by using a solid tank steam sterilization method, wherein the sterilization temperature is more than or equal to 121 ℃, and the sterilization time is more than or equal to 30 min;

vi, after the secondary seed culture tank is sterilized, when the temperature is reduced to 28 ℃, inoculating the primary seed fermentation liquor fermented for 18 hours into a No. 1 1000L secondary seed fermentation tank through an aseptic inoculation system, and performing seed culture by using a seed culture medium, wherein the inoculation amount is 1-2%, the fermentation temperature is 28 ℃, the tank pressure is 0.10Mpa, the fermentation culture is performed for 18 hours under the conditions that the pH is 7.4, the rotation speed of the seed tank is 120r/min for 0-9 hours, and the ventilation volume is 1.2m³Min, after 9h, the rotation speed of the seeding tank is 150r/min, and the ventilation volume is 1.5m³Min, collecting seed liquid after fermentation culture;

vii, accurately weighing the culture medium materials in batches according to the formula, then putting the culture medium materials into a 40000L multiplied by 3 culture medium preparation tank, pumping purified water according to the formula, and stirring for 30 min;

viii, pumping the fermentation medium into a No. 1 100000L fermentation single tank or a No. 1-3 medium liquid supplementing tank, filling the fermentation medium into the tank by a coefficient of 0.7, and sterilizing by using a real tank steam sterilization method, wherein the sterilization temperature is more than or equal to 121 ℃, and the sterilization time is more than or equal to 30 min;

ix, after single-tank sterilization is finished, cooling a culture medium in the tank to 28 ℃ by using chilled water, inoculating the secondary seed fermentation broth fermented for 18h in the No. 1 fermentation tank with 100000L through a sterile inoculation system for fermentation culture, wherein the inoculation amount is 1%, the fermentation temperature is 26 ℃, the tank pressure is 0.03Mpa, the fermentation culture is carried out for 240h under the condition that the pH value is 7, the stirring speed is 130r/min, the ventilation amount is 0.5-0.8 vvm/min, and after 16h, the fermentation tank speed is 150r/min, and the ventilation amount is 1 vvm/min;

x, supplementing the culture medium in real time according to the standard of the culture medium amount in the fermentation period; after the culture medium is complemented and the fermentation time reaches the standard, finishing the fermentation to prepare a mixed solution of endophytic fungi, mycelia and paclitaxel;

the paclitaxel purification method comprises the following steps:

pumping the prepared mixed liquid of the endophytic fungi spores, the mycelia and the paclitaxel into a No. 1-2 40000L fermentation liquid temporary storage tank;

pumping the prepared fermentation liquor into filtrate filtered by a plate-and-frame filter press, pumping into a 4000L/h full-automatic membrane-controlled micro-filtration unit for micro-filtration, and filtering endophytic fungi spores and mycelia to prepare a crude ginkgolide mixed solution;

thirdly, centrifuging and desolventizing the filtered endophytic fungi spores and mycelia by using a PGZ-1000 automatic scraper centrifuge to prepare waste materials such as the taxus chinensis endophytic fungi spores and mycelia;

fourthly, pumping the prepared taxol mixed crude solution into a 4000L/h full-automatic control membrane ultrafiltration concentration unit for concentration to prepare taxol mixed concentrated solution and wastewater;

fifthly, pumping the prepared taxol mixed concentrated solution into a PGZ-1000 automatic scraper centrifuge for centrifugation and desolventizing to prepare a taxol crude product filter cake A;

sixthly, sending the taxol crude filter cake A into an extraction kettle No. 1 of 3000L, adding ethanol solvent with volume fraction of 70-95% of the weight of the extract and active carbon with weight of 0.1 time of the extract for extraction and decoloration, filtering to remove decolorant, and combining filtrates to obtain taxol filtrate;

pumping the paclitaxel filtrate into a 2000L/h all-electric evaporator concentration unit for concentration and solvent recovery to prepare a paclitaxel crude product concentrated solution;

pumping the taxol crude product concentrated solution into a PGZ-1000 automatic scraper centrifuge for centrifugal desolventizing and recovering the solvent to prepare a taxol crude product filter cake B;

conveying the prepared taxol crude product filter cake B into an extraction kettle with the volume of No. 2 and 3000L, extracting with ethyl acetate solvent with the weight of 3 times that of the filter cake B, filtering, and combining the extract liquid to prepare the extract liquid;

pumping the taxol ethyl acetate extract into a 2000L/h full-electric evaporator concentration unit for concentration, and recovering an ethyl acetate solvent to obtain a taxol crude product concentrated solution;

pumping the taxol crude product concentrated solution into a PGZ-1000 automatic scraper centrifuge for centrifugal desolventizing and recovering an ethyl acetate solvent to prepare a taxol crude product filter cake C;

combining the taxol crude product filter cake C and the crystallized mother liquor extract, filling the mixture into a column feed preparation tank of a full-automatic control chromatography system, and dissolving the mixture by using ethanol with the volume fraction of 70-95% to prepare column feed;

the prepared upper column liquid is sent into a full-automatic control HZ-818 macroporous resin column for adsorption; eluting with deionized water and ethanol with the volume fraction of 70-95%, and collecting the effluent and the eluate of column adsorption; adsorbing the eluate serving as upper column liquid by XAD-2 macroporous resin, eluting by deionized water and ethanol with the volume fraction of 70-95%, and collecting the upper column adsorption effluent liquid and the eluate;

subjecting the second macroporous resin to column adsorption of effluent liquid and eluent, respectively pumping into a full-electric evaporator concentration unit of 2000-4000L/h for concentration and solvent recovery to obtain a paclitaxel crude product concentrated solution;

pumping the prepared taxol crude product concentrated solution into a PGZ-1000 automatic scraper centrifuge for centrifugal desolventizing and recovering an ethanol solvent to prepare a taxol crude product filter cake D;

feeding the prepared taxol crude product filter cake D into a 8000L full-automatic control freezing crystallization kettle, dissolving the taxol crude product filter cake D by using a methanol solvent with the volume fraction of 70-95%, crystallizing at the low temperature of-20 ℃, filtering crystals, concentrating a mother solution by using a full-electric evaporator concentration unit, recovering the solvent, and centrifugally desolventizing to prepare taxol crude crystals and a mother solution extract;

⒄, delivering the obtained paclitaxel coarse crystal into a No. 1 or No. 2 8000L full-automatic control crystallization kettle, recrystallizing with 70-95 vol% methanol solvent at-5 deg.C, filtering the crystal, concentrating the mother liquor with 2000L/h full-electric evaporator, recovering the solvent, centrifuging with PGZ-1000 automatic scraper centrifuge, and desolventizing to obtain paclitaxel wet crystal and mother liquor extract;

feeding the prepared paclitaxel wet crystal into a 200L full-automatic control supercritical CO2 drying device for desolventizing, drying and sterilizing to obtain more than or equal to 98% paclitaxel crystal powder 9.62 kg;

⒆, feeding the crystallized mother solution into the step of water pumping, and combining the crystallized mother solution with the paclitaxel crude product filter cake C to prepare upper column solution;

⒇, feeding the taxol crystal powder with the concentration of more than or equal to 98 percent obtained by three batches of fermentation into a batch V-1000 type mixer for mixing to obtain a taxol crystal powder product with the concentration of more than or equal to 98 percent;

example 2

iii, preparing a seed culture medium according to a formula standard, pumping the seed culture medium into a No. 2 500L first-stage seed culture tank, and sterilizing by using a solid tank steam sterilization method, wherein the sterilization temperature is more than or equal to 121 ℃, and the sterilization time is more than or equal to 30 min;

iv, after the first-stage seed culture tank is sterilized, reducing the temperature of a culture medium in the tank to 28 ℃, inoculating the triangular flask fermentation liquor into a No. 2 500L first-stage seed fermentation tank through an aseptic inoculation system, and performing seed culture by using the seed culture medium, wherein the inoculation amount is 1%, the fermentation temperature is 28 ℃, the tank pressure is 0.10Mpa, the fermentation culture is performed for 18 hours under the condition that the pH is 7.2, the rotation speed of the seed tank is 120r/min and the ventilation volume is 0.036m within 0-9 hours³Min, after 9h, the rotation speed of the seeding tank is 180r/min, and the ventilation volume is 0.043m³Min, collecting seed liquid after fermentation culture;

v, preparing a seed culture medium according to a formula standard, filling the seed culture medium into a No. 2 1000L secondary seed fermentation tank, and sterilizing by using a solid tank steam sterilization method, wherein the sterilization temperature is more than or equal to 121 ℃, and the sterilization time is more than or equal to 30 min;

vi, after the secondary seed culture tank is sterilized, when the temperature is reduced to 28 ℃, inoculating the primary seed fermentation liquor fermented for 18 hours into a No. 2 1000L secondary seed fermentation tank through an aseptic inoculation system, and performing seed culture by using a seed culture medium, wherein the inoculation amount is 1-2%, the fermentation temperature is 28 ℃, the tank pressure is 0.10Mpa, the fermentation culture is 18 hours under the conditions that the pH is 7.4, the rotation speed of the seed tank is 120r/min for 0-9 hours, and the ventilation volume is 1.2m³Min, after 9h, the rotation speed of the seeding tank is 150r/min, and the ventilation volume is 1.5m³Min, collecting seed liquid after fermentation culture;

viii, pumping the fermentation medium into a No. 2 100000L fermentation single tank or a No. 1-2 medium liquid supplementing tank, filling the fermentation medium into the tank by a coefficient of 0.7, and sterilizing by using a real tank steam sterilization method, wherein the sterilization temperature is more than or equal to 121 ℃, and the sterilization time is more than or equal to 30 min;

ix, after single-tank sterilization is finished, cooling a culture medium in the tank to 28 ℃ by using chilled water, inoculating the secondary seed fermentation broth fermented for 18h in the No. 2 fermentation tank with 100000L through a sterile inoculation system for fermentation culture, wherein the inoculation amount is 1%, the fermentation temperature is 26 ℃, the tank pressure is 0.03Mpa, the fermentation culture is carried out for 240h under the condition that the pH value is 7, the stirring speed is 130r/min, the ventilation amount is 0.5-0.8 vvm/min, and after 16h, the fermentation tank speed is 150r/min, and the ventilation amount is 1 vvm/min;

the paclitaxel purification method comprises the following steps:

pumping the prepared mixed liquid of the endophytic fungi spores, the mycelia and the paclitaxel into a No. 1-2 30000L fermentation liquid temporary storage tank;

pumping the prepared fermentation liquor into a filtrate filtered by a bag type filter, pumping the filtrate into a 4000L/h full-automatic membrane micro-filtration unit for micro-filtration, and filtering endophytic fungi spores and mycelia to prepare a crude ginkgolide mixed solution;

sixthly, feeding the taxol crude filter cake A into an extraction kettle No. 1 of 3000L, extracting and decoloring for 1-3 times by adding methanol solvent with volume fraction of 70-95% and extract weight being 3 times of that of the extract and activated clay with weight being 0.2 time of that of the extract, filtering to remove a decoloring agent, and combining filtrates to obtain taxol filtrate;

pumping the paclitaxel filtrate into a 2000L/h membrane concentration unit for concentration and solvent recovery to prepare a paclitaxel crude product concentrated solution;

conveying the prepared taxol crude product filter cake B into an extraction kettle with the volume of No. 2 3000L, extracting for 3 times by using dichloromethane solvent with the weight of 3 times that of the filter cake B, filtering, and combining extract liquid to prepare extract liquid;

pumping the taxol extract into a 2000L/h membrane concentration unit for concentration and solvent recovery to obtain taxol crude product concentrated solution;

combining the taxol crude product filter cake C and the crystallized mother liquor extract, filling the mixture into a column feed preparation tank of a full-automatic control chromatography system, and dissolving the mixture by using methanol with the volume fraction of 70-95% to prepare column feed;

the prepared upper column liquid is sent into a full-automatic control HZ-818 macroporous resin column for adsorption; eluting with deionized water and 70-95% methanol by volume fraction, and collecting the effluent and the eluate of column adsorption; adsorbing the eluate serving as upper column liquid by XAD-4 macroporous resin, eluting by deionized water and ethanol with the volume fraction of 70-95%, and collecting the upper column adsorption effluent liquid and the eluate;

subjecting XAD-4 macroporous resin to column adsorption of effluent liquid and eluent, respectively pumping into a 2000-4000L/h membrane concentration unit for concentration, and recovering solvent to obtain taxol crude product concentrated solution;

feeding the prepared taxol crude product filter cake D into a 8000L full-automatic control freezing crystallization kettle, dissolving the taxol crude product filter cake D by using an ethanol solvent with the volume fraction of 70-95%, crystallizing at the low temperature of-20 ℃, filtering crystals, concentrating a mother solution by using a 2000L/h membrane concentration unit, recovering the solvent, and centrifugally desolventizing by using a PGZ-1000 automatic scraper centrifuge to prepare taxol crude crystals and a mother solution extract;

⒄, delivering the obtained paclitaxel coarse crystal into a No. 1 or No. 2 8000L/h full-automatic control crystallization kettle, recrystallizing with 70-95 vol% ethanol solvent at-5 deg.C, filtering the crystal, concentrating the mother liquor with 2000L/h membrane concentration unit, recovering the solvent, centrifuging with PGZ-1000 automatic scraper centrifuge, and desolventizing to obtain paclitaxel wet crystal and mother liquor extract;

feeding the prepared paclitaxel wet crystal into a full-automatic control 200L supercritical CO2 drying device for desolventizing, drying and sterilizing to obtain more than or equal to 98% paclitaxel crystal powder 9.58 kg;

example 3

iii, preparing a seed culture medium according to a formula standard, sequentially pumping the seed culture medium into No. 3, No. 4, No. 5, No. 6, No. 7 and No. 8 500L first-stage seed culture tanks respectively, and sterilizing by using a solid tank steam sterilization method, wherein the sterilization temperature is more than or equal to 121 ℃, and the sterilization time is more than or equal to 30 min;

iv, after the first-stage seed culture tank is sterilized, reducing the temperature of a culture medium in the tank to 28 ℃, sequentially inoculating the triangular flask fermentation liquor into No. 3, No. 4, No. 5, No. 6, No. 7 and No. 8 first-stage seed fermentation tanks respectively through an aseptic inoculation system, performing seed culture by using the seed culture medium, wherein the inoculation amount is 1%, the fermentation temperature is 28 ℃, the tank pressure is 0.10Mpa, the fermentation culture is performed for 18h under the condition that the pH is 7.2, the rotation speed of the seed tank is 120r/min and the ventilation amount is 0.036m within 0-9 h³Min, after 9h, the rotation speed of the seeding tank is 180r/min, and the ventilation volume is 0.043m³Min, collecting seed liquid after fermentation culture;

v, preparing a seed culture medium according to a formula standard, sequentially and respectively filling the seed culture medium into No. 3, No. 4, No. 5, No. 6, No. 7 and No. 8 secondary seed fermentation tanks, and sterilizing by using a solid tank steam sterilization method, wherein the sterilization temperature is more than or equal to 121 ℃, and the sterilization time is more than or equal to 30 min;

vi, after the second-stage seed culture tank is sterilized, when the temperature is reduced to 28 ℃, sequentially inoculating the first-stage seed fermentation liquor fermented for 18 hours into a second-stage seed fermentation tank of No. 3, No. 4, No. 5, No. 6, No. 7 and No. 8L through an aseptic inoculation system, respectively, performing seed culture by using a seed culture medium, wherein the inoculation amount is 1-2%, the fermentation temperature is 28 ℃, the tank pressure is 0.10Mpa, the fermentation culture is performed for 18 hours under the condition that the pH is 7.4, the rotating speed of the seed tank is 120r/min and the ventilation amount is 1.2m for 0-9 hours³Min, after 9h, the rotation speed of the seeding tank is 150r/min, and the ventilation volume is 1.5m³Min, collecting seed liquid after fermentation culture;

viii, sequentially pumping the fermentation culture medium into No. 3, No. 4, No. 5, No. 6, No. 7, No. 8, No.9, No. 10 100000L fermentation single tanks or No. 1-No. 3 culture medium liquid supplementing tanks in batches, filling the fermentation single tanks into the tanks by a coefficient of 0.7, and sterilizing by using a solid tank steam sterilization method, wherein the sterilization temperature is more than or equal to 121 ℃, and the sterilization time is more than or equal to 30 min;

ix, after single-tank sterilization is finished, cooling a culture medium in the tank to 28 ℃ by using chilled water, sequentially and respectively inoculating the secondary seed fermentation liquor fermented for 18h in No. 3, 4, 5, 6, 7 and 8 to No. 3, 4, 5, 6, 7 and 8 100000L fermentation tanks for fermentation culture by using an aseptic inoculation system, wherein the inoculation amount is 1%, the fermentation temperature is 26 ℃, the tank pressure is 0.03MPa, the pH is 7, the fermentation culture is carried out for 240h, the stirring rotation speed is 130r/min, the ventilation amount is 0.5-0.8 vvm/min, and after 16h, the rotation speed of the fermentation tank is 150r/min, and the ventilation amount is 1 vvm/min;

x, supplementing the culture medium in real time according to the standard of the culture medium amount in sequence in the fermentation period; after the culture medium is complemented and the fermentation time reaches the standard, finishing the fermentation to prepare a mixed solution of endophytic fungi, mycelia and paclitaxel;

the paclitaxel purification method comprises the following steps:

the method comprises the steps of sequentially and respectively pumping prepared endophytic fungus spores, mycelia and paclitaxel mixed liquor into a No. 1-No. 2 30000L fermentation liquor temporary storage tank;

pumping the prepared fermentation liquor into a filtrate obtained after centrifugation of an automatic scraper centrifuge, pumping the filtrate into a 4000L/h full-automatic control membrane micro-filtration unit for micro-filtration, and filtering endophytic fungi spores and mycelia to prepare a ginkgolide mixed crude liquid;

thirdly, centrifuging and desolventizing the filtered endophytic fungi spores and the filtered mycelia respectively by a PGZ-1000 automatic scraper centrifuge in sequence to prepare waste materials such as the taxus chinensis endophytic fungi spores and the filtered mycelia;

fourthly, pumping the prepared taxol mixed crude solution into a 4000L/h full-automatic control membrane ultrafiltration concentration unit for concentration respectively according to the sequence to prepare taxol mixed concentrated solution and wastewater;

fifthly, pumping the prepared taxol mixed concentrated solution into a PGZ-1000 automatic scraper centrifuge respectively in sequence for centrifugal desolventizing to prepare a taxol crude product filter cake A;

sixthly, sequentially and respectively feeding the taxol crude filter cake A into an extraction kettle No. 1 of 3000L, extracting and decoloring for 1-3 times by adding activated clay with the weight of 0.3 time into an ethyl acetate solvent with the volume fraction of 3 times the weight of the extract, filtering to remove a decoloring agent, and combining filtrates to obtain taxol filtrate;

pumping paclitaxel filtrate into 2000L/h MVR evaporator concentration unit in sequence for concentration, and recovering solvent to obtain paclitaxel crude product concentrated solution;

pumping the taxol crude product concentrated solution into a PGZ-1000 automatic scraper centrifuge for centrifugal desolventizing and solvent recovery in sequence respectively to prepare a taxol crude product filter cake B;

the method comprises the steps of self-lifting, sequentially and respectively feeding the obtained paclitaxel crude product filter cake B into a No. 2 3000L extraction kettle, extracting for 3 times by using ethyl acetate solvent with the weight 3 times that of the filter cake B, filtering, and combining the extract liquid to obtain the extract liquid;

firstly, sequentially pumping the paclitaxel ethyl acetate extract into a 2000L/h MVR evaporator concentration unit for concentration and solvent recovery to prepare a paclitaxel crude product concentrated solution;

pumping the concentrated solution of the taxol crude product into an automatic scraper centrifuge in sequence for centrifugal desolventizing and solvent recovery to prepare a taxol crude product filter cake C;

combining the taxol crude product filter cake C and the crystallized mother liquor extract, sequentially and respectively filling the mixture into a column feeding liquid preparation tank of a full-automatic control chromatography system, and dissolving the mixture by using ethanol with the volume fraction of 70-95% to prepare column feeding liquid;

selecting the selection, and respectively sending the prepared upper column liquid into a full-automatic control HZ-818 macroporous resin column for adsorption in sequence; eluting with deionized water and ethanol with the volume fraction of 70-95%, and collecting the effluent and the eluate of column adsorption; adsorbing the eluate serving as upper column liquid by XAD-7 macroporous resin, eluting by deionized water and ethanol with the volume fraction of 70-95%, and collecting the upper column adsorption effluent liquid and the eluate;

respectively loading XAD-7 macroporous resin on a column to adsorb effluent and eluent in sequence, pumping into a 2000L/h MVR evaporator concentrator set to concentrate, and recovering solvent to obtain taxol crude product concentrated solution;

pumping the prepared taxol crude product concentrated solution into a PGZ-1000 automatic scraper centrifuge in sequence for centrifugal desolventizing and recovering an ethanol solvent to prepare a taxol crude product filter cake D;

sequentially and respectively feeding the prepared taxol crude product filter cake D into a 8000L full-automatic control freezing crystallization kettle, dissolving the taxol crude product filter cake D with an acetone solvent with the volume fraction of 70-95%, crystallizing at the low temperature of-20 ℃, filtering crystals, concentrating a mother solution by an MVR evaporator concentration unit, recovering the solvent, and centrifugally desolventizing by a PGZ-1000 automatic scraper centrifuge to prepare taxol crude crystals and a mother solution extract;

⒄, sequentially and respectively feeding the obtained paclitaxel coarse crystals into a No. 1 or No. 2 8000L full-automatic control crystallization kettle, recrystallizing with 70-95 vol% acetone solvent at-5 deg.C, filtering the crystals, concentrating the mother liquor by an MVR evaporator set, recovering the solvent, centrifuging and desolventizing by a PGZ-1000 automatic scraper centrifuge to obtain paclitaxel wet crystals and mother liquor extract;

sequentially and respectively sending the prepared paclitaxel wet crystals into a 200L full-automatic control supercritical CO2 drying device for desolventizing, drying and sterilizing to obtain 38.78kg of paclitaxel crystal powder with the concentration of more than or equal to 98%;

⒇, feeding the taxol crystal powder with the concentration of more than or equal to 98 percent obtained by three batches of fermentation into a batch V-1000 type mixer for mixing to obtain the taxol crystal powder product with the concentration of more than or equal to 98 percent.

Example 4

Detection and identification of paclitaxel products

1. Method for evaluating mutagenesis results

First evaluation of TLC

Quantitatively sucking paclitaxel of 0.35mg/L of 5. mu.l, 10. mu.l, 15. mu.l, 20. mu.l, 25. mu.l and 30. mu.l respectively with a microsyringe, spotting on the activated silica gel G plate, developing, taking out, air drying, fumigating with acetic anhydride vapor for 0.5h, taking out, baking at 160 ℃ for 0.5h, performing ultraviolet inspection at 365nm, taking a picture with a digital camera, taking a picture with a gel imager by a quatity one, scanning and analyzing by totallab to obtain the light absorption value of the developed spot of paclitaxel, examining the relation between the sample intake and the light absorption value of the developed spot, and drawing a paclitaxel standard curve. And (3) quantitatively spotting the fermentation sample and the paclitaxel (high and low dose) of each mutant strain on the same silica gel plate, and calculating the content of the total paclitaxel substances by measuring and calculating the content of the sample by totallab after the same method is developed.

Evaluation of HPLC-UV

Paclitaxel standard curve was established using HPLC: 0.85mg of paclitaxel standard substance is weighed, dissolved by ethanol and dissolved to 10 mL. High Performance Liquid Chromatography (HPLC) determination, the chromatographic conditions are as follows: mobile phase, methanol-water-acetonitrile (23: 41: 36), C18 column (4.6mm × 150mm,5 μm), flow rate 1mL/min, detection wavelength 227nm, sample size 20 μ L, column temperature: standard curves were plotted at room temperature, single point. The paclitaxel samples prepared in the first to third examples and the paclitaxel standard were tested by HPLC and had the same time of peak appearance.

The calculation formula of the contents of the paclitaxel and the paclitaxel is as follows:

paclitaxel content in fermentation broth (μ g/L) ═ paclitaxel content in methanol (mg/m L) volume of methanol used to dissolve extract (m L)10⁶Volume of fermentation broth taken at extraction (m L);

2. the paclitaxel obtained in examples 1 to 3 was subjected to ultraviolet spectroscopy, infrared spectroscopy, mass spectroscopy, and nuclear magnetic resonance analysis, and compared with a paclitaxel standard sample, the results were the same, and thus it was found that the paclitaxel prepared according to the present invention has a spectrum highly consistent with that of the purchased standard sample.

3. In vitro biological experiments prove that the taxol sample prepared by the invention has the same killing percentage on breast cancer cells, ovarian cancer cells and taxol standard products. The above-mentioned embodiments only show some embodiments of the present invention, and the description thereof is more specific and detailed, but should not be construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present invention should be subject to the claims.

Claims

1. An industrial preparation process for producing taxol by fermenting a taxus chinensis endophytic fungi mutant is characterized by comprising the following steps:

s5: complex mutagenesis with UV-combined nitrous acid, prepared as described above for about 10⁶Per ml ofThe method comprises the following steps of (1) carrying out ultraviolet mutagenesis according to the method, then sucking 1mL of the mutagenized spore suspension, putting the spore suspension into a large test tube, adding 2mL of acetic acid buffer solution with pH4.5 and 1mL of sodium nitrite solution with 0.1mol/L, keeping the treatment concentration at 0.025mol/L, carrying out heat preservation at 25-26 ℃ for 10-25 min, adding 20mL of disodium hydrogen phosphate solution with 0.07mol/L, PH 8.6.6 to reduce the pH to 6.8 so as to stop the reaction, diluting, coating a flat plate, selecting a bacterial colony, carrying out shaking table fermentation, and carrying out evaluation after the ultraviolet mutagenesis is used;

2. The industrialized preparation process of paclitaxel by fermentation of yew endophytic fungi mutant according to claim 1, which is characterized in that: the strain name is Trichoderma HQ-24 strain; the preservation place is as follows: the preservation number of the China general microbiological culture Collection center (address: No. 3 Xilu No. 1 Beijing of Chaoyang district, Beijing) is CGMCC No. 9251.

3. The industrialized preparation process of paclitaxel by fermentation of yew endophytic fungi mutant according to claim 1, which is characterized in that the preparation and industrial fermentation method of the strain are as follows:

4. The industrialized preparation process of paclitaxel by fermentation of yew endophytic fungi mutant according to claim 3, which is characterized in that:

in the step ix, the inoculation amount is 1-2%, the fermentation temperature is 26 ℃, the tank pressure is 0.03Mpa, the pH is 7.4, the fermentation culture is carried out, the stirring rotation speed is 130r/min, the ventilation rate is 0.6-1.2 vvm/min, and after 16 hours, the fermentation tank rotation speed is 150r/min, and the ventilation rate is 0.8-1.2 vvm/min;

in the step x, the fermentation time is 240 hours;

5. The industrialized preparation process of paclitaxel by fermentation of yew endophytic fungi mutant according to claim 1, characterized in that the paclitaxel purification method is as follows:

6. The industrialized preparation process of paclitaxel by fermentation of yew endophytic fungi mutant according to claim 5, which is characterized in that: in the step 2, the rough filtering equipment is an automatic scraper centrifuge or a bag type filter or a plate and frame type filter press; in the step 2, the capacity of the full-automatic control microfiltration unit is 4000L/h;

in the step 3, the model of the automatic scraper centrifuge is PGZ-1000;

in step 5, the model of the automatic scraper centrifuge is PGZ-1000;

in step 8, the model of the automatic scraper centrifuge is PGZ-1000;

in step 15, the model of the automatic scraper centrifuge is PGZ-1000;

7. The industrialized preparation process of paclitaxel by fermentation of yew endophytic fungi mutant according to claim 5, which is characterized in that:

8. The industrialized preparation process of paclitaxel by fermentation of yew endophytic fungi mutant according to claim 5, which is characterized in that:

in step 6, the decolorant is activated clay or activated carbon;

9. The industrialized preparation process of paclitaxel by fermentation of yew endophytic fungi mutant according to claim 5, which is characterized in that:

10. The industrialized preparation process of paclitaxel by fermentation of yew endophytic fungi mutant according to claim 5, which is characterized in that: