JP5245227B2 - Separation by hydrophobic foaming of hydrophobic compounds - Google Patents

Description

  The present invention relates to a method for separating a hydrophobic compound produced by utilizing a biological reaction in an aqueous medium from the aqueous medium by using bubbles, and is suitable for simplifying a conventional bioproduction process. Can be used for

  Conventionally, utilizing the property of concentrating dissolved or dispersed substances possessed by bubbles, a general foaming, that is, a floating sorting method by generating bubbles (for example, see Patent Document 1), a foam separation method (for example, a patent) Ref. 2), or a pressurized flotation method (for example, see Non-Patent Document 1), etc. was developed to treat wastewater, remove toner, and mine in pulp and other pollutants. It is carried out for the purpose of metal refining and separation of pulverized coal. The nature of the concentration of a hydrophobic substance near the interface of such a bubble is, in principle, dislikes the hydrophobic interaction, that is, the electrical interaction that the hydrophobic substance has in the aqueous solution, and moves away from the hydrophilic group. This is based on the property of gathering at the gas-liquid interface between the aqueous medium and the bubbles (see, for example, Non-Patent Document 2 and Non-Patent Document 3).

  In recent years, the importance of bio-production technology has been recognized for a long time, but in the bio-industry, there is almost no production process using bubbles for recovery and separation of target products. For example, attempts to use bubbles instead of ion exchange resins in amino acid recovery (for example, see Non-patent Document 4) are only slightly observed.

  Conventionally, in the production of biologically useful substances including a hydrophobic substance, an extraction method using an organic solvent is generally used when separating a target substance from a culture solution. For example, in the production of paclitaxel by yew (Taxus cuspidata) cells, it is necessary to extract the paclitaxel using an organic solvent and further purify it. However, since a large amount of organic solvent is used, the recovery cost is not negligible. In addition, there is a concern that the process becomes complicated due to troubles such as extraction or back-extraction, or contamination when the organic solvent leaks into the environment.

JP 51-96702 A JP 2001-170617 A "Freshing Technology Handbook", Water Recycling Promotion Center (issued on May 10, 1993) Chemical Engineering Handbook 6th edition, page 611 (issued by Maruzen Co., Ltd.) Flow 23 (2004) 7-15. Abstracts of the 30th Autumn Meeting of the Chemical Society of Japan (1997, L113)

  It is an object of the present invention to produce a useful metabolite produced by a living cell containing a microorganism or a reaction process using an enzyme in an aqueous medium such as a culture solution or an enzyme reaction solution, that is, a medium containing water or water. Establishing a method that can easily separate the useful hydrophobic metabolites dissolved or dispersed in the organic solvent without performing an extraction operation with an organic solvent or greatly reducing the load of extraction, as well as conventional organic solvents It is an object of the present invention to provide a simple manufacturing means with a small environmental load such as waste liquid treatment, by omitting or minimizing the steps of extraction and back-extraction necessary in the separation method used, avoiding complicated processes and equipment.

  As a result of intensive studies in order to solve the above problems, the present inventors, from a culture solution containing a small amount of paclitaxel produced by yew cell culture, the culture using the hydrophobic interaction with bubbles I learned that it can be separated out of the liquid. This eliminates the need for organic solvent extraction and solvent recovery steps, which are indispensable in the prior art, and a number of complicated subsequent purification steps. I also learned that production costs and the environmental burden associated with production can be reduced because organic solvents are not used or can be used at a minimum.

  Furthermore, it can be widely applied to the separation of organic compounds dissolved or dispersed in an aqueous medium such as a culture solution or enzyme reaction solution of microorganisms, animal cells, or plant cells. It is possible to prevent product inhibition during cultivation by combining the two, and the foaming system, and the equilibrium of the enzyme reaction can be shifted so that a yield higher than the equilibrium reaction rate can be expected. Not only the production of paclitaxel by yew cells, but also various coenzymes such as steroidal compounds and pyrroloquinoline quinone (PQQ) produced by isoflavones, microorganisms or enzymes produced by Kueru (Pueraria lobata) cells Knowing that it can be applied to the separation of various bioproducts, the present invention has been achieved.

That is, the present invention relates to a method for separating a hydrophobic substance from an aqueous medium using bubbles, as shown in the following (1) to (6).
(1) In a method of separating a hydrophobic compound produced by a biological reaction performed in an aqueous medium, after contacting the bubble and the aqueous medium to collect the hydrophobic compound contained in the aqueous medium at the bubble interface, A method for separating a hydrophobic compound, wherein the bubbles are separated from an aqueous medium.
(2) The method for separating a hydrophobic compound according to (1), wherein the biological reaction utilizes microorganisms, animal cells, plant cells, or enzymes.
(3) The method for separating a hydrophobic compound according to (1) or (2), wherein bubbles generated by blowing gas into an aqueous medium are used.
(4) The method for separating a hydrophobic compound according to (1) or (2), wherein bubbles are generated outside the aqueous medium, and the bubbles and the aqueous medium are contacted outside the biological reaction system.
(5) The method for separating a hydrophobic compound according to any one of (1) to (4), wherein millibubbles, microbubbles, or nanobubbles are used as the bubbles.
(6) The method for separating a hydrophobic compound according to any one of (1) to (5), wherein the hydrophobic compound is paclitaxel produced by plant cell culture of Taxus cuspidata.

  In accordance with the present invention, hydrophobic bioproduction useful in various bioproduction processes by foaming with low-cost and easy-to-use gas such as air without using an organic solvent and contacting with the resulting bubbles. The product can be separated from the culture solution or the enzyme reaction solution. In addition, the separation process can be simplified, the risk of environmental pollution caused by organic solvents can be avoided, and the product can be expected to have an effect of proceeding beyond the limits based on product inhibition and reaction equilibrium because the product moves out of the system. I can do it.

The present invention relates to microorganism culture using an aqueous medium, cell culture of animals and plants, or enzyme reaction.
It is suitable when the product dissolved and dispersed in an aqueous medium, particularly when the target product is a hydrophobic substance. Since the amount of the hydrophobic substance collected with respect to the bubbles depends on the size of the bubble surface area, if applied to the separation and production of a hydrophobic substance with a high rare value contained in a small amount in an aqueous medium, it is possible to obtain a multi-stage by organic solvent. Efficient production process can be constructed by omitting the purification process.

  Since the present invention can be widely applied in fermentation culture, cell culture, and enzyme reaction using an aqueous medium, the target objects are various. For example, proteins, polysaccharides, complex polysaccharides, fatty acids such as arachidonic acid and DHA, carotenes such as carotenoids and astaxanthin, terpenes such as hinokitiol, polyphenols such as epigallocatechin and theoflavin, chalcone and isoflavone derived from Ashitaba Flavonoids, saponins such as steroid glycosides and triterpene glycosides, anticancer agents such as paclitaxel (taxol), vitamins such as pyrroloquinoline quinone (PQQ), and other herbal medicine components, among others Particularly preferred examples include hydrophobic substances produced extracellularly.

  Means for producing the target substance include a fermentation production system using microorganisms, a tissue culture system using animal and plant cells, or a hydrolysis reaction, transesterification reaction, and esterification reaction system in an aqueous medium using an enzyme catalyst. The production system described above can be usually carried out in a typical bioreactor system comprising a microbial fermentation tank, a cell culture tank, a medium adjustment tank, a cell separator, an ion exchange tower, a dryer and the like.

In the case of fermentation production, the substance is generally produced in an aqueous solution containing various minerals and additives, which may contain so-called liquid culture media or organic compounds. Although it is difficult to prescribe the reaction conditions during production in a wide range, it is difficult to follow, but general culture procedures are followed. More specifically, as a carbon source, monosaccharides such as glucose and fructose, disaccharides such as sucrose, polysaccharides such as starch and corn starch, waste molasses and pulp waste liquid, oils and fats such as olive oil and paraffin, methanol and ethanol, etc. As long as it can assimilate microorganisms and animal and plant cells, such as alcohols, organic acids such as acetic acid, and ketones such as acetone. As the nitrogen source, inorganic or organic nitrogen sources such as urea, ammonium chloride, ammonium nitrate, ammonium sulfate, tryptone, yeast extract, meat extract, peptone, and malt extract are used, and inorganic salts such as potassium phosphate, magnesium sulfate, and sodium chloride are used.
The culture conditions vary depending on the target species, but the initial pH of the culture solution is 3 to 10, the temperature is 10 to 50 ° C., and the culture is performed in batch or continuously with aeration and stirring as necessary.

  In the case of an enzyme reaction, it can be carried out using the same apparatus as in the case of fermentation production. The aqueous medium in the reaction is preferably water or a medium containing water, and a buffer solution having an optimal composition may be selected and used depending on the type of reaction. In the case where the enzyme reaction is continuously performed, it is preferable to use an immobilized enzyme supported on a carrier such as a bead to facilitate separation of the enzyme and the aqueous medium. In order to increase the reaction rate of the enzyme, metal ions such as Zn, Mn, Fe, and Cu, surfactants, organic solvents, and the like may be used.

In the present invention, the simplest means is to blow air into the same culture tank (reaction tank) in order to separate the target product after completion of cell culture or enzyme reaction. In a system in which aerobic culture is performed with aeration and agitation, bubbles generated by aeration can be used together for separation of the target product.
The size of the bubble is preferably smaller because the size of the gas-liquid contact area affects the amount of collected target product. For example, the millimeter-size and micro-size that have recently made stable generation possible. Alternatively, nano-sized bubbles can be used. A surfactant may be added to the system in order to increase the efficiency of trapping in bubbles, and naturally a gas such as nitrogen other than air may be introduced as necessary. In addition, it is difficult to prescribe | regulate unconditionally conditions, such as above-mentioned ventilation | gas_flowing amount, bubble size, and temperature, It is necessary to carry out according to it, after determining optimal conditions beforehand.

  If cell culture or enzyme reaction is continued in the same culture tank (reaction tank), reaction product inhibition (product inhibition) may occur. In such a case, it is preferable that foaming is performed in a separate apparatus, and the liquid extracted from the culture tank (reaction tank) is transferred to the apparatus and brought into contact with the generated bubbles. If the product collected in the bubbles is separated out of the system by means such as overflowing the weir, the reaction can proceed more than the production inhibition and the equilibrium reaction rate.

The case where the present invention is applied to the production of paclitaxel from yew (Taxus cuspidata) callus by plant cell culture will be further described. Usually, paclitaxel is produced in a batch or continuous callus culture process, a separation process that separates callus cells from the culture solution, a crushing process for separated culture cells, an extraction process using organic solvents, a waste cell separation process, a solvent concentration process, a purification process And a step of extracting and concentrating paclitaxel from the culture solution obtained in the separation step, which is a considerably complicated process (see FIG. 1).
In the present invention, basically, only the culture, bubble separation, washing, and drying steps are required, and complicated process operations related to solvent extraction, concentration, and separation used in the conventional paclitaxel production step are not required, and the process is greatly increased. Can be simplified (see FIGS. 2 and 3). The paclitaxel thus obtained usually has a sufficient chemical purity, but it is preferable that the paclitaxel be purified to a high purity that can be used for pharmaceutical purposes if necessary.

  The present invention will be described in more detail with reference to examples and comparative examples. Of course, the present invention is not limited to only these examples.

Example 1
Separation of paclitaxel by bubbles (cell-free system)
As shown in FIG. 4, an internal glass container (capacity 150 mL) is installed in an external glass container (capacity 650 mL), and further, a glass ball filter and cells for generating bubbles in the internal glass container A cellulosic porous bag was installed to hold the bag.
Place 120 mL of B5 medium (containing 3 g / L of casamino acid and 2 mg / L of α-naphthalene acetic acid) in which paclitaxel is dissolved at a concentration of 0.4 mg / L in an internal glass container, and disinfect the filter at 25 ° C. The paclitaxel was separated out of the system by collecting the foam overflowing from the internal glass container by aeration for 6 hours in the range of 0 to 500 mL per minute, and collecting the foam overflowing from the system, and the paclitaxel was recovered by extraction with dichloromethane. At that time, the wall of the outer container, the rubber stopper, the outer wall of the inner container, and the like were washed with dichloromethane and added to the recovered paclitaxel. In addition, paclitaxel remaining in the B5 medium in the inner glass container was also extracted and recovered using dichloromethane. Thus, when the relationship between the aeration amount and the aeration time and the recovery rate of paclitaxel was determined, the amount of remaining paclitaxel in the B5 medium decreased with time, and 50% or more of the initial amount of paclitaxel decreased with the passage of 2 hours. In 6 hours, 74% or more was removed and separated by bubbles (Table 1).
For paclitaxel in B5 medium, dichloromethane was added to the sampled medium at a ratio of 1: 1, stirred, and then allowed to stand under ice cooling to collect a separated dichloromethane phase. This operation was repeated three times, and the total amount of dichloromethane obtained was concentrated with a centrifugal evaporator, dissolved in 125 μL of methanol, and analyzed by HPLC. The HPLC mobile phase was methanol: water: acetonitrile = 20: 56: 24, and the stationary phase was silica gel having a cyanopropyl group.

Comparative Example 1
When not venting (cell-free)
The same operation as in Example 1 was performed except that air was not blown into the medium. There was no change in the concentration of paclitaxel contained in the medium, indicating that the method using air bubbles for the separation of paclitaxel was very effective (Table 1).

Table 1. Residual rate of paclitaxel in medium (%) and recovery rate after 6 hours (%)
Ventilation air blowing rate (mL / min)
Time (0) (50) (100) (200) (400) (500)
0 hours 100 100 100 100 100 100 100
1 hour 100 50 50 35 33 25
2 hours 100 40 44 20 32 23
4 hours 100 32 31 23 26 15
6 hours 100 26 24 20 12 10
Recovery rate 0 74 76 80 88 90

Example 2
Production volume of paclitaxel when culturing with air bubbles separation Using the apparatus shown in Fig. 5, B5 medium (120 mL) and yew (Taxus cuspidata) cells 1 in a porous cellulosic bag placed in an internal glass container (capacity 650 mL) 0.2 g was added and cultured at 25 ° C. with an aeration rate of 50 ml per minute for 14 days. The paclitaxel concentration in the foam overflowed by aeration and the medium (0.25 mL) were sampled over time to measure the amount of paclitaxel (Table 2).

Table 2. Changes in paclitaxel production over time when cultivated while separating bubbles 0 3 7 10 14
Production (μg) 0 3 4 5 9

Comparative Example 3
The amount of paclitaxel produced when culturing without bubble separation The culture was carried out in the same manner as in Example 2 except that the shaking culture was performed instead of the foaming separation operation, and the production amount of paclitaxel was measured over time. It was shown that the production amount of paclitaxel in the case where the bubbles were not separated was remarkably low, and the production amount could be significantly increased by moving the product out of the system by the bubble separation (Table 3).

Table 3. Changes in paclitaxel production over time when cultured without bubbles separation Days of culture 0 3 7 10 14
Production (μg) 0 2.4 2.3 4 4

  The present invention can be widely applied to an effective method for separating bioproducts and simplification of bioproduction processes.

The conventional bioproduction process by the organic solvent extraction separation method is shown. The bio-production process by foaming separation (culture and separation are the same tank) is shown. The bio-production process by foaming separation (culture and separation are separate devices) is shown. Foam separation in cell-free systems Foam separation in cell culture systems

Claims (5)

In a method for separating paclitaxel produced by a biological reaction using plant cells in an aqueous medium, the plant cells are held and cultured using a porous cellulose bag, and bubbles and an aqueous medium are brought into contact with each other. A method for separating paclitaxel , comprising collecting paclitaxel contained in an aqueous medium at a bubble interface and then separating the bubbles from the aqueous medium. The method for separating paclitaxel according to claim 1, wherein bubbles generated by blowing a gas into the aqueous medium are used. The method for separating paclitaxel according to claim 1 or 2, wherein bubbles are generated outside the aqueous medium, and contact between the bubbles and the aqueous medium is performed outside the biological reaction system. The method for separating paclitaxel according to any one of claims 1 to 3, wherein millibubbles, microbubbles, or nanobubbles are used as the bubbles. The method for separating paclitaxel according to any one of claims 1 to 4, wherein the paclitaxel is produced by plant cell culture of yew (Taxus cuspidata).

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