KR0165548B1 - Method for purification of amino acids nucleic acids and derivatives thereof - Google Patents

Abstract

The present invention relates to a method for effectively separating and purifying crystals, in particular amino acids, nucleic acids or derivatives thereof, from solutions containing both crystal and bacterial cells.

Description

Method for Purifying Amino Acids, Nucleic Acids or Derivatives thereof

1 shows the structure of a liquid cyclone.

2 shows the liquid cyclone connected to the culture tank.

3 shows an apparatus for purifying glutamic acid using two liquid cyclones connected to a tank for crystallization, a belt filter and a reservoir.

The present invention relates to a method for effectively separating and purifying crystals, in particular amino acids, nucleic acids or derivatives thereof, from solutions containing both crystal and bacterial cells.

As a method of separating the crystals in the culture broth or the enzyme reaction solution, filtration or centrifugal precipitation has generally been used until now. However, if cells are contained in the solution being treated, the cells tend to coagulate the filtration cloth during filtration, which requires relatively little capital investment, and at the same time most cells are taken up into the crystals, so Inappropriate. In the centrifugal precipitation represented by the decanter type, crystals can be separated but the selection of cells and adherent mother liquor is insufficient. Therefore, it is necessary to carry out multiple stage separations and further countercurrent washing.

In this case, the loss in the separation of the crystal is unavoidable. Another method is to add an acid or base to the solution that is treated to dissolve the crystals once, remove the cells from the solution and then perform recrystallization.

Liquid cyclones are well known as wet classifiers that rotate a liquid to provide centrifugal force in a fixed circumference and are widely used for the recovery of finely divided materials in minerals, removal of sand particles from pulp, and classification of various starches. come. All of this is due to the application of the liquid cyclone's ability to classify particles with a diameter larger than the critical diameter towards the downstream. In this case, however, the liquid cyclone does not classify particles having a diameter smaller than the critical diameter and such particles are simply classified by the ratio of the liquid in the upstream to the liquid in the downstream.

Applying the above concept, only liquid phase cyclone is used to concentrate the crystal slurry as a solution containing crystals. When the solution containing the cells is supplied to the liquid cyclone, the cells have a diameter smaller than the critical diameter, so there is no difference in concentration between the upper stream and the lower stream of the liquid cyclone, making the separation of the cells impossible.

There has been a need to develop a method for effectively separating crystals from solutions containing crystals and cells in fewer steps and at lower cost.

As a result of extensive investigation to solve the above problems, we apply liquid cyclone to a solution containing both crystals and cells to effectively concentrate the crystals and to separate them by the classification ability of the liquid cyclones, while at the same time It was found that cells having a diameter smaller than the diameter of the crystals, which were considered difficult to apply, could be effectively selected from the concentrated crystal solution.

It downstream streams a crystal slurry containing up to 10% by weight dry cells and 5 to 60% by weight of crystals of amino acids, nucleic acids or derivatives thereof from 10 to 2000 μm in diameter. A liquid cyclone having a diameter capable of sufficiently increasing the concentration of crystals at the side was applied, and if necessary, a countercurrent pressure was applied to the downstream to recover the crystal concentration solution of 30 to 90% by weight of crystal concentration at the downstream and at least 50% of the cells. A method for purifying amino acids, nucleic acids or derivatives thereof according to claim 1, characterized in that screening at the upstream side. In particular, a crystal slurry containing less than 10% by weight based on dry weight of cells having a diameter of 5 μm or less and 5 to 60% by weight of crystals of amino acids, nucleic acids or derivatives thereof having a diameter of 10 to 2000 μm is preferred. To the liquid phase cyclone through the pump. The liquid cyclone used may have a conventional shape as shown in FIG. 1 and has a typical diameter small enough to sufficiently increase the concentration of crystals on the lower stream side. By sorting the feed pressure, the crystals can move towards the downstream because of the centrifugal effect inside the liquid cyclone, thereby increasing the crystal concentration on the downstream. At the same time, because the concentration of crystals concentrated on the downstream side is high, the cells are less affected by the centrifugal effect of the liquid cyclone and migrate mainly to the upstream, although the cells show a state similar to that of the solution. Therefore, crystals and cell solutions can be effectively separated from each other. If necessary, taking into account the leakage of crystals towards the top stream, applying a backflow pressure to the downstream stream also increases the crystal concentration at the downstream stream and accelerates the selection of the cells at the same time.

As noted above, crystals can be effectively separated from solution using liquid cyclones containing three components of crystals, cells and media components.

The crystal concentrate thus obtained is filtered or dehydrated by centrifugation. Therefore, compared to the usual centrifugal precipitation or the like, it is possible to separate good crystals having good selection of cells and having less adherent mother liquor. This is because the cells are selectively removed in the concentrated crystal solution after treatment, and at the same time, microcrystals are selectively removed at the same time, which can cause a reduced rate of filtration or dehydration like cells, while the solution before treatment is filtered by the presence of cells. It is not suitable for use because it causes tangle of.

The crystal slurry applied to the present invention is a slurry obtained from an enzymatic reaction solution of an amino acid, a nucleic acid or a derivative thereof in which a culture solution or crystals are precipitated (eg, guanosine fermentation solution), an acid or a base after completion of fermentation or enzymatic reaction. A slurry containing crystals (e.g. glutamic acid fermentation solution) precipitated by adding thereto, a slurry (e.g., tritopane fermentation solution) containing crystals precipitated by concentration or cooling after completion of the fermentation or enzymatic reaction; All crystal slurries containing crystals of cells and amino acids, nucleic acids or derivatives thereof produced by the cells.

Examples of such crystal slurries include solutions containing various amino acids (e.g., phenylalanine, leucine, isoleucine, glutamine, aspartic acid) or derivatives thereof, as well as solutions containing glutamic acid and tryptophan as described above and the above. Solutions containing cells present are included. The present invention also applies to crystal slurries of nucleosides, nucleotides or derivatives thereof, and also to the aforementioned guanosine obtained by fermentation or enzymatic reaction.

Moreover, the method of the present invention is even more effective when combined with the step of fermentation or enzymatic reaction. By recovering the crystals of amino acids, nucleic acids or derivatives thereof produced by fermentation or enzymatic reactions from the fermentation tanks as they are, the fermentation or enzymatic reactions are intact and at the same time the procedure in the treatment step is simplified, resulting in amino acids, The productivity of the nucleic acid or derivatives thereof can be increased so that the production cost can be reduced.

In order to isolate and recover crystals of amino acids, nucleic acids or derivatives thereof formed during fermentation or enzymatic reaction, the culture broth or reaction solution containing the crystals is recovered continuously or intermittently and the crystals are separated using liquid cyclones. An example of connecting a liquid cyclone with a fermentation tank or a reaction tank is shown in FIG. A concentrated solution can be obtained on the downstream side. By purifying the crystals, higher purity amino acids, nucleic acids or derivatives thereof can be obtained. In the solution on the top stream side, the media components and cells are contained, which can be recycled to fermentation tanks or reaction tanks to be provided back for culture. In another method, the solution also contains a product that is present in the dissolved state, not crystallized, and therefore amino acids, nucleic acids or derivatives thereof from the solution in a conventional manner, such as by crystallization by crystallization or resin method. Can be collected. In addition, when fermentation or enzymatic reactions are completed, crystals can be separated and collected from the culture broth or reaction solution. The product present in the dissolved state can also be collected by using conventional methods alone or in parallel, such as crystallization by concentration or crystallization by a resin method.

According to the method of the present invention, when a solution containing crystals and cells is supplied to a liquid cyclone, the resulting centrifugal effect inside the liquid cyclone moves the crystal toward the downstream and is recovered as a crystal concentrate solution; On the other hand, the cells are less affected by the centrifugal effect and thus show a state similar to that of the solution, but most of the cells are recovered on the upper stream side due to the increased concentration of concentrated crystals on the downstream side. Therefore, the crystal can be effectively separated from the solution.

Separation and purification of crystals using liquid cyclones has advantages in many respects compared to separation and purification using conventional centrifugal machines because the equipment investment is very inexpensive, easy to maintain, the liquid cyclones are compact and have large processing capacity. If it does not require a large scale, it uses only the power of the pump, which saves energy consumption, simplifies the procedure and operation, and facilitates the multi-step process and backwash cleaning.

EXAMPLE

The invention is hereinafter described in more detail by reference to examples.

Example 1

A glutamic acid fermentation solution containing 1% by weight of glutamic acid-producing bacterial cells based on the dry weight of the cells is charged to the crystallization tank 1 shown in FIG. Sulfuric acid is added to the fermentation solution and neutralization crystallization is performed at pH 3.2. The slurry of glutamic acid crystals obtained by crystallization contains 10% by volume of crystals and has a diameter of about 2 to 300 mu m. A slurry containing glutamic acid crystals is fed to the liquid phase cyclone 3 via a pump 2. The liquid cyclone 3 has a conventional shape as a cyclone, is made of ceramic and has a typical diameter of 20 mm in the circumferential region. The feed pressure is set to 5 kg / m ² G and the feed volume per liquid cyclone is 0.72 m ³ / hour. A backflow pressure of 0.5 kg / m ² G is applied to the downstream outlet of the liquid cyclone but not to the upstream. By this operation, crystals of glutamic acid are obtained on the downstream side of the liquid cyclone as a concentrated solution containing 90% by weight of crystals, and at least 90% of the cells in the feed solution are selectively removed. The recovery of crystals on the downstream side of the liquid cyclone is 80%. Next, the obtained crystal concentrate solution is treated with a flat belt filter 4 made by Pannevis. As a result, the average specific filtration resistance (unit: n / kg) is 10 ⁹ at a cake thickness of 30 mm, indicating good filtration properties. In addition, 0.5 kg of wash water per kg of cake precursor weight is used to obtain crystals of glutamic acid with mother solution and 2% of cells adhered to 2.5% of the cake based on the feed solution.

On the other hand, the solution on the upper stream side of the liquid cyclone 3 is stored in the reservoir 5 and then supplied to the liquid cyclone 7 via the pump 6. The liquid cyclone 7 has a shape similar to the liquid cyclone 3 and is made of ceramic and has a typical diameter of 9 mm in the circumferential region. The feed pressure is set to 5 kg / m ² G and the feed volume per liquid cyclone is 0.24 m ³ / hour. The countercurrent pressure does not apply to both the downstream or upstream outlet of the liquid cyclone. Crystals leaking toward the upper stream of the liquid cyclone 3 can be recovered on the lower stream side and recycled to the tank 1 for crystallization. The solution at the upstream side of the liquid cyclone 7 is sent to a process for further purification.

Such a system can be devised to effectively separate the crystals of glutamic acid from a solution containing glutamic acid crystals and glutamic acid-producing bacterial cells.

Example 2

Because of the very low solubility of guanosine, guanosine crystals are already precipitated in guanosine fermentation solution with a guanosine concentration of 2.5 g / dl after completion of the fermentation step. The particle diameter of guanosine crystals is about 2 to 200 μm. A fermentation solution of guanosine containing guanosine-producing bacterial cells is fed to a liquid cyclone similar to the liquid cyclone 7 used in Example 1. The feed pressure is set at 5 kg / m ² G and the feed volume per liquid cyclone is 0.24 m ³ / hour. A backflow pressure of 0.5 kg / m ² G is applied to the downstream stream outlet of the liquid cyclone but not to the overhead stream outlet. By this operation, 70% of guanosine crystals are recovered on the downstream side of the liquid cyclone and selectively provided 80% of the cells in the feed solution. About three times the volume of water was added to the crystal concentrate thus obtained, followed by two washing steps, and the mixture was applied to the liquid cyclone to further selectively and selectively remove impurities in the feed solution containing the coloring material. , A concentrated solution of guanosine crystals with increased cell selectivity above 99% is obtained with a recovery of 50%. Since the absorption of impurities into the crystals precipitated during the fermentation of guanosine is small, the concentrated solution obtained by the above procedure has a degree of purification sufficient to provide as a product as it is after the following separation and drying. In addition, in performing a multi-liquid liquid cyclone procedure, the recovery of crystals can be further increased for performing a multi-fluid countercurrent wash.

As above, the crystals of guanosine can be effectively isolated and purified from the fermentation solution containing guanosine-producing bacterial cells and precipitated guanosine crystals.

Example 3

Tryptophan fermentation solution with a tryptophan concentration of 2 g / dl is cooled to 15 ° C. to effect crystallization. The particle diameter of the tryptophan crystals is about 2 to 150 μm. A fermentation solution of tryptophan containing tryptophan-producing bacterial cells is fed to a liquid cyclone similar to the liquid cyclone 7 used in Example 1. The feed pressure is set at 5 kg / m ² G and the feed volume per liquid cyclone is 0.24 m ³ / hour. A backflow pressure of 0.1 kg / m ² G is applied to the downstream stream outlet of the liquid cyclone but not to the overhead stream outlet. By this operation, about 60% of the tryptophan crystals are recovered on the downstream side of the liquid cyclone and 80% of the cells in the feed volume are selectively removed. The crystal concentrate thus obtained was subjected to the same washing step as in Example 2 twice, to further selectively selectively remove impurities in the feed solution containing the colored material, thereby increasing the cell selectivity to 99% or more. A concentrated solution of tryptophan crystals is obtained with a recovery of 45%. Since the absorption of impurities into the crystals precipitated by cooling the fermentation solution is as small as in the guanosine crystals in Example 2, the concentrated solution obtained by the above procedure is provided as a product as is after the following separation and drying. It has a sufficient degree of purification.

As above, the crystals of tryptophan can be effectively separated and purified from solutions containing tryptophan crystals and tryptophan-producing bacterial cells precipitated by cooling.

Example 4

Brevibacterium flavum AJ 3409 (FERM-BP 662, deposited May 1, 1981) was used on a single plate medium containing 1% yeast extract, 1% peptone, 0.5% sodium chloride and 0.5% glucose. After inoculation to incubated for 24 hours at 31 ℃. 4% glucose in 30 Sakaguchi flasks each with 500 ml volume; 0.1% potassium phosphate, 0.04% magnesium sulfate, 0.001% iron sulfate, 0.2% ammonium sulfate, 0.4% urea, 4 μg / L biotin, 100 μg / L vitamin B ₁ and 0.035% (calculated based on total nitrogen) Add 25 ml of medium (pH 7.0) consisting of digests. After sterilization by heating to 121 ° C. for 15 minutes, the temperature is lowered to room temperature and the Brevibacterium flaboom AJ 3409 cells previously grown on agar plate medium are inoculated onto the sterile medium with platinum wire. Agitation culture broth was prepared by shaking culture at 31 ° C. for 24 hours. In a 20 L fermentation tank, 10% glucose, 0.4% potassium phosphate, 0.06% magnesium sulfate, 0.002% iron sulfate, 5% ammonium sulfate, 8 μg / L biotin, 500 μg / L vitamin B ₁ and 0.056% (based on total nitrogen) Calculated) 10 l of medium (pH 7.0) consisting of soy protein hydrolyzate. After sterilization by heating to 121 ° C. for 20 minutes, the temperature was lowered to room temperature and the passage culture broth previously cultured in the flask was added to the medium, followed by the incubation temperature of 30 ° C., adjusted pH 6.5, 1 / 2vvm of aeration, Incubate under conditions of 600 rpm stirred water and 0.2 kg / cm ² internal pressure. Immediately before the glucose in the culture broth is depleted, the culture tank is replenished with a solution containing 45 g / l glucose (sterilized) to adjust the concentration of glucose in the culture broth to always maintain 0.1 to 1.5%. At the point when glutamine accumulation exceeds its solubility and the crystals of glutamine begin to crystallize in the culture broth, a portion of the culture broth is aseptically taken from the system and passed through a small liquid cyclone to separate the crystals. Then, the culture broth is put back into the culture tank. 100 hours of incubation was obtained by incubating 100 hours in this manner with slurry of glutamine crystals 1.1 kg (16.6% wet amount) and 5.2% glutamine. The culture broth obtained is concentrated to crystallize. The crystals are separated and combined with the crystal slurry obtained upon incubation. Dissolution and crystallization were repeated three times to obtain 1.36 kg of glutamine crystals in high purity.

On the other hand, the culture is carried out for 100 hours under the same conditions without recycling the culture broth to the liquid cyclone and without separating the crystals formed during the culture. As a result, 14.4 L of culture broth containing 9.7% glutamine is obtained. The dissolution and crystallization of the crystals obtained by concentration and crystallization are repeated to purify glutamine. However, it is necessary to repeat dissolution and crystallization four times in order to obtain glutamine crystals having the same purity as that obtained in the foregoing method. As a result, 0.98 kg of high purity glutamine crystals are obtained.

Regarding the two preparation methods of glutamine, the results and characteristics are described in Table 1.

Example 5

Brevibacterium lactofermentum ATCC 21420 was inoculated on an agar plate containing 1% yeast extract, 1% peptone, 0.5% sodium chloride and 0.5% glucose and incubated at 31 ° C. for 24 hours. 30 saggot flasks of 500 ml each, 2% sucrose, 0.1% potassium phosphate, 0.04% magnesium sulfate, 0.001% iron sulfate, 0.001% manganese sulfate, 0.4% ammonium acetate, 0.04% tyrosine, 100 μg / L biotin, 100 μg Add 25 ml of medium (pH 7.0) each consisting of / L vitamin B and 0.2% (calculated based on total nitrogen) soy protein hydrolysate. After sterilization by heating to 121 ° C. for 15 minutes, the temperature is lowered to room temperature and the previously grown Brevibacterium lactofermentum ATCC 21420 cells are inoculated onto the sterile medium with platinum wire. Agitation culture broth was prepared by shaking culture at 31 ° C. for 24 hours. 20% volume fermentation tank contains 10% glucose, 0.15% potassium phosphate, 0.1% magnesium sulfate, 0.001% iron sulfate, 50 μg / L biotin, 2000 μg / L vitamin B and 0.08% (calculated based on total nitrogen) Add 10 L of digested medium (pH 7.0). After sterilization by heating to 121 ° C. for 20 minutes, the temperature was lowered to room temperature and the passage culture broth previously cultured in the flask was added to the medium, followed by a culture temperature of 31 ° C., a controlled aeration of pH 7, 1 / 3vvm, and 400 rpm. Of stirring water and 0.2kg / cm Incubate under conditions of internal pressure. Immediately before the glucose in the culture broth is depleted, the culture tank is replenished with a solution containing 45 g / L glucose (sterilized) to adjust the concentration of glucose in the culture broth at all times between 0.1 and 2.5%. At the time when the accumulation of phenylalanine reaches its solubility, a small amount of phenylalanine crystals is added to the culture broth to promote crystallization of phenylalanine, and a portion of the culture broth is aseptically collected from the system and passed through a small cyclone to separate the crystals. . Then, the culture broth is put back into the culture tank. Incubating for 80 hours in this manner yields 15 l of culture broth containing 0.21 kg (20% wet amount) of slurry of phenylalanine crystals and 2.8% phenylalanine. The culture broth obtained is concentrated to crystallize. The crystals are separated and combined with the crystal slurry obtained upon incubation. Dissolution and crystallization were repeated three times to obtain 0.41 kg of phenylalanine crystals in high purity.

On the other hand, the culture is carried out for 80 hours under the same conditions without recycling the culture broth to the liquid cyclone and without separating the crystals formed during the culture. As a result, 15 L of culture broth containing 3.5% phenylalanine is obtained. Dissolution and crystallization of the crystals obtained by concentration and crystallization are repeated to purify phenylalanine. However, it is necessary to repeat dissolution and crystallization four times in order to obtain phenylalanine crystals having the same purity as that obtained in the aforementioned method. As a result, 0.34 kg of high purity phenylalanine crystals are obtained.

Regarding the two preparation methods of phenylalanine, the results and characteristics are described in Table 2.

Example 6

Bacillus subtilis AJ 11713 (FERM-BP 208, deposited October 26, 1981) was inoculated onto agar plate medium containing 3% soluble starch 0.5% yeast extract and 0.5% peptone and then at 31 ° C. Incubate for 24 hours at. 3% glucose, 0.1% potassium phosphate, 0.04% magnesium sulfate, 0.001% iron sulfate, 0.001% manganese sulfate, 0.3% ammonium sulfate, 0.5% sodium glutamate and 0.065% (based on total nitrogen) Calculated) 1 l of medium (pH 6.7) consisting of soy protein hydrolysate is added. After sterilization by heating at 121 ° C. for 15 minutes, the temperature is lowered to room temperature and the Bacillus subtilis AJ 11713 cells previously grown on agar plate medium are inoculated on sterile medium with platinum wire. Agitation culture broth was prepared by shaking culture at 31 ° C for 30 hours. In a 20 L volume fermentation tank, a medium consisting of 20% glucose, 0.2% potassium phosphate, 0.4% magnesium sulfate, 0.001% iron sulfate, 0.001% manganese sulfate and 0.05% (calculated based on total nitrogen) soy protein hydrolysate (pH 7.0) Add 10 liters. After sterilization by heating to 121 ° C. for 20 minutes, the temperature was lowered to room temperature and the passage culture broth previously cultured in a small fermentation tank was added to the medium, followed by an incubation temperature of 31 ° C., adjusted pH 6.7 and 3 / 4vvm. , 600 rpm agitation and 0.2 kg / cm Incubate under conditions of internal pressure. Immediately before the glucose in the culture broth is depleted, the culture tank is replenished with a solution containing 45 g / l glucose (sterilized) to adjust the concentration of glucose in the culture broth to always maintain 0.1 to 1.5%. At the time when the accumulation of tryptophan exceeds its solubility and the crystals of tryptophan begin to crystallize in the culture broth, detergent is added to the culture broth to facilitate crystallization of tryptophan, and a portion of the culture broth is aseptically taken from the system and The crystals are separated by passing through a small liquid cyclone. Then, the culture broth is put back into the culture tank. In this way 100 hours of incubation yielded 13 liters of culture broth containing 0.28 kg (20% wet amount) of slurry of tryptophan crystals and 1.4% tryptophan. The culture broth obtained is concentrated to crystallize. The crystals are separated and combined with the crystal slurry obtained upon incubation. Dissolution and crystallization are repeated twice to give 0.245 tryptophan crystals in high purity.

On the other hand, the culture is carried out for 100 hours under the same conditions without recycling the culture broth to the liquid cyclone and without separating the crystals formed during the culture. As a result, 13 L of culture broth containing 2.88% tryptophan is obtained. Tryptophan is purified by repeating dissolution and crystallization of the crystals obtained by concentration and crystallization. However, dissolution and crystallization need to be repeated three times in order to obtain tryptophan crystals with the same purity as obtained in the described method. Eventually 0.21 kg of high purity tryptophan crystals are obtained.

With regard to the two preparation methods of tryptophan, the results and characteristics are described in Table 3.

Example 7

Brevibacterium plaboom AJ 3686 (FERM-BP 755, deposited May 1, 1981) was inoculated onto agar plate medium containing 1% yeast extract, 1% peptone, 0.5% sodium chloride and 0.5% glucose. Incubate at 31 ° C. for 24 hours. 30 sagaggar flasks in 500 ml volume each containing 3% glucose, 0.1% potassium phosphate, 0.04% magnesium sulfate, 0.001% iron sulfate, 0.001% manganese sulfate, 0.3% urea, 10 μg / L biotin, 200 μg / L vitamin B and 0.085 % (Calculated based on total nitrogen) Add 25 ml of medium (pH 7.0) each consisting of soy protein hydrolysate. After sterilization by heating to 121 ° C. for 15 minutes, the temperature is lowered to room temperature and the previously grown Brevibacterium flaboom AJ 3686 cells are inoculated on a sterile medium with platinum wire on agar plate medium. Agitation culture broth was prepared by shaking culture at 31 ° C. for 24 hours. 20 L volume fermentation tank in 15% glucose, 0.1% potassium phosphate, 0.04% magnesium sulfate, 0.001% iron sulfate, 1% ammonium sulfate, 50 μg / L biotin, 300 μg / L vitamin B and 0.01% (calculated based on isoleucine) 10 l of medium (pH 7.0) consisting of soy protein hydrolyzate. After sterilization by heating to 121 ° C. for 20 minutes, the temperature was lowered to room temperature and the passage culture broth previously cultured in the flask was added to the medium, followed by a culture temperature of 31 ° C., a controlled pH 7, 1/2 vvm of aeration, 600rpm water and 0.2kg / cm Incubate under conditions of internal pressure. Immediately before the glucose in the culture broth is depleted, the culture tank is replenished with a solution containing 45 g / l glucose (sterilized) to adjust the glucose concentration in the culture broth to always maintain 0.1 to 1.5%. At the time when the accumulation of leucine exceeds its solubility and crystals of leucine begin to crystallize in the culture broth, a portion of the culture broth is aseptically taken from the system and passed through a small liquid cyclone to separate the crystals. Then, the culture broth is put back into the culture tank. Incubation for 60 hours in this way yields 14 liters of culture broth containing 0.24 kg (16.6% wet amount) of slurry of leucine crystals and 2.6% leucine. The culture broth obtained is concentrated to crystallize. The crystals are separated and combined with the crystal slurry obtained upon incubation. Dissolution and crystallization were repeated three times to obtain 0.42 kg of leucine crystals in high purity.

On the other hand, the culture is carried out for 60 hours under the same conditions without recycling the culture broth to the liquid cyclone and without separating the crystals formed during the culture. As a result, 13.7 L of culture broth containing 3.3% leucine is obtained. By dissolving and crystallizing the crystals by concentration and crystallization, leucine is purified. However, it is necessary to repeat dissolution and crystallization four times in order to obtain leucine crystals having the same purity as that obtained in the aforementioned method. As a result, 0.29 kg of high purity leucine crystals are obtained.

With respect to the two preparations of leucine, the results and characteristics are described in Table 4.

Example 8

Bacillus subtilis AJ 11312 (FERM-P 4823) is inoculated on agar plate medium containing 3% soluble starch, 0.5% yeast extract and 0.5% peptone and incubated at 34 ° C. for 24 hours. Small fermentation tank of 2 liter volume in 3% glucose, 0.3% ammonium chloride, 0.1% potassium phosphate, 0.04% magnesium sulfate, 0.001% manganese sulfate, 0.05% yeast extract, 0.5% RNA and 0.12% (calculated based on total nitrogen) ) 1 L of medium (pH 6.4) consisting of soy protein hydrolyzate. After sterilization by heating to 121 ° C. for 15 minutes, the temperature is lowered to room temperature and the Bacillus subtilis AJ 11312 cells previously grown on agar plate medium are seeded on sterile medium with platinum wire. Shaking incubation at 34 ℃ 30 hours to prepare a passage culture broth. In a 20 liter volume fermentation tank, 20% glucose, 0.2% potassium phosphate, 0.15% magnesium sulfate, 0.001% iron sulfate, 0.001% manganese sulfate and 0.31% (calculated based on total nitrogen) soy protein hydrolysate and 0.5% chloride Add 10 L of ammonium medium (pH 6.5). After sterilization by heating to 121 ° C. for 20 minutes, the temperature is lowered to room temperature and the passage culture broth previously cultured in small agar plate medium is added to the medium, followed by a culture vessel of 35 ° C., adjusted pH 6.4, 1 / 2vvm. Capacity, 600 rpm stirring water and 0.2 kg / cm Incubate under conditions of internal pressure. Immediately before the glucose in the culture broth is depleted, the culture tank is replenished with a solution containing 45 g / L glucose (sterilized) to adjust the concentration of glucose in the culture broth to always maintain 0.1 to 1.5%. At the time when the accumulation of guanosine exceeds its solubility and the crystals of guanosine begin to crystallize in the culture broth, a portion of the culture broth is aseptically taken from the system and passed through a small liquid cyclone to separate the crystals. Then, the culture broth is put back into the culture tank. 100 hours of incubation in this manner yields 13.5 L of culture broth containing 0.65 kg (20% wet amount) of slurry of guanosine crystals and 0.4% guanosine. The culture broth obtained is concentrated to crystallize. The crystals are separated and combined with the crystal slurry obtained upon incubation. Dissolution and crystallization were repeated twice to obtain 0.43 kg of guanosine crystals in high purity.

On the other hand, the culture is carried out for 100 hours under the same conditions without recycling the culture broth to the liquid cyclone and without separating the crystals formed during the culture. As a result, 12.5 L of culture broth containing 3.5% guanosine is obtained. Guanosine is purified by repeating dissolution and crystallization of the crystals obtained by concentration and crystallization. However, it is necessary to repeat dissolution and crystallization three times in order to obtain guanosine crystals having the same purity as that obtained in the foregoing method. As a result, 0.28 kg of high purity guanosine crystals are obtained.

Regarding the two preparation methods of guanosine, the results and characteristics are described in Table 5.

Example 9

Pseudomonas spp. ATCC 19121 was inoculated on agar plate medium containing 1% gravy, 1% peptone, 0.5% sodium chloride and incubated at 30 ° C. for 24 hours. 0.3 L medium (pH 7.0) consisting of 0.5% oleic acid, 0.5% soybean oil, 0.1% potassium phosphate, 0.1% magnesium sulfate and 0.1% (calculated based on total nitrogen) soy protein hydrolysate in a 1 L small fermentation tank ). After sterilization by heating to 121 ° C. for 15 minutes, the temperature is lowered to room temperature and Pseudomonas spp. ATCC 19121 cells previously grown on agar plate medium are inoculated on sterile medium with platinum wire. Shaking incubation at 31 ℃ for 20 hours to prepare a passage culture broth. 10 liter volume fermentation tank containing 0.5% fumaric acid, 0.5% oleic acid, 0.5% soybean oil, 0.1% potassium phosphate, 0.1% magnesium sulfate, 0.13% (calculated based on total nitrogen) soy protein hydrolysate (pH 7.0 ) 5 liters. After sterilization by heating to 121 ° C. for 20 minutes, the temperature was lowered to room temperature, and subculture broths previously cultured in a small fermentation tank were added to the medium. And 0.2kg / cm Incubate under conditions of internal pressure. After 20 hours from the start of the culture, 1.5 kg of aspartic acid powder is added continuously. The incubation temperature is maintained at 40 ° C. while maintaining the pH at 5. At the time when the alanine accumulation exceeds its solubility and crystals of alanine begin to crystallize in the culture broth, a portion of the culture broth is aseptically taken from the system and passed through a small liquid cyclone to separate the crystals. Then, the culture broth is put back into the culture tank. In this way, a total of 35 hours was incubated, including 20 hours of initiation, to produce 5 liters of culture broth containing 6.3 kg (20% wet amount) of slurry of alanine crystals and 18% alanine. The culture broth obtained is concentrated to crystallize. The crystals are separated and combined with the crystal slurry obtained upon incubation. Dissolution and crystallization are repeated twice to obtain 1.2 kg of alanine crystals in high purity.

On the other hand, the culture is carried out for 35 hours under the same conditions without recycling the culture broth to the liquid cyclone and without separating the crystals formed during the culture. As a result, 5 L of culture broth containing 28% alanine is obtained. The alanine is purified by repeating dissolution and crystallization of the crystals obtained by concentration and crystallization. However, it is necessary to repeat dissolution and crystallization three times in order to obtain alanine crystals having the same purity as that obtained in the aforementioned method. As a result, 1.1 kg of high purity alanine crystals are obtained.

With regard to the two alanine preparation methods, the results and characteristics are described in Table 6.

As described above, according to the present invention, crystals of amino acids, nucleic acids or derivatives thereof can be efficiently isolated and purified at low cost from a solution containing such crystals, cells and media components.

Claims (9)

A crystal slurry containing up to 10% by weight dry cells and 5 to 60% by weight of crystals of amino acids, nucleic acids or derivatives thereof having a diameter of 5 μm or less and dry weight of the crystals Treatment with liquid cyclone having a diameter capable of sufficiently increasing the concentration and, if necessary, applying a countercurrent pressure toward the downstream, recovering the concentration of the crystal concentrate at a concentration of 30 to 90% by weight from the downstream and producing more than 50% of the cells. The method for purifying amino acids, nucleic acids or derivatives thereof, characterized in that the selection on the upstream side. The purification method according to claim 1, wherein the crystal concentration of the crystal concentration solution in the downstream stream is 60 to 80% by weight. The purification method according to claim 1, wherein the crystal slurry is derived from a culture broth or an enzyme reaction solution, and crystals are formed in the culture broth or an enzyme reaction solution. The method of claim 1, wherein the crystal slurry is derived from a culture broth or an enzyme reaction solution and the crystals are crystallized by adding an acid or a base after fermentation or completion of the enzymatic reaction. The method of claim 1, wherein the crystal slurry is derived from a culture broth or an enzyme reaction solution and the crystal is formed by concentrating or cooling after fermentation or completion of the enzyme reaction. A crystal slurry containing up to 10% by weight dry cells and 5 to 60% by weight crystals of amino acids, nucleic acids or derivatives thereof having a diameter of 5 μm or less and dry weight of crystals Treated with a liquid cyclone having a diameter capable of sufficiently increasing the concentration, and if necessary, applying a countercurrent pressure to the downstream to recover a concentration of 30 to 90% by weight of the crystal concentrate on the downstream and 50% or more of the cells A method for purifying amino acids, nucleic acids, or derivatives thereof, characterized in that the concentrated crystal solution is filtered off or dehydrated by centrifugation after separation on the stream side to obtain crystals with low adhesion to the mother liquor. The purification method according to claim 6, wherein the crystal slurry is derived from the culture broth or the enzyme reaction solution, and the crystal is formed in the culture broth or the enzyme reaction solution. The method of claim 6, wherein the crystal slurry is derived from a culture broth or enzyme reaction solution and the crystal is crystallized by addition of an acid or base after completion of the fermentation or enzymatic reaction. The method of claim 6, wherein the crystal slurry is derived from a culture broth or an enzyme reaction solution and the crystal is formed by concentrating or cooling after fermentation or completion of the enzyme reaction.