JPH05336981A - Production of optically active m-hydroxy acid - Google Patents

Abstract

PURPOSE:To industrially and advantageously obtain the subject compound useful as a synthetic intermediate, etc., for medicines, agricultural chemicals, etc., by culturing a microorganism capable of producing a beta-hydroxy acid together with a porous material, using the resultant immobilized microorganism immobilized in the porous material and miniaturizing the culture vessel. CONSTITUTION:A porous material of a structure having a skin layer comprising small holes of 5-500mum diameter on the surface, holes having 100-1000mum diameter in the interior thereof and small holes having 5-500mum diameter in a partition wall of the holes is added to a SAKAGUCHI flask, etc. A culture medium containing butyric acid or isobutyric acid is then added thereto and a microorganism capable of producing a beta-hydroxy acid [e.g. Candida.rugosa KT8201 (FERM BP-110)] is subsequently inoculated and cultured at 30 deg.C by shaking. The culture solution is then separated and extracted with an organic solvent such as ethyl acetate to industrially and advantageously afford the objective optically active beta-hydroxy acid [e.g. L-(-)-beta-hydroxyiosbutyric acid] to be a synthetic intermdiate for medicines, agricultural chemicals, etc.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a method for producing an optically active β-hydroxy acid. More specifically, it relates to a method for producing an optically active β-hydroxy acid, which has two different functional groups and serves as a useful synthetic intermediate for pharmaceuticals, agricultural chemicals and the like, by using a microorganism immobilized thereon.

[0002]

2. Description of the Related Art In recent years, due to remarkable progress in biochemistry and organic chemistry, various physiologically active substances have been separated and identified or synthesized, and the correlation between their three-dimensional structure and physiological activity has been clarified. Activity has also been confirmed as an important factor. Currently, synthetic research of optically active physiologically active substances is being actively conducted using various methods.
The optically active fatty acid β-hydroxy acid, which is the subject of the present inventors in this patent, has two kinds of functional groups, and by utilizing the difference in the characteristics, it is easily induced to various optically active substances. As a result, it is a group of substances that is both interdisciplinary and industrially attractive.

Economical methods for producing β-hydroxy acids that can be industrially carried out have been vigorously studied, and various β- and β-types of D and L having different stereospecificities are utilized by utilizing metabolic control of microorganisms. -A selective and efficient production method of hydroxy acid has been developed (Japanese Patent Publication No. 59-21599 and Japanese Examined Patent Publication 5).
9-21600, Japanese Patent Publication 60-16235, Japanese Patent Publication 61-12676, Japanese Patent Publication 59-53.
Japanese Patent Publication No. 838, Japanese Patent Publication No. 61-12678, Japanese Patent Publication No. 62-57311, Japanese Patent Publication No. 62-57312, Japanese Patent Publication No. 61-42559, Japanese Patent Publication No. 61-42.
560, JP-B-63-19153, JP-B-62-54474, JP-A-57-65189, JP-B-04-8035, and JP-B-04-803.
No. 6). Among them, for example, _D -β-hydroxyisobutyric acid (hereinafter, sometimes abbreviated as HIBA), which is a raw material of captopril which is a blood pressure lowering agent, has already been industrially produced.

Optically active β-hydroxy acid (hereinafter sometimes simply referred to as β-hydroxy acid), for example, HIBA
In industrial production, fatty acids and alcohols, which are cheap and easily available in large quantities, are used as the raw materials, and the β-hydroxylation method of fatty acids uses the β-oxidase system, which is the main metabolic pathway of fatty acids, and the related branched amino acid metabolic pathway It utilizes an enzyme system that is thought to be common with. In these metabolic pathways, ATP is essential in the main metabolic pathway, and therefore, in order to activate the metabolic system and increase the production rate of β-hydroxy acid, microorganisms such as glucose and glycerin are required under aerobic conditions. It is important to culture while supplementing the energy source. Therefore, as described above, major β-hydroxy acids, such as HIBA, are currently industrially produced by suspension-batch culturing microorganisms whose metabolism is controlled by mutation improvement in an aeration stirred tank.

[0005]

However, since it is difficult to reuse the microorganisms separated from the product (β-hydroxy acid) in the suspension batch culture after the completion of the culture in the suspension batch culture, the β-hydroxy acid production ability is low. It is necessary to discard active microorganisms that have (metabolic activity) and have been raised to a considerably high bacterial cell concentration, and this is an operation that is insufficient as an industrial production technique. . As a technique for repeatedly using microorganisms, it is possible to first immobilize the microorganisms on some carrier, but the general gel encapsulation method using κ-carrageenan, sodium alginate, etc. is (1) gelling agent, immobilization Requires reaction reagents, (2)
It has drawbacks such as low mass transfer rate of particles in substrates and products, (3) aseptic operation is difficult, (4) carrier cannot be reused, and (5) carrier has weak mechanical strength. . Therefore, when utilizing a microbial metabolic system such as the production of β-hydroxy acid, the above-mentioned (2) to (3) are fatal problems, and the application of such a comprehensive immobilization method is Industrially difficult. Therefore, in industrial production of β-hydroxy acid, the above problems (1) to (4), especially the problems (2) and (3), are overcome in order to maximize the effective use of active microorganisms. It has been desired to develop a new immobilization method for β-hydroxy acid-producing microorganisms.

[0006]

Means for Solving the Problems The inventors of the present invention have made extensive studies to solve the above problems. As a result, by culturing the β-hydroxy acid-producing microorganism with a porous material having a skin layer on its surface and relatively high mechanical strength,
The inventors have found that the microorganism can be naturally and aseptically immobilized on the porous material, and an optically active β-hydroxy acid can be produced, and thus the present invention has been completed. Further, the immobilization method of the β-hydroxy acid-producing microorganism in the present invention has a much higher mass transfer rate within the particles of the substrate and the product than the conventional entrapping immobilization method. By adopting, repeated batch operation,
Alternatively, it is possible to industrially produce β-hydroxy acid by continuous operation, and to provide a method for maximally effectively utilizing an active β-hydroxy acid-producing microorganism.

That is, the gist of the present invention is that an .beta.-hydroxy acid-producing microorganism is cultured with a porous material, and the microorganism immobilized on the inside of the porous material is used for production. The present invention relates to a method for producing β-hydroxy acid.

A porous material is used as the carrier in the method of the present invention. As the porous material, one having a skin layer having pores on its surface is preferably used. Further, it is preferable that the inside of the porous material has a structure having pores and small holes in the partition walls of the pores. The diameter of the pores present in the skin layer of the porous material is usually 5 to 500 μm, preferably 10 to 40 μm. If it is smaller than 5 μm, β-
The entry of hydroxy acid-producing microorganisms into the carrier is hindered,
On the contrary, if it is larger than 500 μm, once it enters, it becomes easy to leak. The diameter of the pores present inside the porous material is usually 100 to 1000 μm, preferably 100 to 500 μm.
Is. If it is larger than 1000 μm, it becomes difficult to form colonies. The diameter of the small holes existing in the partition walls of the holes is usually 5 to 500 μm, preferably 10 to 40 μm. 5
If it is smaller than μm, it becomes difficult for β-hydroxy acid-producing microorganisms to pass through, and if it is larger than 500 μm, the formation of colonies is inhibited.

In the present invention, any carrier can be used as the porous material as long as it has such a structure. Specifically, a cellulose porous material, a cellulose-chitin composite porous material, or those having various functional groups added thereto, for example, diethylaminoesterified cellulose porous material, carboxymethylated cellulose porous material,
Typical examples thereof include a diethylaminoesterified cellulose / chitin composite porous material and a carboxymethylated cellulose / chitin composite porous material. In the present invention, although not particularly limited, a cellulose porous material, a cellulose / chitin-based composite porous material and the like are preferable.

The porous body having such a skin layer can be easily manufactured by a known method. For example,
In the case of obtaining a cellulose porous material, it is produced by mixing viscose and a foaming agent such as calcium carbonate, extruding from a nozzle, dropping in a droplet form into an acid bath, and simultaneously performing regeneration and foaming of cellulose. It At this time, by changing the aging conditions of viscose and the conditions of the acid bath, several kinds of porous materials having different pore and small pore structures can be obtained. Usually, the holes are radial, but depending on the state of foaming, they may be continuous bubbles. Even in the case of an open cell, it can be similarly used if it has a small hole in the partition wall and a skin layer on the outer surface. The shape of the porous material having the skin layer ejected from the nozzle in this manner is generally spherical or flattened. Such a shape close to a sphere is very convenient for increasing the filling rate of the porous material in the culture tank during culturing. The diameter of the particles of the porous material is
It can be used in any diameter, but usually 3 to 7
In addition, in order to effectively immobilize the β-hydroxy acid-producing microorganisms in the culture tank, it is desirable that the diameter of the porous material is 1/10 or less of the diameter of the culture tank.

Further, as the porous material, other than cellulose,
For example, when calcium alginate gel is used, it has a skin layer having pores of 5 to 500 μm on the surface, radial or open pores of 100 to 1000 μm inside, and 5 to 500 μm in the partition walls. Any carrier having small pores can be used. This carrier is prepared by adding a foaming agent such as sodium hydrogen carbonate to an aqueous solution of sodium alginate, stirring the mixture, and dropping the resulting foamy liquid into a calcium chloride solution to cause gelation.
At this time, carriers having various pore sizes can be obtained by changing the combination of the concentrations of sodium alginate and sodium hydrogen carbonate. Further, by adjusting the size of the droplets when dropped, carriers with various particle sizes can be prepared. In this case, it is desirable that the particle size be 1/10 or less of the culture tank diameter in order to effectively immobilize the β-hydroxy acid-producing microorganisms in the culture tank.

Yeast is usually used as the β-hydroxy acid-producing microorganism in the present invention. The yeast is not particularly limited and can be used, but typical ones include genus Bullera and Candid.
a) Genus, Cryptococcus genus, Debaryomyces genus, Endomyces
Genus, Hansenula, Pachyso
len genus, Pichia genus, Rhodosporidium (Rho
genus dosporidium, genus Rhodotorula, genus Rhodococcus, Sacchomyces
aromyces), Saccharomycopsis
Genus, Schizosaccharomyces (Schizosaccharomyces) genus, Schwanniomyces (Schwanniomyces) genus, Sporidioburas (Sporidiobulus) genus, Torulopsis (Torulopsis) genus,
Trichosporon genus, Wingea
Yeast of the genus is mentioned. Specifically, Brera Alba (B
ullera alba) IFO1030, Candida parapsilosis
(Candida parapsilosis) IFO0708, IFO1022, Candida pararugosa IFO0966,
KT84015, Candida rugosa
IFO0750, KT8201, KT8202, IFO0591, Candida utilis IFO0396, Cryptococcus Iaurenti
i) IFO0609, Cryptococcu
s terreus) IFO0727, Debaryomyces Hansenyi (Deb
aryomyces hansenii) IFO0032, KT84016, IFO00
26, Endomyces ovetensi
s) CBS192.55, Endomys Tetrasperma (Endomy
ces tetrasperma) CBS765.70, KT84012, Hansenula anomala IFO0120, Hansenula heneicii IFO1477, Pachysolen tannosphilus IFO1
007, Pichia burtonii IFO0844,
KT84013, Pichia farinosa IA
M4327, Pichia membrana efaciens
mbranaefaciens) IAM4904, Rhodosporidium toruloides IFO0559, Rhodotorula rubra IFO0383,
Rhodococcus rhodoch
rous) IFO3338, Saccharomyces cerevisiae (Sacchar
omyces cerevisiae) IFO0735, IAM4274, Saccharomyces rouxii IFO0493, Saccharomyces uvarum IFO0751,
Saccharomycopsis
lipolytica) IFO1542, IFO1545, KT84011, Schizosaccharomyces pombe
IFO0362, Schwanniomyce
s persoonii) IFO1842, Sporidiobulus johnsonni IFO6903, Torulopsis candida IFO0380, Torulopsis gropengiesse
ri) IFO0659, Torulopsis magnoliae
s magnoliae) IFO0705, Trichosporon Beigeri (Tri
chosporon beigelii) ATCC22310, Trichosporon fermentans CBS2529,
Trichosporon pullulans
IFO0114, Trigonopsis barrierbilis
variabilis) IFO0380, Wingea Roberti
a robertsii) IFO1277 and mutants derived therefrom.

Of these, Candida rugosa
KT8201, KT8202, Saccharomycopsis repolitica KT84011, Endomyces tetrasperma KT8401
2, Pichia Burtoni KT84013, Candida Pararugosa KT84015, Debaryomyces Hansenyi KT840
16 has been deposited at the Institute for Microbial Industry, Institute of Industrial Science and Technology, with the numbers shown in Table 1.

[0014]

[Table 1]

In order to obtain the mutant strain of the β-hydroxy acid-producing microorganism of the present invention, natural mutation or artificial mutation is used. Usually, in order to perform efficiently, ultraviolet irradiation and N-methyl-N-nitro are usually used. Treatment with a mutagenesis agent such as -N'-nitrosoguanidine is used, but is not particularly limited to this method.

Further, optically active beta-hydroxy acid produced by these microorganisms, _L - (+) - β- hydroxybutyrate, _L - (+) - β- hydroxy valeric acid, _L -
(+)-Β-hydroxyisobutyric acid, _L -(+)-β-hydroxyisocaproic acid, _L -(+)-β-hydroxypropionic acid, _D -(-)-β-hydroxybutyric acid, _D-
(-)-Β-hydroxyvaleric acid, _D -(−)-β-hydroxyisobutyric acid, _D -(−)-β-hydroxyisocaproic acid, _D -(−)-β-hydroxypropionic acid and the like.

Next, a method for specifically immobilizing a β-hydroxy acid-producing microorganism on a porous material having a skin layer in culture will be described. The culture method and culture tank format for β-hydroxy acid production are appropriately selected depending on the type of microorganism and the type of porous material used. As a culture method,
Although any method such as batch, semi-batch, continuous culture, etc. can be adopted, in the method for producing β-hydroxy acid, glucose concentration as a substrate and β-fatty acid must be kept below a certain concentration. Accordingly, a semi-batch culture in which a substrate is fed, or a continuous culture in which a steady state is created is preferable. As the β-fatty acid as the substrate, one corresponding to the desired β-hydroxy acid is used. For example, isobutyric acid is used when the product is hydroxyisobutyric acid, and butyric acid is used when the product is hydroxybutyric acid. Further, as the culture tank type, any type such as a tower type fermentation tank (bubble tower type, jet tank type, draft tube type), a stirring tank, etc. can be used, but in order to immobilize more efficiently, it is porous A tower type fermenter having a relatively small shearing force acting around the material is preferable.

The culture conditions are not particularly limited,
The temperature is 25 to 45 ° C., the pH is 4 to 9, the culturing time is about 1 to 14 days, and it is carried out under normal conditions. Further, it may be an aerobic condition or an anaerobic condition. The aeration rate when culturing under aerobic conditions is not particularly limited, but is usually about 1 to 10 vvm (a value obtained by dividing the aeration rate per minute by the culture solution amount).

When a bubble column type fermentation tank is used, the porous material is filled as shown in FIG. 1, the porous material is completely immersed in the medium, and the culture is performed by aerating from the lower part of the culture tank. At this time, the medium is
It is better not to be too large with respect to the volume of the porous material. That is, when aerated, the amount necessary and sufficient for the porous material to slightly flow, which is appropriately selected depending on the type of the culture tank and the size of the porous material. On the other hand, about 10 to 20% increase is desirable. If the porous material does not flow at all, it becomes difficult to stir the medium in the tank, mass transfer and oxygen transfer deteriorate, and β
-The efficiency of growth and immobilization of hydroxy acid-producing microorganisms is reduced, which is not preferable.

By the above-mentioned method, the β-hydroxy acid-producing microorganisms form colonies in the pores of the porous material, which are radial or open-celled, and are retained relatively stably. The mechanism of immobilization is that when β-hydroxy acid-producing microorganisms adhere to the pore wall surface and colonies spread from there, and as a result of β-hydroxy acid-producing microorganisms that once entered the pores aggregate and proliferate, It is assumed that it may not be possible to go outside from the small hole. In any case, the size of the pores present in the skin layer, the pores of the radial or open-celled partition walls,
It is important that the pore size is such that the β-hydroxy acid-producing microorganisms can pass through the size of the β-hydroxy acid-producing microorganisms to be immobilized, but movement is restricted to some extent.

In this way, the β-hydroxy acid-producing microorganism can be cultured with the porous material, and the metabolism of the microorganism immobilized in the porous material can be used to produce an optically active β-hydroxy acid. The β-hydroxy acid thus produced can be easily separated and purified from the medium by extraction with an organic solvent such as ether, chloroform or ethyl acetate.

[0022]

EXAMPLES The present invention will be described in more detail by way of examples, which by no means limit the scope of the present invention. Example 1 In a 500 ml Sakaguchi flask, 25 ml of medium (glucose 30 g / L, (NH ₄ ) ₂ HPO ₄ 13 g / L, K
H ₂ PO ₄ 7 g / L, NaCl 100 mg / L, Mg
_{SO 4 · 7H 2 O 1g /} L, ZnSO 4 · 7H 2 O
_{100mg / L, FeSO 4 · 7H} 2 O 90mg /
_{L, MnSO 4 · 4-6H 2 O} 10mg / L, CuS
_{O 4 · 5H 2 O 5mg /} L, yeast extract 3g /
L, further isobutyric acid when the product is hydroxyisobutyric acid, butyric acid 20 g / L when the product is hydroxybutyric acid, pH 7.
5) and 150 spherical cellulose beads having a skin layer and having a diameter of 4 mm (Cellulose beads type II made by Rengo, pore diameter of skin layer: 40 μm, pore diameter: 300 μm, small pore diameter: 40 μm) , After autoclaving,
The strains shown in Tables 2 and 3 were used to inoculate, and the shaking speed was 1
The cells were cultured at 30 oscillations at 00 oscillations / min. When the glucose is exhausted, collect the medium leaving the beads,
An autoclaved fresh medium (the same in composition) was added, and the culture was performed again. The culture was terminated when the substrate was used up. The β-hydroxy acids shown in Tables 2 and 3 were extracted from each medium with ethyl acetate and used for analysis.

On the other hand, microbial cells cultured by shaking in a Sakaguchi flask without beads were entrapped and immobilized on κ-carrageenan beads according to a conventional method and added to a 500 ml Sakaguchi flask together with 25 ml of a fresh medium, and a shaking speed of 100 oscilla.
Cultured at 30 ° C. at tions / min. When the glucose is exhausted, replace the culture medium with a fresh medium, culture again,
The product was extracted in the same manner as above. The extracted β-hydroxy acid was analyzed by gas chromatography manufactured by Hewlett Packard equipped with a capillary column HP-FFAP. 1 each
The concentrations of β-hydroxy acid in the medium at the second and second times are shown in Table 4.
As shown in Table 5 and Table 5, when the bacterial cells were fixed using κ-carrageenan beads, the oxygen supply was poor and the productivity was poor. However, when the cellulose beads were used, good productivity was obtained. Also, it could be used repeatedly.

[0024]

[Table 2]

[0025]

[Table 3]

[0026]

[Table 4]

[0027]

[Table 5]

Example 2 A fermenter (capacity 2 L) shown in FIG. 1 was used. A cellulose carrier having a skin layer (made by Rengo, diameter 4 mm, pore diameter of skin layer: 40 μm, pore diameter: 300 μm, small pore size) was used. Diameter: 40
(800 μm) and 800 ml of the synthetic medium described later was added thereto so that the total volume became 1 L, and the mixture was cultured to produce β- _D -hydroxyisobutyric acid. The strain is Candida rugosa K
T8501, medium composition: glucose 40 g / L, isobutyric acid 20 g / L, (NH ₄ ) ₂ HPO ₄ 13 g / L, KH ₂
PO ₄ 7 g / L, NaCl 100 mg / L, MgSO
_{4 · 7H 2 O 1g / L} , ZnSO 4 · 7H 2 O 10
_{0mg / L, FeSO 4 · 7H} 2 O 90mg / L, M
nSO ₄・ 4-6H ₂ O 10 mg / L, CuSO ₄・
5H ₂ O 5 mg / L, thiamine hydrochloride 1.8 mg / L,
Biotin is 1 mg / L. First, 30 ° C., ventilation amount 2.
After performing batch culture at 5 L / min, when glucose was exhausted, a 20% glucose solution and a 20% isobutyric acid solution were flown so that the concentration of glucose was 0.02 g / L or less and the concentration of isobutyric acid was 3 g / L. Was added to produce β- _D -hydroxyisobutyric acid. Table 6 shows the production rate per fermentor when the medium was replaced and the production was repeated every 24 hours. As a result, it can be seen that the cellulosic beads having the skin layer maintain a high production rate even at the second and third times because the bacterial cells are less likely to fall off.

[0029]

[Table 6]

Example 3 In the same manner as in Example 2, continuous production of β- _D -hydroxybutyric acid was carried out. As the carrier, a Rengo cellulose carrier having a skin tank (pore diameter of skin layer: 40 μm,
Diameter of pores: 300 μm, diameter of small pores: 40 μm) was used. The strain was Candida rugosa KT8201, and the medium composition used was the same as in Example 2. First, batch culture was performed at 30 ° C. and an aeration rate of 2.5 L / min, and when glucose was exhausted, 20% glucose solution and 20%
A butyric acid solution was fed while maintaining glucose at a concentration of 0.02 g / L or less and butyric acid at a concentration of 3 g / L, and β- _D -hydroxybutyric acid was produced while withdrawing the same amount of medium as the fed amount. FIG. 2 shows the change over time in the concentration of β- _D -hydroxybutyric acid in the effluent after the start of continuous operation. Thus, it was confirmed that β- _D -hydroxybutyric acid, which is an aerobic process, can be efficiently produced even in continuous culture.

[0031]

INDUSTRIAL APPLICABILITY According to the production method of the present invention, it becomes possible to effectively utilize β-hydroxy acid-producing microorganisms repeatedly, and thus the productivity of optically active β-hydroxy acid is improved and the culture tank for production is improved. Miniaturization is realized. Also,
This is an industrially advantageous method because the produced β-hydroxy acid can be easily separated from the microorganism, and the load in the post-treatment process such as the purification of β-hydroxy acid can be reduced.

[Brief description of drawings]

FIG. 1 shows a schematic view of the case where a bubble column type fermenter is used in the present invention.

FIG. 2 shows the concentration of β- _D -hydroxybutyric acid in the effluent and changes with time when β- _D -hydroxybutyric acid was continuously produced using the method of the present invention.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Internal reference number FI Technical display location C12R 1:72) (C12P 7/42 C12R 1:78) (C12P 7/42 C12R 1:84) (C12P 7/42 C12R 1: 865) (C12P 7/42 C12R 1:85) (C12P 7/42 C12R 1:88)

Claims (5)

[Claims] 1. A method for producing an optically active β-hydroxy acid, which comprises culturing a β-hydroxy acid-producing microorganism with a porous material, and producing using the microorganism immobilized in the porous material. Method. 2. The porous material has a structure having a skin layer having pores on the surface thereof, and having pores therein and small pores in partition walls of the pores. The manufacturing method described. 3. The diameter of the pores present in the skin layer of the porous material is 5 to 500 μm, the diameter of the pores is 100 to 1000 μm,
The manufacturing method according to claim 2, wherein the diameter of the small holes present in the partition walls of the holes is in the range of 5 to 500 µm. 4. The β-hydroxy acid is _L -(+)-β-
Hydroxybutyric acid, _L -(+)-β-hydroxyvaleric acid,
_L -(+)-β-hydroxyisobutyric acid, _L -(+)-β
-Hydroxyisocaproic acid, _L -(+)-β-hydroxypropionic acid, _D -(-)-β-hydroxybutyric acid,
_D -(-)-β-hydroxyvaleric acid, _D -(−)-β-
The production method according to any one of claims 1 to 3, which is hydroxyisobutyric acid, _D -(-)-β-hydroxyisocaproic acid, or _D -(-)-β-hydroxypropionic acid. 5. The β-hydroxy acid-producing microorganism is a brera.
(Bullera), Candida, Cryptococcus, Debaryomyces
ces, Endomyces, Hansenula
senula), Pachysolen, Pichia
hia genus, Rhodosporidium genus, Rhodotorula genus, Rhodococcus
genus coccus, genus Saccharomyces, genus Saccharomycopsis, genus Schizosaccharomyces, schwaniomyces
wanniomyces), Sporidiobulus
Genus, Torulopsis, Trichosporon
genus chosporon), microorganisms belonging to the genus Wingea,
Alternatively, it is a mutant strain derived from them.
4. The method according to any one of 4 above.