JPH0471494A - Production of alpha-hydroxy acid - Google Patents

Abstract

PURPOSE:To obtain the subject compound useful as an intermediate for medicines and agricultural chemicals at a low cost by reducing an inorganic salt of an alpha-keto acid sparingly soluble in water with a microorganism, etc. CONSTITUTION:An inorganic salt of an alpha-keto acid (e.g. 2-keto-4methylpentanoic acid) sparingly soluble in water is reduced with a microorganism [e.g. Streptococcus faecalis (IFO 12964)] or an enzyme [Staphyloccocus epideromidis (DSM 20343)] to afford the objective compound.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Field of Application) The present invention relates to a method for producing α-hydroxy acids. α-Hydroxy acids are important as intermediates for various medicines and agrochemicals, and there is a particular need for efficient and inexpensive methods for producing their optically active forms.

(Prior Art) Various methods are known for producing α-hydroxy acids, especially their optically active forms, by reducing α-keto acids with microorganisms or enzymes, but none of them are industrial production methods. (For example, JP-A-57-198096, JP-A-57-198096,
-198097, JP-A-63-32492, JP-A-63
-32493. Japanese Patent Publication No. 59-74983, Special Publication No. 61-
11591, JP-A-60-12975, JP-A-62-1
00286, JP 2-39893, JP 1-211
494 etc.).

The cause of this is substrate inhibition or product inhibition, and the concentration of the substrate α-keto acid and the accumulated concentration of α-hydroxy acid are only 1% at most, and the production efficiency is low.
This is because the cost increases. There is also a coenzyme NADH
Alternatively, NADPH is also expensive, and it is not easy to incorporate a regeneration system for continuous reaction.In order to solve these problems, various glue actors have been devised, but they have not yet been put to practical use. Nae et al., Bioindustry 5 (4), 261-268 (1988), Simo
nE, S, PlanteR, & Whitesides
G.

M,,Appl,Biochem,Biotech,.

22.169-179 (1989)].

(Problem to be Solved by the Invention) In view of the above-mentioned situation, the problem of the present invention is to industrially produce α-hydroxy acids, which are important as intermediates for various medicines and agrochemicals, by a simple and efficient method. An object of the present inventors is to provide a method for dramatically increasing the accumulated concentration of α-hydroxy acids when reducing α-keto acids with enzymes or microorganisms.6 (Means for solving the problem) The present inventors As a result of intensive investigation into a simple and practical method for achieving high product accumulation concentrations, we found that if α-keto acids and/or α-hydroxy acids are present in the form of sparingly soluble salts, substrate inhibition and product inhibition will be minimal. The present invention was completed by discovering that this problem does not occur. That is, according to the present invention, when reducing an α-keto acid with a microorganism or an enzyme to form an α-hydroxy acid,
By converting the α-keto acid into an inorganic salt that is poorly soluble in water, or/and using the inorganic salt as a neutralizing agent for the α-keto acid,
Substrate inhibition and product inhibition, which are disadvantages peculiar to enzyme reactions, can be avoided, and the accumulated concentration of the product α-hydroxy acid can be dramatically improved.

The α-keto acids used in the present invention include 2-keto 4-
Methylpentanoic acid, 2-ketobutyric acid, 2-getobentanoic acid, 2-methylbutyric acid, 2-ketohexanoic acid, 2-keto3-
Methylpentanoic acid, 2-getooctanoic acid, 2-keto-3
-mercapto-10pionic acid, 2-keto-4-(methylmercap1-)-butyric acid, 2-keto-3-phenylpropionic acid, 2-keto-4-phenylacetic acid, benzoylformic acid,
Phenylpyruvic acid etc. can be used.

The α-keto acid only needs to be sparingly soluble in water, and examples include divalent metal salts of α-keto acids, more specifically alkaline earth metals such as calcium, barium, and strontium salts.

In addition, as a neutralizing agent, any agent that forms a water-soluble salt with α-keto acid or α-hydroxy acid can be used, but for example, neutralizing agents that form divalent metal ions can be used. Alkaline earth metal salts such as calcium, barium, strontium etc. Specific neutralizing agents include those of the metals mentioned above, ie, calcium carbonate, barium carbonate, strontium carbonate, and the like.

As the enzyme used in the present invention, any enzyme that reduces α-keto acid to α-hydroxy acid can be suitably used. For example, monolactate dehydrogenase derived from bovine heart, enzyme derived from microorganism, etc. is Staphylococcus epidemidis (Staphylococcus epidemidis)
sep+derol-dis) DSH203430
Iconostoc mesenteroides (Leuconos)
tocnesenteroides) DSH2034
D-lactate dehydrogenase from Lactobacillus 3
Lacto-bacillus casei
) D-2-hydroxyisocaproate dehydrogenase from DSH20008, Lactobacillus core 77
, :L-sus (Lactobacillus conf
L-2-hydroxyisocaproate dehydrogenase derived from DSH20196, Lactobacillus
Lactobacillus c-
urvatus) D-7 nderate dehydrogenase derived from DSH2001. Coenzyme NADH
The enzymes used to regenerate the
Examples include formic acid dehydrogenase derived from DSH92 and alcohol dehydrogenase derived from baker's yeast. As an enzyme for regenerating NADPH, Leuco-nostoc ncsenter
Examples include glucose-6-phosphate dehydrogenase derived from P. oides). These enzymes can be suitably used as commercially available enzymes, or may be natural enzymes isolated from living organisms, chemically modified enzymes, or enzymes mutated through protein engineering. It may also be immobilized on acrylamide, calcium alginate, or the like.

In addition, microorganisms such as Streptococcus
Streptococcus faeca
lis) IFo 12964, Lactobacillus casei I
FO12004, Leucons 1~Tsuku mesenteroides subspecies Textlanicum (Leuco
nostoc nesente-roides 5ub
sp, dextranicu > IFO3349,
Leucomes l-ivy Textlanicum (Leucon)
ostoc dextranicun) IFO334
7. Leuconostoc mesenteroides (Leu-c
onostoc 11esenteroides) A
HU 1067, Lactobacillus 1 rankrum (Le
uconostoc plantarul) AHU
Examples include 3070.

Any strain of these microorganisms can be suitably used, such as a wild strain, a mutant strain, or a recombinant strain induced by genetic techniques such as cell fusion or genetic manipulation.

In addition, microorganisms with IFO numbers are designated by the Fermentation Research Institute (Foundation).
IFO) Fermentation of Li5t of Cultures
, 8th edition, Volume 1 (1988),
It can be obtained from F0. The microorganisms with AHU numbers are the Japan Microorganism Strain Conservation Federation (JFCC) fermentation Ca
Talogue of Cultur'es, No. 4
(1987) and is available from the Faculty of Agriculture, Hokkaido University.

For culturing the microorganisms used in the present invention, any material that allows the microorganisms to grow can be suitably used. Specifically, glucose, fructose, sucrose, dextrin, starch, etc. can be suitably used. Carbon sources include sugars, alcohols such as sorbitol, ethanol, and chrycerol, fumars, FR acids and their salts such as citric acid, acetic acid, and propionic acid, hydrocarbons such as paraffin, or mixtures thereof, and ammonium sulfate and ammonium nitrate. Inorganic nitrogen sources such as yeast extract, malt extract, peptone, meat extract and the like can be used alone or as a mixture as the nitrogen source. In addition, inorganic salts,
Nutrient sources used in normal culture, such as trace metal salts and vitamins, can be mixed and used as appropriate. Further, if necessary, a factor that promotes the growth of microorganisms, a factor that increases the ability to produce the target compound of the present invention, or a substance that is effective in maintaining the pH of the culture medium can also be added. As for the culture method, the pH is 3.
°0-9.5, preferably 5-8, culture temperature 20-4
5°C, preferably 25 to 37°C, anaerobically or aerobically, under conditions suitable for the growth of the microorganism.
Cultivate for a period of time, preferably about 12 to 72 hours.

As a method of reduction reaction, α-keto acid or α-keto acid to be reduced
When a bacterial cell culture after culturing in the presence of a goto acid salt, a gas collected therefrom, or a processed product thereof is allowed to act on an α-keto acid or an α-keto acid salt, or when a purified enzyme and its processed product are used. It is possible to implement either of the following. The treated bacterial cells here include, for example, crushed bacterial cells using a homogenizer, those treated with acetone, freeze-drying, etc., and those that have been subjected to these treatments or the above-mentioned treatments, for example. It also includes substances immobilized by known methods such as polyacrylamide gel method, sulfated polysaccharide gel method (carrageenan gel method), alginate gel method, and agar gel method.

When collecting bacterial cells from a bacterial culture after culturing microorganisms for reaction, collect the bacterial cells by a method such as centrifugation, and then add them to an appropriate buffer solution, either as is or after washing with water or physiological saline. The mixture is suspended, and an α-keto acid or a divalent metal salt thereof, or a carbonate of a divalent metal salt is added and reacted.

During this reaction, it may be better to add a carbonate source such as glucose or sucrose as an energy source.

When adding a free α-keto acid as a substrate, it may be added together with a carbonate of a divalent metal.

When using a purified enzyme, an enzyme that reduces α-keto acids,
α-keto acid as a substrate, or its divalent metal salt, in the case of a free acid, a carbonate of a divalent metal, and NADH as a coenzyme.
Alternatively, NADPH (the appropriate one is selected depending on the enzyme) is added, and for industrially advantageous implementation, an enzyme for recycling the coenzyme and a substrate for the recycling enzyme are added and the reaction is carried out. The amount of enzyme is preferably such that the ratio of the activity of the NAD H or NADPH recycling enzyme to the activity of the substrate α-keto acid reductase is 1:0.1.
It is used in an amount such that the ratio is ˜1:5. NADH
As the combination of the recycling enzyme and its substrate, formate dehydrogenase/formic acid or its alkali metal salt system, and alcohol dehydrogenase/ethanol system are advantageous, and as the combination of the NADPH recycling enzyme and its substrate, glucose- 6-phosphate dehydrogenase/glucose-6-phosphate or an alkali metal salt thereof is preferably used.

The reaction mixture is as commonly used for enzymatic reactions.
pH in the range 3-9, preferably pH 5-8, temperature 1
1 in the range of 0 to 60°C, preferably 20 to 40°C.
The concentration of the substrate added is 0.1 to 20%, preferably 0.5 to 10%, for about 120 hours under stirring or standing still.
%, but it may be prepared in one batch, divided into parts, or continuously. The concentration of the neutralizing agent to be added may be such that the necessary amount is added to maintain a pH suitable for the reaction. The amount of coenzyme depends on the amount of enzyme used, but preferably 0.01 to 10iH in the shell.
Add about HgH.

The α-hydroxy acid produced by the reaction is collected by α-
Since most hydroxy acids form insoluble salts,
It is efficient to acidify the reaction-completed solution with a mineral acid to convert the α-hydroxy acid into a free acid, and then extract it with a suitable organic solvent. After extraction, it can be easily produced by conventional methods such as various column chromatography, crystallization, and activated carbon treatment.

(Examples) Hereinafter, the present invention will be specifically explained using Examples, but the present invention is not limited only to these Examples.

The quantitative determination of the reaction products in the examples was carried out by high performance liquid chromatography using a reversed phase column (column: N
ucleosil 10C18+64.6m1x250
nl, mobile phase: 40 nH potassium phosphate buffer pH
3,0/acetonitrile=4=1, flow rate: 1, O
nl/nin, detection: 254ni).

In addition, the optical purity was measured by high performance liquid chromatography using an optical resolution column (column: Daicel Chemical Industries, Ltd. Chiralpak WH1φ4.6nrt x2
50u, mobile phase: 0.25nHCuSO4/acetonitrile=4:Li rate: 1, Onl/nin, detection: 254r+n).

Example 1, Comparative Example 1 (R)-2-hydroxy-4-
Production of phenyl acetic acid Glucose 8%, yeast extract 1%, MnSO4.4-5
2.6L of 2000m1 of leased land consisting of 5H2O10pp
After sterilization, put it in a mini jar (manufactured by Marubishi Bioengine),
teuconos tcmesenteroides
5ubsp, dextranicun IFO334
9 was inoculated and cultured at 30° C. and 1100 rpi for 17 hours. A pH electrode was attached to the 4 cultured inocula, and the pH was adjusted to 6.5 to 7.0 with 25% NaOH before culturing. After the culture is completed, the bacterial cells are separated by centrifugation, and the bacterial cell concentration is 0D66.
It was suspended in distilled water so that o was 40. This is 200
Pour the same 2.6 m each into a mini jar and add 40 m.
% glucose 20C) nl, 6% 2-keto-4-phenylbutyric acid 400 nl < Neutralized with KOH, pH 7°0
(adjusted) was added.

For culture tank A, 2% CaCO3 was added, and for culture tank B, a p)l electrode was attached and 25% Na OH was added.
3 each while keeping the T pH between 6.5 and 7.0.
The reaction was carried out at 0°C and 1100 rpm. (Final concentration: 2-keto-4-phenylbutyric acid 3%, glucose 10%, bacterial cell concentration 0D66o-Reaction results are shown in Table 1.

Example 2 Production of 2-hydroxy-4-phenylbutyric acid 8% glucose, 1% yeast extract, 1% peptone, CaCO
35%, HnSO4-5~6H2010ppn, Fe
SO4・7H2010001, N a CI 10DD
Pour 100 ml of Il into a 500 ml Erlenmeyer flask, and after sterilizing it, teuconostoc dex-
tranicun IFO3347, Leuconos
toc nesenteroides 5ub-3D,
dextranicun IFO3349, teuc
onostoc IIeSenterOld-es A
HU 1067 was inoculated. After culturing at 30°C for 40 hours,
Bacterial cells were separated by centrifugation and suspended in distilled water.

Final concentration of bacterial cell concentration 0D66o-10, glucose 10%
, Ca CO31%, 2-keto-4-phenylbutyric acid 4.
Adjust the reaction solution so that it is 5% and the reaction solution volume is 2 ml.
The reaction was carried out in a φ21 mm test tube at 30° C. for 43 hours. The results are shown in Table 2.

Claims (1)

[Claims] 1. A method for producing α-hydroxy acid characterized by reducing an inorganic salt of an α-keto acid that is sparingly soluble in water using a microorganism or an enzyme. 2. A process for producing an inorganic salt of an α-keto acid that is sparingly soluble in water. A method for producing α-hydroxy acid characterized by reduction with microorganisms or enzymes in the presence of