JPH09131194A - Production of alpha-hydroxycarboxylic acid or alpha-hydroxyamide with microorganism - Google Patents

Abstract

PROBLEM TO BE SOLVED: To efficiently obtain the subject compound useful as a raw material for medicines, agricultural chemicals, etc., from an aldehyde and hydrogen cyanide or an α-hydroxynitrile by the action of a microorganism having a nitrilase or nitrile hydratase activity, while constantly maintaining the concentrations of the reagents. SOLUTION: This α-hydroxycarboxylic acid or α-hydroxynitrile of formula III (X is amide, carboxyl) is produced from the corresponding aldehyde of formula I [R is a (substituted) alkyl, a (substituted) alkenyl, a (substituted) cycloalkyl, a (substituted) alkoxy, a (substituted) aryl, etc.], and hydrogen cyanide, or from the corresponding α-hydroxynitrile of formula II by the action of a microorganism having a nitrilase or nitrile hydratase activity in an aqueous medium. Therein, the concentration of the aldehyde and/or the concentration of the α- hydroxynitrile in the reaction solution are detected, and the aldehyde and the hydrogen cyanide or the α-hydroxynitrile are supplied on the basis of the detection results to control the concentrations in constant ranges.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to microbial α-
It relates to a method for producing a hydroxy acid or α-hydroxyamide. In particular, the optically active α-hydroxyamide and α
-Hydroxy acid is industrially important as a raw material for the synthesis of various medical and agricultural chemicals.

[0002]

2. Description of the Prior Art Regarding the production of α-hydroxy acids by microorganisms, the genera Alcaligenes, Pseudomonas and Rhodops are known.
Eudomonas), Corynebacterium
Genus, Acinetobacter genus, Bacillus (B
acillus) genus, Mycobacterium genus,
Rhodococcus spp., Candid
a) Method using microorganisms of genus, Nocardia (Nocardia), etc. (JP-A-2-84198, 3-224496, 3-277292, etc.), Nocardia (Nocardia), Bacillus
Genus, Brevibacterium genus, Aureobacterium genus, Pseudomonas (Pse
genus udomonas, genus Caseobacter, genus Alcaligenes, Acinetobacter
tobacter genus, Enterobacter genus, Arthrobacter genus, Escherichia
genus richia, genus Micrococcus, genus Streptomyces, flavobacterium (F
genus lavobacterium, genus Aeromonas, genus Mycoplana, cellulomonas
Genus, Erwinia Genus, Candida
Genus, Bacteridium, Aspergillus
(Aspergills) genus, Penicillium (Penicillium) genus, Cochliobolus (Cochliobolus) genus, Fusarium (Fusarium)
Genus, Rhodopseudomonas, Rhodococcus, Corynebacterium
Acterium), Microbacterium
Method using microorganisms such as genus, genus Obsumbacterium, genus Gordona, etc. (JP-A-4-9949)
No. 5, No. 4-99496, No. 4-99497, No. 4-218385,
5-95795, 5-21987, 5-192189, 6-237789
No. 6, No. 6-284899, No. 7-213296, etc.) are known.

On the other hand, regarding the production of α-hydroxyamides by microorganisms, Rhodococcus spp, Corynebacterium spp, Pseudomonas spp, Arthrobacter spp.
Genus, Alcaligenes, Bacillus
genus lus, genus Bacteridium, genus Micrococcus, Brevibacterium
erium, Nocardia and other microorganisms (JP-A-4-222591, JP-A-5-192189, JP-A-7-213296)
No.) etc. are known.

[0004]

However, when α-hydroxynitrile is enzymatically hydrolyzed or hydrated with nitrilase or nitrile hydratase to produce α-hydroxy acid or α-hydroxyamide,
There is a problem that the enzyme is inactivated in a short time, and it has been difficult to obtain a high concentration of α-hydroxy acid or α-hydroxyamide with high productivity. In addition, α-
Using aldehyde and hydrocyanic acid, which are more economical than hydroxy nitrile, α-hydroxy acid or α-hydroxy acid in the reaction solution is used.
When the concentration of hydroxyamide was increased, the reaction rate decreased, and there was a problem that the reaction did not proceed.

[0005]

As a result of intensive studies to solve these problems, the present inventors detected the aldehyde concentration and / or α-hydroxynitrile concentration in the reaction solution and It was found that by keeping the aldehyde concentration and / or the α-hydroxynitrile concentration in a certain range, deactivation of the enzyme can be prevented and a high concentration of α-hydroxy acid or α-hydroxyamide can be obtained with high productivity, The present invention has been reached.

That is, according to the present invention, by the action of a microorganism having a nitrilase or nitrile hydratase activity, the aldehyde and hydrocyanic acid represented by the following general formula (1) or the following general formula (2) in an aqueous medium by the action of the microorganism. ) Corresponding to α-hydroxy acid or α represented by the following general formula (3)
-When producing a hydroxyamide, the concentration of an aldehyde and / or the concentration of α-hydroxynitrile in the reaction solution is detected, and aldehyde and hydrocyanic acid or α- is added to the reaction solution.
An α-hydroxy acid or α-hydroxy acid characterized in that the aldehyde concentration and / or α-hydroxynitrile concentration in the reaction solution is kept within a certain range by continuously and / or intermittently supplying hydroxynitrile. The gist is a method for producing an amide.

[0007]

[Wherein R represents a substituted or unsubstituted alkyl group, a substituted or unsubstituted alkenyl group, a substituted or unsubstituted cycloalkyl group, a substituted or unsubstituted alkoxy group, a substituted or unsubstituted aryl group, A substituted or unsubstituted aryloxy group, a substituted or unsubstituted saturated or unsaturated heterocyclic group, and X represents an amide group or a carboxyl group. ]

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION In the present invention, it is difficult to know the actual concentration of aldehyde, hydrocyanic acid, α-hydroxynitrile, and optionally added sulfite ion because they have an equilibrium relationship and a reaction relationship in the reaction solution. . Therefore, the aldehyde concentration and / or the α-hydroxynitrile concentration in the reaction solution detect an apparent value. Specifically, the reaction solution is collected while the reaction is in progress, and if necessary, diluted and solid-liquid separated to analyze the resulting solution. As an analytical method,
Liquid chromatography is preferred, but gas chromatography can also be used. The aldehyde concentration and / or α-hydroxynitrile concentration in the reaction solution is usually 0.01 mM to 1000 mM, preferably 0.05.
The supply amount of aldehyde or α-hydroxynitrile is controlled so as to be mM to 200 mM, more preferably 1 mM to 50 mM. At the final stage of the reaction, the supply of aldehyde or α-hydroxynitrile is stopped to reduce the aldehyde concentration and / or α-hydroxynitrile concentration in the reaction solution.

When aldehyde and hydrocyanic acid are used as the raw materials, the cyan concentration in the reaction solution is usually detected by detecting the cyan concentration with a cyan ion sensor or the like.
Prussic acid is supplied so as to have a concentration of 300 mM, preferably 0.1 mM to 100 mM, and more preferably 1 mM to 50 mM.

In general, aldehydes have the property of binding to proteins and deactivating enzyme activity. Therefore, the concentration of aldehydes and / or α-hydroxynitrile in the reaction solution is detected and the reaction solution in the reaction solution is detected. It is considered that the suppression of the enzyme inhibitory action by keeping the aldehyde concentration and / or the α-hydroxynitrile concentration within a certain range can be applied to all microbial reactions involving aldehyde in principle. That is, the aldehyde of the present invention, hydrocyanic acid and α-
In the method for producing an acid or amide from hydroxynitrile, the microorganism used is not particularly limited as long as it has the ability to produce the acid or amide, so-called nitrilase or nitrile hydratase activity.

The microorganism having nitrilase activity that can be used in the present invention is Pseudomonas.
Genus, genus Alcaligenes, genus Acinetobacter, genus Caseobacter
Genus, genus Corynebacterium, genus Brevibacterium, Nocardia
ardia genus, Rhodococcus genus, Gordona (G
ordona), Arthrobacter, Bacillus, Aureobacter
ium genus, Enterobacter genus, Escherichia genus, Micrococcus
Genus, Streptomyces, Flavobacterium, Aeromona
s), Mycoplana, Cellulomonas (Ce
llulomonas genus, Erwinia genus, Candida genus, Bacteridium genus, Aspergillus genus, Penicillium (Penicil)
lium, Cochliobolus, Fusarium and Rhodopseudo
monas) and other microorganisms.

Specific examples include the following microorganisms. Pseudomonas sp. BC13-2 (No. 3319), BC15-2 (No. 3320).
SK 13 (Microtechnical Research Institute No. 3325), SK31 (Microtechnical Research Co., Ltd. 11310), SK87 (Microtechnical Research Co., Ltd. 11311), Pseudomonas synxanta IAM 12356, Alcaligenes sp. BC12-2 (Microbiology Research Institute No. 11263),
BC20 (Microtechnical Research Institute No. 11264), BC35-2 (Microtechnical Research Article No. 3318), Acinetobacter bacter sp. BC9-2 (Microtechnical Research Article No. 3317), Kaseoba sp. BC4 (Microtechnical Institute No. 3316), BC23 (Microtechnical Microbiology No. 11261), Corynebacterium nitrilophilus AT
CC 21419, Brevibacterium acetylicam
cum) IAM 1790, Brevibacterium helvolum (hel
volum) ATCC11822, Nocardia sp. N-775 (Ministry of Industrial Science and Technology No. 4447), Nocardia Asteroides (aster)
oides) IFO 3384, Nocardia Calcarea (calcare
a) KCCA0191, Nocardia polychromogenes (polyc
hromogenes) IFM 19, Rhodococcus sp. SK70 (Microtechnical Research Institute No. 11304), SK92 (Microtechnical Research Article No. 3324),
HR11 (Microbiology Research Institute No. 11306), Rhodococcus rhodochrous ATCC 12674, ATCC 19140
, ATCC 33258, Rhodococcus erythropolis (er
ythropolis) IFM 155, same IFO 12320, same IFO 12538,
The same IFO 12540, Gordona Terrae MA-1 (FERM
BP-4535), Arthrobacter sp.SK103
11300), HR1 (Ministry of Industrial Science, Microbial Law No. 3323), HR4
(Microtechnology Research Institute, No. 11302), Arthrobacter oxydans IFO 12138, Bacillus subtilis (sub)
tilis) ATCC 21697, Bacillus licheniformis (lic
heniformis) IFO 12197, Bacillus megaterium (meg
aterium) ATCC 25833, Aureobacterium testaceum IAM 1561, Enterobacter sp. S
K12 (Ministry of Fine Arts, Article 3322), Escherichia coli (col
i) IFO 3301, Micrococcus luteus ATC
C 383, Micrococcus varians IAM 109
9, Micrococcus roseus IFO 3768, Streptomyces griseus IFO 3355, Flavobacterium sp. SK150 (Microtechnology Research Institute No. 11645),
Flavobacterium flavescens ATC
C 8315, Aeromonas punctata IFO 132
88, Mycoplana dimorpha ATCC 4297,
Cellulomonas fimi IAM 12107, Erwinia
Herbicola IFO 12686 and Candida guilliermondii IFO 0566. Each of the above microorganisms is described in the above publication.

On the other hand, the microorganisms having nitrile hydratase activity which can be used in the present invention include Rhodococcus spp.
There are microorganisms of the genus Corynebacterium, Pseudomonas, Arthrobacter, Alcaligenes, Bacillus, Bacteria, Micrococcus, Brevibacterium and Nocardia.

Specific examples include the following microorganisms. Rhodococcus sp. HT40-6 (FERM BP-5
231), Rhodococcus rhodochrous ATCC 33278, Rhodococcus erythropolis IFO 12320, Corynebacterium nitrilophilus ATCC 21419, Pseudomonas
sp. SK87 (Microtechnical Research Institute No. 11311), Arthrobacter sp. HR1 (Microtechnical Research Article No. 3323) and Alcaligenes sp. BC16-2 (Microtechnical Research Article No. 3321). Each of the above microorganisms is described in the above publication.

In the aldehyde represented by the general formula (1) and the α-hydroxyniryl represented by the general formula (2) of the present invention, in the formula, R is a substituted or unsubstituted alkyl group, a substituted or unsubstituted alkyl group. Alkenyl group, substituted or unsubstituted cycloalkyl group, substituted or unsubstituted alkoxy group, substituted or unsubstituted aryl group, substituted or unsubstituted aryloxy group, substituted or unsubstituted saturated or unsaturated heterocycle It is represented by a group, and the aldehyde, hydrocyanic acid and α-hydroxynilyl are in dissociation equilibrium in the reaction solution.

The heterocyclic group includes nitrogen as a hetero atom,
Examples include those containing at least one of oxygen and sulfur.
Further, examples of the substituent include an alkyl group, an alkoxy group, an acyl group, an aryl group, an aryloxy group, a halogen such as chlorine and bromine, a hydroxy group, an amino group, a nitro group, and a thiol group.

Specifically, examples of the aldehyde include acetaldehyde, propionaldehyde, n-butyraldehyde, n-pentylaldehyde, n-hexylaldehyde, n-heptylaldehyde, β-hydroxy-α, α-dimethylpropionaldehyde. , Acrolein, methacrylaldehyde, 2-chloroacetaldehyde, 3-methylthio-propionaldehyde, 2-
Examples of phenyl aldehyde, or substituted products thereof, and those having an aromatic or heterocyclic ring include benzaldehyde, 2-thiophene aldehyde, 2-pyridine aldehyde, 2-pyrrole aldehyde, 2-furaldehyde, 2
-Naphthyl aldehyde, a substitution product thereof, or the like. Regarding cyanide used, cyanide such as soda cyanide and potassium cyanide can be used.

Examples of the α-hydroxy nitrile include lactonitrile, α-hydroxy-n-butyronitrile, α-hydroxy-n-pentyronitrile, α-hydroxy-n-hexylonitrile and α-hydroxy-n.
-Heptylonitrile, α-hydroxy-n-octylonitrile, α, γ-dihydroxy-β, β-dimethylbutyronitrile, acrolein cyanohydrin, methacrylaldehyde cyanohydrin, 3-chlorolactonitrile, 4-methylthio- α-hydroxybutyronitrile,
α-hydroxy-α-phenylpropionyl, or substituted products thereof, and those having an aromatic or heterocyclic ring include mandelonitrile, 2-thiophenealdehyde cyanohydrin, 2-pyridinealdehyde cyanohydrin, 2 -Pyrrole aldehyde cyanohydrin, 2-furaldehyde cyanohydrin, 2-naphthyl aldehyde cyanohydrin, or these substitution products etc. can be mentioned.

In order to reduce the inhibition of the enzyme by aldehyde, it is effective to add sulfite ion such as sulfite or acid sulfite. The salt is sodium salt, potassium salt, ammonium salt or the like, and the addition amount thereof may be in the range of 1 to 1000 mM in the reaction solution.

When a microorganism having a stereospecific nitrilase or nitrile hydratase in the reaction is used, or a treated product thereof (enzyme, immobilized enzyme, immobilized bacterium) is produced, an α-hydroxy acid or α-hydroxyamide is produced. 50% or more of the starting materials, that is, 50% or more of the raw materials can be converted into one of the optically active substances, so that it is extremely advantageous to use the optically active α-hydroxy acid or α without undergoing the optical resolution and racemization steps. A hydroxyamide can be obtained.

Next, embodiments of the present invention will be described.
The hydrolysis or hydration reaction is carried out in a medium such as water or a buffer solution by adding a mixture of an aldehyde represented by the general formula (1) and hydrocyanic acid or an α-hydroxynitrile represented by the general formula (2) to microorganisms. It is carried out by contacting the body or treated cells (crushed cells, crude / purified enzyme, immobilized cells / enzyme, etc.). At this time, in the present invention, the aldehyde concentration and / or the α-hydroxynitrile concentration in the reaction solution is detected so that the aldehyde concentration and / or the α-hydroxynitrile concentration in the reaction system are kept within a certain range. , Aldehyde and hydrocyanic acid, or α
-Hydroxynitrile is fed continuously and / or intermittently.

The aldehyde concentration and / or α-hydroxynitrile concentration in the reaction solution are as described above. The amount of microorganisms used for the substrate is 0.00 as dry cells.
It is an amount equivalent to 1 to 5.0% by weight. The reaction temperature is generally freezing point to 50 ° C, preferably 5 to 30 ° C. When the solubility of these α-hydroxynitriles in an aqueous medium is extremely small, 0.1 to 5.0% by weight of a surfactant such as Triton X-100 or Tween 60, or methanol, or The reaction can be efficiently performed by adding ethanol, dimethylsulfoxide, or the like.

The obtained α-hydroxy acid or α-hydroxyamide can be isolated by a known method such as concentration, ion exchange, electrodialysis, extraction, crystallization, etc. of the reaction solution from which insoluble matter such as cells has been removed. Can be done using.

[0025]

EXAMPLES Next, the present invention will be described in more detail by way of examples, but the present invention is not limited to these examples.

Example 1 50 mM containing 1000 mM sodium sulfite in a reactor
A phosphate buffer solution (pH 8.0) was charged, and the temperature was adjusted to 30 ° C. Gordona terrae MA-1 strain was OD in this solution.
_It was suspended so that ₆₃₀ = 4.2. The molar ratio of the raw material sodium cyanide aqueous solution and benzaldehyde is 1.0: 1.
The ratio was controlled so that it became 0 before supply. The cyan ion detector was used to detect the cyan ion concentration in the reaction solution, and the supply amount of the aqueous solution of sodium cyanide was controlled so that the output of the cyan ion detector was −220 mV to −224 mV.
During the reaction, the reaction solution was sampled and filtered, the filtrate was diluted 10 times with water, and the benzaldehyde concentration was measured by liquid chromatography. The ratio of the ratio control between the aqueous solution of sodium cyanide and benzaldehyde was adjusted so that the concentration of benzaldehyde in the reaction solution was 10 mM to 15 mM. After the reaction was carried out for 22 hours, the concentration of R-mandelic acid in the reaction-completed liquid was 10.5%, and the optical purity was 99.0%.
It was ee. The yield of R-mandelic acid based on the supplied benzaldehyde was 97.0%.

Example 2 The same operation as in Example 1 was carried out except that prussic acid was used in place of the aqueous solution of sodium cyanide in Example 1. After the reaction was carried out for 22 hours, the concentration of R-mandelic acid in the reaction-completed liquid was 14.2%, and the optical purity was 99.0% ee. The yield of R-mandelic acid based on the supplied benzaldehyde was 97.0%.

Example 3 A reactor was charged with 20 mM phosphate buffer (pH 8.5) and the temperature was raised to 10 ° C. Rhodococcus sp.
The HT40-6 strain was suspended so that OD ₆₃₀ = 4.2. After starting material benzaldehyde was added to the solution and dissolved in 30 mM, the starting material hydrocyanic acid and benzaldehyde were supplied at a controlled ratio of 1.0: 1.0.
The cyan ion detector detects the cyan ion concentration in the reaction solution, and the output of the cyan ion detector is -145 mV ~
The supply amount of the aqueous solution of sodium cyanide was controlled so as to be −150 mV. During the reaction, sample the reaction mixture and add 1 with water.
It was diluted to 0 times and filtered, and the filtrate was measured for benzaldehyde concentration by liquid chromatography. The ratio of the ratio control of the sodium cyanide aqueous solution and the benzaldehyde was adjusted so that the concentration of benzaldehyde in the reaction solution was 35 mM to 40 mM. After the reaction was performed for 66 hours, the concentration of mandelamide in the reaction-terminated liquid was 32%, and the optical purity of S-mandelamide was 77% ee. The yield of mandelamide based on the supplied benzaldehyde was 95%. In addition, mandelamide was crystallized in the reaction completed liquid.

Example 4 A reactor was charged with a 50 mM phosphate buffer solution (pH 8.0) containing 100 mM of sodium sulfite, and the temperature was raised to 30 ° C. Gordona Terrae MA-1 strain was OD ₆₃₀ =
Suspended to 4.2. The raw material mandelonitrile was supplied at a predetermined flow rate. During the reaction, the reaction solution was sampled and filtered, the filtrate was diluted 10 times with water, and the benzaldehyde concentration was measured by liquid chromatography. The concentration of benzaldehyde in the reaction solution is 10 mM to 1
The flow rate of mandelonitrile was corrected to be 5 mM. After the reaction was carried out for 22 hours, the concentration of R-mandelic acid in the reaction-terminated liquid was 12.5%, and the optical purity was 77% ee. The yield of R-mandelic acid based on the supplied benzaldehyde was 95%.

Comparative Example 1 A reactor was charged with a 50 mM phosphate buffer solution (pH 8.0) containing 100 mM sodium sulfite, and the temperature was raised to 30 ° C. Gordona Terrae MA-1 strain was OD ₆₃₀ =
Suspended to 4.2. The raw material aqueous solution of sodium cyanide and benzaldehyde were supplied while controlling the ratio so that the molar ratio was 1.0: 1.0. The cyan ion detector was used to detect the cyan ion concentration in the reaction solution, and the supply amount of the aqueous solution of sodium cyanide was controlled so that the output of the cyan ion detector was −220 mV to −224 mV. After the reaction was carried out for 22 hours, the concentration of R-mandelic acid in the reaction-terminated liquid was 5.0%, and the optical purity was 97% ee. The yield of R-mandelic acid based on the supplied benzaldehyde was 9
3%.

[0031]

According to the present invention, the hydrolysis and hydration reaction of α-hydroxynitrile can be carried out while keeping the aldehyde concentration in the reaction, which is considered to be one of the causes of enzyme inhibition, low at all times. It can be stably maintained, thus allowing high concentration accumulation of the acid or amide produced.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical display location C07C 235/34 9547-4H C07C 235/34 (C12P 7/42 C12R 1:01) (C12P 13 / 02 C12R 1:01) C07M 7:00

Claims (4)

[Claims] 1. An aldehyde and hydrocyanic acid represented by the following general formula (1) or an α-hydroxynitrile represented by the following general formula (2) in an aqueous medium by the action of a microorganism having a nitrilase or nitrile hydratase activity. To produce a corresponding α-hydroxy acid or α-hydroxyamide represented by the following general formula (3), the aldehyde concentration and / or α-hydroxynitrile concentration in the reaction solution are detected to obtain an aldehyde in the reaction solution. And hydrocyanic acid or α-hydroxynitrile are continuously and / or intermittently supplied to maintain the aldehyde concentration and / or the α-hydroxynitrile concentration in the reaction solution within a certain range. A method for producing an α-hydroxy acid or an α-hydroxyamide. [Wherein, R is a substituted or unsubstituted alkyl group, a substituted or unsubstituted alkenyl group, a substituted or unsubstituted cycloalkyl group, a substituted or unsubstituted alkoxy group, a substituted or unsubstituted aryl group, a substituted or unsubstituted A substituted aryloxy group, a substituted or unsubstituted saturated or unsaturated heterocyclic group, and X represents an amide group or a carboxyl group. ] 2. The method for producing α-hydroxy acid or α-hydroxyamide by the microorganism according to claim 1, wherein the aldehyde concentration and / or α-hydroxynitrile concentration in the reaction solution is detected by liquid chromatography. 3. The aldehyde concentration and / or α-hydroxynitrile concentration in the reaction solution is 0.01 mM to 100.
The method for producing α-hydroxy acid or α-hydroxyamide by the microorganism according to claim 1, which is maintained at 0 mM. 4. The method for producing α-hydroxy acid or α-hydroxyamide by the microorganism according to claim 1, wherein sulfite ion is present in the reaction solution.