JPH05219972A - Biological production method of α-hydroxyisobutyric acid amide - Google Patents

Abstract

PURPOSE:To efficiently convert alpha-hydroxyisobutylonitrile into the subject compound by a microorganism and simultaneously enable high concentration accumulation of the subject compound into a reaction liquid. CONSTITUTION:alpha-Hydroxyisobutylonitrile is hydrated in the presence of acetone using a microorganism [e.g. Rhodococcus rhodochrous J-1 (FERM P-1478) to provide the objective compound. The concentration of acetone is normally 0.5-50wt.% concentration of alpha-hydroxyisobutylonitrile is preferably 0.1-10wt.%.. The microorganism is preferably used in an amount of 0.01-5wt.%, expressed in terms of dried bacterial cell. Furthermore, the reaction is preferably carried out at pH 6-8 and 2-30 deg.C.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a biological process for producing α-hydroxyisobutyric acid amide. α-Hydroxyisobutyric acid amide is not only a compound useful as a raw material for organic synthesis, but can also be converted to methyl methacrylate, which is important as a raw material monomer for methacrylic resin, by dehydration after methyl esterification. It is a useful compound.

[0002]

2. Description of the Related Art As a method for producing α-hydroxyisobutyric acid amide by microbiologically hydrating α-hydroxyisobutyronitrile, it is possible to utilize a microbial cell or an enzyme. (See Japanese Patent Application Laid-Open No. 60-78939), and there is no specific example of the microorganism, and its hydration activity is unknown.

[0003]

The present inventors have found that in a method of producing α-hydroxyisobutyric acid amide from α-hydroxyisobutyronitrile in a microbial manner, an enzymatic activity for hydrating a nitrile to an amide, so-called When the reaction using various microorganisms having nitrile hydratase activity was examined, reaction inhibition and enzyme deactivation appeared under normal reaction conditions, and the reaction did not proceed smoothly. Therefore, in order to solve this problem, the present invention has been completed by discovering that reaction conditions have been earnestly studied, and as a result, addition of acetone to the reaction system has greatly improved both the reaction rate and the storage property.

That is, the present invention is a method for microbially hydrating α-hydroxyisobutyronitrile to convert it into α-hydroxyisobutyric acid amide, characterized by allowing acetone to coexist in the reaction system. A biological process for the production of hydroxyisobutyric acid amide.

It is not clear what kind of mechanism of action of acetone the effect of the present invention is, but α-hydroxyisobutyronitrile ← → acetone + in an aqueous medium.
The dissociation equilibrium of hydrocyanic acid leans toward α-hydroxyisobutyronitrile due to the addition of acetone and the concentration of hydrocyanic acid in the reaction system decreases, or a trace amount of liberated hydrocyanic acid is rapidly supplemented by acetone in excess. It is considered that this is because the decrease in the activity of the hydrating enzyme caused by hydrocyanic acid was alleviated. However, the present invention is not limited to such an action mechanism. In addition, since acetone was effective against various nitrile hydratase-active bacteria, this action effect was not a phenomenon observed only with limited nitrile hydratase, but was sensitive to hydrocyanic acid of various bacteria. It seems to be commonly found in various nitrile hydratase.

The microorganism used in the present invention may be any microorganism as long as it has the ability to hydrate α-hydroxyisobutyronitrile and convert it to α-hydroxyisobutyric acid amide.
For example, bacteria of the genus Pseudomonas and the genus Rhodococcus can be mentioned. In particular,
Rhodococcus rhodochr-ou
s) J-1 [Ministry of Fine Arts, Article 1478], Pseudomonas
Chlorophyll (Pseu-domonas chlororaphis) B23 (Microtechnical Research Institute No. 187) can be mentioned, and their mycological properties are described in JP-A-2-470 and JP-B-59-37951, respectively. There is.

Next, a general embodiment of the present invention will be described. For culturing the microorganism used in the present invention, usually,
A medium containing an assimilable carbon source, a nitrogen source, and inorganic nutrients necessary for the growth of microorganisms is used. For example, glucose, glycerol, sucrose are used as carbon sources, yeast extract, taste liquid, peptone, sodium glutamate, ammonium sulfate as nitrogen sources, and monopotassium hydrogen phosphate, dipotassium hydrogen phosphate, chloride as inorganic nutrient sources. Examples include magnesium, ferric chloride, ferrous sulfate, and magnesium sulfate. Further, in the early or middle stage of the culture, nitriles (isobutyronitrile, propionitrile, etc.) at concentrations that do not significantly inhibit growth, amides corresponding to these nitriles, lactams (ε-caprolactam, γ-butyrolactam, etc.) It is preferable to add, for example, higher enzyme activity. Cultivation may be carried out under aerobic conditions at a pH of 4 to 10, a temperature of 20 to 40 ° C., and a culturing time of about 1 to 4 days while controlling to a range suitable for each microorganism.

In the hydration reaction, the above culture solution, cells separated from the culture solution, or a treated product of the cells (crushed cell bodies, extracted enzyme, immobilized cells, enzyme, etc.) is treated with acetone in an aqueous medium. It is carried out by contacting with α-hydroxyisobutyronitrile in the coexistence. The acetone concentration varies depending on the type of microorganism used, the concentration of α-hydroxyisobutyronitrile as a substrate, the reaction temperature, and the pH, but it is usually 0.5 to 50.
The optimum concentration may be selected within the range of weight%. The concentration of the substrate α-hydroxyisobutyronitrile is 0.1-10
% By weight, the amount of microorganisms used is 0.01-5% by weight in terms of dry cells
And the reaction is pH 4-10, preferably 6-8, temperature
It is carried out at a freezing point to 50 ° C, preferably 2 to 30 ° C. The reaction may be carried out while sequentially adding α-hydroxyisobutyronitrile, which decreases with the progress of the reaction, to the reaction solution.

[0009]

INDUSTRIAL APPLICABILITY According to the present invention, α-hydroxyisobutyronitrile can be efficiently converted into α-hydroxyisobutyroamide over a long period of time, and a high concentration of the product can be accumulated in the reaction solution. is there.

Isolation of α-hydroxyisobutyric acid amide from the reaction mixture is carried out by removing the bacterial cells by centrifugation or the like and then concentrating,
It can be carried out by utilizing a known method such as extraction or crystallization. Hereinafter, the present invention will be described more specifically with reference to Examples, but the present invention is not limited to the Examples.
Unless otherwise specified,% in the examples means% by weight.

[0011]

【Example】

Example 1 (1) Culturing A 500 ml Erlenmeyer flask containing 100 ml of the following medium was inoculated with 1 ml of Pseudomonas chlororaffis B23 strain previously cultured in the same medium, and shake culture was carried out at 25 ° C. for 4 days.

[0012]

(2) Hydration reaction The composition of the reaction solution is as follows: acetone concentration 0 to 17.4%, α-hydroxyisobutyronitrile concentration 2%, cell concentration OD ₆₃₀ (63
(Absorbance at 0 nm) = 2, pH7, 5
The reaction was performed at 4 ° C for 4 hours.

(3) Analysis The amount of α-hydroxyisobutyric acid amide formed was determined by high-performance liquid chromatography [Shodex ODS F-511A column] after the reaction was completed by centrifuging the reaction solution to remove bacterial cells. ], The eluent 0.2M phosphate buffer (pH 2) was used.
The absorbance was detected at 08 nm to quantify α-hydroxyisobutyric acid amide. The results are shown in Table-1.

[0015]

[Table 1]

Example 2 (1) Culture In a 500 ml Erlenmeyer flask containing 100 ml of the following medium, 5 ml of Rhodococcus rhodochrous J-1 strain previously cultured in the same medium was used.
After inoculation, shake culture was performed at 30 ° C. for 4 days.

Medium composition Glucose 1.5% Sodium glutamate 0.75% Monopotassium hydrogen phosphate 0.05% Dipotassium hydrogen phosphate 0.05% Magnesium sulfate heptahydrate 0.05% Yeast extract 0.1% ε-caprolactam 0.5%

(2) Hydration reaction The composition of the reaction solution is as follows: acetone concentration 0 to 34.8%, α-hydroxyisobutyronitrile concentration 2%, cell concentration OD ₆₃₀ = 16.
The reaction solution was prepared at pH 6 and reacted at 30 ° C. for 20 hours.

(3) Analysis The amount of α-hydroxyisobutyric acid amide produced was quantified in the same manner as in the examples. The results are shown in Table-2.

[0020]

[Table 2]

Example 3 (1) Culture The same as in Example 1.

(2) Hydration reaction The composition of the reaction solution was such that the concentration of acetone was 5.8%, the concentration of α-hydroxyisobutyronitrile was 2%, and the cell concentration was OD ₆₃₀ = 10.
It was prepared so that α-hydroxyisobutyronitrile was continuously added dropwise at a rate of 2% every 30 minutes, and the pH was adjusted to 7
Then, the reaction was carried out at 5 ° C. for 6 hours, and the analysis was carried out in the same manner as in Example 1. As a result, it was confirmed that the reaction was carried out almost quantitatively and 30.4% of α-hydroxyisobutyric acid amide was produced. ..

Claims (3)

[Claims] 1. A method for converting α-hydroxyisobutyronitrile into α-hydroxyisobutyric acid amide by microbiologically hydrating α-hydroxyisobutyronitrile, wherein acetone is made to coexist in the reaction system. Biological manufacturing method. 2. The microorganism is Pseudomonas.
The genus or Rhodococcus genus.
A method for biologically producing the described α-hydroxyisobutyric acid amide. 3. The concentration of acetone coexisting in the reaction system is
The method for producing α-hydroxyisobutyric acid amide according to claim 1, which is 0.5 to 50% by weight.