JPS58209987A - Microbial preparation of aromatic carboxylic acid amide - Google Patents

Abstract

PURPOSE:To prepare an aromatic carboxylic acid amide from an aromatic nitrile, in high efficiency, by the action of microorganism belonging to Corynebacterium genus or Nocardia genus. CONSTITUTION:A microbial strain belonging to Corynebacterium genus or Nocardia genus and capable of hydrolyzing an aromatic nitrile, e.g. Corynebacterium N-771 strain (FERM-P No.4445), Nocardia N-775 strain (FERM-P No.4447), etc. is made to react with an aromatic nitrile such as benzonitrile, nicotinonitrile, etc. at 6-10pH and 0-40 deg.C.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing aromatic carboxylic acid amide from aromatic nitrile by the action of microorganisms. For more information, see Corynebacterium spp.
The present invention relates to a method for producing an aromatic carboxylic acid amide from an aromatic nitrile by the action of a microorganism belonging to the genus Corynebacterium or Genus Nocardia and having the ability to hydrate an aromatic nitrile.

Aromatic carboxylic acid amides are useful compounds as raw materials for fine chemicals such as pharmaceuticals and agricultural chemicals.

In particular, pyridine-3-carboxylic acid amide and cotinic acid amide) are components of the vitamin B complex, and are important compounds used in pharmaceuticals, food additives, feed additives, and the like.

There are a number of known methods for producing aromatic carboxylic acid amides, including the old method using aromatic carboxylic acids as raw materials and the more recent method of catalytically hydrating aromatic nitriles using an alkaline earth metal catalyst. However, it has problems such as the preparation of the catalyst, the separation and purification of the produced amide due to the production of by-products, and the like.

In order to solve these problems, the two inventors.

We focused on a method using microorganisms as a method for producing aromatic carboxylic acid amides from aromatic nitrile under mild conditions and with high selectivity.

An attempt to obtain aromatic carboxylic acid amide from aromatic nitrile using microorganisms has been made in JP-A-51-86186, but this method is still unsatisfactory as an industrial production method due to the large amount of microorganisms used relative to the substrate. It's not something I like.

Under these circumstances, the present inventors searched for a highly productive and highly active bacterial strain targeting a wide range of microorganisms, and as a result, a new strain of the genus Corynebacterium (Qenus Coryneba) was discovered.
cterium) or Nocardia (QenusN
The present invention was achieved by discovering that microorganisms belonging to the genus A. ocardia efficiently produce aromatic carboxylic acid amides from aromatic nitriles.

That is, 2 the present invention is directed to Corynebacterium spp.
An aromatic carboxylic acid produced by a microorganism characterized in that it produces an aromatic carboxylic acid amide from a nitrile by the action of a microorganism belonging to the genus Corynebacterium or Genus Nocardia and having the ability to hydrolyze an aromatic nitrile. This is a method for producing amide.

The aromatic nitriles used in the present invention include:

For example, benzene-based nitriles such as benzonitrile and cotinonitrile in 3-cyanopyridine).

A pyridine-based nitrile such as 4-cyanopyridine,
These may be selected from any source, but
Industrially, it is advantageous to produce them by ammoxidation of the corresponding aromatic methyl substituted product.

In addition, one microorganism used in the present invention is the genus Corynebacterium or the genus Nocardia.
It is a bacterium or actinomycete that belongs to 2 and has the ability to produce aromatic carboxylic acid amide from aromatic nitrile.
for example.

Corynebacterium genus N-771 strain (Cory
nebacterium SP ・N-771) C
Microtechnical Research Institute No. 4445], Corynebacterium spp. N
-774 strain (Corynebacterium, S
P aN-774) (Feikoken Bacterial Serial No. 4446) and Nocardia sp. N-775 strain (Nocardia
S P ・N-775) (Fiber Engineering Laboratory No.4447
No.] etc. can be cited as suitable examples. The mycological properties of these strains are disclosed in the above-mentioned Japanese Patent Publication No. 17918/1983. usually.

Although one type of these bacterial strains is used, a mixture of two or more types of bacterial cells may be used, and furthermore, a strain other than the above-mentioned bacterial strains having a similar effect may be used in combination.

Next, two general embodiments of the present invention will be described.

(1) Reaction method The production of aromatic carboxylic acid amide in the present invention is specifically carried out using, for example, the above-mentioned microorganisms that have been cultured in large quantities in advance in a suitable medium, 9 isolated bacterial cells, or these bacterial cells or isolated and extracted from these bacterial cells. Immobilization obtained by immobilizing enzymes, etc. by various methods such as carrier binding method, crosslinking method, entrapment method, etc. - A single bacterial cell or immobilized enzyme, etc. is immobilized with aromatic nitrile in water, physiological saline, or other aqueous medium. This can be carried out by producing and accumulating an aromatic carboxylic acid amide through contact, and then isolating and collecting this.

In addition, methods for culturing microorganisms in the presence of nitrile,
Alternatively, the amide can also be produced by adding nitrile to the culture solution after culturing the microorganism.

(2) Preparation of bacterial cells The bacterial cells used in the present invention are prepared by culturing them in advance in large quantities in an appropriate medium, collecting them, and washing them.

The medium used at this time may be either synthetic or natural, as long as it contains adequate amounts of carbon sources, nitrogen sources, inorganic salts, and other growth-promoting substances. Carbon sources include glucose and maltose, and nitrogen sources include ammonium sulfate and ammonium chloride.
Yeast extract as an organic nutritional source.

Meat extract, malt extract, peptone, etc. and phosphates, magnesium, potassium as inorganic nutritional sources.

Zinc, iron, manganese, etc. are commonly used.

These media have a pH of 6 to 9. After preparing it, it is sterilized by heating etc. Next, the bacteria are inoculated and cultured with aeration for 1 to 5 days at a temperature of 20 to 35C, preferably 25 to 30C.
Culture under aerobic conditions such as shaking culture. After the culture is completed, the bacterial cells are separated by centrifugation and then mixed with water or PH.
Wash with buffer solution 6 to 9.

Immobilization of bacterial cells can be carried out according to conventional methods using polyacrylamide gel entrapment method, etc., if necessary.

(3) Reaction conditions The reaction medium was water containing aromatic nitrile as a raw material;
Although an aqueous medium such as physiological saline or a buffer solution can be used, water is preferred in view of separation of the product after one reaction. The concentration of aromatic nitriles in the reaction medium is 0.1-20w
t%, preferably 0.5 to 10 wt%, and there is no problem even if it is not completely dissolved in one reaction medium. Further, a surfactant may be used in combination as a solubilizing agent. The concentration of bacterial cells used in the reaction is usually in the range of 0.05 to 10 wt%. The pH of the reaction system is 6-10. Preferably between 7 and 9.

The reaction temperature is 0 to 40C2, preferably 5 to 301R. It is preferable to adjust the concentrations of substrate and bacterial cells so that the reaction usually completes in 10 minutes to 8 hours. Excessive reaction results in the by-product of aromatic carboxylic acid and must be avoided.

(4) Recovery To separate and recover the aromatic carboxylic acid amide from the reaction mixture, 1. For example, after separating (immobilized) bacterial cells from the reaction mixture by filtration or centrifugation, 1.
It may be distilled off under reduced pressure at a temperature of 0C or lower and then dried to absolute dryness, but conventional separation methods such as extraction with an organic solvent such as ethyl acetate may also be used.

According to the method of the present invention, since side reactions are almost prevented, highly pure aromatic carboxylic acid amide can be obtained.

Higher quality products can be obtained by purification using conventional purification methods such as conventional exchange resin treatment or recrystallization.

Typical examples of the nine methods of the present invention are shown below.

The present invention will be explained in more detail, but the present invention is not limited to these examples in any way.

Example 1゜(1) Production of bacterial cells Glucose 101μ, peptone 5μ, yeast extract 3? μ
, and malt extract 3? Contains μ.

A 100-liter medium adjusted to pH 7.2 was dispensed into a 500 tl Erlenmeyer flask and sterilized under pressure at 120°C for 15 minutes.

One platinum loop of Corynebacterium N-774 strain was inoculated into this medium and cultured with shaking at 301Z' for 3 days to produce total bacterial cells. The cells were separated from the culture solution by centrifugation and washed twice with phosphate buffer of pH 7.2. The water content of the washed bacterial cells thus obtained was about 80%.

(Il1 reaction 4.07 of the above-mentioned washed bacterial cells were mixed with 100 ml of an aqueous solution containing 5 ml of 3-cyano-pyridine and adjusted to pH 8.5K with ammonia.
The mixture was added to P and reacted with stirring for 1 hour in a 30c water tank.

After the completion of the reaction, a part of the reaction solution was analyzed by high performance liquid chromatography, and it was found that it contained 5.86% (conversion rate 100%) of nicotinic acid amide, unreacted 3-cyanopyridine, and nicotinic acid as a side reaction product. was not recognized.

Bacterial cells were removed from this reaction solution by centrifugation in an OC, water was then distilled off at less than 501 T, and further vacuum drying yielded 5.87 white crystals, which were identified as nicotinic acid amide from the IR spectrum etc. It was done. The melting point of this nicotinic acid amide is 127-130 C, and the yield is 1.
It was 00%.

Example 2 (1) Production of bacterial cells Bacterial cells were produced in exactly the same manner as in Example 1 (■) except that Nocardia F%N-775 strain was used. The water content of the washed bacterial cells was about 85%.

(U) Reaction above washed bacterial cells IF! - P containing 1% benzonitrile
Add H8,5(1) aqueous solution 100FI to 1 at 3oC.
The reaction was stirred for 1 hour.

After the reaction is complete, a portion of the reaction solution is analyzed using gas chromatography.
Analysis revealed that it contained 1.17% (conversion rate 100%) of benzamide, and that unreacted benzonitrile and benzoic acid as a side reaction product were not observed.

After centrifugation to remove the bacterial cells from this reaction solution in OC, extraction with heated benzene iooy- resulted in 1.
White crystals of 02y- were obtained.

It was identified as benzamide based on the IR spectrum etc. The melting point of this benzamide is 126-1281:.

The yield was 86.8%.

Examples 3 to 4 Washed bacterial cells 1i prepared in the same manner as in Example 1 were mixed with PL (8
, 5 and reacted for 1 hour at 30C with stirring.

After the reaction was completed, a portion of the two reaction solutions was analyzed by high performance liquid chromatography, and the results shown in the following table were obtained.

Patent applicant: Nitto Chemical Industry Co., Ltd. Representative Masahiko Nanami

Claims (1)

[Claims] Genus coryneba
cterium) or Genus
Nocardia). 1. A method for producing an aromatic carboxylic acid amide using a microorganism, which comprises producing a corresponding aromatic carboxylic acid amide from a nitrile by the action of a microorganism having the ability to hydrate the aromatic nitrile.