JP2926350B2 - Microbial production of nicotinic acid - Google Patents

Description

The present invention relates to a method for producing nicotinic acid by a microorganism.
More specifically, the present invention relates to a method for producing nicotinic acid by a microorganism, which comprises subjecting 3-cyanopyridine to a hydrolysis reaction caused by the action of a microorganism belonging to a specific genus, and separating and obtaining the produced nicotinic acid. Nicotinic acid is known to function as a coenzyme in vivo, and nicotinic acid derivatives are used in pharmaceuticals and the like.

[Conventional technology and problems]

At present, nicotinic acid is produced by a method via aldehyde collidine obtained by a Reppe reaction between acetaldehyde and ammonia (see Ger. Offen. 2,046,556) or 3-cyanopyridine obtained by ammoxidation of β-picoline. (Ger.)
pat.828,246).

However, these methods have problems until the production of by-products and purification steps become complicated, and furthermore, development of a new and industrially advantageous production method has been desired.

On the other hand, as a method for converting 3-cyanopyridine to nicotinic acid using a microorganism having an activity of hydrolyzing nitrile, Rhodococcus rhodochros species (Appl. Environ. M.
ic-robiol. 54 1030-1032 (1988)), Nocardia rhodochrous species (J. Gen. Microbiol. 134 1099-1108).
(1988)).
The production method using these microorganisms is considered to be a more industrially advantageous production method because it can react under mild conditions and has less reaction by-products than the chemical production method. However, these microorganisms have only been subjected to basic experiments, and have not been studied for industrialization.

[Means for solving the problem]

The present inventors have intensively searched for a more effective new microorganism for the purpose of industrial production, and as a result, the genus Agrobacterium, Acinetoba (Acinetoba)
cter) genus, Microbacterium genus,
Cellulomonas, Cytoferga (Cy)
tophaga), Obesumbacteriu
m) A microorganism belonging to the genus or the genus Streptomyces having high 3-cyanopyridine hydrolysis activity was found, and the present invention was completed.

That is, the present invention relates to a method for producing nicotinic acid, which converts 3-cyanopyridine to nicotinic acid by a hydrolysis reaction caused by the action of a microorganism, wherein the microorganism is a genus of Agrobacterium, Acinetobacter (A)
genus cinetobacter, Microbacteri
um), Cellulomonas, Cytophaga, Obesumu
bacterium) or Streptomyce (Streptomyce)
s) A method for producing nicotinic acid by using a microorganism belonging to the genus and having the ability to hydrolyze the nitrile.

Examples of the microorganism used in the present invention include, for example, Agrobacterium tumefa
ciens) IAM 1037, Agrobacterium rhizogenes IFO 14554, Acinetobacter calcoaceti (Acinetobacter calcoaceti)
cum) IFO 12552, Microbacterium lacticum (Mi
crobacterium lacticum) IAM 1640, Cellulomonas fimi IAM 12107, cytofaga
sp. (Cytophaga sp.) ATCC 29474, Ovesumbacterium proteus ATCC 12
841, Streptomyces griseus
griseus) IFO 3355 and the like.

As a medium for culturing these microorganisms, a medium in which an amide or a nitrile compound is added to a medium in which these microorganisms can grow is usually used. For example, glucose, sucrose, glycerol, etc. as a carbon source, peptone, meat extract, yeast extract, amino acids, various inorganic, organic acid ammonium salts, etc., other inorganic salts, trace metal salts, vitamins, etc. are required as a nitrogen source. A medium obtained by adding an amide or a nitrile compound to a medium appropriately added according to the necessity is used.

The amide and nitrile compound to be added include aliphatic such as isobutyronitrile, isobutylamide, propionitrile, propionamide, methacrylonitrile and methacrylamide, aromatic such as benzonitrile and benzamide, 3-cyanopyridine and nicotinic acid. Examples include heterocyclic compounds such as amides.

The cultivation is performed at pH 6-10, preferably 7-9, at a temperature of 15
It is carried out aerobically at ~ 37 ° C, preferably at 25-30 ° C for 1-7 days.

In the present invention, as a method for producing nicotinic acid from 3-cyanopyridine, for example, a culture solution of a microorganism obtained by culturing as described above or a suspension of cells obtained by centrifugation or the like can be used. A method of adding a cyanopyridine, a method of adding a substrate to a suspension of a treated cell (eg, a crushed cell, a crude enzyme, a purified enzyme, etc.) or a cell or a treated cell immobilized by a conventional method. In addition, there is a method in which a substrate is added to a culture solution at the time of culturing a microorganism and a reaction is performed simultaneously with the culturing.

Although the substrate concentration in the reaction solution is not particularly limited,
The range is preferably from 1 mM to 1 M, and the substrate can be added to the reaction solution at once or in portions. The reaction is preferably performed at a reaction temperature of 5 to 50 ° C and a pH of 4 to 10.
The reaction time varies depending on the concentration of the substrate, the concentration of the bacterial cells, and other reaction conditions, but is usually completed in 10 minutes to 48 hours.

The recovery of nicotinic acid from the reaction solution can be performed by a conventional method. Since most of the accumulated nicotinic acid is an ammonium salt, for example, cells are removed from the reaction solution by centrifugation or the like, ammonia is removed by acid treatment or the like, and then recovered and purified by extraction, recrystallization, or the like. be able to.

〔Example〕

Next, the present invention will be described with reference to examples, but the present invention is not limited to these examples. Nicotinic acid was analyzed by high performance liquid chromatography (Showa Denko Shode).
x F-411A column).

Example 1 Polypeptone 0.5%, yeast extract 0.2%, glucose 0.
5%, K ₂ HPO ₄ 0.05%, KH ₂ PO ₄ 0.05%, MgSO ₄・ 7H ₂ O 0.05
%, 0.042 mM CoCl ₂ , 0.042 mM FeSO ₄ , 0.042 mM NiCl ₂ ,
_{0.042mM MnCl 2, 0.042mM CuSO 4,} 0.042mM ZnSO 4, 0.042
A washed strain prepared by shaking each strain shown in Table 1 at 30 ° C. for 48 hours in a medium containing mM Na ₂ Na ₂ MoO ₄ , 0.2% propionitrile, and 0.2% propionamide was treated with 10 mM 3-cyano. The suspension was suspended in 50 mM potassium phosphate dry solution (pH 7.2) in 0.5 ml containing pyridine and reacted at 25 ° C. for 26 hours. After completion of the reaction, the cells were removed by centrifugation and the supernatant was examined. As shown in Table 1, production of nicotinic acid was observed.

Example 2 Polypeptone 0.5%, yeast extract 0.2%, glucose 0.
5%, K ₂ HPO ₄ 0.05%, KH ₂ PO ₄ 0.05%, MgSO ₄・ 7H ₂ O 0.0
5%, 0.042 mM CoCl ₂ , 0.042 mM FeSO ₄ , 0.042 mM NiCl ₂ ,
_{0.042mM MnCl 2, 0.042mM CuSO 4,} 0.042mM ZnSO 4, 0.042
Each of the strains shown in Table 2 was incubated at 30 ° C. for 48 hours in a medium containing mM Na ₂ MoO ₄ , 0.2% isobutyronitrile and 0.2% isobutyramide.
The washed cells prepared by shaking culture for 10 hours are mixed with 0.5 ml of 50 mM potassium phosphate buffer solution (pH: 10 mM) containing 10 mM 3-cyanopyridine.
The suspension was reacted at 25 ° C for 26 hours. After the reaction was completed, the cells were removed by centrifugation and the supernatant was examined.
As shown in Table 2, generation of nicotinic acid was observed.

Claims (1)

(57) [Claims] 1. A method for producing nicotinic acid, which converts 3-cyanopyridine to nicotinic acid by a hydrolysis reaction under the action of a microorganism, wherein the microorganism is a genus of Agrobacterium or Acinetobact.
er), Microbacterium, Cellulomonas, Cytofaga (Cyto)
phaga), Obesumbacterium
A method for producing nicotinic acid using a microorganism, which comprises using a microorganism belonging to the genus or Streptomyces and having the ability to hydrolyze the nitrile.