JPH0440899A - Biological production of alpha-hydroxy-4-methylthiobutyramide - Google Patents

Abstract

PURPOSE:To produce alpha-hydroxy-4-methylthiobutyramide industrially and advantageously under a mild condition of normal temperature and normal pressure by using a bacterium having hydrating activity of alpha-hydroxy-4- methylthiobutronitrile. CONSTITUTION:alpha-Hydroxy-4-methylthiobutyronitrile is converted into alpha- hydroxy-4-methylthiobutyramide by hydrating reaction by action of bacterium to produce alpha-hydroxy-4-methylthiobutyramide. A bacterium belonging to the genus Rhodococcus, Corynebacterium, Pseudomonas, Arthrobacter of Alcaligenes, capable of hydrating alpha-hydroxy-4-methylthiobutronitrile, is used as the bacterium. The reaction condition is 0.01-50 wt.% amount of the cells used and 0.1-70 wt.% alpha-hydroxy-4-methylthiobutronitrile concentration wherein a substrate may be not completely dissolved in the reaction medium. The reaction is carried out at 5-60 deg.C at pH 4-11 for 0.5-100 hours.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to a method for producing α-hydroquine-4-methylthiobutylamide using microorganisms. α-hydroxy-4-
Methylthiobutylamide can be converted to the corresponding acid by chemical and biological hydrolysis reactions, but the resulting U-
Hydroxy-4-methylthiobutyric acid is a feed additive that is added to feed for livestock, especially poultry, as a substitute for methionine to compensate for the lack of sulfur-containing amino acids.

[Conventional technology and problems]

Industrial methods for producing amides include hydrating acrylonitrile using reduced copper as a catalyst, as shown in the example of acrylamide, and hydrating the corresponding nitrile in the presence of a dilute alkali and ketones, as shown in the example of producing α-aminoamide. A method of hydrolyzing the compound (see JP-A-57-158743) is known.

On the other hand, microbiological methods for producing amides from nitriles have also been proposed, such as a method using microorganisms of the genus Corynebacterium or Nocardia
-17918 Publication], Reference - Method using a microorganism of the genus Domonas [see Japanese Patent Publication No. 59-37951], and method using a microorganism of the genus Rhodococcus [JP-A-61]
-162193, 62-91189, JP-A-2-
470], etc. However, these methods do not mention anything about the production of α-hydroxy-4-methylthiobutyramide from α-hydroxy-4-methylthiobutyronitrile. In addition, a method using microorganisms of the genus Bacillus, Hacteridium, Micrococcus or Brevibacterium [JP-A-51
No. 86186], α-hydroxy-T-methylthiobutyronitrile is mentioned as one of the substrates, but it is unclear what activity the microorganism exhibits toward this substrate.

[Means for solving problems]

Under these circumstances, the present inventors developed α-hydroxy-4-
As a result of our intensive search for microorganisms that have high hydration activity toward methylthiobutyronitrile, we found that microorganisms of the genus Rhodococcus, Corynebacterium, Pseudomonas, Arthrohactor, and alkaline earth metals have α-hydroxy-4- The present invention was completed based on the discovery that the present invention has the ability to hydrate methylthiobutyronitrile and produce α-hydroxy 4-methylthiobutyramide.

That is, the present invention provides α-hydroxy-4-methylthiobutyronitrile through a hydration reaction caused by the action of microorganisms.
In the method for producing α-hydroxy-4-methylthiobutylamide, which is converted into α-hydroxy-4-methylthiobutylamide, the microorganism is Rhodococcus
ccus), Corynebacterium (Coryneba
C ter i us), Pseudomonas (Pseudomonas)
domonas), Arthrobacter (Arthro)
bacter genus or Alcaligenes
α-hydroxy-4-
This is a biological production method of methylthiobutylamide.

The microorganism used in the present invention is specifically Rhodococcus rho
dochrous) ATCC33278, Rhodococcus erythropolis (Rhodococcus ery
throρO order 5) IFO12320, Corynebacterium nitrilophyllus
um n1trilophilus) ATCC214
19, Pseudomonas sp, 5K87 (Feikoken Bacteria No. 11311), Arthrohactor sp, HRI
(Feikoken Bibori No. 11301) and Alcaligenes S
ρ, BCl2-2 (Feikoken Bibori No. 11276)
Examples include. Moreover, these mutant strains can also be used. These microorganisms, Corynebacterium nitrilophilus ATCC 33278, Corynebacterium nitrilophilus ^TCC 21419, and Rhodococcus erythropolis rF012320, are known microorganisms, and each is a member of the American Type Collection (A
TCC) and the Institute for Fermentation Research (IFO). The other microorganisms were newly isolated from the natural world by the present inventors, and have been deposited at the Institute of Microbial Technology, Agency of Industrial Science and Technology under the above deposit numbers, and their mycological properties are as follows. That's right.

5K87 and) Il? Strain I BCl2-2 Strain N = Not tested The above mycological properties were determined from Bergey's Manual
of Systematic Bacteriology
y, 1986, 5K87 belongs to the genus Pseudomonas, HRI belongs to the genus Arthrobacter, and BCl2-2 belongs to the genus Alcaligenes.
s) were identified as bacteria belonging to the genus.

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

A nitrile compound such as isobutyronitrile, propionitrile, benzonitrile, etc., or an amide compound such as urea, isobutyramide, etc. suitable for each microorganism is added as an activity inducer to the microorganism culture medium used in the present invention, In addition, if necessary, activity promoting substances such as iron and cobalt ions may be coexisting. Carbon sources include normally assimilated glucose and glycerol, nitrogen sources include ammonium nitrate and ammonium sulfate, and inorganic nutrients include may be cultured using a medium containing magnesium chloride, ferric chloride, etc., and it is also possible to use these mediums to which natural medium such as yeast extract or meat extract is added.

Culture conditions are aerobic, pH 4-10, temperature 20-5.
The temperature may be selected within the range of 0°C, and the culture period may be from 1 to 10 days until the activity is maximized.

For the hydration reaction, collect bacterial cells cultured on a liquid medium or plate medium, prepare treated bacterial cells such as immobilized bacterial cells, crude enzyme, and immobilized enzyme if necessary, and use n-hexane. , α-hydroxy-4- dissolved in a suitable solvent such as ethyl acetate.
A two-phase system reaction of methylthiobutyronitrile with water, a buffer, etc., or a reaction by directly mixing α-hydroquine-4-methylthiobutyronitrile into a suspension of bacterial cells or a bacterial cell-treated product. I can do it.

The reaction conditions are a bacterial cell usage amount of 0.01 to 50% by weight, α
-Hydroxy-4-methylthiobutyronitrile concentration 0.
From 1 to 70% by weight, the substrate may not be completely dissolved in the reaction medium.The reaction temperature is from 5 to 60 °C, preferably from 10 to
The reaction pH at 40°C is 4 to 11, preferably 6 to 10, and the reaction is carried out for 0.5 to 100 hours.

Further, the consumed amount of α-hydroxy-4-methylthiobutyronitrile may be replenished continuously or intermittently.

Thus, α-hydroxy-4-methylthiobutyronitrile is converted and accumulated to the corresponding amide.

The product can be isolated using known methods such as concentration, extraction, crystallization, and distillation.

〔Effect of the invention〕

The present invention utilizes a microorganism having hydration activity for α-hydroxy-4-methylthiobutyronitrile.
α-Hydroquine-4-methylthiobutylamide can be produced under mild conditions of normal pressure.

α-Hydroxo-4-methylthiobutylamide is a compound useful as a raw material for the production of α-hydroxy-4-methylthiobutyric acid, which is useful as a feed additive, and the present invention provides an advantageous method for producing α-hydroxy-4-methylthiobutyric acid. It can be applied to methods.

[Experiment example]

EXAMPLES Next, the present invention will be explained in more detail with reference to Examples, but the present invention is not limited to these Examples.

Example 1 (1) Cultivation The bacterial cells shown in Table 1 were cultured under the following conditions.

) Medium C unit: What/V) Glycerol 2% Yeast extract 0.3% Potassium phosphate 0.68% Disodium phosphate 0.71% Sodium sulfate 0.28% Magnesium chloride 0.04% Calcium chloride 0 ．． OO4% manganese sulfate 4XlO-'% iron chloride
6X10-'% Zinc Sulfate 3X10-
'% agar 1.8% pH 7.5 In addition, isobutyronitrile (IBN) 0 as an inducer
．． 2%, α-chloropropionitrile (CPN) 0.0
5% or 0.02% Hensonitrile (BN) was added.

In addition to the above-mentioned inducer, the culture medium of Corynebacterium nitrilophilus also contains 10 μ/l of CoCl□・6H2.
0 was added as an activity promoter.

Ii) Culture Conditions One platinum loop of bacterial cells was taken from the slanted medium, spread on the above plate medium, and cultured under aerobic conditions at 30°C for 48 hours.

(2) Hydration reaction Cells were collected from the plate and centrifuged, and each cell was washed three times with 0.05 M phosphate buffer (pl+ 7.5). The precipitated bacterial cells were resuspended in 1.5 i of the same buffer solution, α-hydroxy 4-methylthiobutyronitrile was added at a final concentration of 1005 M, and the reaction was carried out with shaking at 25°C for 20 hours. After the reaction is complete, bacteria are removed from each reaction solution by centrifugation, and α-hydroxy-4-methylthiobutylamide in the centrifuged supernatant is determined by liquid chromatography.
(h Ram; 5HODEX 00SF511A, carrier: 0, 2 M H3PO4, 30°C1 monitor;
208 nm).

The results are shown in Table 1.

Table 1 (2) 200mM was added to the bacterial cell suspension prepared in the same manner as in Hydration Reaction Example 1.
α-Hydroxy-4-methylthiobutyronitrile was added thereto and reacted at 25°C for 20 hours with shaking. Next, 200 mM of substrate was added again and the reaction was continued for an additional 20 hours. The bacterial cells were removed by centrifugation, and α-hydroxy-4-methylthiobutylamide produced in the reaction solution was quantified by the method shown in Example 1, and the results are shown in Table 2.

Table 2 Example 2 (1) Culture Rhodococcus rhodochrous ATCC33278 strain was cultured in the same manner as in Example 1.

Claims (1)

[Claims] The hydration reaction of α-hydroxy-4-methylthiobutyronitrile by the action of microorganisms
α-Hydroxy-4 converted to methylthiobutylamide
- In the method for producing methylthiobutylamide, the microorganism belongs to the genus Rhodococcus, the genus Corynebacterium, the genus Pseudomonas, the genus Arthrobacter, or the genus Alcaligenes, A method for biologically producing α-hydroxy-4-methylthiobutylamide, characterized in that it has the ability to hydrate the nitrile.