JPH04365491A - Production of 4-halo-3-hydroxybutylamide - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention]

0001

FIELD OF THE INVENTION The present invention relates to a method for producing 4-halo-3-hydroxybutyramide. Since 4-halo-3-hydroxybutyramide has two different functional groups, it is useful as a synthetic raw material for various physiologically active substances such as carnitine and 2,3-dihydroxybutanoic acid, pharmaceuticals, and agricultural chemicals. It is.

0002

[Prior Art and Problems] Generally, as a method for producing amides by hydration of nitriles, a method using a mineral acid such as hydrochloric acid or an alkali is known. However, these methods have problems such as complicated steps and a decrease in yield due to the production of the corresponding acid from the nitrile. There is also no example of the production of 4-halo-3-hydroxybutyramide. In addition, various biological methods are known for producing the corresponding amides by hydrating nitriles through the action of nitrile hydratase. Until now, there has been no knowledge of the existence of an enzyme that produces halo-3-hydroxybutyramide.

0003

SUMMARY OF THE INVENTION The present inventors have conducted intensive studies on a method for producing 4-halo-3-hydroxybutyramide from 4-halo-3-hydroxybutyronitrile. Acts on lonitrile4
A microorganism having a nitrile hydratase that produces -halo-3-hydroxybutyramide was discovered, and the present invention was completed.

That is, the present invention aims at converting 4-halo-3-hydroxybutyronitrile into 4-halo-3-hydroxybutyramide through the action of nitrile hydratase having the activity of converting 4-halo-3-hydroxybutyronitrile into 4-halo-3-hydroxybutyramide. This is a method for producing 4-halo-3-hydroxybutyramide, which is characterized by producing 4-halo-3-hydroxybutyramide from lonitrile.

[0005]

DETAILED DESCRIPTION OF THE INVENTION Nitrile hydratase as used in the present invention is an enzyme that catalyzes the reaction of adding water to the nitrile bond of 4-halo-3-hydroxybutyronitrile, and according to the international enzyme classification, It is an enzyme belonging to lyase.

[0006] The microorganism having nitrile hydratase used in the present invention is, for example, Arthlobacter (Arth).
(robacter), Brevibacterium (Brev)
ibacterium), Caseobacter (Caseobacter)
genus Corynebacterium (Cory
genus nebacterium, Pseudomonas (Pse
udomonas) or Rhodococcus (Rhod
It is a microorganism belonging to the genus Arthrobacter globiformis.
robacter globi-formis) IFO
12138, Brevibacterium herborum (B
revibacterium helvolum) AT
CC 11822, Corynebacterium flaves
s-cens) IAM 1642, Rhodococcus
Erythropolis (Rhodococcus eryth)
ropolis) IFO 12540 and IFO
12539 etc. These strains are each included in the American Type Culture Collection (AT
CC), the Institute for Fermentation Research (IFO), and the Institute of Applied Microbiology (IAM), the University of Tokyo.

[0007] Furthermore, Rhodococcus rhodochrous J-1 (see Japanese Patent Application Laid-open No. 2-470) [Feikoken Article No. 1478], which Yamada et al. isolated from soil, and Earth, which the present inventors newly isolated from soil, Lobacter s
p. SK103 [Feikoken Bacteria No. 11300], Caseobacter sp. BC23 [Microtechnology Research Institute No. 112
No. 61], Pseudomonas sp. BC15-2
[Feikoken Article No. 3320], Pseudomonas sp.
SK31 [Feikoken Bacteria No. 11310], Pseudomonas sp. SK87 [Microtechnology Research Institute No. 1131
No. 1], Pseudomonas sp. SK13 [Feikoken Article No. 3325], Rhodococcus sp. SK70
[Feikoken Bacteria No. 11304], Rhodococcus s
p. HR11 [Feikoken Bacteria No. 11306] and Rhodococcus sp. SK49
No. 303]. These bacteria have been deposited at the Institute of Microbial Technology (Feikoken) of the Agency of Industrial Science and Technology under the above deposit numbers, and their mycological properties are as follows.

(1) SK103 strain morphology
Pleomorphic bacillus gram staining
+ spores

-Motility
-oxidase
-catalase
+ro
d-coccus cycle
+ Elongation of peripheral cells of the colony
Growing under anaerobic conditions without permission
- Cell wall diamino acids
Lysine glucholyl test - (acetyl type) sugar composition of cell wall arabinose
-galactose
-quinone type
MK-9 (H2)

000
9] (2) BC23 strain morphology
Pleomorphic bacillus gram staining
+ spores

-Motility
-oxidase
-catalase
+ro
d-coccus cycle
+ Elongation of surrounding cells
Growing under anaerobic conditions without permission
- Cell wall diamino acid meso-diaminopimelic acid glucoryl test -(
acetyl type) cell wall sugar composition arabinose
+galactose
+quinone type
MK-8 (H2)

001
0] (3) BC15-2 strain morphology
bacillus gram staining
−Spores
−
Motility
+ flagellum
polar hair oxidase
+ catalase
+OF
O

(4)
SK31 strain form
bacillus gram staining
−Spores
−
Motility
+ flagellum
polar hair oxidase
+ catalase
+OF
O

(5)
SK87 strain form
bacillus gram staining
−Spores
−
Motility
+ flagellum
polar hair oxidase
+ catalase
+OF
O

(6)
SK13 strain form
bacillus gram staining
−Spores
−
Motility
+ flagellum
polar hair oxidase
+ catalase
+OF
O

(7)
SK70 strain form
Pleomorphic bacillus gram staining
+ spores

-Motility
-oxidase
-catalase
+ro
d-coccus cycle
- Elongation of peripheral cells of the colony
Growing under anaerobic conditions without permission
-Diamino acid in cell wall meso-glycolyl diaminopimelate test + (glucoryl type) sugar composition in cell wall arabinose
+galactose
+quinone type
MK-8 (H2)

001
5] (8) HR11 strain morphology
polymorphic bacillus gram staining
+ spores

-Motility
-oxidase
-catalase
+ro
d-coccus cycle
- Elongation of peripheral cells of the colony
Growing under anaerobic conditions without permission
- Cell wall diamino acids
Meso-glycolyl diaminopimelate test + (glycolyl type) Sugar composition of cell wall Arabinose
+galactose
+quinone type
MK-8 (H2)

001
6] (9) SK49 strain morphology
Pleomorphic bacillus gram staining
+ spores

-Motility
-oxidase
-catalase
+ro
d-coccus cycle
- Elongation of peripheral cells of the colony
Growing under anaerobic conditions without permission
- Cell wall diamino acid meso-diaminopimelic acid glucoryl test + (glucoryl type) cell wall sugar composition arabinose
+galactose
+quinone type
MK-8 (H2)

001
7] The above mycological properties are summarized in Bergey's Manual of Systematic Bacteriology (Bergey's Manual of Systematic Bacteriology).
's manual of systematic b
acteriology) Vol. 2 (1986)
When searching according to
-2, SK31, SK87 and SK13 strains were identified as bacteria belonging to the genus Pseudomonas, and strains SK70, HR11 and SK49 were identified as bacteria belonging to the genus Rhodococcus.

[0018] As the medium composition for culturing the microorganisms producing the nitrile hydratase used in the present invention, any medium can be used as long as these microorganisms can grow. For example, sugars such as glucose, fructose, sucrose, and maltose as carbon sources,
Organic acids such as acetic acid and citric acid, alcohols such as ethanol and glycerol, peptone,
In addition to natural nitrogen sources such as meat extracts, yeast extracts, protein hydrolysates, and amino acids, various inorganic and organic acid ammonium salts can be used.In addition, inorganic salts, trace metals,
Vitamins and the like are used as needed. On this occasion,
4-chloro-3- to induce higher enzyme activity
Various nitrile compounds such as hydroxybrylonitrile, propionitrile, isobutyronitrile, and benzyl cyanide, and various amide compounds such as 4-chloro-3-hydroxybutyramide, propionamide, and isobutyramide can also be added to the medium. Extremely useful. The above-mentioned microorganisms may be cultured by a conventional method, for example, at pH 4-
10, aerobically at a temperature of 10 to 45°C 10 to 18
Incubate for 0 hours. Cultivation can be carried out either in liquid culture or solid culture.

4-Halo-3-hydroxybutyramide can be obtained by reacting 4-halo-3-hydroxybutyronitrile with an enzyme, using the microbial culture solution obtained as described above or centrifugation. A method of adding a substrate to the suspension of the obtained bacterial cells, a method of adding a substrate to the obtained bacterial cell suspension, a method of adding a substrate to the obtained bacterial cell suspension, a method of suspending a bacterial cell processed material (for example, a bacterial cell crush material, a bacterial cell extract, etc.), or a method of suspending bacterial cells fixed by a conventional method or a bacterial cell processed material, etc. a method of adding a substrate to a liquid;
There is a method in which a substrate is added to the culture solution during culturing of microorganisms and a reaction is carried out simultaneously with the culturing. The substrate concentration in the reaction solution is not particularly limited, but is 0.1 to 10 (W/V).
%, and the substrate can be added to the reaction solution all at once or in portions. The reaction temperature is 5
It is desirable to carry out the reaction at a temperature of ~50°C and a reaction pH of 4 to 10. Although the reaction time varies depending on the substrate concentration, bacterial cell concentration, and other reaction conditions, it is generally preferable to set the conditions so that the reaction is completed in 1 to 120 hours.

The 4-halo-3-hydroxybutyramide thus produced and accumulated in the reaction solution can be collected and purified using known methods. For example, 4-halo-3-hydroxybutyramide can be obtained by removing bacterial cells from the reaction solution using a method such as centrifugation and then concentrating it. Further, it can be further purified by crystallization using benzene or the like.

[0021]

[Examples] The present invention will be specifically explained below with reference to Examples, but the present invention is not limited to these Examples. Example 1 (1) Culture i) Use of medium A Dispense 5 ml of medium A with the following composition into test tubes, 12
After sterilization at 0° C. for 15 minutes, 5 (W/V)% aqueous solutions of isobutyronitrile and isobutyramide were sterilized using a membrane filter, and 100 μl each was added. This medium was inoculated with the bacterial strains shown in Table 1, and incubated at 30℃ for 72 hours.
A shaking culture was performed for hours.

Medium A Glucose 0.5%
KH2 PO4 0.05%
K2 HPO4 0.05%
MgSO4 ・7H2 O 0.05% yeast extract 0.2% polypeptone 0.5% MgCl2
0.04% KCl
0.004%MnS
O4 0.4×10-3
%FeCl3 0.6×
10-5% ZnSO4
0.3×10-4%

ii) Use medium B. After sterilizing the following medium at 120°C for 15 minutes,
Propionitrile was added to the mixture at a ratio of W/V)% and poured aseptically into a plate with an inner diameter of 95 mm to prepare an agar medium. This medium was inoculated with the bacterial strains shown in Table 1 and incubated at 30℃ for 7 days.
Static culture was performed for 2 hours.

Medium B Glycerol 2% KH2
PO4 0.68%Na2
HPO4 0.71%Na2
SO4 2mM MgCl2
0.04% KCl
0.004%MnSO
4 0.4×10-3%
FeCl3 0.6×1
0-5%ZnSO4 0
．． 3 x 10-4% agar
2% pH 7.5

(2) Reaction The bacteria obtained by culturing according to the above method were collected by centrifugation, and then added to 50mM phosphate buffer (pH 7.2) 1.5
After washing with 50mM phosphate buffer (pH 7) containing 88mM 4-chloro-3-hydroxybutyronitrile.
．． 2) 1 ml was added and reacted at 20°C for 24 hours. 4-chloro-3-hydroxybutyramide in the reaction solution is
ODS column (Inerts) manufactured by Gascro Kogyo Co., Ltd.
Analyzed by HPLC using il ODS-2). The results are shown in Table-1.

[0026]

[Table 1]

Claims (2)

[Claims] Claim 1: Through the action of nitrile hydratase having the activity of converting 4-halo-3-hydroxybutyronitrile into 4-halo-3-hydroxybutyramide, 4-halo-3-hydroxybutyronitrile is
-halo-3-hydroxybutyronitrile to 4-halo-
A method for producing 4-halo-3-hydroxybutyramide, which comprises producing 3-hydroxybutyramide. 2. The nitrile hydratase is derived from the genus Arthrobacter, the genus Brevibacterium, the genus Caseobacter, the genus Corynebacterium, the genus Pseudomonas and the genus Rhodo Coccus (Rhodococcus) genus The method for producing 4-halo-3-hydroxybutyramide according to claim 1, which is derived from a microorganism belonging to any one of the following.