JPH0260594A - Production method of α-acetamido-β-hydroxybutyric acid ester - 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 [Industrial Application Field] The present invention relates to a method for producing α-acetamido-β-hydroxybutyrate by treating α-acetamido-acetoacetate with a specific mold.

[Prior Art] α-acetamido-β-hydroxybutyric acid ester is a useful raw material for producing L-threonine.

Conventionally, such butyrate esters have been microbiologically evaluated as 'fJ! For example, α-acetamide-acetoacetic acid ester can be prepared using Saccharomyces waxi (Saccharomyces waxi).
myces rouxii). CHe l v, Ch im, Act a
,, 70° 232 (1987)) [Problems to be Solved by the Invention] However, this method has a low yield of the target product of about 32%, so it is not satisfactory as a practical manufacturing method, and the yield is low. There is a need for the development of sophisticated methods.

[Means for Solving the Problems] As a result of intensive research in order to solve the above problems, the present inventors have found that α-acetamido-acetoacetate contains Altenaria sp., Aspergillus sp., Aureobasodium sp., Cephalosporium sp. We have found that α-acetamido-β-hydroquinobutyric acid ester can be produced with good yield when at least one mold selected from the genus Emericelopsis, Fusarium, Gibberella, Hypomyces, Rhodoceptria, and Trichoderma is used. The present invention has now been completed.

The starting material α-acetamide-acetoacetate used in the present invention has the general formula CCH3C0CHCOOR)NHC.
In the compound represented by OCI-I3, R is usually an alkyl group. Examples of the alkyl group include those having about 1 to 10 carbon atoms, such as methyl, ethyl, propyl, butyl, amyl, hexyl, hebutyl, octyl, and the like.

In addition, the mold used in the present invention is Alternaria maxi (I F O8
984], Aspergillus soja x
illus 5 ojae) CT FO4389], Aureobumpium pullulans (Aureobasi
CI Fo 4
464), Cepha (7 Suborium chrysogenum (
Cephalosporium chrysogenu
n) CIFO30055), Emerycellopsis humicola
) (IF 0 8518), Fusarium sumbucinum
(ATCC24384), Gibberella fujikuroi (Gi
bberella rujikuroi) (ATC
C14164), Hypomyces rosellus (Ilypom
yces rosellus) (AT CC+6
240), aDoseptria S P (Rhodose
ptoriasp,) CATCC11833:l, Trichoderma vird
e) CI FO5720] and the like. α−
For the reaction with acetamide-acetoacetate, mold is dispersed in an aqueous system (water, physiological saline, buffer solution, culture medium, etc.), sugar is added as an energy source, and then the ester is added to the mixture at 10 to 70°C, preferably 20°C. 0 at ~40°C.
Shaking for 1 to 150 hours, preferably 1 to 40 hours,
The reaction may be carried out by stirring or standing still.

PI-(of the system) is suitably 3 to IO, preferably 4 to 8.

In addition, a method such as that of immobilizing mold separately and making it work is adopted. As the reaction method, a batch method or a continuous method in which immobilized microorganisms are packed in a tube or column and α-acetamidoacetoacetic ester is allowed to flow down can be adopted.

As a medium for such a reaction, not only water but also an organic solvent compatible with water, such as a mixed water/organic solvent system such as alcohol or acetone, is used. It is of course necessary to select an organic solvent that is not harmful to microorganisms.

α-acetamido-acetoacetate is added to the system as it is or after being dissolved or dispersed in an organic solvent. The concentration of the ester in the system is usually 0.01 to 30% by weight, preferably 1 to 10% by weight. Further, it is advantageous that the weight ratio (dry weight) of the ester and mold is 1 or less, preferably about 0.001 to 0.8.

There is no problem in allowing sugars such as glucose and other nutrients to coexist during the reaction. Such sugars and other nutrients can be added at any stage of the reaction, and can be carried out in bulk, continuously or in portions. Furthermore, an esterase inhibitor such as phenylmethylsulfonyl fluoride may be added as appropriate.

After the reaction is complete, separate the mold using conventional methods such as centrifugation.
The liquid is extracted using an organic solvent such as ether, carbon tetrachloride, or benzene.

α-acetamido-β-hydroxybutyric acid ester is obtained by distilling off the solvent from the extract.

[Function] [Effect] In the present invention, α-acetamido-β-hydroxybutyric acid ester can be obtained in a high yield of 50% or more. It is useful as an intermediate for

[Example code] Next, the method of the present invention will be explained in detail by giving examples.

Example 1 ~ IO Sabouraud medium (40 g glucose, peptone I09, water I
QSP)-16) 5 ml was placed in a test tube, one platinum loop of the specified mold was inoculated, and cultured with shaking at 30°C for 72 hours to obtain a seed culture solution.

Next, 100 ml of a medium having the same composition was placed in a 500 ml Sakaro flask, 5 ml of the seed culture was inoculated, and cultured with shaking at 30° C. for 72 hours. After culturing, collect and collect PH7 of 0.1
Wash the cells once with molar phosphate buffer (wet).
4.5? I got it.

10 ml of the same buffer was placed in a 30 ml tube, and 49 bacterial cells were added thereto to produce a bacterial suspension.

Subsequently, 1% (w/v) of α-acetamide-acetoacetate was added to the bacterial suspension, and a shaking reaction was performed at 30° C. for 24 hours.

After the reaction, the substrate and product are extracted with ethyl acetate, and the ethyl acetate is distilled off using a rotary evaporator to obtain the target product (
α-acetamide-β-hydroxybutyric acid ester) was obtained.

The target product was analyzed by high performance liquid chromatography to determine the yield.

These results are shown in the table.

The measurement conditions for high-speed liquid a are as follows.

Column・0PP-50461111110-eluate;
CH2ON/H, 0 (1/9) flow rate; 0, 5 m
l/lN1n detection, R1 5m1L

Claims (1)

[Claims] α-acetamide-acetoacetate and at least one mold selected from the genus Altenaria, Aspergillus, Aureobasidium, Cephalosporium, Emeryseropsis, Fusarium, Gibberella, Hypomyces, Rhodoseptria, and Trichoderma. 1. A method for producing α-acetamido-β-hydroxybutyric acid ester.