JPS63283591A - Production of l-alpha-amino-epsilon-caprolactam - Google Patents

Abstract

PURPOSE:To produce L-alpha-amino-epsilon-caprolactam efficiently and simply, treating N-formyl-D,L-alpha-amino-epsilon-caprolactam with a culture mixture of a bacterium belonging to the genus Bacillus. CONSTITUTION:An aqueous solution of N-formyl-D,L-alpha-amino-epsilon-caprolactam is treated with a culture solution of a bacterium such as Bacillus brevis (FERM P-9072) belonging to the genus Bacillus, capable of hydrolyzing N-formyl-L-alpha- amino-epsilon-caprolactam and producing L-alpha-amino-epsilon-caprolactam, a culture cell or a treated material thereof at pH about 6-11 and at <=about 70 deg.C.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a method for producing L-α-amino-ε-caprolactam (hereinafter α-amino-ε-caprolactam is abbreviated as ACL).

[Problems to be solved by conventional technology and invention]

L-ACL is used as an optical resolution agent or as a raw material for the production of lysine using L-ACL hydrolase, but its production method involves cyclization of L-lysine,
Since optical resolution of L-ACL is a complicated method, there has been a demand for the development of a simpler manufacturing method.

Therefore, the present inventors investigated N-formyl-D, L-α-amino-ε-caprolactam, which can be produced in large quantities and at low cost as an industrial raw material, or can be synthesized relatively easily from L-ACL and formic acid in acetic anhydride. (Hereinafter, N-formyl α
-Amino-epsilon-caprolactam (abbreviated as f-ACL)), and as a result of intensive studies to develop a method for producing L-ACL using this substance as a raw material, the present invention, which has remarkable effects, was achieved.

[Means for solving problems]

The present invention involves culturing a microorganism belonging to the genus Bacillus and having the ability to hydrolyze f-L-ACL to produce L-ACL, that is, a microorganism having f-L-ACL amidohydrolase. Or use the treated product to act on f-D, L-ACL, f-L-ACL
is hydrolyzed to produce L-ACL, and L- is extracted from the reaction solution.
The present invention provides a method for producing L-ACL, which comprises separating and collecting ACL.

An example of the microorganism used in the present invention is Bacillus brev, which was isolated by the present inventors.
is) Microtechnical Research Institute No. 9072 is mentioned.

The taxonomic properties of this microorganism are shown in Table 1.

These properties are described in the Purging Aids Manual of Determinative Bacteriology (BergeV's
) random of Determinative
According to BaCteriOIOQV) 8th edition <1974) (hereinafter referred to as the manual), the microorganism is Bacillus brcvis.
It was concluded that it belongs to

Margin Table 1 Below: To obtain a culture of the microorganism of the present invention, f-L-
ACL or f-D, L-ACL is contained as an inducing agent, and the microorganism of the present invention is cultured.

The inducing agent can be added either by adding it to the medium in advance or by culturing it in a normal medium and adding it at an appropriate time. The form of the medium may be either liquid or solid, and the culture method may be static culture, shaking culture, or aerated agitation culture, but liquid culture using aerated agitation culture is industrially convenient. The culture temperature is 10 to 45°C, preferably 30 to 40°C.

It is desirable to maintain the pH of the medium during culture near neutrality.

When carrying out the method of the present invention, f-
Microorganisms that have the ability to hydrolyze L-ACL and produce L-ACL are cultured for 1 to 3 days using the method described above, and the culture solution is used as is or the bacterial cells are collected from it by centrifugation, filtration, etc. f suspended in water or M% solution
-D, in addition to an aqueous solution of L-ACL, a reaction for producing L-ACL is performed. Prior to the reaction, treatment such as physically crushing the bacterial cells or lysing them using a surfactant or the like has the effect of smoothing the progress of the reaction.

f-D and L-ACL used in the method of the present invention are D
, L-ACL can be obtained by dissolving L-ACL in formic acid, adding acetic anhydride dropwise thereto, adding water and drying under reduced pressure.

The concentration of raw materials f-D and L-ACL in the reaction solution in the present invention is 1 to 30% at the start of the reaction, but preferably 5 to 15%. Depending on the progress of the reaction, raw materials can be added all at once or in portions to increase the final L-ACL concentration. If ACL racemase activity is observed in the bacterial cells,
By adding hydroxylamine hydrochloride to the enzyme reaction solution at a concentration depending on the amount of bacterial cells used, the by-product of D-ACL can be suppressed.

The pH of the reaction is usually kept at 6-11, particularly preferably at 8-9. The reaction temperature is usually 70° C. or lower and is appropriately selected depending on the required time.

Furthermore, the enzyme activity can be increased by including a manganese salt, particularly preferably manganese chloride, in the reaction solution.

For collection, first, the reaction solution is sterilized using, for example, a centrifuge, and then passed through an acidic ion exchange resin column.
CL is adsorbed, washed, eluted with ammonia, etc., the eluate is concentrated, and L-ACL is crystallized, or after ammonia is completely removed, hydrochloric acid is added to neutralize it, and L-ACL is concentrated under reduced pressure. It can also be crystallized as ACL hydrochloride. If some lysine is mixed in, the lysine may be removed using, for example, a basic ion exchange resin before crystallization.

After passing the reaction solution through an acidic ion-exchange resin column, the microorganism of the present invention is applied again to the reaction solution that flows out without being adsorbed to the column, and L-ACL is collected in the same manner. can be raised. By performing the reaction not only once but multiple times in this manner, L-ACL can be produced with good yield.

〔Example〕

Hereinafter, the present invention will be explained in more detail with reference to Examples.

Example 1) Synthesis of f-D, L-ACL 12.8 g of commercially available glue, L-ACL, was added to 200 ml of 98
After dissolving in ice water with stirring in % HCOOH, 5%
1 g of acetic anhydride was added dropwise over about 30 minutes. After stirring for a further hour at room temperature, 200 ml of water were mixed in.

This reaction solution was concentrated under reduced pressure, and the solidified residue was recrystallized using ethyl acetate-ethanol (1:1). Yield is 7
It was 0-85%.

2) Culture of bacteria Bacillus brevis
) (Feikoken Bibori No. 9072) with 0.5% f-
D, L-ACL, 0.5% glucose, 0.2% NaH
2PO4・2H20,0,3% to 2 HP 04.0-
05%M g S 0.4 ・7 H20,0,002
%MnCl2・4H20,0,05% yeast extract (p
6 ml of H7゜2) medium was sterilized at 121°C for 15 minutes and then cultured with shaking at 30°C for 8 hours. The bacterial cells were collected by centrifugation, washed with water, frozen at -20°C, and then thawed at room temperature.

3) Enzyme reaction Add 10% f-D, L-ACL-50mM phosphate buffer (pH 8,0)-1mM hydroxylamine to the bacterial cells to make a total of 0.5ml, and incubate at 30°C for 4 hours to react. Ta. Figure 1 shows the course of the reaction.

To quantify the produced L-ACL, add 1 μm of acetone-dried bacterial cells of Cryptococcus laurentii to the reaction solution and
-The total amount of ACL was converted to L-lysine, and this L-lysine was quantified using an automatic amino acid analyzer to determine the amount of L-ACL.

Here, acetone-dried bacterial cells were produced by the following method.

Cryptococcus laurentii (cryptococcus +aur) increases L-ACL hydrolysis enzyme production
Entii) (Feikoken Bibori No. 709) was mixed with 0.5% glucose, 1% DL-ACL-HCl, and 0.1% KH.
2PO4,0.05%MgSO4・7H20.0.00
2%MnCl2・4H20,0,05% yeast extract (
pt17.2) in 300 ml of medium at 30°C.
After culturing for 20 hours, the cells were collected by centrifugation, washed twice with water, and then dehydrated with acetone to obtain acetone-dried cells.

After 4 hours of reaction, the lysine content was 21.84 mg/m
l, and the molar conversion of f-D, L-ACL to L-ACL was calculated to be 23.3%.

4) L-ACL extraction 30 ml of the enzyme reaction solution sterilized by centrifugation was added to approximately 40 ml of
ACL was adsorbed on a column of strongly acidic ion exchange resin "Diaion PK228".

After further washing with 150 ml of distilled water, the adsorbed ACL was eluted with 5% ammonia water.

The eluate was collected in 2 ml portions using a fraction collector.
Both parts were subjected to thin layer chromatography (n-proper tool:
The presence or absence of ACL was determined using 28% ammonia water = 2:1 as a developing solvent and a thin layer of silica gel, and 50 tubes containing ACL, totaling 100 ml, were obtained. Furthermore, this solution was applied to a strongly basic ion exchange resin (“Diaion PA318”) column to adsorb trace amounts of lysine, and the eluate was
The purity of CL was increased. This eluate was transferred to an eggplant-shaped flask and concentrated under reduced pressure in a water bath at about 40°C to remove ammonia.''The contents were dissolved again in 10 ml of distilled water, and IN hydrochloric acid was added dropwise to neutralize.

When a part of 123■ obtained by drying this solution under reduced pressure was subjected to infrared absorption analysis and elemental analysis, it was confirmed that it was ACL as shown in Figures 2 and 3 and Table 2. Ta. Figure 3 shows the infrared absorption spectrum of commercially available standard L-ACL, and Figure 4 shows the L-ACL extracted in this example.
The infrared absorption spectrum of ACL is shown.

Furthermore, it was confirmed by thin layer chromatography that it was completely degraded by L-ACL hydrolase, and the optical purity was 99.4 as determined by high performance liquid chromatography.
%was. The compound extracted from the above results is L-AC
It was concluded that L.

Table 2 5) Enzyme activity Add 1% f-D to 2 ml of the cultured cells in item 2> above.

L-ACL-100mHTris-HCIII street solution (p
118.0>-4mM hydroxylamine-5mM manganese chloride was added to make a total of 1ml, and the mixture was incubated at 40°C for 3 hours to react. f-D, L-ACL to L-
The molar conversion to ACL was 43.0%.

On the other hand, when the bacterial cells of item 2) were incubated with 20 m HEDTA for 30 minutes and subjected to enzymatic reaction, the activity disappeared completely, but after washing with water, when 50 ml manganese chloride was added to the enzyme reaction solution, the activity was restored.

6) Thermostability of enzymes Disperse 1 ml of the cultured cells in item 2) above in 18.0>0.5 ml of 50 mM phosphoric acid solution (1).
After incubating at 0.40.50.60.70.80.90°C for 30 minutes each, enzyme activity was measured.
A decrease in activity was observed at temperatures above 40°C. However, 1%f-
D, 50% phosphoric acid WJI solution containing L-ACL < pits
, 0) After dispersing in 0.5 ml and incubating at each of the above temperatures for 30 minutes, washing with water and measuring enzyme activity,
A decrease in activity was first observed at 70°C.

[Effects of the Invention] According to the method of the present invention, L-ACL can be produced simply and efficiently.

[Brief explanation of drawings]

FIG. 1 shows the relationship between the reaction time and the amount of L-lysine produced. FIG. 2 shows the infrared absorption spectrum of standard L-α-amino-ε-caprolactam, and FIG. 3 shows the infrared absorption spectrum of L-α-amino-ε-caprolactam obtained in the present invention. Patent applicant Toshi Co., Ltd. Figure 1 0 0 0 0 0 0 0 0
o 0':.

Claims (1)

[Claims] (1) A culture, bacterial cells, or the like of a microorganism that belongs to the genus Bacillus and has the ability to hydrolyze N-formyl-L-α-amino-ε-caprolactam to produce L-α-amino-ε-caprolactam. A method for producing L-α-amino-ε-caprolactam, which comprises allowing a treated product to act on N-formyl-D,L-α-amino-ε-caprolactam.