JPS6062991A - Preparation of l-amino acid - Google Patents

Abstract

PURPOSE:To collect a converted L-amino acid in high yield, by treating an L- acyl-DL-amino acid with acylase produced by a fungus belonging to the genus Cryptococcus. CONSTITUTION:A fungus capable of producing acylase, belonging to the genus Cryptococcus, Candida, Trichosporon, is cultivated in a liquid medium. This cultivation is usually carried out by aerated spinner culture at 30-35 deg.C at 6- 8pH. The culture is directly used as an enzyme source, or a culture such as a cell-free extracted solution obtained by grinding the separated mold or mold, or separated acylase is used as the enzyme source. Namely, this acylase is made to act on an N-acyl-DL-amino acid at 30-35 deg.C at 6.0-7.0pH, it is asymmetrically hydrolyzed, to give an L-amino acid (e.g., L-methionine). In the asymmetric hydrolysis, the presence of a metal ion such as Co ion in the reaction solution promotes the reaction, and the reaction can be completed in 3-20hr.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing L-amino acids by allowing acylase of microbial origin (hereinafter referred to as "acylase") to act on N-acyl-DL-amino acids.

Amino acids, especially essential amino acids, are one of the most important amino acids for human and animal nutrition, and are therefore extremely useful for the food and feed industries.

Conventionally, as a method for producing L-amino acids from N-acyl-DL-amino acids using microorganisms, there has been proposed a method in which acylases produced by Aspergillus spp., Penicillium spp. -22580 Publication A method in which acylase produced by bacteria belonging to the genus Pafmicola, Muscle, Peciloseces, Sporotrium, Bysochlamys, Chrysosporium, Gilmaniera, Monascus, and Cherabia is allowed to act at 50°C or higher. (Japanese Patent Publication No. 5B-16877) and the genus Aspergillus, the genus Penicillium, the genus Achromobacter', and the genus Pseudomonas.

A method of using acylase produced by bacteria belonging to the genus Micrococcus, alkaline earth metals, and Streptomyces (Japanese Patent Publication No. 37277/1983) is known.

The present inventors discovered that a microorganism that was not previously known to produce acylase produces acylase, and that L-amino acids are recovered from N-acyl-DL-amino acids by the action of such acylases. The present invention was achieved by discovering that it can be produced efficiently.

That is, the present invention allows acylase produced by microorganisms belonging to the genus Cryptococcus, Candida, Trichosporon, or Proteus to act on N-acyl-DL-amino acids to convert them into L-amino acids and collect them. This is a method for producing b-amino acids, which is characterized by the following.

Specific examples of microorganisms that produce acylase in the present invention are as follows.

(1) Cryptococcus laurentii (Crypt
oooccus 1aurentii) Fiber Technology Research Institute No. 709 TORAY 2001 (2) FAI Research Institute No. 710 TORAM 2002 (3) Candj-αah
umicola) Microtechnical Research Institute No. 715 TOEtAY
2008 (4) The same No. 717 TORAM2010 (5) The same No. 719 TORA/Y20
20 (6) Trichospon cutaneum (Trj-chosp
oroncutaneum) Microtechnical Research Institute No. 214 TORAY 2 0 0 7 'j7) Microtechnology Research Institute No. 725 TORAY 2 o
35 (8) Proteus retgelii
ttgeri) ATOCi 29944 These acylase producers are cultured in a liquid medium. Cultivation is usually carried out by aeration and agitation, at a temperature of 30 to 30℃.
It is cultured by performing seed culture for 24 to 48 hours and main culture for 10 to 24 hours at 5°C and pH 6 to 8.

Acylase is also produced in a medium commonly used for main culture, i.e., a medium containing glucose and ammonium chloride as the nitrogen source and nitrogen source, respectively, but DL-
Acrase is similarly produced by straining α-amino-ε-caprolactam.

ft is produced when main culture is carried out using N-acyl-D-mophenylalanine, N-acyl-L-phenylalanine, N-7flu-DL-mefonine, or N-aflu-L-methionine as the sole nitrogen source. Acylase activity is improved several times.

Once the culture is completed, most of the produced acylase remains in the bacterial cells.

When this acylase is used in the reaction, the culture mentioned above may be used as it is, isolated bacterial cells, or a processed product such as a cell-free extract obtained by disrupting the bacterial cells, or isolated acylase or these. An immobilized product obtained by immobilizing the enzyme or bacterial cells can be used as an enzyme source.

The properties of the acylase obtained here are as follows.

1. Action: Acts on N-acyl-DL-amino acids to asymmetrically hydrolyze them to produce L-amino acids. 2. Substrate specificity. Acts only on 2-N-acyl-L-amino acids and decomposes them, producing L-amino acids. produced, but has no effect on N-r acyl-D-amino acids.

The asymmetric hydrolysis reaction is carried out at 60-50°C, preferably at 35-40°C.
pH 6.0 to 7.0 under electric current at 5°C, preferably pH
6,4 to &8.

When performing an enzymatic reaction, the substrate concentration is usually 1 to 50 wt%.
However, it is preferably 3 to 15 wt%. The amount of bacterial cells is 1 to 50 wt% of the substrate, preferably 5 to 50 wt% in terms of dry bacterial cells.
It is 15wt%. When using a culture, the OD of the culture
The amount of bacterial cells calculated from the values is as described above.

Although the reaction time varies depending on the amount of bacterial cells and the substrate concentration e, the asymmetric hydrolysis reaction D is normally completed in 10 to 100 hours. At this time, if metal ions such as cobalt, manganese, copper, etc., preferably cobalt ions, are added to the reaction solution, one reaction is accelerated and completed in 5 to 20 hours. The amount of metal ions added is [105
~0.5 mmol, preferably 11-15 mmol
It is l.

The microorganism of the present invention does not racemize or decompose L-amino acids produced by asymmetric hydrolysis.

N-acyl-L-amino acids as substrates to be asymmetrically hydrolyzed include N-acetyl-L-glycine, N-acetyl-L-phenylalanine, N-acetyl-L-methionine, and N-α-acetyl-L- Lysine, N-7 cetyl-L-leucine, N-acetyl-L-isoleucine, N
-acetel-L-glutamic acid, N-pensoyl-L-
Examples include glycine. Particularly preferred is 11N-7 cetyl-L-phenylalanine%N-7 cetyl-L-methionine. The acylase of the present invention is N-acyl-D-amino, and therefore serves as a raw material for the enzyme reaction of the present invention.

In addition to the above-mentioned N-acyl-cycloamino acids (L-form),
The corresponding N-acyl-DL-amino acid (DL form) can be used.

The L-amino acid can be collected from the asymmetric hydrolysis reaction solution by conventional methods such as the ion exchange resin method, @ray method, and organic solvent addition method.

The present invention will be explained below with reference to Examples.

In the examples, the specific activity i unit is ゛1 per hour.
It shows the activity of producing μmol of L-amino acids. unit
/mg indicates the activity per mg of bacterial cells used in the asymmetric hydrolysis reaction.

Example 1 10ml of the following seed culture medium was placed in a test tube, and after sterilization and cooling,
Inoculate Cryptococcus laurentii No. 709 (TORAM 200 +) at the first platinum, and
Seed culture was performed by shaking reciprocally for 24 hours at 0 to 35°C.

Seed culture medium composition Nigelcose 296, polypeptone 1l
L596. Yeast extract 11%, aqueous solution (PH 7,0) Next, IGQmji of the main culture medium having the following composition was dispensed into 14 Erlenmeyer flasks and sterilized. Add all of the above seed culture solution to this medium and incubate at 30-35°C for 10 hours at about 20 O
Main culture was performed by shaking at rpm.

Main culture medium composition Nigelcose 1.2%, KH2PO40
2% + Mg5OslQ 2%, MnQlg -4f (g 00.02%, corn steep liquor 02%, DL-α-amino-ε-prolactam 1%, aqueous solution (PH 7,0) After completion of cultivation, culture Centrifuge the solution at approximately +DODOG,
Dilute the obtained bacterial cells approximately 10 times with ion-exchanged water to 3 B
ml, 00 (i116H20o, add 48 g, adjust pH to 6.9t!
I strained it. Shake at 38°C and after 24 hours, the produced L-
The amount of methionine was 2sqsgc (yield 91%).

Example 2 108 t of N-acetyl-DL-phenylalanine was added with 100 ml of ion-exchanged water, the pH was adjusted to 6.8 t with KOH, and 200,060 g of coc12.6H was added to prepare a substrate solution. To this substrate solution was added 10 md of diluted bacterial cells similar to those in Example 1, and the mixture was shaken at 40°C for 24 hours.

After the reaction was completed, the reaction solution and the bacterial cells were separated, and then the reaction solution was passed through an ion exchange resin 5KIB CH type column (Mitsubishi Kasei Ji).The effluent was concentrated, and the resulting residue was filtered from water. It was recrystallized to obtain 4.15 g (yield: 83%) of N-acetyl-D-phenylalanine. The optical rotation was as follows.

5 (α), = -37,17' CC = [1,60Hxo)
Next, a 3% aqueous NH4OH solution was passed through the column, the effluent was concentrated, and the resulting residue was recrystallized from water.

5.2 g (yield a 596) of L-phenylalanine was obtained. The optical rotation was as follows.

(α) = -35 @ (0 = 1.59 FIzO) Example 3 200 mJi' of ion-exchanged water was added to 20 g of N-acetyl-DL-phenylalanine, and the pH was 6,
8 and used as a substrate solution. Example 1
20 m4 of the same diluted bacterial cells were added and shaken at 40°C. L-7 after reaction for 90 hours = #alanine production t bar 6, q q g (yield, 192.64%).

Example 4 Among the culture conditions of Example 1, DL-
1% of α-amino-ε-caprolactam was changed to 1.4% of N-acetyl-DL-methionine, and cultivation was carried out under the same conditions except for that, to obtain 1% diluted bacterial cells.N- Add 1 ooml of ion exchange water to 10 g of ace-f A, -D L -phenylalanine, and add OH'''QPt (&8
After adjusting the temperature, 200.06 g of coal 66H was added to prepare a substrate solution. Add 10 m4 of the above diluted bacterial cells to this substrate solution.
was added and shaken at 40°C. After 8 hours, the amount of L-phenylalanine produced was 54 g (90%).

Example 5 Culture was carried out under the same conditions as in Example 1. After the culture was completed, the culture solution was centrifuged at about 10,000 G, and the obtained bacterial cells were diluted to about 596% with ion-exchanged water.

Dissolve 1 g of N-acetyl-DL-phenylalanine in ion-exchanged water, add 2 ml of the diluted bacterial cells above, and sterilize with KOH.
H was adjusted to ZO to make the total volume 10 m4. This reaction solution was shaken at 40°C for 722 hours. The results are shown in Table 1.

Example 6 DL-α of the main culture medium composition among the culture conditions of Example 1
-Amino-ε-caprolactam 1% in NH401 (L
42%, but culturing was carried out under the same conditions. Shortly after the cultivation was completed, the cells were strained in the same manner as in Example 5 to obtain 5% diluted bacterial cells. The same reaction as in Example 5 was carried out using this diluted bacterial cell.

The results are shown in Table 1.

Example 7 In Example 6t, 42% of NH401IIL was modified to 1.6% of N-acef#-D L-phenylalanine, and the culture was carried out under the same conditions except for the reaction under the same conditions. The results are shown in Table 1.

Table 1 Examples 8-13 Table 2? The microorganism shown above was cultured in the same manner as in Example 4. After culturing, 1ml of culture solution! 2 mJ of a 10% aqueous solution of N-acetyl-DL-phenylalanine,
0oC11116FIz OO, 25% aqueous solution α5mI
Add I and KOE! ''After adjusting to QPH48.

The mixture was shaken at 50°C for 1 hour and the specific activity was measured.

The results are shown in Table 2.

Table 2 Example 14 The following minimal (Davis) medium for Escherichia coli 'Erm1 was put into a test tube, and after sterilization and cooling, Proteus letgelii AT
The first platinum inoculation of OC29944 was carried out at 30-35℃ for 24 hours.
The mixture was shaken back and forth for hours, and seed culture was performed.

Minimum (Davi8) medium for Escherichia coli 1 culture medium composition:
KH2po4 (1.7%, ni11 (2P○402%,
MgSO47F1200.011N, (NH4)280
4 0.1%, trisodium citrate dihydrate 0.05
%.

Glucose 112% bacteria. Add all of the above seed culture solution to this medium, and
Main culture was carried out at 55°C for 24 hours with approximately 20 Orpm''Q rotational shaking.

After culturing, add 1 ml of culture solution to N-acetyl-DIL.
-Phenylara=y, 2 ml of 1096 aqueous solution.

0o011-6H200, 25% aqueous solution 0.5 m/ was added and adjusted to KO+ ('11" PEl 7.0, then 50°C, 1
Time swing 284 &? unit/mg.

Patent applicant Higashi Shikikai Co., Ltd.

Claims (3)

[Claims] (1) Allowing acylase produced by microorganisms belonging to the genus Cryptococcus, Candida, Trichosporon, or Proteus to act on N-acyl-DL-amino acids, converting them into one L-amino acid, and collecting the amino acids. A method for producing L-amino acids, characterized by: (2) The method according to claim 1, wherein the N-acyl-DL-amino acid is N-acylphenylalanine. (3) The method according to claim 1, wherein the N-acyl-DL-amino acid is N-acylmethionine.