JPS62270A - Novel microorganism - Google Patents

Abstract

PURPOSE:To convert 5-substituted hydantoin and N-carbamoylamino acid to respective corresponding L-amino acids, in high efficiency, by using a novel microbial strain belonging to Arthrobacter genus. CONSTITUTION:A novel microbial strain belonging to Arthrobacter genus and capable of converting 5-substituted hydantoin and N-carbamoylamino acid to respective corresponding L-amino acids [Arthrobacter sp. DP-B-1001 (FERM-P 8190)] is cultured in a proper medium, and the obtained culture product, culture liquid, microbial cell, treated cell, etc., is used as an enzyme source and made to react with 5-substituted hydantoin or N-carbamoylamino acid.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to Ar/', Arthrobacter
The present invention relates to a novel microorganism that belongs to the genus Erl and has the ability to convert 5f-converted hydantoin and N-carbamoyl amino acid to the corresponding L-amino acid.

In the present invention, unless otherwise specified, the compounds consisting of 5-substituted hydantoin and N-carbamoyl amino acid are:
Each term collectively refers to D-form, 5-form, and DL-form.

[Conventional technology and problems]

Conventionally, a method for enzymatically converting DL-5 substituted hydantoins into L-amino acids was disclosed in Japanese Patent Publication No. 1385-1385.
Many patent applications have been filed since No. 0, and in particular, as a method for producing aromatic amino acids, Flavobacterium aminogenes
A method is known in which FERM-3133 and its mutant strains FERM-3134 and FERM-3135 are made to act on a 5-substituted hydantoy.

As a method for enzymatically converting 47'1-1L-N-carbamoyl amino acid to L-amino acid, L- or 1-j)
Corynebacterium scepedonicum (DL-N-carbamoylmethionine)
A method is known in which L-N-carbamoylmethionine-1L-methionine is converted by reacting with L-N-carbamoylmethionine-1L-methionine (Japanese Patent Publication No. 55-29678). However, only 5 bodies can be converted by this method, and the D body is converted into L-
It cannot be converted to methionine.

Furthermore, in the conventional enzymatic method for producing L-amino acids, it is necessary to use different types of microorganisms when using a 5-substituted hydantoin as a raw material and when using an N-carbamoyl amino acid as a raw material. Folds/toin and N-
No microorganism has been found that can act on both carbamoyl amino acids and convert them into the corresponding L-amino acids.

Therefore, when using DL-5-substituted hydantoins as a raw material, it is necessary to remove the DL-N-carbamoyl amino acid produced as a by-product during the production of the raw material.
When carbamoyl amino acids are used as raw materials, measures are taken to prevent D-N-carbamoyl amino acids from being mixed into the reaction system, or complicated steps are required to separate and racemize the D-N-carbamoyl amino acids remaining after the reaction. I had no choice but to adopt. Furthermore, if DL-N-carbamoyl amino acid is used as a water-soluble salt, the concentration of the reaction solution can be increased and the reaction should be carried out advantageously. Since there were no microorganisms to obtain, it was impossible to simplify the procedure.

[Means for solving problems]

While researching the decomposition mechanism of penta-substituted hydantoins by microorganisms, the inventors found that newly isolated microorganisms from soil cleave hydantoin compounds and produce amino acids when L-N- Carbamoyl amino acids and D-N
-This microorganism has previously unknown abilities because it produces carbamoyl amino acids and the amino acids that are successively produced are in the L form.

They discovered this and completed the present invention.

That is, the present invention relates to a new bacterial species, Arthrobacter sp., which belongs to the genus Arthrobacter and has the ability to convert 5-substituted hydantoins and N-carbamoyl amino acids into their respective L-amino acids.
sp, ) DP-B-1001 (Feikoken Bibori No. 8190).

Arthrobacter sp. DP-B-1001 is a microorganism newly isolated by the present inventors from the soil of Nikko City, Tochigi Prefecture, and has the following mycological properties.

From the above results, the mycological properties of the DP-B-1001 strain are as follows: (11 Gram staining is slightly positive;
They change with the course of culture and show characteristic polymorphism in terms of morphology, (2) they do not produce spores, and (3) the basic amino acid in their cell walls is lysine.

Based on these properties, Percy's Manual of Determinative Bacteriology (Berg.
ey's Manual of Determinat
ive BaCteri-ology) 8th edition, strain DP-B-1001 was determined to be a bacterium belonging to the genus Arthrobacter. However, even if a further search is performed regarding species included in the genus Arthrobacter, no description of bacterial species matching the DP-B-1001 strain can be found.

According to the 8th edition of the Bird's Manual of Determinative Pacteriology, Arthrobacter
Microorganisms belonging to the genus are classified into Schiff species based on their vitamin auxotrophy. The point that the DP-B-1001 strain requires only biotin is that Arthrobacter globi 7 ormis (
Arthrobacterglobiformis)
However, the GC content of the DNA is 59.3% and it is 1.
) Arthrobacter globi 7 ormis 60-64.4
% range and clearly different. Furthermore, D P -B
The ability of the -1001 strain to act on both 5-substituted folds/toin and N-carbamoyl amino acids and convert them into the corresponding L-amino acids is also a feature of the microorganism of the present invention.

Therefore, the present inventors recognized strain DP-B-1001 as a new bacterial species belonging to the genus Arthrobacter, named it Arthrobacter sp. Deposited.

This Arthrobacter sp. DP-B-1001 is
For example, it was isolated in soil by the method described in Examples below.

Arthrobacter sp. DP-B-10 of the present invention
In order to produce L-amino acids using 01, for example, a culture obtained by culturing this in an appropriate medium is used as an enzyme source to produce L-amino acids.
- Act on carbamoyl amino acids or 5fi-converted folds/toin.

As the culture medium, a conventional culture medium containing a carbon source, a nitrogen source, inorganic salts, etc. can be used. As a carbon source,
For example, sugars such as glucose, fructose, sucrose, and maltose; sugar alcohols such as glycerin and mannitol; and organic acids such as hexamaric acid and citric acid can be used as appropriate. As a nitrogen source, in addition to natural organic nitrogen sources such as meat extract, yeast extract, polybet/corn staple liquor, etc.
Inorganic ammonia sources such as ammonium chloride, ammonium sulfate, ammonium phosphate and ammonium fumarate,
Ammonium salts of organic acids such as ammonium citrate can be used. In addition, examples of inorganic salts include sodium phosphate, potassium phosphate, magnesium sulfate,
Sodium chloride, potassium chloride, ferrous sulfate, manganese sulfate, etc. can be used. Furthermore, organic salts such as cobalt acetate can also be used as appropriate. In addition, DP-B-10
For strain 01, even higher activity can be obtained by adding 0.05 to 0.2% by weight of DL-5-indolylmethylhydantoy/ to the medium.

Cultivation can be carried out according to a conventional method, for example, the pH of the medium is adjusted to a range of 5 to 9, preferably 6 to 8.5, and after inoculating the strain, the culture is carried out at about 20 to 35°C, preferably 26°C.
A method of culturing at a temperature of ~30° C. with aeration and stirring for 16 to 72 hours is adopted.

The culture obtained in this way can be used as an enzyme source as it is, but it can also be used as a culture solution, a crude enzyme preparation, a purified enzyme preparation, as well as bacterial cells collected from the culture solution or freeze-dried cells, for example. It can also be used in the form of processed products such as acetone-dried bacterial cells, crushed bacterial cells, and washed bacterial cells. Furthermore, the bacterial cells or treated bacterial cells can be immobilized on, for example, polyacrylamide, calcium alginate, carrageenan, photocrosslinked resin, etc., by a known method.

Further, the reaction of converting N-carbamoyl amino acids and/or 5-substituted hydantoins into the corresponding L-amino acids is carried out, for example, by allowing these compounds and the above-mentioned culture to coexist in an aqueous medium.

In this case, due to the unique ability of the microorganism of the present invention, the N-carbamoyl amino acid and the 5-substituted hydantoin can be converted into the corresponding L-amino acids, regardless of whether they are in the 0-form, L-form, or DL-form. can. Therefore, as a raw material compound for producing L-amino acids using the microorganism of the present invention, N-
0 bodies of carbamoyl amino acids or 5-substituted carbamoyl amino acids,
A mixture of L-form, DL-form, or any combination thereof can be used. Note that the N-carbamoyl amino acid can also be subjected to the reaction in the form of a water-soluble salt such as its sodium salt.

The reaction is suitably carried out in the range of pH 6 to 11, preferably pH 6.5 to 9.5. To adjust the pH, commonly used buffers such as phosphate buffer and ammonium buffer can be used. The reaction temperature is suitably adjusted to a range of 10 to 50°C, preferably 30 to 40°C.

Note that adding inorganic ions such as iron, manganese, and cobalt or reducing substances such as sodium sulfite to the reaction solution, or blowing nitrogen gas into the reaction solution may increase the reaction rate or stabilize the enzyme activity of the culture. Can give favorable results. Furthermore, it becomes possible to recover the bacterial cells from the reaction solution and reuse them in the reaction. Inorganic ions are preferably added at a concentration of 0.1 to 10 mM.

Furthermore, even if the amount of 5-substituted hydantoin added to the reaction solution exceeds its solubility, the reaction will not be hindered because the insoluble components will be dissolved as the reaction progresses and will be sequentially converted to L-amino acids. Furthermore, since the N-carbamoyl amino acid can be added in the form of a water-soluble salt, the raw material concentration can be kept high. Therefore, when carrying out a reaction by flowing a raw material solution into a reactor filled with immobilized bacterial cells in a column, it is possible to use not only 5-substituted hydantoin but also a water-soluble salt of N-carbamoyl amino acid as a raw material, which allows for sterilization. Operations can be carried out easily and efficiently at high concentrations, including when carrying out.

The reaction is thus completed in several hours to about 80 hours, and the 5-substituted hydantoin and N-carbamoyl amino acid used in the reaction are completely converted to L-amino acids and accumulated in the reaction solution. The produced L-amino acid can be easily separated and purified by using conventional crystallization methods, ion exchange resin methods, and other known methods as appropriate. Particularly when the reaction is carried out at a high concentration, the L-amino acid can be easily obtained as crystals by cooling the reaction solution and adjusting the pH.

In addition, it can be confirmed that the obtained amino acid is the desired L-amino acid using a bioassay method using Leuconostoc mesenteroides, high performance liquid chromatography, and an amino acid analyzer.

[Effect]

Arthrobacter sp. DP-B-10 of the present invention
As shown in the test examples below, 01 has the effect of converting 5-substituted folds/toin and N-carbamoyl amino acids into the corresponding L-amino acids, regardless of whether they are iD, 5, or DL.

〔Effect of the invention〕

Since the novel microorganism of the present invention has the above-mentioned ability, if it is used in the production of L-amino acids, the productivity can be dramatically improved. That is, (1) D-, which could not be used due to lack of suitable microorganisms,
N-carbamoyl amino acids can also be used as starting materials for the expansion of the corresponding L-amino acids.

(2) Since there is no need to remove DL-N-carbamoyl amino acid, which is produced as a by-product when chemically producing 5-substituted folds/toin, from the reaction system, DL-5-substituted folds/toin can be easily and inexpensively obtained industrially. L-amino acids can routinely be produced from a mixture of toy/ and DL-.N-carbamoyl amino acids in a one-step reaction. Therefore, conventional DL-
L-amino i using 5 as a starting material!
! ! 2. Measures to prevent the contamination of DN-carbamoyl amino acids into the DL-5 substituted folds/toin that were necessary in the manufacturing process, and to separate and racemize the DN-carbamoyl amino acids remaining after the completion of the reaction. Cumbersome and complicated steps are completely unnecessary, the production process is extremely simplified, and the desired L-amino acid can be produced in high yield.

(3) Since the reaction can be carried out using a water-soluble salt of DL-N-carbamoyl amino acid as a raw material using an immobilized cell column, the production process can be easily made continuous.

〔Example〕

Next, examples and test examples will be given and explained.

Example 1 Arthrobacter sp. DP-13-tooi
The strain was isolated from soil in Nikko City, Tochigi Prefecture as a result of screening using the following method.

Soil about 0.5? The suspension was suspended in sterilized water, diluted, spread on a tryptone agar plate, and cultured at 28°C to obtain bacteria. The bacteria obtained in this way were treated with glucose 1o P/
A. Yeast extract 5? /ノ、Polypeptone 5 L? /13
, Meat Extract 2 f-/43, MgSO4・71 (,OO
,4f-/A,FeS,O4・7H,OO,01t/
13, inoculated into a liquid medium consisting of Mn5O1-5H,OO,01'//-13 and adjusted to pH 7.0,
The cells were cultured with shaking at ℃ for 2 days to obtain bacterial cells. There are 10 bacterial bodies
t/43 N-carbamoyl amino acids (e.g. DL-N
-carbamoyltributophane) or 5-substituted hydantoin/(e.g. DL-5-indolylmethylhydantoin)
was added to 0.2 M ammonium buffer (pH 9, OJ) and incubated at 37°C for 24 hours. After incubation, L-amino acids in the buffer supernatant were analyzed in a bioassay using Leuconostoc mesenteroides P-60. As measured by thin layer chromatography using silica gel method and silica gel (L/-), this strain was found to have particularly high amino acid production activity.

Test Example 1 (1) Cultivation of Inoculum The same liquid medium as used in Example 1 was dispensed into a 500 ml Erlenmeyer flask for 200 days, and after sterilization, Arthrobacter sp. DP-B-1001 strain obtained in Reference Example 1 was injected. A platinum loop was inoculated and cultured with shaking at 28°C for 24 hours.

(2) Main culture, bacterial cell preparation Add 0.15-5 to the same liquid medium as used in Example 1.
- Prepare a medium supplemented with indolylmethylhydantoin, inoculate 4 ml of the culture solution obtained in +1), and incubate at 28°C.
The cells were cultured with shaking for 24 hours. From culture solution, 18000G
The microbial cells were collected by centrifugation for 10 minutes and washed once with physiological saline to obtain wet microbial cells, which were used as washed microbial cells for use in enzyme reactions.

(3) 10 washed bacterial cells obtained in enzyme reaction (2)? o, 8 M NH40H-NHl (J
(pH 8, O), 4 mM Fe504-7H, O and 4 mM MnSO4.5H, O buffer solution 25
rnl was added, and further as a raw material ■Dzyl) Hida/Toin 10y-1 ■DL-5-(4'-Hydroxybenzyl)
Hydantoin li, QDL-5-indolylmethylhydantoin 1%, QDL-5-methylmercaptoethylhydantoin 1? , CIDL-5-isopropylhydantoin I P, 0DL-5-inbutylhydantoin 1?
were each suspended and kept at 37°C for 48 hours by blowing in a N2 stream.

After the reaction was completed, the bacterial cells were removed by centrifugation, and the amino acids in the centrifuged supernatant were quantified in the same manner as in Example 1. As a result, L-amino acids were accumulated in the amounts shown in Table 2 below.

Below margin Table 2 *Same as Table 1.

that's all

Claims (1)

[Scope of Claims] 1. Belongs to the genus Arthrobacter and contains a 5-substituted hydantoin and an N-carbamoyl amino acid with the corresponding L-
A new bacterial species Arthrobacter sp. DP-B-1001 (Feikoken Bibori No. 8190) that has the ability to convert into amino acids.