JPH05168486A - Production of l-citrulline - Google Patents

Abstract

PURPOSE:To obtain L-citrulline by an extremely simple method by infecting a lactic acid bacterium belonging to the genus Pediococcus and having an arginine.deiuminase pathway with a bacteriophage and subsequently treating L-arginine with the product. CONSTITUTION:A lactic acid bacterium belonging to the genus Pediococcus and having an arginiine.deiminase pathway is infected with a bacteriophage to lyse the bacterium. L-Arginine is treated with the L-Arginine is treated with the lysed bacterium to produce L-citrulline, and the L-citrulline is collected from the product.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a method for producing L-citrulline, which is a type of amino acid, and more specifically to L-arginine which is obtained by infecting a specific lactic acid bacterium with Bacteria phage and lysing it. The present invention relates to a method for producing citrulline.

[0002]

2. Description of the Related Art Conventionally, the enzyme arginine deiminase which decomposes L-arginine to produce L-citrulline is
For example, the genus Pseudomonas,
It is known to be separated and collected from the genus Mycoplasma and the like [Kakimoto T. et al., FEBS Lett., Vol. 19, pp. 166-168. 1971); Shimke RT, Method of Enzymology, (Meth. Enzymol.), Volume 17A, pages 310-313 (1970), JP-A-2-35081]. .. That is, L-citrulline can be produced by acting the enzyme on L-arginine.

Further, a method is known in which a bacterium having an arginine deiminase activity of the genus Streptococcus is cultivated to collect the bacterium, and the bacterium having permeabilized cell membrane is subjected to L-arginine by subjecting the bacterium to toluene treatment. [E.L. Oginsky,
Journal of Biological Chemistry (J. Biol. Chem.), Volume 198, 791
-797 (1952)]. However, these methods have a drawback that complicated operations such as separation and collection of arginine deiminase, collection of bacteria, and treatment with toluene are required.

[0004]

DISCLOSURE OF THE INVENTION The present invention has been made for the purpose of eliminating such drawbacks and producing L-citrulline by an extremely simple method.

[0005]

Means for Solving the Problems The present inventors have conducted various studies to achieve the above-mentioned object, and as a result, arginine
It was found that L-citrulline can be easily produced by acting L-arginine by infecting and lysing a specific lactic acid bacterium having a deiminase pathway with a bacteriophage, and based on this finding, the present invention was completed. I arrived.

[0006] That is, the present invention is a lactic acid bacterium belonging to the genus Pediococcus and having an arginine deiminase pathway, which is lysed by infecting a bacteriophage.
-Acting on arginine to produce L-citrulline,
L characterized by collecting L-citrulline from this
-A method for producing citrulline.

The present invention will be described in detail below. The lactic acid bacterium used in the present invention may be any strain as long as it belongs to the genus Pediococcus and has the arginine deiminase pathway. This arginine deiminase pathway is known in, for example, bacteria belonging to the genus Lactococcus and Pseudomonas and is schematically shown as follows [Raymond Cunin et al.
in et al. ), Microbiological Review (Microbiol. Rev.), Volume 50, Volume 3
14-352 (1986)].

Arginine deiminase pathway Note: ADI; arginine deiminase OTCase; ornithine carbamoyl transferase

That is, in the arginine deiminase pathway, arginine is arginine deiminase (A
It is converted to citrulline by DI), and this citrulline becomes ornithine and carbamoyl-phosphate by OTCase. Some of the present inventors have found that the arginine deiminase pathway is also present in some strains of lactic acid bacteria.

The lactic acid bacterium belonging to the genus Pediococcus of the present invention and having the arginine deiminase pathway is, for example, Pediococcus halofilus which is one of soy sauce lactic acid bacteria.
s) IAM 1693 (IFO 12172) and the like. The presence or absence of the arginine-deiminase pathway is determined to be “present” when the “conversion ability (conversion rate) of arginine to ornithine” is 90% or more. Furthermore, the conversion rate was 5 ° C at 5 ° C in the medium described in the table below.
After static culture for a day, and conversion of arginine to ornithine, a high speed amino acid analyzer made by Hitachi (Biofluid analysis method) L-8
It is determined by quantifying with the 500 form.

Table Yeast extract 0.3 g / 100 ml Polypeptone 0.5 Dipotassium hydrogen phosphate 0.5 Sodium thioglycolate 0.1 NaCl 5.0 Glucose 0.23 Arginine (hydrochloride) 0.32

Arginine, which belongs to the genus Pediococcus
The bacteriophage used to infect and lyse a lactic acid bacterium having a deiminase pathway may be any bacteriophage as long as it can lyse the microorganism. Then, the bacteriophage contains 0.5% salt if necessary.
~ 20% (W / V) except that it can be separated according to the medium and method used for the detection and separation of ordinary bacteriophage (Sadajiro Uemura, Aidarai "Fermentation and Microorganisms II", No. 75- See "Phage Experimental Method" on page 92, published by Asakura Shoten, October 10, 1945).

For example, in order to search for a bacteriophage that lyses a lactic acid bacterium belonging to the genus Pediococcus and having the arginine deiminase pathway and to lyse the lactic acid bacterium, a sample in which the presence of bacteriophage is presumed or wild lactic acid bacterium is abundant The soy sauce moromi, which is considered to be growing in the soil, is filtered to obtain a moromi sap sample, which is filtered through a membrane filter with a pore size of 0.2 to 0.4 μm to completely remove the microorganisms and to sterilize it containing bacteriophage. A filtrate is obtained. Next, this is diluted with 0 to 20% (W / V) saline (the same salt concentration as the collected sample) in multiple stages, and a part thereof, for example, 0.5 ml, and a specific lactic acid bacterium (indicator bacterium) are cultured. 0.5 ml of the liquid was mixed, and a part of the mixed liquid, for example, 100 μl, was uniformly applied on a pre-prepared agar plate medium, and according to an ordinary agar culture method of lactic acid bacteria, under anaerobic conditions [Gas Pak (Becton -Manufactured by Dickson) or multi-layer culture method], the cells are cultured at 20 to 30 ° C for 2 to 6 days. If there is a bacteriophage that infects the applied indicator bacterium, lytic plaques (Plaques) will appear depending on the dilution step. Therefore, the bacterium is picked up and mixed with the indicator bacterium again to obtain an agar plate. By culturing, bacteriophages that infect the indicator strain can be isolated from the plaques that have regenerated.

Then, a lactic acid bacterium belonging to the genus Pediococcus and having an arginine deiminase pathway is infected,
As the bacteriophage used for lysing,
For example Pediococcus halophilus IAM 169
3 (IFO 12172) is used, ΦK169
3 and the like. The bacteriophage ΦK169
3 is separated and collected by the method shown below, for example. That is, soy sauce moromi, which is presumably rich in wild lactic acid bacteria, is filtered to obtain moromi sap, which is filtered through a membrane filter with a pore size of 0.2 μm to completely remove microorganisms and contain bacteriophage. A sterile filtrate was obtained. Next, this was diluted with 15% (W / V) saline in multiple stages, and 0.5 ml of it was diluted with Pediococcus halophilus IAM 1693 (I) of the host (indicator).
FO 12172) was mixed with 0.5 ml of a culture solution, and 100 μl of this mixture was prepared in advance, and an MRS medium (Difco 08 manufactured by Difco, Inc.) containing 15% salt and 1.5% agar.
81-01-3) on a flat plate, and according to the usual agar culture method of lactic acid bacteria, under anaerobic conditions [Gas Pak
(Manufactured by Becton-Dickson)], 25
After culturing at 4 ° C. for 4 days, lytic plaques appeared depending on the stage of dilution. When this is mixed with the above-mentioned host again and subjected to liquid stationary culture at 30 ° C. for 3 days, it is completely lysed, so this lysate is filtered through a membrane filter with a pore size of 0.2 μm to completely remove microorganisms and to contain bacteriophage. A sterile filtrate was obtained and used as the ΦK1693 bacteriophage solution.

The bacteriophage Φ thus isolated
K1693 has the following properties. Morphology: It has an isometric head with a width of about 80 nm and a contractile tail with a length of 120 to 180 nm and a width of about 25 nm. Specific gravity: about 1.505 Plaque form: 3-5 mm in diameter, transparent

Next, in the method of the present invention, a lactic acid bacterium belonging to the genus Pediococcus and having an arginine deiminase pathway [hereinafter also referred to as ADI (+) lactic acid bacterium). ] Lysed with the above bacteriophage
-The method of acting on arginine is not particularly limited, and any method may be adopted as long as arginine deiminase acts on L-arginine. For example, (1)
A method of reacting with L-arginine by culturing the ADI (+) lactic acid bacterium in an L-arginine-containing liquid, inoculating this with the bacteriophage, and lysing the bacterium;
(2) A method in which the ADI (+) lactic acid bacterium is previously cultivated, the obtained culture is inoculated with the bacteriophage, and the lysed bacterium is allowed to act on L-arginine.

When the method (1) is adopted, for example, in order to culture the ADI (+) lactic acid bacterium in the L-arginine-containing liquid, 0.1 to 10% of L-arginine is used as a hydrochloride in the medium. (W / V), preferably 0.5-5%
(W / V) Ordinary medium and culturing method for culturing lactic acid bacteria are used except that it is contained. The nitrogen source of the medium may be any available nitrogen compound or any containing nitrogen compound, such as yeast extract, peptone, meat extract, gelatin, corn steep liquor, amino acid, soybean or wheat koji leaching solution. More than one nitrogen source is used. One or more appropriate inorganic salts such as manganese, phosphoric acid, potassium, magnesium and calcium are appropriately added to the nitrogen source, and if necessary, a carbon source necessary for the growth of the fungus, such as sugars, various organic substances, inorganic substances, vitamins, etc. Is preferably used as the medium, and if necessary, 0.5 to 20% (W / V) of sodium chloride may be added.

A liquid culture method is preferable for the culture, and static culture is preferably performed. At this time, the inoculation amount of the ADI (+) lactic acid bacterium is preferably such that the number of bacteria in the medium is about 10 ⁶ / ml. Then, it is cultured at 20 to 30 ° C, preferably 25 to 32 ° C for 10 to 35 hours. Further, the pH during culture is suitably 6.5 to 8. L thus obtained
-The above-mentioned bacteriophage is inoculated into a culture of arginine-containing ADI (+) lactic acid bacterium, and the inoculation amount of the bacteriophage at this time is 10 ⁵ to 10 ⁷ bacteriophages / ml in the medium. It is preferable that And 20 to 35 ° C., preferably 25
When cultivated at ~ 32 ° C, lysis is completed in 1 to 3 days, and further at 20 to 35 ° C, preferably 25 to 32 ° C, 1 to 1
After culturing for 0 days, preferably 3 to 6 days, L-citrulline is produced and accumulated in the culture. In this method, L-arginine may be contained in the entire amount at the start of culturing the ADI (+) lactic acid bacterium, or may be divided and contained during the lactic acid bacterium culturing period.

In addition, for example, the method of the above (2), that is, the ADI (+) lactic acid bacterium is previously cultivated, and the obtained culture is inoculated with the bacteriophage and lysed, and L
-In the method of acting on arginine, the ADI
For the culture of the (+) lactic acid bacterium, the same medium and culture method as described above may be used except for the L-arginine content. At this time, the concentration of L-arginine in the medium is preferably 0.1 to 1% (W / V) as its hydrochloride in order to further enhance the activity of arginine deiminase. The resulting culture was then inoculated with the bacteriophage, 20-35
The lactic acid bacterium is lysed by culturing at 1 ° C. for 1 to 3 days. The inoculation amount of the ADI (+) lactic acid bacterium and the bacteriophage at this time may be the same as in the case of (1) above. L-arginine was added to this lysed product in an amount of 0.1 to 20
% (W / V), preferably contained at a concentration of 0.5 to 10% (W / V), and allowed to act at 20 to 40 ° C. for 1 to 10 days to produce and accumulate citrulline.

As described above, a method for collecting L-citrulline from the L-citrulline content obtained by reacting ADI (+) lactic acid bacterium with a bacteriophage to act on L-arginine is as follows. An appropriate combination of a conventional method, for example, an ion-exchange resin method for the filtrate obtained by filtration and other known separation and purification methods is used.
Thus, the substance produced by the method of the present invention,
As a result of measuring infrared absorption spectrum, elemental analysis, melting point, nuclear magnetic resonance spectrum, optical rotation, etc., it was found to be in agreement with pure L-citrulline [Sadtler Infrared
Grating Standard Spectr
a, Vol. 25, 24569K (1972),
The Merck Index, Elements
Edition, 364 (1989), etc.].

[0021]

INDUSTRIAL APPLICABILITY According to the present invention, L-citrulline can be easily produced by an extremely simple method in which a lactic acid bacterium having an arginine deiminase pathway is lysed with a bacteriophage and allowed to act on L-arginine. is there.

[0022]

EXAMPLES Examples will be shown below. In the examples, L-citrulline was quantified using a high-speed amino acid analyzer (Biofluid analysis method) L-8500 manufactured by Hitachi [Hitachi, Technical Data>.
LC No. 82, 10 (1986)]. Example 15% salt (W / V) and L-arginine (hydrochloride) in MRS medium (Difco 0881-01-3).
Containing 2.5% of 2.4% (W / V) (pH 7) in 500 ml of pedinococcus halophilus IAM 1 having the arginine deiminase pathway
5 ml of a preculture solution containing 10 ⁸ cells / ml of 693 (IFO 12172) was inoculated and statically cultured at 30 ° C. for 15 hours. Then, to this, the bacteriophage ΦK16
1 ml of bacteriophage solution containing 10 ⁸ cells / ml of 93
When inoculated with and cultivated statically at 30 ° C., lysis was completed after 2 days, and culturing was continued for another 4 days, whereby the content of L-citrulline in the culture was 0.7% (W / V). Generated and accumulated.

This L-citrulline-containing culture was centrifuged to obtain a supernatant liquid, which was passed through a strongly acidic ion exchange resin Amberlite 120 (H type) to adsorb L-citrulline. Next, L-citrulline was eluted with 3% aqueous ammonia, and the eluate was concentrated under reduced pressure, cooled, and allowed to stand to precipitate L-citrulline crystals. The crystals were collected and dried to give L-citrulline crystals (1.73 g).
Got

 ─────────────────────────────────────────────────── ─── Continuation of front page (72) Inventor Masaoki Sasaki 339 Noda, Noda City, Chiba Prefecture Kikkoman Corporation (72) Inventor Takanao Matsudo 339 Noda City, Noda City, Chiba Prefecture (72) Inventor Nami Fukuda 339, Noda, Noda, Chiba Prefecture Kikkoman Corporation (72) Inventor Kinji Uchida 339, Noda, Noda City, Chiba Prefecture Kikkoman Corporation

Claims (1)

[Claims] 1. A lactic acid bacterium belonging to the genus Pediococcus, which is lysed by infecting a lactic acid bacterium having an arginine deiminase pathway with a bacteriophage, is lysed to act on L-arginine to produce L-citrulline, and from this L-citrulline is produced. A method for producing L-citrulline, which comprises: