JP2000069988A - Strain for producing epsilon-poly-l-lysine and production of epsilon-poly-l-lysine using the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject new strain for producing ε-poly-L-lysine, etc., useful for a toiletry article, cosmetic, food additive medicine, agrochemical, etc., comprising Streptomyces sp. SP-72 strain, etc., capable of producing ε-poly- L-lysine. SOLUTION: New Streptomyces sp. SP-72 strain (FERM P-16,810) capable of producing ε-poly-L-lysine or its variant has high safety and specific physical properties due to high cation content and is useful for a toiletry article, cosmetic, feed additive, medicine, agrochemical, food additive, electronic material, etc., and for producing ε-poly-L-lysine. The new strain is obtained by suspending soil in sterilized physiological saline, shaking the suspension at 30 deg.C, allowing the suspension to stand, then diluting its supernatant liquid with physiological saline, applying the diluted solution to a medium for separating an actinomycete, culturing the bacterium at 28 deg.C for 1-2 weeks, measuring the amount of polylysine produced and screening so as to separate and collect a bacterium capable of producing a large amount of a polylysine from soil at Tagamachi Inukami District in Shiga Prefecture.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a strain producing ε-poly-L-lysine and a method for producing ε-poly-L-lysine using the same.

[0002]

2. Description of the Related Art ε-poly-L-lysine is a polymer compound in which an amino group at the ε-position of L-lysine is bonded to a carboxylic acid group of an adjacent L-lysine by an amide bond. ε-poly-L-lysine is a polymer of L-lysine, which is an essential amino acid, and therefore has high safety, and has high cation content and thus has unique physical properties. Therefore, toiletries, cosmetics, feed additives, medicines, pesticides, food additives,
Applications such as electronic materials can be expected. In particular, in the field of food additives, attention has been paid to natural additives.

As a method for producing ε-poly-L-lysine,
culturing a strain producing ε-poly-L-lysine in a medium,
A method for collecting ε-poly-L-lysine from the obtained culture is known. As the strain used in such a method, Streptomyces albulus subspecies lysinopolymeras (Streptomyces albulus subsp.
lysinopolymerus) No. 346-D strain (Microtechnical Laboratory No. 3834)
(Japanese Patent Publication No. 59-20359), 11011A-1 strain which is an S-aminoethyl-L-cysteine resistant mutant of No. 346-D strain (Microtechnical Research Laboratories No. 1109) (Japanese Patent Publication No. 3-42070) No.), No.
50833 strain, which is a plasmid amplification mutant of 346-D strain (Microtechnical Research Institute, No. 1110) (Japanese Patent Publication No. 3-42075, Japanese Patent Publication No. 6-755)
No. 01), B2, a mutant of 11011A-1 strain, which is resistant to high concentrations of S-aminoethyl-L-cysteine.
1021 strain (FERM BP-5926) (Japanese Patent Application Laid-Open No. Hei 9-73057) and Streptomyces nour
sei) (FERM P-9797) (JP-A-1-87090) is known.

[0004]

SUMMARY OF THE INVENTION An object of the present invention is to provide an ε-
It is an object of the present invention to provide a novel strain which produces a large amount of poly-L-lysine and a method for producing ε-poly-L-lysine with a high yield by a fermentation method using the strain.

[0005]

Means for Solving the Problems As a result of the screening, the present inventors have found a novel strain producing ε-poly-L-lysine in a remarkable amount, and have completed the present invention.

That is, the present invention relates to Streptomyces sp. Which produces ε-poly-L-lysine. SP-72 strain (FERM
P-16810) or a mutant thereof (hereinafter, also referred to as the strain of the present invention).

[0007] The present invention also provides a method for producing ε-poly-L-lysine, which comprises culturing the strain of the present invention in a liquid medium and collecting ε-poly-L-lysine produced and accumulated in the culture solution. A production method (hereinafter, also referred to as the production method of the present invention) is provided.

The strain of the present invention is preferably Streptomyces sp. SP-72 strain (FERM P-16810).

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION <1> Strain of the Present Invention The strain of the present invention has a property of producing ε-poly-L-lysine (hereinafter, referred to as polylysine). Producing polylysine means producing polylysine in a recoverable amount. In addition, the strain of the present invention may be, for example, a production medium (2.0
% Glycerol, 2.0% citric acid / H ₂ O (pH 4.5), 1.0
% (NH ₄ ) ₂ SO ₄ , 0.2% L-lysine.HCl), when cultured at 30 ° C., the amount of polylysine in the culture solution decreases when Streptomyces albulus IFO 14147 strain is cultured under the same conditions. Even during the period (usually after the sixth day of culturing), the amount of polylysine in the culture solution is reduced or not reduced as compared with the IFO 14147 strain. Furthermore, the preferred strain of the present invention has the property that the amount of polylysine in the culture solution is not substantially reduced during the same period. Furthermore, since the strain of the present invention does not produce a melanin-like pigment (soluble pigment) as described below, there is also a practically preferable property that decolorization and purification of polylysine are facilitated when the strain of the present invention is used for production of polylysine. Have. The amount of polylysine in the culture solution can be measured by the method described in Example 1 (1) (c) below.

[0010] Among the strains of the present invention, Streptomyces s
p. The SP-72 strain (hereinafter referred to as the SP-72 strain) is described in Example 1 below.
Was isolated from soil using the productivity of polylysine as an index by the screening shown in (1).

The mycological properties of the SP-72 strain are as follows. (1) Morphological properties The results of microscopic observation of aerial mycelia and basal mycelia of the SP-72 strain grown on yeast extract / malt extract agar medium (ISP Medium 2) at 28 ° C for 1-2 weeks are as follows. Show. Branching method and morphology of sporulating hyphae: spiral
l). Spore surface structure and size: Spores are circular or elliptical and have a size of about 0.7-1.1 μm, and their surface structure is spiny.

(2) Growth state on various media The properties on various media shown in Table 1 below are 28
It is an observation result after culturing at 2 ° C. for 2 weeks.

[0013]

[Table 1]

(3) Physiological properties Liquefaction of gelatin: positive. Cell wall composition: For the type of diaminopimelic acid in the cell wall composition, the method of Staneck et al. (See Applied Microbiology Vol. 28, p. 226 (1974))
As a result of the analysis, the product was of L and L type. Assimilation of various carbon sources (on Prideham Gottlieb agar medium): L-arabinose-D-xylose-D-glucose + D-fructose + L-rhamnose-D-galactose + sucrose-raffinose + D-mannitol + i- Inositol + salicin-melibiose + Note) +: assimilate,-: do not assimilate. Production of melanin-like pigment (tyrosine agar): none.

The SP-72 strain is determined to belong to the genus Streptomyces based on the mycological properties, particularly the morphological properties and the type of diaminopimelic acid in the cell wall. Bergey's Manual of Systematic Bacteriolog
y) and E.I. B. EB Shirling and D.S.
D. Gottlieb's International Journal of Systematic Bacteriology
(Intern. J. Syst. Bacteriol.) Volume 18 pages 69-189
(1968), Vol. 19, pp. 391-512 (1969) and 2
2, 265-394 (1972), searching for related bacterial species, no species with the same mycological properties were found, and polylysine-producing bacteria belonging to the known genus Streptomyces. Have different properties. From the above, the strain of the present invention was recognized as a new strain belonging to the genus Streptomyces, and Streptomyces sp. It was named SP-72 strain. This Streptomyces sp. The SP-72 strain was deposited with the Ministry of International Trade and Industry at the National Institute of Advanced Industrial Science and Technology on May 19, 1998, and has been assigned the accession number FERM P-16810.

The strain of the present invention also includes a mutant strain of the SP-72 strain as long as it has the property of producing polylysine at least as high as that of the SP-72 strain (preferably, the above-mentioned preferable properties of the strain of the present invention). You. The mutant strain of the SP-72 strain means a polylysine-producing mutant strain or a spontaneous mutant strain of the SP-72 strain that can be induced by mutating the SP-72 strain.

Mutants of the SP-72 strain can be obtained by subjecting the SP-72 strain to a mutant strain obtained by mutagenizing the cells or a spontaneous mutant strain of the SP-72 strain, using the properties of producing polylysine as an index. Obtained by screening. Examples of the mutagenesis treatment include ultraviolet irradiation and treatment with a mutagen such as N-methyl-N-nitro-N'-nitrosoguanidine. Screening using the property of producing polylysine as an index is described, for example, in Example 1 below.
Can be performed by the method described in (1).

<2> Production method of the present invention The production method of the present invention is characterized by culturing the strain of the present invention in a liquid medium and collecting polylysine produced and accumulated in the culture solution. The strain of the present invention is preferably the SP-72 strain.

The liquid medium may be any as long as it contains a carbon source, a nitrogen source, inorganic salts and other nutrients. Examples of the carbon source include glucose, fructose, glycerin, starch and the like.
10% (w / v) is preferred. Examples of the nitrogen source include yeast extract, peptone, casein hydrolyzate, organic compounds such as amino acids, and inorganic ammonium salts such as ammonium sulfate, and the content is 0.1 to 5% (w / v).
Is preferred. The liquid medium preferably contains glucose or glycerin as a carbon source and ammonium sulfate or yeast extract or peptone as a nitrogen source. Examples of the inorganic salt include those which give phosphate ion, potassium ion, sodium ion, magnesium ion, zinc ion, iron ion, manganese ion, nickel ion, sulfate ion and the like.

The culture can be carried out under aerobic conditions by shaking culture, stirring culture, or the like. Culture temperature is 20-40 ° C
Is preferred. The pH of the medium is preferably 3 to 9. The culture period is usually 1 to 10 days, but in the strain of the present invention,
The culture can be continued for a longer period. During the culture, a carbon source and a nitrogen source may be sequentially added. Also, L-
Lysine is preferably added to the liquid medium, and the amount is usually 0.1 to 2% (w / v).

By such culturing, polylysine is produced and accumulated in the culture solution. The polylysine produced and accumulated in the culture solution can be collected by removing cells from the culture solution by centrifugation or filtration, and isolating polylysine from the obtained cell-removed solution by a known method. Specifically, for example, the cell-removing solution is removed through a column of an anion exchange resin to remove most of the impurities, and further purified and concentrated through a column of a cation exchange resin. Polylysine is obtained by crystallization from the obtained concentrated liquid with an organic solvent such as acetone or ethanol.

[0022]

The present invention will be specifically described below with reference to examples.

[0023]

[Example 1] Acquisition of polylysine-producing bacteria (1) Screening of polylysine-producing bacteria (a) Separation method from soil 1 g (wet weight) of soil is suspended in sterilized saline (10 ml) and shaken at 30 ° C. (At 200 rpm for 10 minutes) and after standing (30 minutes), the supernatant was diluted with physiological saline, and the diluted solution was applied to an actinomycete separation medium (medium 1). 28 ℃
For 1 to 2 weeks, and the grown actinomycete colonies were transferred to a yeast extract-malt extract agar medium (medium 2), isolated and stored.

(B) Production of polylysine The isolated and obtained actinomycetes were first grown sufficiently at 30 ° C. for 1 to 3 days on a growth medium (medium 3).
The medium was aseptically removed by centrifugation to collect the cells. next,
The collected cells were suspended in a production medium (medium 4) and incubated at 30 ° C for 2 hours.
The cells were cultured with shaking for days.

(C) Detection of polylysine The isolated / acquired strain was cultured in a production medium (medium 4), and then the culture was centrifuged to remove the cells, and polylysine was detected in the supernatant. Detection of polylysine was performed by Itzhak
i) (Analytical Biochemistry (Analytical
Biochemistry), vol. 50, p. 569). That is, 0.5 ml of culture supernatant, 1 mM methyl orange and 50 mM
After mixing with 2 ml of an aqueous solution of NaH ₂ PO ₄ —Na ₂ HPO ₄ (pH 7.0) and leaving at room temperature for 30 minutes, the resulting polylysine-methyl orange complex was removed by centrifugation, and the absorbance (470 nm) of the supernatant was measured. The measurement was performed, and the amount of polylysine in the culture solution was determined from a standard curve prepared using a polylysine solution having a known concentration.

The compositions of the above culture media 1 to 4 are shown in Tables 2 to 5 below. In the table, “%” is% (w / v).

[0027]

Table 2 Medium 1 Medium for actinomycetes separation (Glycerol-Czapek medium) Glycerol 0.3% NaNO ₃ 0.2% K ₂ HPO ₄ (pH 7.0) 0.1% KCl 0.05% MgSO ₄・ 7H ₂ O 0.05% FeSO ₄・7H ₂ O 0.001% Agar 1.5% Cycloheximide 50μg / ml Nystatin 50μg / ml

[0028]

Table 3 Medium 2 Yeast extract-Malt extract agar medium (ISP Medium)
2) Yeast extract 0.4% Malt extract 1.0% Glucose 0.4% Agar 1.5% pH 7.2

[0029]

[Table 4 Medium 3 0.5% growth medium Glycerol 2.0% yeast extract _{MgSO 4 · 7H 2 O 0.05%} KH 2 PO 4 -Na 2 HPO 4 (pH 6.8) 1 / 50M

[0030]

Table 5 Medium 4 Production medium Glycerol 2.0% Citric acid / H ₂ O (pH 4.5) 2.0% (NH ₄ ) ₂ SO ₄ 1.0% L-Lysine / HCl 0.2%

As a result of screening polylysine-producing bacteria from the soil by the above-mentioned screening method, polylysine-producing bacteria that produce a large amount of polylysine were isolated and obtained from soil in Taga-cho, Inukami-gun, Shiga Prefecture.

(2) Bacteriological properties of the obtained strain The morphological properties, growth state on various media and physiological properties of the isolated / acquired strain were examined, and the above properties were found.

This strain was determined to be of the genus Streptomyces based on its morphological characteristics, diaminopimelic acid type in the cell wall, and the like. In addition, a known polylysine-producing bacterium, Streptomyces albulus No. 3
The strain 46-D (described in Japanese Patent Publication No. 59-20359) was recognized as a novel strain in that it was distinguished by the assimilation of raffinose and melibiose and the productivity of melanin pigment (soluble pigment). This strain is Streptomyces sp. SP-7
Named 2 strains, deposited at the Research Institute of Biotechnology and Industrial Technology, Ministry of International Trade and Industry on May 19, 1998
FERM P-16810 has been granted.

(3) Confirmation of SP-72 Strain Product After culturing the SP-72 strain in the production medium (medium 4), the culture filtrate was precipitated and recovered with methanol (40-60% fraction). Next, the collected methanol precipitate fraction is applied to a gel filtration column (Supe
rdex 75pg), and it was confirmed that the purified product was electrophoretically uniform. Next, the purified product was hydrolyzed with hydrochloric acid, and the hydrolyzate was subjected to amino acid analysis.
The amino acid analysis method was to convert the amino group of the hydrolyzate into DABS (dimethylaminoazobenzenesulfonyl-) in advance, and analyzed using liquid chromatography (aminochrome amino acid analysis system).

As a result of amino acid analysis, it was confirmed that the product of strain SP-72 was a polypeptide having lysine as the only constituent amino acid, ie, polylysine.

The molecular weight of polylysine produced by the SP-72 strain was measured by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and found to be about 4000. Furthermore, polylysine produced by the SP-72 strain was degraded by a protease that hydrolyzes an ε-polyamide bond (Protease A derived from Aspergillus oryzae; Amano Pharmaceutical). On the other hand, the enzyme trypsin, which hydrolyzes α-polylysine bonded to α-polyamide, did not hydrolyze.

When the antibacterial activity of polylysine produced by the SP-72 strain was examined by a paper disk method, it was found that Bacillus brevis (IFO 3331), a gram-positive bacterium, and Escherichia coli K-12 (IFO 3301), a gram-negative bacterium. Str
It had the same antibacterial activity as polylysine from eptomyces albulus No. 346-D strain.

[0038]

Example 2 Polylysine productivity of SP-72 strain SP-72 strain was grown on a growth medium (medium 3) in a Sakaguchi flask (100 ml).
After culturing at 30 ° C for 36 hours using a medium (500 ml volume),
The cells were collected by centrifugation. The collected cells were washed once with physiological saline, transferred to a production medium (medium 4), and cultured at 30 ° C. using a Sakaguchi flask (100 ml medium / 500 ml volume). Polylysine (ε) in the culture supernatant collected during this culture
-PL) The concentration was measured by the method described above. The pH was also measured. It was confirmed that no melanin-like pigment (soluble pigment) was produced.

Further, Streptomyces albulus IFO 14147
The same operation was performed using a strain (same as the No. 346-D strain described in JP-B-59-20359) and compared with the SP-72 strain.

FIG. 1 shows the results. In a 6-day culture, St
The reptomyces albulus IFO 14147 strain produced 1.9 g / l of polylysine, while the SP-72 strain showed very good productivity of 3.6 g / l. Also, Streptomyces albulus IFO 141
In 47 strains, polylysine was degraded by further culturing after 6 days, but in the SP-72 strain, polylysine was not degraded.

[0041]

According to the present invention, a strain with high productivity of polylysine is provided. According to the method for producing polylysine using this strain, polylysine can be produced in high yield, and the production cost and purification cost of polylysine can be reduced as compared with the conventional method. In particular, when this strain is used, no degradation of polylysine in long-term culture is observed, which is advantageous for industrial production.

[Brief description of the drawings]

FIG. 1 shows a polylysine production curve.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification FI FI Theme Court II (Reference) C12R 1: 465) (72) Inventor Yoshinori Inoue 2500 Yasakacho, Hikone City, Shiga Prefecture Shiga Prefectural University Faculty of Engineering ( 72) Inventor Hideo Hirohara 2500 Yasaka-cho, Hikone-shi, Shiga F-term in Shiga Prefectural University Faculty of Engineering 4B064 AG01 CA04 DA01 DA10 DA11 DA20 4B065 AA50X BA16 BA22 CA24 CA41 CA44 CA50

Claims (4)

[Claims] A streptomyces sp. Producing ε-poly-L-lysine. SP-72 strain (FERM P-16810) or a mutant thereof. 2. Streptomyces sp. Producing ε-poly-L-lysine. SP-72 strain (FERM P-16810). 3. Streptomyces sp. Producing ε-poly-L-lysine. The SP-72 strain (FERM P-16810) or a mutant strain thereof is cultured in a liquid medium, and ε-poly-L-lysine produced and accumulated in the culture solution is collected.
-A method for producing poly-L-lysine. 4. A Streptomyces sp. Producing ε-poly-L-lysine. The SP-72 strain (FERM P-16810) was cultured in a liquid medium, and ε-poly-L- produced and accumulated in the culture solution.
A method for producing ε-poly-L-lysine, which comprises collecting lysine.