JPH03143398A - Production of epsilon-poly-l-lysine - Google Patents

Abstract

PURPOSE:To obtain the title compound useful as article for toiletry, cosmetic, food additive, etc., by varying a specific strain belonging to the genus Streptomyces into a variant having resistance to analog substance of L-lysine and culturing the variant in a medium. CONSTITUTION:Streptomyces albulus subsp. lysinopolymerus strain is varied into a variant [preferably variant 11011A-1 strain (FERM BP-1109) having resistance to glycine added to S-aminoethyl-L-cystine of Streptomyces albulus subsp. lysinopolymerus No346-D strain] having resistance to analog substance (preferably substance to which S-aminoethyl-L cystine is added) of L-lysine. The prepared variant is cultured in a medium and the objective compound is collected from the formed substance.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Field of Application) The present invention relates to a method for producing epsilon-poly-luricin (hereinafter abbreviated as εPL).

(Prior art and its problems) As shown in the structural formula below, εPL is a compound in which the agono group at the ε position of L-lysine is bonded to the carboxylic acid of the adjacent I-lysine through an amide bond. It is a high molecular compound.

+1J”-(CHz) t-coco +NH(CH2
) 4-C) ICO+n0 The substance is a polymer of the essential amino acids ■, -lysine, so it is highly safe, and has a high cation content, so it has unique physical properties. Therefore,
Utilizing these properties, it can be expected to be used in toiletry products, cosmetics, feed additives, medicines, agricultural chemicals, food additives, electronic materials, etc.

Conventionally, the substance was εPL belonging to the genus Streptomyces.
The producing bacterium, Streptomyces alplus subsp.
es albulus 5ubsp, lysinop
olymerus), 346-D strain (Feikoken Bacterial Serial No. 3
No. 834) in a medium, and the resulting culture is separated and purified (Japanese Patent Publication No. 59-20359).

However, with the strain of this previous application, at most 0 per 11 culture solutions.
．． The productivity of εPL was only about 5 g, and therefore the production cost was high, preventing the wide use of this substance.

The present inventors obtained a strain that produces a significant amount of εPL, conducted repeated research with the aim of producing a large amount of εPL using this strain, and arrived at the invention described below.

(Means for Solving the Problems) The present invention is directed to Streptomyces alplus subsp.
esalbulus 5ubsp, Iysinopo
lymerus) strain into a mutant strain that is resistant to L-lysine analog substances, the resulting mutant strain is cultured in a medium, and epsilon-poly-lymerus is produced and accumulated in the culture solution, which is collected. 8.
This is a method for manufacturing PL.

An analog substance of L-lysine is S-aminoethyl-L-
Preferably, cysteine or this S-atanoethyl-L-cysteine is added with one or more substances selected from I,-threonine, glycine, L-homoserine, and I,-methionine.

The mutant strain is Streptomyces alplus subsp. rhizinovolimerus N [L346-ri strain S-
A mutant strain 11011A-1 (Kikoken Jokyo No. 1109) that is resistant to aminoethyl-L-cysteine with glycine added is preferred.

The present invention will be explained in detail below.

First, the method for obtaining the bacterial strain of the present invention will be described. A mutant strain having resistance to an analog substance of L-lysine, for example, an S-ξnoethyl-L-cysteine ml mutant strain is obtained, for example, by the following method.

Streptomyces alplus subsp.
Tris-maleate buffered spores of Lysinovolimelas strain N1346-D? & (pH 9,0) and to this is added N-methyl-N-nitro-No-nitrosoguanidine.

After shaking this, the spores are collected using a centrifuge, washed with sterile water, inoculated into a medium, and cultured with shaking to grow the bacteria. Dilute the culture medium containing bacteria (hereinafter referred to as culture solution).

Next, S-aminoethyl-L cysteine, or glycine, I4-threonine, L-homoserine, L-
One or more methionine amino acids are selected and added to an agar medium having the same composition as the above medium.

At that time, S-atanoethyl L-
Cystine, or SA〈noethyl-L-cysteine and agar medium 0.2 to 5 per ml to the same concentration.
The above-mentioned amine acid is added to a concentration of 1 mg, preferably 1 mg. The above culture dilution solution is applied to this agar medium. After keeping this agar ftf fabric warm:
! The strain grown as Kuchi Niichi is S-aminoethyl-L-
It is a cysteine-resistant mutant strain. At this time, the strain grown on the agar medium to which only S-aminoethyl-■,-cysteine was added was the resistant mutant strain 81512, and the strain grown on the agar medium to which glycine was added to S-aminoethyl-L-cysteine. is the resistant mutant strain 11011A1 (Feikoken Jokyo No. 1109), and the strain grown on an agar medium containing S-aminoethyl-L-cysteine and L-threonine is the resistant mutant strain 81502. .

Moreover, a plasmid amplifiable mutant strain is obtained, for example, by the following method. Streptomyces alplus subsp. rhizinobolimelas strain 346-D, or
After the S-aminoethyl-L-cysteine resistant mutant strain is inoculated into a medium and cultured with shaking, chloramphenicol is added and the culture is continued. The bacteria are collected by centrifugation, washed, and then spread on an agar medium. After static culture, Staphylococcus aureus
), and the strain with a large staphylococcal growth inhibition zone is the target strain that produces high ξF-amplifying cPL, that is, the positive-amplifying mutant strain 50833. Stock (Feikoken Joyori No. 111O). Among these mutant strains, lloIIAl strain and 5
The mycological properties of strain 0833 are as follows.

(1) Morphological properties The results of microscopic observation of the aerial and basal hyphae of the 11011A-1 strain and the 50833 strain grown on a sucrose/nitrate agar medium at 30°C for 10 days are shown below.

■ Branching method and morphology of spore-prone hyphae: simple branching, closed spiral (closed 5 pira+) ■ Number of spores: two to several dozen ■ Surface structure and size of spores: Spores are circular or oval in shape, and the size is approx. 1.2 to 1.5μ, and its surface structure is Spiny.

■ The presence or absence of flagellated spores, sclerotia, and sporangia is not observed.

■ Spore stalk settlement position: Aerial mycelium 2 (2) Growth status on various media The properties on the following various media are 10% at 30°C.
These are the observation results after culturing for ~14 days.

mouth) 50833 stocks (Feikoken Joyori 1st No. 0) physiological properties 1011A Physiology of 1 strain and 50833 strain The properties are as follows.

■ Growth temperature range Approximately 15-40℃.

Optimum temperature for growth: Around 30'C.

■ Liquefaction of gelatin, hydrolysis of starch and peptonization of skimmed milk; all positive ■ Coagulation of skimmed milk: negative ■ Formation of melanin-like pigment On tyrosine agar medium, brown pigment is produced.

■ Cell wall composition Cell wall composition! Regarding the type of diaminopimelic acid in y, the method of Becker et al. [Applied Microbiology Vol. 13, p. 236 (1965)
As a result of the analysis conducted by [Reference], it was found to be type L.

(4) Assimilation of various carbon sources (on Bridham-Gottlieb agar medium) L-arabinose D-xylose D-glucose + D-fructose + L-rhamnose D-galactose + sucrose raffinose D-mannitol + i-inositol -1 Salicin's Note〉＋；Assimilate, -do not assimilate.

As described above, the mycological properties of the mutant strain are similar to those of the original strain, Stojib I. Myces alplus subsp. rhizinovolimerus fk346-D.

Next, εPL is produced by the method of the present invention using the mutant strains obtained by these methods. Note that % in the text indicates weight (g)/volume (mffi)% unless otherwise specified.

First, the obtained mutant strain is inoculated into a medium and cultured, and the produced and accumulated εPL is separated and purified from the culture solution.

The medium may be any medium as long as it contains a carbon source, a nitrogen source, inorganic salts, and vitamins, but preferably contains grapes @ 5% or glycerin 5% as a carbon source, and ammonium sulfate, ■ or L as a nitrogen source. -It is better to use lysine or peptone. A carbon source and a nitrogen source may be added sequentially during the cultivation. p
)L is allowed to drop to pH 4.0 at the initial stage of culture, and then raised to pH 4 with an alkali such as sodium hydroxide aqueous solution.
, 0 may be maintained. After removing bacterial cells from the culture solution using a centrifuge or filter, the filtrate is purified and decolorized, and then concentrated. εPL is crystallized from the concentrated solution using an organic solvent such as acetone or ethanol.

(Effects of the Invention) According to the present invention, a Streptomyces alplus subsp. lysinovolimeras strain is mutated into a mutant strain having resistance to an analog substance of L-lysine, and the mutant strain is cultured. Since the productivity is improved and a significant amount of εPL can be produced than using the same strain, the production cost of εPL can be significantly reduced compared to the conventional method.

(Example> Hereinafter, the present invention will be described in detail with reference to examples.

Example I Obtaining a S-atanoethyl-L-cysteine resistant strain: Streptomyces alplus xabsp.
Streptomyces alb
ulus 5ubsp, lysinopolymer
A platinum loop of spores of the t'h 346-D strain was suspended in 5 ml of Tris-maleic acid buffer (pH 9,0), and N-methyl-N-nido0-N''-nitrosoguanidine was added to this. The spores were added to a concentration of 1.5 nw/m#. After shaking at 30°C for 30 minutes, the spores were collected using a centrifuge and washed with sterile water.
! 5%, ammonium sulfate 1%, yeast extract 0.5%,
Potassium dihydrogen phosphate heptahydrate 0.136%, disodium hydrogen phosphate heptahydrate 0.158%, magnesium sulfate heptahydrate 0.05%, zinc sulfate heptahydrate 0.0
04%, ferrous sulfate heptahydrate 0.003%, pH 6.8
medium (hereinafter referred to as the first medium) 5mA! The bacteria were inoculated and cultured with shaking at 30°C day and night to grow the bacteria.

The culture solution was mixed with MS solution (I precept is magnesium sulfate heptahydrate 0.05%, sodium chloride 0.5%, Tween 80
0.05%) and dilute 500 times. Next, this diluted culture solution was mixed with S-aminoethyl-L-cysteine at a concentration of 2 per ml of agar medium, or S-aminoethyl-L-cysteine at a concentration of 2 per ml of agar medium and 1 cysteine per ml of agar medium. The mixture was coated on an agar medium having the same composition as the first medium described above, to which glycine or L-threonine was added at a concentration of 1. This agar medium was incubated at 30'C for 48 hours to grow as a colony to obtain an S-aminoethyl-L cysteine resistant mutant strain.

Among these, one strain, strain 81512, is in the agar medium to which only S-A-adinoethyl-L-cysteine has been added. One strain in the agar medium containing S-aminoethyl-L-cysteine and glycine was 1101.1 A1 strain (Feikokenjoyori No. 11).
No. 09). ■ To S-aminoethyl-L-cysteine
, - one strain in an agar medium supplemented with threonine is 8150
There are 2 stocks.

Production of εPL: One platinum loop of S-aminoethyl-L-cysteine resistant strain 81512 was inoculated into medium 5-, which had the same composition as the first medium, and cultured with shaking at 30°C for 8 days. After completion of the culture, the concentration of εPL in the culture solution was measured by the method of Rtzhaki.

The results are shown in Table 1.

Examples 2 and 3 S-aminoethyl-L-cysteine resistant mutant strain 8151
Instead of 2 stocks, S-Apical noethyl-L cysteine →-
Glycine-resistant mutant strain 1], Ol], A1 strain (Feikoken Jokyo No. 1109) (Example 2), S~75 Noethyl-cysteine + L-threonine-resistant mutant strain 815
The cells were cultured in the same manner as in Example 1, except that the 02 strain (Example 3) was used, and the concentration of cl)L was measured in the same manner.

The results are shown in Table 1.

Example 4 Obtaining a positive amplification mutant strain: The S-A-saturated-L-cysteine resistant mutant strain obtained in Example 1 was added to 5 ml of a medium having the same composition as the first medium described in Example 1. to be inoculated.

After culturing this with shaking at 30°C for 2 days, chloramphenicol was added at a concentration of 50 to 500, preferably 100, per 11 V of the culture solution, and the culture was continued for an additional 5 to 10 hours, preferably 8 hours. continue.

After centrifuging and collecting the bacterial cells and washing them with sterile water or physiological saline, add 1 part of agar to the same 1 IJ 1 medium as the first medium.
．． Bacteria are spread on an agar medium supplemented with 7%.

After static culture at 30°C for 8 days, Staphylococcus (Stap
hylococcus aureus) and cultured overnight, the strain with a large staphylococcal growth inhibition zone is a plasmid-amplifiable εPT, high-producing strain. One of these strains is strain 50833 (Feikoken Joyori No. 1110).

Production of εPL: Cultivation was performed in the same manner as in Example 1, except that the obtained plus multidomain amplification mutant strain 50833 was used, and the concentration of εPL was measured in the same manner.

The results are shown in Table 1.

Comparative Example 1 S-agonoethyl-L-cysteine resistant mutant strain 8151
εPL
The concentration of was measured in a similar manner.

The results are shown in Table 1.

Table 1 Number εPL Productivity Example 1 0.67 2 0.88 3 0.72 4 1.80 Comparative Example 1 0.20 (g/e) Example 5 Same composition as the first medium described in Example 1 Medium 1.51
0.05% by volume of a polyoxyalkylene glycol derivative antifoaming agent was added to 50 m of a culture solution prepared by pre-cultivating the S-adenoethyl L-cysteine resistant mutant strain 11011A-1.
j! inoculated, 600 rpm, air flow rate 26/a+in.
, cultured at 30°C.

After 24 hours, 5% glucose and 1% ammonium sulfate were added aseptically. After the pH was lowered, 6N sodium hydroxide was added to prevent the pH from falling below 4.0 (with automatic and continuous control using the controller). After culturing, remove the bacterial cells using a centrifuge. εPL in the culture solution was treated with anion exchange resin IRA-402 and cation exchange resin IRC-
50 and purified with activated carbon Carporafine 50w to obtain the results shown in Table 2.

Comparative Example 2 S-aminoethyl-L-cysteine resistant mutant strain 1101
1A-1 strain, Streptomyces alplus subsp. rhizinovolimerus NCl346
- Cultured in the same manner as in Example 5 except for using the D strain,
The product was purified in the same manner and the results shown in Table 2 were obtained.

Table 2 Number Yield N (g) Purity (%) Example 5
4. ．． 77 99.9 Comparative example 2 0.56 97.8

Claims (3)

[Claims] (1) Streptomyces albulus subsp. lysinopolymerus (Streptomyces
lbulussubsp. lysinopolymers
us) A bacterial strain is mutated into a mutant strain that is resistant to an analog substance of L-lysine, the resulting mutant strain is cultured in a medium, and epsilon-poly-L-lysine is produced and accumulated in the culture medium. Epsilon, which is characterized by collecting
Method for producing poly-L-lysine. (2) The analog substance of L-lysine is S-aminoethyl-L-cysteine or this S-aminoethyl-L-cysteine.
- The manufacturing method according to claim 1, wherein one or more substances selected from L-threonine, glycine, L-homoserine, and L-methionine are added to cysteine. (3) The mutant strain is Streptomyces albulus subsp.
ycesalbulussubsp. lysinopo
lymerus) No. 11011A-1 strain, a mutant strain resistant to glycine added to S-aminoethyl-L-cysteine of strain 346-D (Feikoken Joyori No. 1109)
2. The manufacturing method according to claim 1, which is