JPH0342070B2 - - Google Patents

Description

[Detailed description of the invention]

(Industrial Application Field) The present invention relates to a strain that improves the productivity of epsilon-poly-L-lysine (hereinafter abbreviated as εPL) and produces it in significant amounts. (Prior art and its problems) εPL is a polymer compound in which the amino group at the ε position of L-lysine is bonded to the carboxylic acid of the adjacent L-lysine through an amide bond, as shown in the structural formula below. It is. The substance is a polymer of L-lysine, which is an essential amino acid, so it is highly safe and has unique physical properties because it has a high cation content. Therefore, these properties can be expected to be used in toiletry products, cosmetics, feed additives, medicines, agricultural chemicals, food additives, electronic materials, etc. Conventionally, the substance has been used to produce Streptomyces albulus subsp., an εPL-producing bacterium belonging to the genus Streptomyces.
lysinopolymerus) No. 346-D strain
No. 3834) is cultured in a medium, and the resulting culture is isolated and purified (Special Publication No. 59-20359).
issue). However, the strain of this prior application had a productivity of only about 0.5 g of εPL per culture solution at most, and therefore the production cost was high, which prevented the widespread use of this substance. The present inventors obtained a strain that produces a significant amount of εPL,
We have conducted extensive research with the aim of producing large amounts of εPL using this, and have arrived at the invention described below. (Means for Solving the Problems) The present invention provides a mutant strain that improves the productivity of εPL and produces a significant amount of εPL, which is obtained by mutating a strain that produces εPL. Furthermore, the present invention is characterized by using this mutant strain, culturing it in a medium, producing and accumulating a significant amount of εPL in the culture solution, and collecting it. The mutant strain is a strain that produces a significant amount of εPL, and is preferably a mutant strain of Streptomyces albulus subsp. lysinopolymerus No. 346-D strain that is resistant to L-lysine analog substances. The analog substance of L-lysine is S-aminoethyl-L-cysteine, or one or more selected from L-threonine, glycine, L-homoserine, and L-methionine to this S-aminoethyl-L-cysteine. Preferably, a seed substance is added. The present invention will be explained in detail below. First, the method for obtaining the bacterial strain of the present invention will be described. L-
A mutant strain resistant to a ricin analog substance, for example, a mutant strain resistant to S-aminoethyl-L-cysteine, is obtained, for example, by the following method. Spores of Streptomyces albulus subsp. Rhizinopolymerus No. 346-D strain were suspended in Tris-maleate buffer (PH9.0),
To this is added N-methyl-N-nitro-N'-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, L-cysteine, and
- One or more amino acids selected from threonine, L-homoserine, and L-methionine are added to an agar medium having the same composition as the above medium. At that time, S-aminoethyl-
L-cysteine, or S to the same concentration
-Aminoethyl-L-cysteine and agar medium 1ml
The above-mentioned amino acids are added at a concentration of 0.2 to 5 mg, preferably 1 mg. The above culture dilution solution is applied to this agar medium. After keeping this agar medium warm, the strain that grew as a colony is an S-aminoethyl-L-cysteine resistant mutant strain. At this time, the strain grown on the agar medium to which only S-aminoethyl-L-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 was the resistant mutant strain 81512. The resistant mutant strain 11011A-1 (Feikoken Jokyo No. 1109) is the resistant mutant strain 81502, and the strain grown on an agar medium containing S-aminoethyl-L-cysteine and L-threonine is the resistant mutant strain 81502.
It is a stock. Among these mutant strains, the mycological properties of strain 11011A-1 are as follows. (1) Morphological properties At 30℃ on sucrose/nitrate agar medium.
The results of microscopic observation of aerial mycelia and basal mycelia of strain 11011A-1 grown for 10 days are shown below. Branching method and morphology of spore-forming hyphae: Simple branching, closed spiral Number of spores: Several dozen Surface structure and size of spores: Spores are circular or oval in shape, and the size is approximately 1.2~
It is 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: on aerial mycelia (2) Growth status on various media The following properties on various media are 30.
These are the observation results after culturing at ℃ for 10 to 14 days.

[Table] (3) Physiological properties The physiological properties of the 11011A-1 strain are as follows. Growth temperature range: approximately 15-40℃. Optimum temperature for growth: around 30℃. Liquefaction of gelatin, hydrolysis of starch and peptonization of skimmed milk: All positive Coagulation of skimmed milk: Negative Production of melanin-like pigments Produces a brown pigment on tyrosine agar. Cell Wall Composition The type of diaminopimelic acid in the cell wall composition was analyzed by the method of Becker et al. [see Applied Microbiology Vol. (4) Assimilation of various carbon sources (on Pridham-Gotzlieb agar medium) L-arabinose - D-xylose - D-glucose + D-fructose + L-rhamnose - D-galactose + seuucrose - raffinose - D-mannitol + i -Inositol + Salicin - Note) +: Assimilated, -: Not assimilated. As described above, the mycological properties of the mutant strain of the present invention are similar to those of the original strain Streptomyces albulus subsp. lysinopolymerus No.
The mycological properties are similar to those of the 346-D strain. Next, εPL is produced by the method of the present invention using the mutant strains obtained by these methods. In addition, % in the sentence
Unless otherwise specified, weight (g)/volume (ml)%
shows. First, the obtained mutant strain is inoculated into a medium and cultured, and the εPL produced and accumulated is separated and purified from the culture solution. The medium may be any medium as long as it contains a carbon source, nitrogen source, inorganic salts, and vitamins, but preferably contains 5% glucose or 5% glycerin as a carbon source, and ammonium sulfate or L-lysine as a nitrogen source. Or something containing peptone is better. A carbon source and a nitrogen source may be added sequentially during the cultivation. The pH may be allowed to drop to 4.0 in the early stage of culture, and then maintained at 4.0 with an alkali such as an aqueous sodium hydroxide solution. After removing bacterial cells from the culture solution using a centrifuge or filter, the filtrate is purified and decolorized, and then concentrated. Crystallize εPL from the concentrated solution using an organic solvent such as acetone or ethanol. (Effects of the Invention) The mutant strain of the present invention has the ability to improve the productivity of εPL and produce it in a significant amount, and by culturing the mutant strain, it is possible to improve the productivity of εPL, and by culturing the mutant strain, it is possible to improve the productivity of εPL. Since εPL can be produced in significant quantities, the production cost of εPL can be significantly reduced compared to conventional methods. (Examples) Hereinafter, the present invention will be described in detail with reference to Examples. Example 1 Obtaining S-aminoethyl-L-cysteine resistant strain: Streptomyces albulus subsp. lysinopolymerus
lysinopolymerus) No. 346-D strain in tris-maleate buffer (PH).
9.0) N-methyl-N-nitro-N'-nitrosoguanidine was added to the suspension to a concentration of 1.5 mg/ml. After shaking this at 30°C for 30 minutes, the spores were collected using a centrifuge and washed with sterile water. Salt 0.136%, Disodium monohydrogen phosphate 12 hydrate 0.158%, Magnesium sulfate 7 hydrate
0.05%, zinc sulfate heptahydrate 0.004%, ferrous sulfate heptahydrate 0.003%, pH 6.8 medium (hereinafter referred to as the first medium) 5 ml was inoculated and cultured with shaking at 30°C overnight. Bacteria were grown. The culture solution is diluted 5000 times with MS solution (composition: 0.05% magnesium sulfate heptahydrate, 0.5% sodium chloride, 0.05% Tween 80). Then,
This diluted culture solution was mixed with S-aminoethyl-L-cysteine at a concentration of 2 mg per ml of agar medium, or with S-aminoethyl-L-cysteine at a concentration of 1 mg per ml of agar medium.
It was applied to 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 . This agar medium was kept at 30° C. for 48 hours to grow as a colony, and an S-aminoethyl-L-cysteine resistant mutant strain was obtained. Among these, one strain in the agar medium to which only S-aminoethyl-L-cysteine was added is strain 81512.
One strain in the agar medium prepared by adding glycine to S-aminoethyl-L-cysteine is strain 11011A-1 (Feikoken Joki No. 1109). S-Aminoethyl-
One strain in the agar medium containing L-cysteine and L-threonine is strain 81502. Production of εPL: One platinum loop of S-aminoethyl-L-cysteine resistant strain 81512 was inoculated into 5 ml of a medium having 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 Itzhaki. The results are shown in Table 1. Examples 2 and 3 S-aminoethyl-L-cysteine resistant mutants
Instead of the 81512 strain, S-aminoethyl-L-cysteine + glycine-resistant mutant 11011A-1 strain (Feikoken Jokyo No. 1109) (Example 2), S-aminoethyl-L-cysteine + L-threonine Culture was performed in the same manner as in Example 1, except that the resistant mutant strain 81502 strain (Example 3) was used, and the concentration of εPL was measured in the same manner. The results are shown in Table 1. Example 4 Obtaining a plasmid-amplifiable mutant strain: The S-aminoethyl-L-cysteine resistant mutant strain obtained in Example 1 was transferred to the first strain described in Example 1.
Inoculate 5 ml of a medium with the same composition as the medium. After culturing this with shaking at 30°C for 2 days, 50 to 500 mg of chloramphenicol was added per culture solution.
It is preferably added to a concentration of 100 mg, and the culture is continued for an additional 5 to 10 hours, preferably 8 hours. The cells are collected by centrifugation, washed with sterile water or physiological saline, and then plated on an agar medium with the same composition as the first medium plus 1.7% agar. After statically culturing at 30°C for 8 days, a regular agar medium containing Staphylococcus aureus was overlaid and cultured overnight.
It is a high producing strain. One of these strains is strain 50833 (Feikoken Joyori No. 1110). Production of εPL: Cultured in the same manner as in Example 1 except that the obtained plasmid amplification mutant strain 50833 was used.
The concentration of εPL was also measured in a similar manner. The results are shown in Table 1. Comparative example 1 S-aminoethyl-L-cysteine resistant mutant strain
Instead of strain 81512, Streptomyces albulus subsp. rhizinopolymerus No.
The cells were cultured in the same manner as in Example 1, except that the 346-D strain was used, and the concentration of εPL was measured in the same manner. The results are shown in Table 1.

[Table] Example 5 Medium with the same composition as the first medium described in Example 1
Add 0.05% by volume of polyoxyalkylene glycol derivative antifoaming agent to 1.5, and add S-aminoethyl-
Inoculate 50 ml of culture solution pre-cultured with L-cysteine resistant mutant strain 11011A-1, and heat at 600 rpm and aeration rate 2.
/min., and cultured at 30°C. After 24 hours, 5% glucose and 1% ammonium sulfate were added aseptically. After the pH decreased, 6N sodium hydroxide was added under automatic and continuous control using a pH controller to prevent the pH from falling below 4.0. After culturing, the bacterial cells were removed using a centrifuge, and the εPL in the culture solution was purified using anion exchange resin IRA-402, cation exchange resin IRC-50, and activated carbon Carborafine 50W to obtain the results shown in Table 2. Comparative example 2 S-aminoethyl-L-cysteine resistant mutant strain
The cells were cultured in the same manner as in Example 5, except that Streptomyces albulus subsp. Lysinopolymerus No. 346-D strain was used instead of the 11011A-1 strain, and the results were purified in the same manner as in Table 2. I got it.

【table】

Claims (1)

[Claims] 1. Streptomyces albulus subsp. lysinopolymerus
lysinopolymerus) strain, which produces epsilon-poly-L-lysine that is resistant to L-lysine analog substances. 2. The L-lysine analog substance is S-aminoethyl-L-cysteine, or a type selected from L-threonine, glycine, L-homoserine, and L-methionine to this S-aminoethyl-L-cysteine. Or, the strain according to claim 1, which is added with two or more kinds of substances. 3 The strain that produces epsilon-poly-L-lysine is Streptomyces albulus subsp.
lysinopolymerus) No. 346-D strain, which is resistant to S-aminoethyl-L-cysteine and glycine added to the mutant strain 11011A-1 (Feikoken Jokyo No. 1109). The strain according to item 1.