JPH0342075B2 - - Google Patents

Description

[Detailed description of the invention]

(Industrial Application Field) The present invention relates to a method for producing epsilon-poly-L-lysine (hereinafter abbreviated as εPL). (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 in 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 providing a method for producing large amounts of εPL using this, and have arrived at the invention described below. (Means for Solving the Problems) The present invention uses epsilon-polysiloxane, which is a plasmid containing a gene involved in the production of εPL, using L-lysine as a raw material and amplifying a plasmid containing a gene involved in the production of εPL. - Production of εPL, which comprises culturing a bacterial strain that produces L-lysine in a medium containing L-lysine or L-lysine and sugars, producing and accumulating εPL in the culture solution, and collecting the εPL. It is the law. The strain that produces εPL is a strain that produces a significant amount of εPL, and Streptomyces albulus
Subspecies Rhizinopolymeras No.346-D
A plasmid amplified mutant strain in which the plasmid of the strain is amplified is preferred. The present invention will be explained in detail below. A plasmid-amplified mutant strain is obtained by performing a treatment to amplify a plasmid, and is obtained, for example, by the following method. Streptomyces albulus subsp. lysinopolymerus No. 346-D strain or S-aminoethyl-L-cysteine resistant strain was inoculated into the medium, cultured with shaking, chloramphenicol was added, and further cultured. Continue. The bacteria are collected by centrifugation, washed, and then spread on an agar medium. After static culture, a regular agar medium containing Staphylococcus aureus was overlaid, and the resulting staphylococcus strains with a large growth inhibition zone were plasmid-amplified εPL high-producing strains. An example of such a plasmid amplified mutant strain is strain 50833 (Feikoken Joyori No. 1110).
The mycological properties of the 50833 strain are as follows. (1) Morphological properties At 30℃ on sucrose/nitrate agar medium.
The results of microscopic observation of the aerial and basal hyphae of strain 50833 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 spores is not observed. Spore stalk settlement position: on aerial mycelia (2) Growth status on various media The properties on the following various media are different from each other.
These are the observation results after culturing at 30°C for 10 to 14 days.

【table】

[Table] (3) Physiological properties 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 pigment Produces a brown pigment on tyrosine agar medium. 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, plasmid amplified mutant strains
The mycological properties of strain 50833 are similar to those of the original strain Streptomyces albulus subsp. lysinopolymerus No. 346-D. Next, εPL is produced by the method of the present invention using the mutant strain obtained by this method. Note that the percentages in the text are weight (g)/volume (ml)% unless otherwise specified. First, the obtained mutant strain was treated with L-lysine or L-lysine.
- Inoculate and culture in a medium supplemented with lysine and sugars, and separate and purify εPL produced and accumulated from the medium containing the culture (hereinafter referred to as culture solution). The medium may be any medium as long as it contains a carbon source, a nitrogen source, an inorganic salt, and pitamine, but preferably contains 5% budo sugar or 5% glycerin as a carbon source, and ammonium sulfate or peptone as a nitrogen source. It's good to include something. L-lysine and saccharides may be added at the early or late stage of the culture, but preferably in the middle stage, when the pH begins to fall. L-lysine to be added is used in the range of 0.05 to 2% of the entire culture solution as L-lysine monohydrochloride, but preferably 0.5% of L-lysine monohydrochloride is added. One or more types of saccharides selected from the group of saccharides such as glucose, sucrose, maltose, starch, and lactose, and glycerin are used in an amount of 0.5 to 5% of the entire culture solution, but preferably glucose is used. It is best to add 2.5%. In the case of sequential addition, sugars and L-lysine are added when the sugar concentration in the culture solution falls below a predetermined percentage. For example, when the glucose concentration falls below 0.1%, it is preferable to add 2.5% glucose and 0.5% L-lysine. In continuous addition, the concentration of sugars, such as glucose, in the culture solution is adjusted to 1%, and the concentration of L-lysine is adjusted to 1%.
For example, add glucose solution and L- to maintain the concentration at 0.2%.
It is better to pass ricin solution through the culture tank and drain the culture solution. Furthermore, an antifoaming agent may be added to the culture solution. 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 passed through an anion exchange resin column to remove most of the impurities, then purified through a cation exchange resin column, decolorized with activated carbon, and concentrated. Add an organic solvent such as alcohol or acetone to the concentrate to crystallize εPL. (Effects of the Invention) According to the present invention, when culturing a mutant strain of a bacterial strain that produces εPL, L-lysine or L-lysine is added to the culture solution.
- By adding lysine and sugars, productivity can be improved and εPL can be produced in significant amounts. Therefore, 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 1 Obtaining S-aminoethyl-L-cysteine resistant strain: Streptomyces albulus subsp.
Seeds lysinopolymerus (Streptomyces)
lysinopolymerus) No. 346-D strain in Tris-maleic acid buffer (PH).
9.0) N-methyl-N-nitro-N'-nitrosoguanidine was added to the suspension at a concentration of 1.5 mg/ml. This was done for 30 minutes at 30°C.
After shaking, the spores are collected using a centrifuge.
Washed with sterile water, glucose 5%, ammonium sulfate 1%, yeast extract 0.5%, monopotassium dihydrogen phosphate heptahydrate 0.136%, disodium monohydrogen phosphate dodecahydrate 0.158%, magnesium sulfate heptahydrate. salt
0.05% zinc sulfate heptahydrate, 0.004% ferrous sulfate heptahydrate, 0.003% ferrous sulfate heptahydrate, pH6.8 medium (hereinafter referred to as the above medium) 5 ml was inoculated and cultured with shaking at 30°C overnight. Bacteria were grown. The culture solution was 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 above medium to which glycine or L-threonine was added at a concentration of . This was incubated at 30° C. for 48 hours and grown as a colony to obtain an S-aminoethyl-L-cysteine resistant strain. Acquisition of plasmid amplified mutant strain: S-aminoethyl-L thus obtained
- Inoculate the cysteine-resistant strain into 5 ml of a medium with the same composition as the above medium. After culturing this with shaking at 30°C for 2 days, 50 to 500 mg of chloramphenicol was added to each 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 applied to an agar medium containing the same composition as the above medium plus 1.7% agar. After statically culturing at 30°C for 8 days, an ordinary agar medium containing Staphylococcus aureus was overlaid, and the resulting staphylococcus strains with a large growth inhibition zone were determined to be plasmid-amplified εPL high-producing strains. be. One of these strains is strain 50833 (Feikoken Joyori No. 1110). Production of εPL: The same composition as the above medium plus L-lysine 1
One platinum loopful of plasmid amplified mutant strain 50833 was inoculated into 5 ml of a pH 6.8 medium supplemented with 0.5% hydrochloride.
The cells were cultured with shaking at ℃ for 8 days. After completion of the culture, the concentration of εPL in the culture solution was measured by the method of Itzhaki. The production amount of εPL per culture solution was 1.85 g. Comparative Example 1 Culture was performed in the same manner as in Example 1, except that Streptomyces albulus subsp. Lysinopolymerus No. 346-D strain was used instead of the plasmid amplified mutant strain 50833, and the concentration of εPL was Measured using the same method. The production amount of εPL per culture solution was 0.16 g. Example 2 Plasmid amplified mutant strain 50833 was added to a medium 1.5% with the same composition as the above medium and 0.05% by volume of a polyoxyalkylene glycol derivative antifoaming agent.
50 ml of the culture solution in which the strain had been precultured was inoculated, and cultured with aeration and stirring at 30°C for 8 days. When PH starts to decrease, glucose 2.5%, L-lysine monohydrochloride 0.5%
was added aseptically. From then on, the glucose concentration was adjusted to 2.5% to prevent the glucose concentration in the culture solution from falling below 2%.
% were added sequentially aseptically. After PH decreases, PH is 4.0
Add 6N sodium hydroxide to a pH not below
It was added under continuous automatic control using a controller. After culturing, the bacterial cells were removed using a centrifuge, and the concentration of εPL in the culture solution was measured by the method of Itzhaki. The production amount of εPL per culture solution was 20.3 g. Comparative Example 2 Culture was performed in the same manner as in Example 2, except that Streptomyces albulus subsp. Lysinopolymerus No. 346-D strain was used instead of the plasmid amplification mutant strain 50833, and the concentration of εPL was Measured using the same method. The production amount of εPL per culture solution was 0.20 g. Example 3 Plasmid amplified mutant strain 50833 was added to a medium 1.5% with the same composition as the above medium and 0.05% by volume of a polyoxyalkylene glycol derivative antifoaming agent.
Inoculate the strain into 50 ml of pre-cultured culture solution, and incubate at 600 rpm.
Culture was carried out at 30°C with an aeration rate of 2/min. After 24 hours, the pH started to decrease, so the glucose concentration in the culture solution was adjusted to 1%, and the L-lysine concentration was increased to 1%.
Glucose solution and L-lysine solution were passed through the culture tank so as to maintain the concentration at 0.2%, and the culture solution was discharged. 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 Carbo Rhine 50W, and crystallized with alcohol, resulting in a purity of 99.9% by weight. ,
A yield of 5.02 g of εPL was obtained.

Claims (1)

[Scope of Claims] 1. Streptomyces albulus subsp. lysinopolymerus
albulus subsp.lysinopolymerus) strain to produce epsilon-poly-L-lysine using chloramphenicol to amplify a plasmid containing the gene involved in the production of epsilon-poly-L-lysine using L-lysine as a raw material. Epsilon-poly-L-lysine is produced and accumulated in the culture solution by culturing a bacterial strain containing L-lysine in a medium supplemented with L-lysine, and the epsilon-poly-L-lysine is collected.
Method for producing L-lysine. 2 The strain that produces epsilon-poly-L-lysine is Streptomyces albulus subsp.
50833, a plasmid amplified mutant strain of No. 346-D strain (Kaikokenjoyokyo)
1110) according to claim 1. 3 Streptomyces albulus subsp. rhizinopolymerus
albulus subsp.lysinopolymerus) strain to produce epsilon-poly-L-lysine using chloramphenicol to amplify a plasmid containing the gene involved in the production of epsilon-poly-L-lysine using L-lysine as a raw material. epsilon-poly-L-lysine, which is characterized by culturing a bacterial strain containing L-lysine and saccharides in a medium to which epsilon-poly-L-lysine is produced and accumulated in the culture solution, and collecting the epsilon-poly-L-lysine. manufacturing method. 4. The manufacturing method according to claim 3, wherein L-lysine and saccharide are added sequentially. 5. The manufacturing method according to claim 3, wherein the addition of L-lysine and saccharide is continuous addition. 6 The strain that produces epsilon-poly-L-lysine is Streptomyces albulus subsp.
50833, a plasmid amplified mutant strain of No. 346-D strain (Kaikokenjoyokyo)
1110) according to claim 3.