JPH0767675A - Production of epsilon-poly-l-lysine - Google Patents

Abstract

PURPOSE:To smoothly obtain an epsilon-poly-L-lysine 2-24 in polymerization degree in a high yield. CONSTITUTION:An epsilon-poly-L-lysine 2-24 in polymerization degree can be obtained by using the resting microbes or crude enzyme liquor of Streptomyces albulus subsp. lysinopolymerus as the epsilon-poly-L-lysine-productive strain.

Description

Detailed Description of the Invention

[0001]

The present invention relates to ε-having a degree of polymerization of 2-24.
It relates to a method for producing poly-L-lysine. More specifically, ε-poly-L-lysine having a degree of polymerization of 25 or more is an ε-producing bacterium, Streptomyces albus subsp.
Seeds Lysino Polymerase ( Streptomyces albulus sub
resting cells of St. sp. lysinopolymerus or Streptomyces albulus sub sp. lysinopolymer
us )) The degree of polymerization is characterized by degrading with a crude enzyme solution of 2).
24 relates to a method for producing ε-poly-L-lysine.

[0002]

2. Description of the Related Art Epsilon-poly-L-lysine having a degree of polymerization of 25 to 35 is known to have an antibacterial activity against various fungi such as Gram-positive bacteria, Gram-negative bacteria, fungi and yeasts. And is used as a food preservative based on such properties. Further, since it forms a gel at room temperature by the reaction with milk whey protein, it has been proposed to be used as a physical property improving agent for foods.

However, in the gelation of milk whey protein, a gel having practically satisfactory physical properties cannot be prepared unless ε-poly-L-lysine is added at a high concentration to a high-concentration protein suspension, and However, a long reaction time is required. Therefore, a low degree of polymerization of about 5 to 24 ε-
A method has been proposed in which a gel can be prepared in a short time by using poly-L-lysine, which has necessary and sufficient physical properties even when the protein concentration and ε-poly-L-lysine concentration are low. However, the low degree of polymerization used for this is ε-
Conventionally, poly-L-lysine has been obtained by a hydrolysis reaction with an acid or an alkali, but it is very difficult to control the hydrolysis reaction and to purify the product after the hydrolysis by a method using a hydrolysis reaction with an acid or an alkali. In addition, there is a problem that the degree of polymerization is difficult to control and the degree of polymerization distribution is widened.

There is also known a method of hydrolyzing ε-poly-L-lysine using a neutral protease produced by the genus Aspergillus (JP-A-4-287693), but the time required for hydrolysis is known. Since it requires a long time of 24 to 40 hours and the reaction conditions are limited to around pH 7, when the hydrolysis reaction is performed, the substrate ε-poly-L is used.
-Because lysine exhibits a strong basicity, it binds to an enzyme protein, or ε-poly-L-lysine, which is a hydrolyzate and has a degree of polymerization of 2 to 24, also binds to an enzyme protein to give a desired degree of polymerization of 2 to The yield of 24 ε-poly-L-lysine may be significantly reduced.

[0005]

DISCLOSURE OF THE INVENTION The inventors of the present invention have earnestly studied a method for easily and efficiently obtaining ε-poly-L-lysine having a degree of polymerization of 24 or less, which is highly effective in improving the physical properties of foods. As a result, ε-poly-L-lysine having a degree of polymerization of 25 to 35 or ε-poly-L-lysine having a degree of polymerization of not less than ε-poly-L-lysine, which is a ε-poly-L-lysine producing bacterium, is Streptomyces albus Streptomyces albulus sub sp. Lysinopolymerus
The present invention was completed by finding that ε-poly-L-lysine having a degree of polymerization of 24 or less can be easily and efficiently obtained by hydrolyzing the resting microbial cells or the crude enzyme solution. As is clear from the above description,
An object of the present invention is to provide a method for easily and efficiently producing ε-poly-L-lysine having a degree of polymerization of 24 or less. In addition, ε-poly-L-lysine is represented by the following chemical formula 1.

[Chemical 1]

[0006]

The present invention has the following constitution. (1) ε-poly-L-lysine having an average degree of polymerization of 25 or more is ε
-Streptomyces, which is a poly-L-lysine-producing bacterium
Albrus Subspace Seeds LysinoPolymeras ( St
of reptomyces albulus sub sp. lysinopolymerus ) characterized by being decomposed by resting cells of reptomyces albulus sub sp.
A method for producing poly-L-lysine. (2) ε-poly-L-lysine having an average degree of polymerization of 25 or more is ε
-Streptomyces, which is a poly-L-lysine-producing bacterium
Albrus Subspace Seeds LysinoPolymeras ( St
of reptomyces albulus sub sp. lysinopolymerus ) with a degree of polymerization of 2 to 24 characterized by being decomposed by a crude enzyme solution
A method for producing poly-L-lysine. (3) Streptomyces albulus s u b s
(p. lysinopolymerus ) crude enzyme solution is a crude enzyme solution containing ε-poly-L-lysine degrading enzyme, ε-
A method for producing poly-L-lysine.

Ε-having a degree of polymerization of 25 or more used in the present invention
Poly-L-lysine is, for example, described in JP-A-59-20359, Streptomyces albus subsp. Streptomyc
es albulus sub sp. lysinopolymerus ) is cultured in a medium, and ε-poly-L-lysine is separated from the obtained culture,
It can be obtained by collecting. Most of the raw material ε-poly-L-lysine may have a degree of polymerization of 25 or more, and even if it has only a single degree of polymerization,
It may be a mixture of various degrees of polymerization. It may also contain a small amount of ε-poly-L-lysine having a degree of polymerization of 24 or less.

The ε-poly-L-lysine-producing bacterium used in the present invention is Streptomyces albulus (Streptomyces albulus).
subsp. lysinopolymerus), preferably Streptomyces albus subsp.
Seeds LysinoPolymerus (Streptomyces albulus sub
sp. lysinopolymerus) No. Strain 346-D (Microtechnical Research Institute No. 3834) or Streptomyces albula subspides lysinopolymeris (Streptom
yces albulus sub sp. lysinopolymerus) No. 110
11A-1 strain (Ministry of Industrial Technology Article 1109).

Streptomyces albulus Streptomyces albulus
sub sp. lysinopolymerus) No. 346-D strain and No. The 11011A-1 strain has the following mycological properties. (1) Morphological properties No. 1 grown on sucrose / nitrate agar medium at 30 ° C. for 10 days. 346-D strain and No. 11011A
The results of observing the aerial hyphae and basal hyphae of the -1 strain with a microscope are shown below. Branching method and morphology of sporulating hyphae: simple branching, closed spiral Number of spores: several tens Surface structure and size of spores: spores are circular or elliptic and size is about 1.2- 1.5 μ and its surface structure is spiny. Presence or absence of flagella spores, sclerotium and sporangia is not observed. Position of spore stalk: on aerial mycelium

(2) Growth state on various media The properties on the following various media are 10 at 30 ° C.
It is an observation result after culturing for 14 days. The results are shown in Tables 1 and 2.

[0011]

[Table 1]

[0012]

[Table 2]

(3) Physiological properties No. Strain 346-D (Mikoken Kenjoyori No. 3834) and No. 11011A-1 strain (Ministry of Fine Arts, Article 1109)
Has the following physiological properties. Growth temperature range: about 15-40 ° C. Optimal growth temperature: around 30 ° C. Liquefaction of gelatin, hydrolysis of starch and peptization of skim milk: All positive. Coagulation of skim milk: Negative. Formation of melanin-like pigment: A brown pigment is formed in tyrosine medium. Cell wall composition: Regarding the type of diaminopimelic acid in the cell wall composition component, the method of Becker et al. [Applied Macrobiology Vol. 13, p. 236 (1
965))], and it was L, L type.

(4) The assimilability of various carbon sources is shown in Table 3.

[Table 3]

In the present invention, ε-poly-L-lysine having a degree of polymerization of 2 to 24 can be produced by using any of these strains.

The method using resting cells is a method in which a hydrolysis reaction is carried out by using a bacterium whose growth is suppressed. The resting cells used in the hydrolysis reaction are ε-poly-L.
- is a lysine-producing bacterium Streptomyces albulus-Sabusupi - Cities -'s Rijinoporimerasu (Streptomyces
albulus sub sp. lysinopolymerus ) is cultivated and the fungus is grown sufficiently. After this, the bacteria are recovered by centrifugation,
The collected bacteria are contained in a sodium phosphate buffer solution or sodium phosphate solution of about 0.1M, which is 2 to 3 times the weight of the wet cells.
It can be obtained by repeatedly washing 2-3 times with a physiological saline buffer solution or the like.

The above-mentioned hydrolysis reaction of ε-poly-L-lysine using resting cells is performed with ε having a polymerization degree of 25 or more as a raw material.
-The poly-L-lysine is preferably carried out in the form of an aqueous solution or dispersion. The hydrolysis reaction is carried out by suspending the obtained bacterial cells in a phosphate buffer solution (pH 5 to 9) to obtain ε with a polymerization degree of 25 or more.
-Add poly-L-lysine to the liquor. After this, 30 ℃,
The hydrolysis reaction is performed for 5 to 20 hours with stirring at about 300 rpm. The reaction time of the hydrolysis reaction depends on the required εε
Since it depends on the degree of polymerization of poly-L-lysine, the reaction solution is sampled over time during the reaction to obtain the desired degree of polymerization ε.
-It is desirable to analyze the proportion of poly-L-lysine by a paired ion chromatography method or the like to confirm the status of the hydrolysis reaction and take measures such as continuing or stopping the reaction. Generally, the reaction is carried out until about 80% by weight or more of ε-poly-L-lysine having a polymerization degree of 25 or more disappears.

The crude enzyme solution to be used in the present invention is prepared by the method of producing ε-poly-L-lysine, Streptomyces albus subsp.
omyces albulus sub sp. lysinopolymerus ) is cultivated to fully grow the fungus. Thereafter, the bacteria are collected by centrifugation or the like. The collected bacteria are suspended in a buffer solution, the cells are crushed by ultrasonic waves, and the crushed suspension is removed to obtain a crude enzyme solution. This crude enzyme solution may be partially purified by dialysis, ultrafiltration, nucleic acid removal, or the like. In addition, this crude enzyme solution may be used after being immobilized on a carrier or the like. The crude enzyme solution prepared by this method has a protein concentration of about 1 to 5 mg / ml.

The hydrolysis reaction of ε-poly-L-lysine with this crude enzyme solution is carried out in an aqueous solution prepared by dissolving ε-poly-L-lysine having a polymerization degree of 25 or more, which is a raw material, in water or water dispersed in water. It is preferably carried out in the form of a dispersion. The conditions of the hydrolysis reaction differ depending on the activity of ε-poly-L-lysine degrading enzyme in the obtained crude enzyme solution and the required degree of polymerization of ε-poly-L-lysine, but generally, pH 5-9,
The reaction temperature is about 28 to 40 ° C., and the reaction time is preferably 5 to 20 hours. The reaction is generally terminated until about 80% by weight or more of ε-poly-L-lysine having a polymerization degree of 25 or more disappears.

Ε-Poly-L- having a degree of polymerization of 25 or more
Lysine is hydrolyzed by the hydrolysis reaction by the resting cells of the present invention or the hydrolysis reaction by the crude enzyme solution to gradually become a low polymerization degree ε-poly-L-lysine having a polymerization degree of 24 or less. At this time, when the hydrolysis is excessively performed, the amino acid finally becomes L-lysine, and the desired ε-poly-L-lysine having a polymerization degree of 2 to 24 cannot be obtained or even if it is obtained. The yield is low. Therefore, in the hydrolysis reaction, the reaction solution is sampled over time during the hydrolysis reaction to obtain the desired degree of polymerization of 2-24 ε-poly-
The ratio of L-lysine was measured by paired ion chromatography.
It is desirable to conduct analysis by the method and formation of a complex with methyl orange to confirm the status of the hydrolysis reaction, and to take measures such as continuing and stopping the reaction. The paired ion chromatography method can measure the amount of ε-poly-L-lysine present in each residue of L-lysine, but the analysis takes time. In addition, the method by complex formation with methyl orange can quantify ε-poly-L-lysine by the method of Itzaki, and the degree of polymerization is about 10 or less ε-.
It is a method that can be easily measured by utilizing the fact that poly-L-lysine does not form a complex with methyl orange.

Usually, as described above, the hydrolysis reaction is terminated when about 80% by weight or more of the raw material ε-poly-L-lysine having a polymerization degree of 25 or more is hydrolyzed. Is good. The method for stopping the hydrolysis reaction is to deactivate the enzyme by heating or other suitable method,
Alternatively, in the case of resting cells, the reaction can be stopped by adding hydrochloric acid or the like to make the pH acidic (pH 4 or less) and then removing the cells by centrifugation or filtration. When the reaction is stopped by heating, it is generally 80-
It is better to heat to 100 ° C. In this case, ε-poly-L-lysine having a low degree of polymerization, which is an object, is modified by heating,
It is hardly affected by disassembly.

Next, Streptomyces albula subspase seeds, which is an ε-poly-L-lysine-producing bacterium.
LysinoPolymerus (Streptomyces albulus sub sp. Lys
inopolymerus) 11011A-1 strain
No. 09) using a crude enzyme solution will be described. First, Streptomyces albulus
The sub sp. lysinopolymerus) 11011A-1 strain (Microtechnology Research Institute No. 1109) was cultured under ε-poly-L-lysine production conditions, and the cells were collected by centrifugation. The cells are crushed by an ultrasonic crusher and centrifuged to obtain a crude enzyme solution (protein concentration 6 mg / ml). Degree of polymerization 2
5 to 35 of ε-poly-L-lysine at a concentration of 10 mg / ml
Phosphate buffer or citrate buffer (p
H5-9) Dissolve in 3 ml, add 2 ml of crude enzyme solution, and add 3
The hydrolysis reaction is performed at 0 ° C. After the reaction was carried out for 5 to 20 hours and the reaction was stopped by heating, about 90 to 99% by weight of ε-poly-L-lysine used as a raw material was hydrolyzed to give a polymerization degree of 2 to 24. A product containing about 80% by weight or more of ε-poly-L-lysine is obtained.

Then, the above reaction solution is simply dried by spray drying, freeze drying, or any other suitable drying method to obtain a polymerization degree of 2
A powder containing a large amount of .about.24 .epsilon.-poly-L-lysine is obtained. Further, in order to obtain a product with a higher degree of purity, the reaction solution containing ε-poly-L-lysine having a polymerization degree of 2 to 24 obtained above is subjected to an ion exchange method, an ultrafiltration method, a gel. By using a separation means such as a filtration method, the target ε-poly-L-lysine having a degree of polymerization of 2 to 24 can be separated and recovered as a mixture containing only one having an arbitrary degree of polymerization or a polymer having an arbitrary degree of polymerization. .

Unlike the hydrolysis reaction using an acid or an alkali, the method of the present invention produces ε-poly-L-lysine having a degree of polymerization of 2 to 24, in which the distribution of the degree of polymerization is easy to control and salt is not contaminated. can do. Further, unlike the reaction by the neutral protease produced by Aspergillus, it is possible to react in a pH range of 5 to 9,
In particular, since the reaction can be carried out in acidic conditions (pH 5 to 6), there is no adsorption to enzyme proteins or bacterial cells and formation of a complex, and a high polymerization yield of ε-of the desired polymerization degree of 2 to 24 is obtained.
Poly-L-lysine can be produced.

In the present invention, the method for measuring the degree of polymerization of ε-poly-L-lysine is preferably the paired ion chromatography method. For example, using a reversed-phase chromatography column (L-Colum: manufactured by Chemicals Inspection Association: internal diameter 4.6 mm, length 26 cm) as an eluent, 10
Aqueous solution (solution A) containing 10 mM sodium hydrogen phosphate, 100 mM sodium perchlorate, 10 mM sodium octane sulfonate, and 10 mM sodium hydrogen phosphate.
A 50% by volume aqueous solution of acetonitrile (B solution) containing 0 mM sodium perchlorate and 10 mM sodium octanosulphonate is used. An aqueous solution of ε-poly-L-lysine was applied to the column, and the concentration of the solution B was linearly increased from 50% by weight to 75% by weight in a mixed solution of the solutions A and B for 40 minutes. Elute at a flow rate of 0.0 ml / min. This eluate is detected by an ultraviolet absorption spectrum having a wavelength of 215 nm. As a result, ε-poly-L-lysine becomes L
-Detected as peaks separated for each lysine residue, whereby the average degree of polymerization of ε-poly-L-lysine provided can be determined.

[0026]

EXAMPLES The details will be described below using test examples and examples of the present invention. The present embodiment does not limit the present invention at all. Example 1 Epsilon-poly-L-lysine-producing bacterium Streptomyces albus subspides lysinopolymerase ( Strept
omyces albulus sub sp. lysinopolymerus ) No. 11
The 011A-1 strain (Mikoritsukenjoyori No. 1109) was inoculated into the medium shown in Table 1 and shake-cultured at 30 ° C. for 48 hours. After culturing, the bacterial cells were collected by centrifugation (10,000 g for 15 minutes), and washed three times with 0.1 M sodium phosphate / saline buffer (pH 7) three times the weight of the wet bacterial cells, and then washed. , 10 M sodium phosphate / physiological saline buffer solution was suspended to obtain a bacterial cell suspension. Then,
100 mg of ε-poly-L-lysine having a degree of polymerization of 25 to 35 and 2 ml of the above-mentioned cell suspension (OD660 nm: 6.6) were added to 8 ml of 0.1M sodium phosphate buffer (pH 7), and 30 ° C., 300 rpm. The reaction was carried out by shaking for 10 hours. After completion of the reaction, 0.04 M hydrochloric acid was added and centrifugation was carried out to remove bacterial cells. The liquid is spray-dried to give a powder of 92.7 m.
g was obtained. This powder is dissolved in water and paired ion chromatography using a column for reversed phase chromatography (column: L-colum: manufactured by Chemicals Inspection Society: internal diameter 4.6 m).
m, length 25 cm). Then 10 mM sodium hydrogen phosphate, 100 mM sodium perchlorate, 10 m
Aqueous solution containing M octanceulfonsan sodium (A
Solution) and 10 mM sodium hydrogen phosphate, 100 mM sodium perchlorate, 10 mM sodium octanosulphonate in 50% by volume aqueous solution (solution B) in a mixed solution of solutions A and B for 40 minutes. Elution was performed at a flow rate of 1.0 ml / min with a concentration gradient in which the concentration of solution B increased linearly from 50% to 75%. This eluate was detected by an ultraviolet absorption spectrum method with a wavelength of 215 nm. FIG. 1 shows ε-poly-L-lysine before the reaction, and FIG. 2 shows a chromatogram of the sample after the reaction. As a result, the powder obtained in Example 1 contained about 70% by weight of ε-poly-L-lysine having a degree of polymerization of 2 to 24.

[0027]

[Table 4]

Example 2 Streptomyces albulus sub sp. Lysinopolymerus N, which is an ε-poly-L-lysine-producing bacterium
o. The 11011A-1 strain (Mikori Kenjojo No. 1109) was inoculated into the medium shown in Table 1 and shake-cultured at 30 ° C. for 72 hours. After culturing, centrifuge the cells (10,000g1
5 minutes). 5 g of the collected wet cells was suspended in 20 ml of 0.1 M sodium phosphate buffer (pH 7), sonicated at 5 ° C. for 20 minutes, and centrifuged (10,000 g for 20 minutes). 0.1 to the supernatant
The solution was dialyzed against M sodium phosphate buffer and centrifuged again (10,000 g for 20 minutes), and the supernatant was used as a crude enzyme solution (protein concentration 6.98 mg / ml). 50 mg of ε-poly-L-lysine having a degree of polymerization of 25 to 35
And 3 ml of the above crude enzyme solution were dissolved in 2 ml of 0.1 M sodium citrate buffer (pH 5). Reaction is 30 ℃, 8
The shaking was performed at 0 rpm. 0.5 ml of the reaction solution was sampled at the third and sixth hours of the reaction, and the degree of polymerization was examined by the paired ion chromatography method according to Example 1. The results are shown in FIGS. 3 and 4. After the hydrolysis reaction for 12 hours, the reaction solution is subjected to heat treatment to stop the reaction, followed by centrifugation (10.
(00G for 2 minutes), the supernatant was spray-dried to obtain 60 mg of powder. The obtained powder was dissolved in water and subjected to a pad ion chromatography under the conditions based on Example 1. The result is shown in FIG. As a result, Example 2
The powdery product obtained in step 1 contained about 60% by weight of ε-poly-L-lysine having a degree of polymerization of 2 to 24.

Example 3 Streptomyces albulus sub sp. Lysinopolymerus N, which is an ε-poly-L-lysine-producing bacterium
o. The medium described in Table 1 was inoculated with the strain 346-D (Microtechnical Laboratory No. 3834), and shake culture was performed at 30 ° C for 48 hours. After culturing, the bacterial cells were collected by centrifugation (10,000 g for 15 minutes), and washed three times with 0.1 M sodium phosphate / saline buffer (pH 7) three times the weight of the wet bacterial cells, and then washed. , 10 M sodium phosphate / physiological saline buffer solution was suspended to obtain a bacterial cell suspension. Then, 100 mg of ε-poly-L-lysine having a degree of polymerization of 25 to 35 and 2 ml of the above-mentioned cell suspension (OD660 nm: 6.6) were added to a sodium citrate buffer solution (pH 5), and the mixture was added at 30 ° C. for 3 days.
The mixture was shaken at 00 rpm for 10 hours to carry out the hydrolysis reaction. After completion of the reaction, 0.04 M hydrochloric acid was added and centrifugation was carried out to remove bacterial cells. The liquid is spray-dried and powder 93.5 mg
Got This powder was dissolved in water and subjected to paired ion chromatography using a column for reverse phase chromatography under the conditions according to Example 1 to obtain ε with a degree of polymerization of 2 to 24.
-Poly-L-lysine content was measured. As a result, in the powder obtained in Example 3, ε-poly-having a degree of polymerization of 2 to 24 was added.
About 80% by weight of L-lysine was contained.

Example 4 Streptomyces albulus sub sp. Lysinopolymerus N, which is an ε-poly-L-lysine-producing bacterium
o. The medium described in Table 1 was inoculated with the strain 346-D (Microtechnical Laboratory No. 3834), and shake culture was carried out at 30 ° C. for 72 hours. After culturing, the bacterial cells were collected by centrifugation (10,000 g for 15 minutes). 5 g (wet weight) of the recovered cells were suspended in 20 ml of 0.1 M sodium phosphate buffer (pH 7), ultrasonically disrupted at 5 ° C. for 20 minutes, and centrifuged (10,000 g for 20 minutes), and then 0. After dialysis against 1M sodium phosphate buffer, centrifugation (10,000 g for 20 minutes) was performed, and the obtained supernatant was used as a crude enzyme solution (protein concentration 6.01 mg / ml). 50 mg of ε-poly-L-lysine having a degree of polymerization of 25 to 35
And 3 ml of the above crude enzyme solution were dissolved in 2 ml of 0.1 M sodium phosphate buffer (pH 7). The reaction is 30 ° C, 80
It was carried out under the shaking condition of rpm. 90 ° C after reacting for 12 hours
After 10 minutes of heat treatment to stop the reaction, centrifuge (1
(10,000 g for 2 minutes), the supernatant was spray-dried to obtain 67 mg of powder. The obtained powder was dissolved in water and subjected to paired ion chromatography under the conditions according to Example 1. As a result, in the powdery product obtained in Example 4, ε-poly-L-lysine having a degree of polymerization of 2 to 24,
The content was about 58% by weight.

COMPARATIVE EXAMPLE 1 50 mg of ε-poly-L-lysine having a degree of polymerization of 25 to 35 was dissolved in 5 ml of 0.1 M sodium phosphate buffer (pH 7), and added to Denateam AP [Nagase Sangyo Co., Ltd .: 5 mg of a neutral protease produced by Aspergillus oryzae ] was added and the hydrolysis reaction was carried out at 40 ° C. for 12 hours, and then the reaction solution was heated at 90 ° C. for 10 minutes to stop the reaction, followed by centrifugation. (10,000g
After 2 minutes), the supernatant liquid is spray-dried to obtain 51 m of powder.
g was obtained. The obtained powder was dissolved in water and subjected to paired ion chromatography under the conditions according to Example 1. This chromatogram is shown in FIG. Further, as a result, the powdery product obtained in Comparative Example 1 had a degree of polymerization of 2 to
It contained about 38% by weight of 24 ε-poly-L-lysine.

Comparative Example 2 5 mg of Denazyme AP was added to a solution prepared by dissolving 50 mg of ε-poly-L-lysine having a degree of polymerization of 25 to 35 in 5 ml of 0.1 M sodium citrate buffer (pH 5) to give 40
After carrying out a hydrolysis reaction at ℃ for 12 hours,
The reaction was stopped by heating at 10 ° C. for 10 minutes, and after centrifugation (10,000 g for 2 minutes), the supernatant was spray-dried to obtain 52 mg of powder. The obtained powder was dissolved in water and subjected to paired ion chromatography under the conditions according to Example 1. This chromatogram is shown in FIG. 7. Further, as a result, it was confirmed that the powdery product obtained in Comparative Example 2 did not contain ε-poly-L-lysine having a polymerization degree of 2 to 24 at all, and the hydrolysis reaction did not proceed. Was given.

Comparative Example 3 5 ml of hydrochloric acid was added to an aqueous solution prepared by dissolving 50 mg of ε-poly-L-lysine having a degree of polymerization of 25 to 35 in 5 ml of water, and the mixture was heated to 100 ° C.
After performing a hydrolysis reaction for 3 hours, the mixture was neutralized by adding a 6N sodium hydroxide aqueous solution. After cooling, the liquid was spray-dried to obtain 3.5 g of powder. The obtained powder is mixed with 10 m of water.
Dissolve in 1 and add 6N sodium hydroxide solution
To 9. Add 3 times the amount of methanol to this solution,
The precipitate was removed with glass fiber-filter paper. The filtrate was spray dried to obtain 1.2 g of powder. The obtained powder was dissolved in water and subjected to paired ion chromatography under the conditions according to Example 1. As a result, about 2.2% by weight of ε-poly-L-lysine having a degree of polymerization of 2 to 24 was contained in the powdery product.
About 37% by weight of .about.24 .epsilon.-poly-L-lysine had been lost to the salt.

[0034]

The method of the present invention is different from the hydrolysis reaction using an acid or an alkali, and the distribution of the degree of polymerization is easily controlled, and the contamination of salts is small, and the degree of polymerization of ε-poly-L-lysine is 2 to 24. Can be manufactured efficiently and easily. Further, unlike the reaction by the neutral protease produced by Aspergillus, the reaction can be carried out in the pH range of 5 to 9, particularly in the acidic (pH 5 to 6) range, so that the enzyme protein and the bacterial cells can be obtained. It is possible to produce ε-poly-L-lysine having a desired degree of polymerization of 2 to 24 in a high yield without the adsorption of the above and formation of a complex.

[0035]

[Brief description of drawings]

FIG. 1 shows paired ion chromatography of a liquid containing ε-poly-L-lysine having a degree of polymerization of 25 to 35, and the eluate at the time of elution was detected by an ultraviolet absorption spectrum at a wavelength of 215 nm. It is a figure which shows a chromatogram.

FIG. 2 is a chromatograph in which the liquid obtained by dissolving the hydrolyzate obtained in Example 1 in water was subjected to paired ion chromatography, and the eluate when eluted was detected by an ultraviolet absorption spectrum at a wavelength of 215 nm. It is a figure which shows a gram.

FIG. 3 is a reaction time 3 in the hydrolysis reaction in Example 2.
It is a figure which shows the chromatogram which detected the eluate at the time of 215-nm wavelength ultraviolet-absorption spectrum by making the reaction liquid of the time subject to paired ion chromatography.

FIG. 4 shows a reaction time of 6 during the hydrolysis reaction in Example 2.
It is a figure which shows the chromatogram which detected the eluate at the time of 215-nm wavelength ultraviolet-absorption spectrum by making the reaction liquid of the time subject to paired ion chromatography.

FIG. 5 is a chromatograph in which the liquid obtained by dissolving the hydrolyzate obtained in Example 2 in water was subjected to paired ion chromatography, and the eluate at the time of elution was detected by an ultraviolet absorption spectrum at a wavelength of 215 nm. It is a figure which shows a gram.

FIG. 6 is a chromatograph in which the solution obtained by dissolving the hydrolyzate obtained in Comparative Example 1 in water was subjected to paired ion chromatography, and the eluate when eluted was detected by an ultraviolet absorption spectrum at a wavelength of 215 nm. It is a figure which shows a gram.

FIG. 7 shows a solution obtained by dissolving the powder obtained after the reaction of Comparative Example 2 in water was subjected to paired ion chromatography, and the eluate when eluted was detected by an ultraviolet absorption spectrum at a wavelength of 215 nm. It is a figure which shows the performed chromatogram.

 ─────────────────────────────────────────────────── --- Continuation of the front page (72) Inventor Takashi Oshiro 1-152, Kitazono, Tottori City, Tottori Prefecture (72) Inventor Shoaki Kamibayashi 128-2, Kitayama, Koyamacho, Tottori City, Tottori Prefecture (72) Inventor Jun Hiraki 10-2, Otosetsu-cho, Kanazawa-ku, Yokohama-shi, Kanagawa Prefecture (72) Inventor Kako Mizokami 10-2-2101, Otsusetsu-cho, Kanazawa-ku, Yokohama-shi, Kanagawa

Claims (3)

[Claims] 1. Streptomyces albulus sub sp. Lysinopolymeru, which is an ε-poly-L-lysine-producing bacterium, containing ε-poly-L-lysine having an average degree of polymerization of 25 or more.
s ) Degradation by resting cells, degree of polymerization 2
24. A method for producing 24-poly-L-lysine. 2. Streptomyces albulus subsp . Lysinopolymeru, which is an ε-poly-L-lysine-producing bacterium, of ε-poly-L-lysine having an average degree of polymerization of 25 or more.
s ) is decomposed by the crude enzyme solution, and the degree of polymerization is 2 to
24. A method for producing 24-poly-L-lysine. 3. Streptomyces albulus subsp.
The crude enzyme solution of us sub sp. lysinopolymerus ) is ε-poly-
The method for producing ε-poly-L-lysine according to claim 2, which is a crude enzyme solution containing L-lysine degrading enzyme.