JP3525190B2 - Strain producing ε-poly-L-lysine in remarkable quantity and method for producing ε-poly-L-lysine using the same - Google Patents

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a strain producing a large amount of ε-poly-L-lysine (hereinafter referred to as εPL) and a method for producing εPL by a fermentation method using the strain. Since εPL is a polymer of L-lysine which is an essential amino acid, it has high safety, and since it has a high cation content, it has unique physical properties. Therefore, applications such as toiletry products, cosmetics, feed additives, medicines, agricultural chemicals, food additives, electronic materials, etc. can be expected. In particular, in the field of food additives, it has attracted attention as a natural product-based additive.

[0002]

2. Description of the Related Art Conventionally, as a method of producing εPL by fermentation, Streptomyces albus subspices lysinopolymeris, which is an εPL-producing strain belonging to the genus Streptomyces isolated from nature.
albulus subsp.lysinopolymerus) No. 346-D strain (Microtechnology Research Institute No. 3834) is cultivated in a medium, and ε is extracted from the obtained culture.
A method for separating and purifying PL is known (Japanese Patent Publication No.
59-20359 publication). In addition, the Streptomyces albus subspides lysinopolymeris No. 346-D strain was mutated to a mutant strain having resistance to S- (2-aminoethyl) -L-cysteine which is an analog substance of L-lysine. The resulting mutant strain, Streptomyces
There is also known a method of culturing Albrus lysinopolymeris 11011A-1 strain (Mikoken Kenjoyori No. 1109) in a medium, separating and purifying from the resulting culture to obtain the εPL (Japanese Patent Publication No. 42070 and Japanese Patent Publication No. 3-78998). However, in order to produce inexpensive εPL that can be used for various purposes, the production amount of εPL and the yield with respect to sugar are still insufficient even if the 11011A-1 strain is used. It has been desired to develop an improved strain having a higher yield and an industrially inexpensive and efficient process for producing εPL using the improved strain.

[0003]

DISCLOSURE OF INVENTION Problems to be Solved by the Invention The present inventors have conducted extensive studies to obtain an εPL-producing strain having a higher εPL-producing ability than the conventional εPL-producing strain or its mutant strain. As a result, a high concentration of 20 mg / ml or more of S- (2-aminoethyl) -L-cysteine (hereinafter referred to as AEC)
It was found that a mutant strain resistant to E. coli becomes a strain capable of producing a large amount of εPL, and by culturing the strain aerobically in a medium, a large amount of εPL can be produced. We have found what we can do, and completed this research based on these findings. As is clear from the above description, the object of the present invention is that the εPL productivity is higher than that of the conventional εPL-producing strain or its improved strain.
And a method for producing εPL at low cost and in a large amount using the strain.

[0004]

The present invention comprises the following technical means. (1) S- (2-aminoethyl at a high concentration of 20 mg / ml or more , obtained by mutating a microorganism of Streptomyces albulus species capable of producing ε-poly-L-lysine. ) A strain producing a large amount of ε-poly-L-lysine having resistance to -L-cysteine. (2) Streptomyces albulus subsp. L
It was obtained by mutating ysinopolymerus) 11011A-1 strain (Microtechnology Research Institute No. 1109) and has a high concentration of 20 mg / ml or higher S- (2
B21021 strain (deposition number FERM P-15193) that produces a significant amount of ε-poly-L-lysine having resistance to -aminoethyl) -L-cysteine. (3) A microorganism belonging to the genus Streptomyces that is resistant to high concentrations of 20 mg / ml or more of S- (2-aminoethyl) -L-cysteine and has the ability to produce ε-poly-L-lysine. Is aerobically cultivated in a medium, and ε-poly-L-lysine produced and accumulated in the culture solution is collected, which is a method for producing ε-poly-L-lysine. (4) The ε-poly-L-lysine, which is characterized in that the B21021 strain described in the above item 2 is aerobically cultured in a medium, and ε-poly-L-lysine produced and accumulated in the culture solution is collected. Manufacturing method.

AEC used in the present invention is a structural analogue (analog substance) of L-lysine, and L-lysine means
It is a compound having a different structure except that the carbon atom at the 4-position is replaced with a sulfur atom. Addition of the AEC to the medium causes growth inhibition of the bacterium, but the combination of L-threonine further clearly shows the growth inhibition.

The 11011A-1 strain disclosed in Japanese Examined Patent Publication No. 3-42070 is an εPL-producing strain having resistance to AEC, which is an analog substance of L-lysine, but has a concentration of 2 mg / ml of A.
Screening was carried out in the presence of EC, and the growth range was only when the AEC concentration was at most 5 mg / ml, and no growth was shown when the AEC concentration was 10 mg / ml or more.

The improved strain of the present invention is a microorganism belonging to the genus Streptomyces which has been screened in the presence of AEC at a concentration of 10 mg / ml or more and has a resistance level capable of growing even at an AEC concentration of 40 mg / ml. There are some differences in mycological properties from the parent strain 11011A-1.

As a parent strain used for obtaining such an improved strain, a microorganism belonging to the genus Streptomyces that produces εPL can be used, but Streptomyces alblas lysinopolymeris 11011A-1 strain (Microtechnology Research Institute) No. 11
No. 09) is preferable.

[0009] The mutation treatment in the present invention, of the genus Streptomyces to produce εPL microorganisms (strain), 20mg / ml or more
It refers to a treatment for mutating to an improved strain resistant to the above high concentration of AEC. As a method for such a mutation treatment, N-methyl-N'-nitro-N-nitrosoguanidine (hereinafter referred to as NTG) is used. , 0.1 mg to 3 m per 1 ml of buffer solution
added so as to have a concentration of g and contacting the strain with the NTG for 10 minutes to 2 hours, or an irradiation dose of ultraviolet rays of 10
Examples include a method of irradiating the strain at 0 to 1000 J / cm ² , a method of treating with a drug such as 5-bromouracil, and other conventional chemical or physical mutation treatment methods.

A method for screening the strains subjected to mutation treatment is to inoculate on a minimal agar medium supplemented with AEC of 10 mg or more per 1 ml of the medium and L-threonine added to 1 mg per ml of the medium. A method of collecting a strain growing on the medium can be mentioned.

The present invention will be described in detail below. One of the specific methods for obtaining the improved strain of the present invention, which is an AEC high-concentration resistant strain, is a method of producing εPL, Streptomyces alblas subspices lysinopolymeris 11011A-1.
The spores of the strain are suspended in Tris-maleic acid buffer solution (pH 6.0), and NTG is added to the buffer solution so as to be 1.5 mg per mL of the buffer solution, followed by contact at 37 ° C. for 60 minutes. Then, the spores were collected by centrifugation and the phosphate buffer solution (0.05M,
After washing with pH 7.0), liquid nutrient medium (glucose 5
%, Ammonium sulfate 1%, yeast extract 0.5%, K ₂ HPO _{4
0.08%, KH 2 PO 4 0.136} %, MgSO 4 · 7H 2 O 0.05%, ZnSO
_{4 · 7H 2 O 0.004%,} FeSO 4 · 7H 2 O O.003% pH6.8, however,
% Means g / dl%) and culture overnight. After culturing, the cells were collected by centrifugation and washed with phosphate buffer (0.05 M, pH 7.0), and then the agar medium (glucose) containing 20 mg of AEC per 1 ml of the medium and 1 mg of L-threonine per 1 ml of the medium (glucose). 5%, ammonium sulfate 1%, K ₂ HPO ₄ 0.08%, KH ₂ PO _{4
0.136%, MgSO 4 · 7H 2} O 0.05%, ZnSO 4 · 7H 2 O 0.004%,
_{FeSO 4 · 7H 2 O O.003%} pH6.8, 1.5% agar, however, percent
g / dl%), and cultured at 30 ° C. for 3 to 4 days, and the grown colonies are collected. The AEC high-concentration-resistant strain thus obtained was evaluated for the productivity of εPL, and the strain having the highest productivity was Streptomyces albus subspides lysinopolymeris B21021 strain (deposit number FERM P-15193). Following this are the B22107 and B22201 strains.

The degree of resistance of the obtained improved strain, AEC high-concentration resistant strain, to AEC is measured as follows.
That is, AEC and L of each concentration described in Table 1 below
-Comparing the degree of resistance by applying the resistant strain to each of the minimal agar media (above) containing 1 ml of threonine per mg of the culture medium, culturing at 30 ° C for 2 to 7 days, and observing the growth with the naked eye. To do. The results are shown in Table 1. The parent strain 11011A-1 grows at an AEC concentration of 5 mg / ml, but does not grow at a concentration of 10 mg / ml, whereas the high-concentration-resistant strain B21021 strain has an AEC concentration of 5 mg / ml. Even at 40 mg / ml, growth is observed. Thus, the improved strain of the present invention can be clearly distinguished from the parent strain in that it has resistance to high concentrations of AEC.

[0013]

[Table 1]

Next, the bacteriological properties of the B21021 strain are shown below. (1) Morphological properties 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 elliptical in size Is about 1.2 to 1.5 μ, and its surface structure is spiny. Presence or absence of flagella, sclerotium, and sporangium: Not observed. Position of spore stalk: on aerial hyphae (2) Culture properties on various media The properties on various media in Table 2 are 10 to 30 at 30 ° C, respectively.
It is an observation result after culturing for 14 days.

[0015]

[Table 2]

(3) Physiological properties Growth temperature range: about 15-40 ° C. Optimal growth temperature: around 30 ° C. Liquefaction of gelatin, hydrolysis of starch and peptone of skim milk Coagulation of positive skim milk: negative Coagulation of melanin-like pigment A brown pigment is formed on tyrosine agar medium. Cell Wall Composition Regarding the type of diaminopimelic acid in cell wall composition components, the method of Becker et al. [Applied Microbiology, Vol. 13, p236].
(1965)], it was L and L type. (4) Utilization of carbon source (Pridham Gottlieb agar medium) L-arabinose-D-xylose-D-glucose + D-fructose + L-rhamnose-D-galacto -Su + Sucrose-Raffinose-D-Mannitol + Inositol + Salicin + +: Used,-: Not used

As described above, one of the improved strains of the present invention
The bacteriological properties of the B21021 strain, which is one of them, is 11011A-1 which is the parent strain.
There are some differences with the mycological properties of the strains. For example, in terms of availability of carbon source, the 11011A-1 strain cannot utilize salicin, but the B21021 strain does. Also in the culture properties, on sucrose nitrate nitrate agar, 11011A-1 strain gray-brown aerial mycelium grows,
B21021 strain does not grow. 11011A-1 on nutrient agar
The strain grows abundantly in white aerial hyphae, but in the B21021 strain, aerial hyphae grow only slightly. In addition, the soluble dye is 1101
Strain 1A-1 is also found on glucose asparagine agar and glycerol asparagine agar, whereas strain B21021 is not found on both media. Thus, the B21021 strain is a parent strain in terms of mycological properties as well.
It can be clearly distinguished from the 011A-1 strain.

In order to produce εPL using the obtained improved strain, the improved strain is inoculated into a medium and cultivated, and εPL generated and accumulated from the culture solution is separated and purified. As the medium, any medium can be used as long as it appropriately contains a carbon source, a nitrogen source, an inorganic substance and other nutrients. The carbon source is not limited as long as the improved strain such as glucose, fructose, glycerin and starch can be assimilated, and the content thereof is 1 to 5% (% is g / dl%). Is preferred. As a nitrogen source,
Any of peptone, casein hydrolyzate, amino acid, inorganic ammonium salt and the like may be used, but ammonium sulfate is preferable. The content of nitrogen source is 0.2-2% (% is
g / dl%) is preferred. A carbon source and a nitrogen source may be sequentially added during the culture. Examples of the inorganic substance include phosphate ion, potassium ion, sodium ion, magnesium ion, zinc ion, iron ion, manganese ion, nickel ion, and sulfate ion. In addition, yeast extract
When it is contained in an amount of 0.1 to 0.5% (% is g / dl%), the growth of the bacterium is improved and favorable results are obtained in the production of εPL.

The culture is carried out by shaking culture, stirring culture, etc. under aerobic conditions. The culture temperature is preferably 25 to 35 ° C. The pH of the medium is preferably around neutrality (pH 6 to 8), but the pH decreases with the growth of the bacterium after the start of culturing. When the pH drops to 4, alkali is added to maintain the pH at 4. Ammonia water is preferable as the alkali to be added, but sodium hydroxide, potassium hydroxide or the like may be used. Usually, εPL is accumulated in the culture medium in 1 to 7 days.

After the cells are removed from the above culture solution by centrifugation or a filter, the cell removal solution is purified, decolorized and concentrated. ΕPL can be obtained by crystallizing the concentrated solution with an organic solvent such as acetone or ethanol.

[0021]

EXAMPLES The present invention will be described in more detail by way of examples.
The production of εPL in the culture medium can be measured by Itzhaki.
Et al. Analytical Biochemistry
hemistry), 50, 569, (1972). That is, after centrifuging the culture solution to remove the bacterial cells, 2 ml of the supernatant (εPL: 0 to 200 μg) and 2 ml of a 1 mM methyl orange aqueous solution were mixed and allowed to stand at room temperature for 30 minutes,
The resulting εPL-methyl orange complex is removed by centrifugation, and the absorbance of the supernatant water at 465 nm is measured to determine the amount of εPL in the culture solution. In addition,% in Examples and Comparative Examples is weight (g) / volume (dl)% unless otherwise specified.

Example 1 Glucose 5%, yeast extract 0.5%, ammonium sulfate 1%, K ₂ HPO ₄ 0.08%, KH ₂ PO ₄ 0.136%, MgSO ₄ .7H ₂ O 0.
_{05%, ZnSO 4 · 7H 2} O 0.004%, FeSO 4 · 7H 2 O 0.003%, pH
Prepare 2 liters of 6.8 medium and use a 3 liter volume jar.
And obtained the improved strain of Streptomyces albula subspides lysinopolymeris B2.
Inoculate 100 ml of the pre-cultured solution of 1021 strain, 30 ℃, 700rpm
Aerobic culture was carried out for 72 hours at an aeration rate of 3 l / min. However, the pH was adjusted to 4 by using 10% aqueous ammonia. Further, glucose and ammonium sulfate were sequentially added when the residual concentration decreased. Table 3 shows the amount of εPL production and the yield based on sugar after 72 hours of culture. Here, the yield with respect to sugar (%) is a value represented by (εPL production amount / glucose consumption amount) × 100.

Example 2 Instead of Streptomyces alblas subspices lysinopolymeris B21021 strain, Streptomyces alblas subspices lysinopolymeris B2
Culturing was carried out in accordance with Example 1 except that strain 2107 was used, and the amount of εPL in the culture solution was measured. Table 3 shows the amount of εPL production and the yield based on sugar after 72 hours of culture.

Example 3 Instead of the Streptomyces alblas subspices lysinopolymeris B21021 strain, the Streptomyces alblas subspices lysinopolymeris B2 was used.
Culture was carried out in accordance with Example 1 except that strain 2201 was used, and the amount of εPL in the culture solution was measured. Table 3 shows the amount of εPL production and the yield based on sugar after 72 hours of culture.

Comparative Example 1 Same as Example 1 except that the parent strain Streptomyces alblas subspices lysinopolymeris 11011A-1 was used in place of the Streptomyces alblas subspices lysinopolymeris B21021 strain. I went. Table 3 shows the amount of εPL production and the yield based on sugar after 72 hours of culture.

Example 4 Glucose 5%, yeast extract 0.5%, ammonium sulfate 1%, K ₂ HPO ₄ 0.08%, KH ₂ PO ₄ 0.136%, MgSO ₄ .7H ₂ O 0.
_{05%, ZnSO 4 · 7H 2} O 0.004%, FeSO 4 · 7H 2 O 0.003%, pH
Prepare 2 liters of 6.8 medium and use a 3 liter volume jar.
And obtained the improved strain of Streptomyces albula subspides lysinopolymeris B2.
Inoculate 100 ml of the pre-cultured solution of 1021 strain, 30 ℃, 700rpm
Aerobic culture was carried out for 168 hours at an aeration rate of 3 l / min. However, the pH was adjusted to 4 by using 10% aqueous ammonia. Further, glucose and ammonium sulfate were sequentially added when the residual concentration decreased. Table 4 shows the εPL production amount and the sugar yield after 168 hours of culture.

Example 5 Instead of Streptomyces alblas subspices lysinopolymeris B21021 strain, Streptomyces alblas subspices lysinopolymeris B2
Culture was carried out in accordance with Example 4 except that strain 2107 was used, and the amount of εPL in the culture solution was measured. ΕPL after 168 hours of culture
The production amount and the yield based on sugar are shown in Table 4.

Example 6 In place of the Streptomyces alblas subspices lysinopolymeris B21021 strain, Streptomyces alblas subspices lysinopolymeris B2
Culture was carried out in accordance with Example 4 except that 2201 strain was used, and the amount of εPL in the culture solution was measured. ΕPL after 168 hours of culture
The production amount and the yield based on sugar are shown in Table 4.

Comparative Example 2 According to Example 4, except that the parent strain of Streptomyces alblas subspices lysinopolymeris 11011A-1 was used in place of the Streptomyces alblas subspices lysinopolymeris B21021 strain. I went. Table 4 shows the εPL production amount and the sugar yield after 168 hours of culture.

Example 7 Regarding the culture broth cultivated in Example 4 for 168 hours, the culture broth was centrifuged to remove the cells, and the pH was adjusted to 7.5. Then, IRC-50 (cation exchange resin) and IRA were used. -40
2 (anion exchange resin) and XT-1006 (cation exchange resin) were separated and purified with ion exchange resins, and concentrated with a reverse osmosis membrane (RO) to obtain εPL. The yield is shown in Table 5.

Comparative Example 3 The culture broth cultivated for 168 hours in Comparative Example 2 was centrifuged to remove the cells in the same manner as in Example 7, and the pH was adjusted to 7.5.
After adjusting to, IRC-50 (cation exchange resin),
IRA-402 (anion exchange resin), XT-1006
(Cation exchange resin) was separated and purified with each ion exchange resin, and concentrated with a reverse osmosis membrane to obtain εPL. The yield is shown in Table 5.

As is clear from the results of Table 3, Table 4 and Table 5, both the B21021 strain produced εPL and the yield based on sugar were 11
It can be seen that the strain is remarkably increased as compared with that of the 011A-1 strain.

[0033]

EFFECT OF THE INVENTION The improved strain of the present invention having a high concentration tolerance of AEC has the ability to produce εPL with high production and high yield, and by using the production strain, high productivity and high yield can be obtained. It is possible to manufacture εPL.

[0034]

[Table 1]

[0035]

[Table 2]

[0036]

[Table 3]

[0037]

[Table 4]

[0038]

[Table 5]

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI C12R 1: 465) (56) References JP-A-63-49075 (JP, A) JP-A-63-49097 (JP, A) JP-A-3-143398 (JP, A) (58) Fields investigated (Int.Cl. ⁷ , DB name) C12N 1/00-1/38 C12P 13/00-13/24 JSTPlus (JOIS) BIOSIS / WPI (DIALOG)

Claims (4)

(57) [Claims] 1. Streptomyces albulu having the ability to produce ε-poly-L-lysine.
s) species of microorganisms obtained by mutation treatment and having a high concentration of ε-poly-L-lysine having resistance to high concentrations of 20 mg / ml or more of S- (2-aminoethyl) -L-cysteine. The strain to produce. 2. Streptomyces albulus su
bsp. lysinopolymerus) 11011A-1 strain
No.), a high concentration of S of 20 mg / ml or more
B21021 which produces a significant amount of ε-poly-L-lysine having resistance to-(2-aminoethyl) -L-cysteine
Strain (deposit number FERM P-15193). 3. Resistant to a high concentration of 20 mg / ml or more of S- (2-aminoethyl) -L-cysteine, and ε
-A microorganism belonging to the genus Streptomyces having a poly-L-lysine-producing ability is aerobically cultured in a medium, and ε-poly-L-lysine produced and accumulated in the culture solution is collected. Process for producing ε-poly-L-lysine. 4. The B21021 strain according to claim 2 is cultivated aerobically in a medium, and the ε-poly-L-lysine produced and accumulated in the culture medium is collected. Manufacturing method.