JPH0568537A - Microbial strain of producing epsilon-poly-l-lysine - Google Patents

Abstract

PURPOSE:To obtain a microbeal strain producing epsilon-PL from Streptomyces albus subsp. lysinopolymerus, a strain of Streptomyces albus by multiplying a plasmid containing a gene taking part in biosynthesis of epsilon-PL by using chloramphenicol. CONSTITUTION:The objective strain to produce epsilon-poly-L-lysine(epsilon-PL) is obtained by multiplying a plasmid containing a gene taking part in biosynthesis of epsilon-PL by treating a strain of Streptomyces albus subsp. lysinopolymerus with chloramphenicol.

Description

Detailed Description of the Invention

[0001]

This invention relates to epsilon-poly-L-.
The present invention relates to a strain that produces a significant amount of lysine (hereinafter abbreviated as εPL).

[0002]

2. Description of the Related Art εPL is a polymer compound in which the amino group at the ε-position of L-lysine is bonded to the carboxylic acid of adjacent L-lysine by an amide bond, as represented by the following structural formula.

[0003]

[Chemical 1]

Since the substance is a polymer of L-lysine which is an essential amino acid, it is highly safe and has a high cation content, and therefore has unique physical properties. Therefore, by utilizing these properties, applications such as toiletry products, cosmetics, feed additives, medicines, agricultural chemicals, food additives, electronic materials, etc. can be expected. Conventionally, this substance belongs to the genus Streptomyces.
Streptomyces albul, a PL-producing bacterium, Streptomyces albulus subspecies
It is obtained by culturing us subsp. lysinopolymerus) No. 346-D strain (Microtechnology Research Institute, No. 3834) in a medium and separating and purifying from the obtained culture (Japanese Patent Publication No. 59-203).
59).

[0005]

However, in the strain of this prior application, εPL of about 0.5 g per liter of culture solution is used at most.
Therefore, the production cost was high and the widespread use of the substance was hindered. The present inventors have obtained a strain that produces a large amount of εPL, conducted research for the purpose of producing a large amount of εPL using this, and arrived at the invention described below.

[0006]

The present invention is a plasmid containing a gene involved in the biosynthesis of εPL using chloramphenicol from Streptomyces albulus subsp. Lysinopolymerus strain. Is a strain that produces εPL that is amplified.

A strain producing εPL of the present invention is
Streptomyces Alblas Subspecies
A plasmid-amplified mutant strain of Lysinopolymeris No. 346-D strain is preferred. This mutant strain is a strain obtained by subjecting the above-mentioned bacterium to a treatment for amplifying a plasmid, and a plasmid-amplified mutant strain 50833 strain (Microtechnology Research Institute No. 1110) is obtained by chloramphenicol treatment.

The present invention will be described in detail below. First, the method for obtaining the strain of the present invention will be described. Streptomyces
Albrus Subspecies Lizino Polymeras No.
The 346-D strain or the S-aminoethyl-L-cysteine resistant mutant strain is inoculated into the medium and shake-cultured, and then chloramphenicol is added to continue the culture. The cells are collected by centrifugation and washed, and then the cells are applied to the agar medium.
After static culture, Staphylococcus aureus
Strains with a large growth-inhibiting circle of Staphylococcus, which were produced by superimposing normal agar medium containing the
It is the 0833 strain (Microtechnical Laboratory Article No. 1110). 508
The mycological properties of the 33 strains are as follows.

(1) Morphological Properties The results of microscopic observation of aerial hyphae and basal hyphae of strain 50833 grown on sucrose / nitrate agar medium at 30 ° C. for 10 days are shown below. Branching method and morphology of sporulating hyphae: simple branching, closed spiral Number of spores: dozens Surface structure and size of spores: spores are circular or oval in size, approximately 1.2 to 1.5 μ, and its surface structure is Spiny. Presence or absence of flagella spores, sclerotium and sporangia is not observed. Location of spore stalk: On aerial mycelium.

(2) Growth state on various media The properties on the following various media are 10 to 14 at 30 ° C., respectively.
It is an observation result after culturing for a day. (3) Physiological properties The physiological properties of strain 50833 are as follows. Growth temperature range about 15-40 ℃. Optimal growth temperature: around 30 ° C. Liquefaction of gelatin, hydrolysis of starch and peptonization of skim milk: All positive Skim milk coagulation: Negative Formation of melanin pigment A brown pigment is formed on tyrosine agar medium. Cell Wall Composition The type of diaminopimelic acid in the cell wall composition components was analyzed by the method of Becker et al. [See Applied Microbiology, Vol. 13, p. 236 (1965)], and it was L or L type.

(4) Assimilation of various carbon sources (pridham
(On Gottlieb agar medium) L-arabinose-D-xylose-D-glucose + D-fructose + L-rhamnose-D-galactose + sucrose-raffinose-D-mannitol + i-inositol + salicin-Note) +: Assimilate. ,-: Do not assimilate.

As described above, the mycological properties of the mutant strain of the present invention are based on the original strain Streptomyces albus subspecies lysinopolymeris No. 346-.
It is similar to the mycological properties of strain D. Next, using the obtained mutant strain, εPL is produced by the method of the present invention. In the text,% means weight (g) / volume (ml)% unless otherwise specified.

First, the obtained mutant strain is inoculated into a medium and cultured, and εPL generated and accumulated from the culture solution is separated and purified. The medium may be any medium as long as it contains a carbon source, a nitrogen source, an inorganic salt and vitamins, but preferably contains 5% glucose or 5% glycerine as a carbon source and ammonium sulfate or L- as a nitrogen source. Those containing lysine or peptone are preferable. A carbon source and a nitrogen source may be sequentially added during the culture. pH is p at the beginning of culture
The pH may be allowed to fall to H 4.0, and then the pH may be maintained at 4.0 with an alkali such as an aqueous sodium hydroxide solution. After removing the bacterial cells from the culture solution with a centrifuge or a filter, the filtrate is purified and decolorized, and then concentrated. From the concentrated solution, use an organic solvent such as acetone or ethanol to
Crystallize PL.

[0014]

INDUSTRIAL APPLICABILITY The mutant strain of the present invention has the ability to produce a large amount of εPL, and by culturing the mutant strain, a large amount of εPL can be produced as compared with the case of using a known strain. ..

[0015]

EXAMPLES The present invention will be described in detail below with reference to examples. Example: Acquisition of a plasmid amplification mutant: Streptomyces albus subspecies lysinopolymeris (Strep
tomyces albulus subsp. lysinopolymerus) No. 346
-A platinum loop of spores of strain D was suspended in 5 ml of Tris-maleic acid buffer (pH 9.0), and N-methyl-N-nitro-N'-nitrosoguanidine was added thereto to a concentration of 1.5 mg / ml. Was added. After shaking this at 30 ° C for 30 minutes, the spores were collected by a centrifuge and washed with sterilized water. Glucose 5%, ammonium sulfate 1%, yeast extract 0.5
%, Monopotassium dihydrogen phosphate heptahydrate 0.136%, disodium monohydrogen phosphate dihydrate 0.158%, magnesium sulfate heptahydrate 0.05%, zinc sulfate heptahydrate 0.004%, ferrous sulfate -Inoculate 5 ml of a 7-hydrate salt 0.003%, pH 6.8 medium (hereinafter referred to as the first medium) and shake culture at 30 ° C overnight.
The fungus was grown.

The culture solution is diluted 500 times with MS solution (composition: magnesium sulfate heptahydrate 0.05%, sodium chloride 0.5%, Tween 80 0.05%). This diluted culture is then added to S-aminoethyl-L-cysteine to a concentration of 2 mg per ml of agar, or S-aminoethyl-L-cysteine to this concentration and 1 mg per ml of agar. It was applied to an agar medium having the same composition as the above-mentioned first medium, to which glycine or L-threonine was added so as to have a concentration. This agar medium was incubated at 30 ° C for 48 hours to grow as a colony, and S-aminoethyl-L
-A cysteine resistant mutant was obtained.

The S-aminoethyl-L-cysteine resistant mutant strain thus obtained is inoculated into 5 ml of a medium having the same composition as the above-mentioned first medium. After shaking culture at 30 ° C. for 2 days, chloramphenicol is added so that the concentration of chloramphenicol is 50 to 500 mg, preferably 100 mg, per liter of the culture solution, and the culture is further continued for 5 to 10 hours, preferably 8 hours. .. Centrifuge to collect the cells, wash with sterile water or saline, and then add agar to the medium of the same composition as the first medium.
Spread the bacteria on the agar medium containing 1.7%.

After static culture at 30 ° C. for 8 days, ordinary agar medium containing Staphylococcus aureus was overlaid and further cultured overnight. A strain with a large growth-inhibiting circle of staphylococci produced a high plasmid amplification εPL. It is a production stock. One of these is 50833 (Mikoken Kenjoyori No. 1110)
Is. Production of εPL: 5 ml of the same composition as the first medium was inoculated with 1 platinum loop amount of the plasmid amplification mutant strain 50833 strain,
The culture was carried out at 30 ° C. for 8 days with shaking. After the culture was completed, the concentration of εPL in the culture solution was measured by the Itzhaki method.

The results are shown in Table 1. Comparative Example Streptomyces Alblas Subspecies
Culturing was carried out in the same manner as in Example except that the Lysinopolymeris No. 346-D strain was used, and the εPL concentration was measured in the same manner. The results are shown in Table 1.

[0020]

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Claims (2)

[Claims] 1. A Streptomyces albulu (Streptomyces albulu)
s subsp. lysinopolymerus) A strain producing epsilon-poly-L-lysine, which is obtained by amplifying a plasmid containing a gene involved in biosynthesis of epsilon-poly-L-lysine using chloramphenicol. 2. A strain producing epsilon-poly-L-lysine is a Streptomyces albulus subspecies.
The strain according to claim 1, which is a plasmid-amplified mutant strain 50833 strain of sp. lysinopolymerus) No. 346-D strain (Mikiko Kenjoyori No. 1110).