JPH066061B2 - Method for producing ε-polylysine and ε-polylysine-producing bacterium - Google Patents

Description

The present invention relates to a novel method for producing ε-polylysine and ε-polylysine.
It relates to a polylysine-producing bacterium.

(Prior Art and its Problems) ε-Polylysine has the following structural formula:
It is a polymer of lysine, in which the amino group at the ε-position of L-lysine is linearly linked to the carboxyl group of adjacent L-lysine by a peptide bond.

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 thus has unique physical properties. Therefore, by utilizing these properties, applications such as toiletry products, cosmetics, feed additives, agricultural chemicals, medicines, food additives, electronic materials, etc. are being developed.

This ε-polylysine is used for Streptomyces albrus subspecies lysinopolymeris (Streptomyces
It is already known that it can be obtained by culturing albulus subsp.lysinopolymerus) No. 346 (JP-A-53-7).
No. 2896).

This Streptomyces albrus subspecies lysinopolymeris has an assimilation ability with respect to D-glucose, D-galactose and D-mannitol,
It does not have the ability to assimilate sucrose. Therefore, when culturing this strain to produce ε-polylysine, D-glucose or glycerol has been used as a carbon source of the medium. However, since D glucose and glycerol are expensive, ε-polylysine obtained is expensive. Therefore, a method for producing ε-polylysine using an inexpensive carbon source has been required.

[Means for solving problems]

The present inventors have arrived at the present invention as a result of searching for a strain that produces ε-polylysine by assimilating sucrose, particularly molasses as a carbon source in order to obtain inexpensive ε-polylysine. That is, the present invention is characterized by ε-polylysine-producing bacterium belonging to Streptomyces noursei, and culturing this bacterium in a medium, and separately collecting ε-polylysine from the resulting culture.
It is a method for producing polylysine.

The Streptomyces norusei strain of the present invention can be applied to the Institute of Microbial Science and Technology of the Agency of Industrial Science and Technology for application number 9797 (F
Deposited as ERM P-9797).

In the method of the present invention, the medium used for culturing this bacterium,
To reduce production costs, it is preferable to utilize sucrose and / or molasses as carbon source.

It has not been known so far that the Streptomyces norsei strain produces ε-polylysine.

The mycological properties of this strain are as follows.

(1) Morphological properties The results of microscopic observation of aerial hyphae and basal hyphae 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: several dozen Surface structure and size of spores: spores are round or oval in size, approximately 1.2-1.5 μ,
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 condition on various media The properties on the following media are 10 to 14 at 30 ℃.
It is an observation result after culturing for a day.

(3) Physiological properties The physiological properties of this strain 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 Coagulation of skim milk: Negative Formation of melanin pigment A brown pigment is formed on tyrosine agar medium.

(4) Assimilation of various carbon sources (on Pridham-Gottlieb agar medium) L-arabinose-D-xylose-D-glycose + D-fructose + L-rhamnose-D-galactose + sucrose + raffinose-D-mannitol + Salicin − Note) +: Assimilate, −: Not assimilate.

Based on the above-mentioned mycological properties, this bacterium is considered to belong to Streptomyces norsei.

The culturing method in the method for producing ε-polylysine of the present invention basically follows the culturing method of general microorganisms, but the aerobic culturing method using a liquid medium is usually advantageous.

A synthetic medium, a semi-synthetic medium or a natural medium is used as the medium used for the culture, and glucose, fructose, glycerol, sucrose, molasses, liquefied starch, etc. are used as the carbon source of the medium. From the economical point of view, it is advantageous to use molasses. As the nitrogen source, yeast extract, meat extract, casein hydrolyzate, peptone, ammonium sulfate, ammonium phosphate, ammonium chloride and the like are used. Inorganic salts such as sodium hydrogen phosphate, potassium dihydrogen phosphate, calcium carbonate, ferrous sulfate, magnesium sulfate, copper sulfate, zinc sulfate, manganese chloride and magnesium chloride are also added if necessary. As a defoaming agent, soybean oil, higher alcohols, silicon compounds and the like are also added as appropriate.

A suitable culture temperature is around 30 ° C. Culture time is 24-100
Time is appropriate. The pH during the culture is not adjusted at first and is allowed to fall, and the pH is kept between 3.5 and 5.0, preferably to pH 4.0, and then maintained at that pH with an aqueous solution of sodium hydroxide or an aqueous solution of ammonium hydroxide.

The ε-polylysine thus accumulated in the culture broth is centrifuged or filtered to remove the strain, and then separated and purified from the filtrate by a conventional means used for the production of general fermentation products. That is, vacuum concentration, freeze-drying,
Purified polylysine can be obtained by using means such as solvent extraction, ion exchange resin treatment, activated carbon treatment, and crystallization alone or in any combination or repeatedly.

(Effects of the Invention) The method of the present invention can produce ε-polylysine using Streptomyces knorsei. In addition, various carbon sources can be used in the medium without being limited to D-glucose and glycerol, and since ε-polylysine can be produced particularly in an inexpensive molasses medium, the production cost can be significantly reduced as compared with the conventional method. be able to.

(Example) Hereinafter, the present invention will be specifically described based on Examples.

Waste molasses 200g (reducing sugar 25.1%, pure sugar content 30.2%), ammonium sulfate 10g, yeast extract 5g, magnesium sulfate (7
Hydrate) 0.5 g, sodium monohydrogen phosphate (12 hydrate) 1.5
Dissolve 8 g and 1.36 g of potassium dihydrogen phosphate in water to 1000
m. Adjust pH to 6.8 with NaOH solution.

100 m of this medium is poured into seven 500 m Sakaguchi flasks and sterilized at 120 ° C. for 20 minutes. Each of them is inoculated with 1 platinum loop of a slope culture of Streptomyces norsei, and cultured by shaking at 30 ° C for 20 minutes.

The above culture medium 1.8 is poured into a mini jar fermenter (made by Iwashiya Co., Ltd.) having a volume of 3.0, and sterilized at 120 ° C. for 20 minutes. This is inoculated with the above-mentioned shaking culture of Streptomyces norsei. Temperature 30 ℃, stirring 600rpm, aeration rate 3.0 / min
(1 atm), the pH is allowed to start to fall, and when the pH reaches 4.0, the pH is adjusted to 4.0 with a 6N sodium hydroxide aqueous solution and cultivated for 40 hours.

After the culture is completed, the culture is filtered to remove the bacterial cells. The filtrate is adjusted to pH 8.5 with aqueous sodium hydroxide solution and the precipitate formed is filtered off. The amount of this filtrate is 1.8 including the washing liquid,
The amount of ε-polylysine in this was 17.5 g.

This filtrate was used as a cation exchange resin Amberlite IR-50.
It is passed through a column filled with (H ⁺ type) to adsorb polylysine. The column is washed with 0.2N acetic acid and then eluted with 0.1N hydrochloric acid. Neutralize the eluate with aqueous sodium hydroxide solution to pH 6.
Set to 8. 10 g of powdered activated carbon is added, and the mixture is stirred at room temperature for about 1 hour to decolorize it. Activated carbon is filtered off. The filtrate
Concentrate at 35 ° C. The same amount of methanol is gradually added to the concentrated liquid with stirring, and then three times the amount of acetone is gradually added with stirring in the same manner. After the addition of acetone is completed, stirring is continued for 1 hour. The precipitate formed was filtered off with suction, washed once with acetone, and then dried under reduced pressure to obtain 5.5 g of white powdery polylysine hydrochloride. The purity of this product was 99.1% as measured by the methyl orange method.

Claims (3)

[Claims] Claims: 1. Strepto
A method for producing ε-polylysine, which comprises culturing an ε-polylysine-producing bacterium belonging to myces noursei) in a medium and separating and collecting ε-polylysine from the resulting culture. 2. The method according to claim 1, wherein the carbon source of the medium is selected from sucrose and molasses. 3. Streptomyces Noursei ((Strept
omyces noursei) Microorganism Research Institute, No. 9797), an ε-polylysine-producing bacterium.