CN1161455C - Bacterial strain and production method for producing a large amount of ε-poly-L-lysine - Google Patents

Abstract

The present invention provides a strain which has higher capacity for generating epsilon-poly-L-lysine than that of a conventional epsilon-poly-L-lysine producing strain and can improve the production rate of the epsilon-poly-L-lysine, or an improve strain thereof and a method for largely producing the epsilon-poly-L-lysine by using the strain. According to the method for producing the epsilon-poly-L-lysine, microbes which belong to small streptomyces albus and have high capacity for generating epsilon-poly-L-lysine are mutagenized so as to obtain a strain which can be used for largely producing the epsilon-poly-L-lysine and have resistance to S-(2-aminoethyl)-L-cysteine with the concentration of 10 mg/ml or higher than 10 mg/ml, the strain is cultivated in a culture medium under a good weather condition, and the epsilon-poly-L-cysteine formed in the culture medium is collected.

Description

Bacterial strain and production method for producing a large amount of ε-poly-L-lysine

field of invention

The present invention relates to a bacterial strain producing ε-poly-L-lysine (hereinafter referred to as εPL) in large quantities and a method for producing εPL by fermentation using the bacterial strain.

Since εPL is a polymer of the essential amino acid L-lysine, it is safe and has unique physical properties due to its high cationic content. Therefore, it is expected to be used in cosmetics, beauty products, feed additives, pharmaceuticals, insecticides, food additives, electronic equipment and the like. Especially in the field of food additives, it has drawn attention as a natural additive.

Background technique

As a method for fermentatively producing εPL, there is known a method (approved Japanese Patent Laid-Open No. 59-20359) comprising culturing Streptomyces parvus lysinopolymerus subspecies 346-D (FERM P-3834) in a culture medium, which strain is The εPL-producing strain isolated from nature, belonging to the genus Streptomyces, was then isolated and purified from the culture medium. There is also another known method (approved Japanese patent publication 3-42070 or approved Japanese patent publication 3-78998), which comprises mutagenizing Streptomyces parvus lysinopolymerus subspecies 346-D to transform it into L-lysine analogue S-(2-aminoethyl)-L-cysteine has a resistant mutant, and the resulting mutant is cultivated in a medium, that is, Streptomyces parvus lysinopolymerus subspecies 11011A-1 (FERMBP-1109), and then isolate and purify εPL from the medium.

In order to produce inexpensive εPL suitable for various uses, even the use of the above-mentioned 11011A-1 strain is not satisfactory in terms of εPL yield and glucose conversion efficiency. Therefore, there is a need to obtain strains with improved ability to produce εPL, and a method for industrially producing εPL inexpensively and efficiently using the above strains.

The inventors have conducted extensive studies in an attempt to obtain strains or mutants thereof having higher εPL production ability than conventional εPL producing strains. As a result, it has been found that a mutant resistant to S-(2-aminoethyl)-L-cysteine (hereinafter referred to as AEC) at a concentration of 10 mg/ml or higher is likely to be capable of producing εPL in large quantities A strain that can produce a large amount of εPL by aerobically culturing it in a medium. Based on these findings, the present invention has been accomplished.

Based on the above, the object of the present invention is to provide a strain with higher εPL production ability or an improved strain thereof, compared with conventional εPL producing strains, so that the yield of εPL can be increased; Methods.

Contents of the invention

The present invention includes following technical contents:

1). A bacterial strain capable of producing ε-poly-L-lysine in large quantities, which is obtained by mutagenizing microorganisms belonging to Streptomyces parvus and having the ability to produce ε-poly-L-lysine. S-(2-aminoethyl)-L-cysteine at a concentration of 10 mg/ml or higher was resistant.

2). The bacterial strain B21021 (preservation number FERM BP-5926) capable of producing a large amount of ε-poly-L-lysine, which was obtained through the detection of Streptomyces parvus lysinopolymerus subspecies 11011A-1 strain (FERM BP-1109) Obtained by mutagenesis treatment, it is resistant to S-(2-aminoethyl)-L-cysteine at a concentration of 10 mg/ml or higher.

3). A method for producing ε-poly-L-lysine comprising aerobically culturing in a culture medium S-(2-aminoethyl)-L- Microorganisms resistant to cysteine and capable of producing ε-poly-L-lysine collected ε-poly-L-lysine formed and accumulated in the medium.

4). The method for producing ε-poly-L-lysine, comprising aerobically culturing the B21021 strain described in 2) above in a culture medium, and collecting the ε-poly-L-lysine formed and accumulated in the culture medium .

AEC used in the present invention is a structural analogue (analogue) of L-lysine, which differs from L-lysine only in that a sulfur atom replaces a carbon atom at the 4-position. Addition of AEC to the medium resulted in inhibition of microbial growth, which was more pronounced and intense when used together with L-threonine.

The strain 11011A-1 disclosed in Examined Japanese Patent Laid-Open No. 3-42070 is an εPL producing strain resistant to AEC, an analogue of L-lysine, which was selected in the presence of AEC at a concentration of 2 mg/ml , can only grow at a concentration of 5 mg/ml or lower AEC, and cannot grow at an AEC concentration of 10 mg/ml or higher.

The improved bacterial strain of the present invention is obtained by screening under the condition of AEC concentration of 10 mg/ml or higher, belongs to the microorganism of the genus Streptomyces, and can still grow even under the condition of AEC concentration of 40 mg/ml. In addition, the bacteriological characteristics of the improved strain were partially different from the parental strain 1101A-1.

Microorganisms belonging to the genus Streptomyces capable of producing εPL can be used as parent strains for obtaining improved strains. Among them, Streptomyces parvus lysinopolymerus subspecies 11011A-1 bacterial strain (FERM BP-1109) is preferred.

"Mutage treatment" herein refers to treatment that causes mutations in microorganisms (strains) belonging to the genus Streptomyces capable of producing εPL, thereby obtaining improved strains resistant to AEC at a concentration of 10 mg/m1 or higher. Examples of mutagenesis treatment methods include adding N-methyl-N'-nitro-N-nitrosoguanidine (hereinafter referred to as NTG) to a concentration of 0.1 to 3 mg per ml of buffer solution, contacting the strain with NTG for 10 minutes to The 2-hour method; the method of subjecting the strain to ultraviolet radiation of 100 to 1000 J/cm ² ; the method of treating the strain with chemicals such as 5-bromouracil; and other conventional chemical or physical mutagenic treatment methods.

An example of a screening method for a mutagenized strain includes inoculating the strain on a minimal agar medium to which AEC has been added to a concentration of 10 mg or more and L-threonine to a concentration of 1 mg per ml of the medium, and collected in cultured strains grown in the base.

The present invention is described in detail below.

An illustrative example of obtaining a strain of the present invention resistant to high concentrations of AEC is as follows. Spores of the εPL-producing strain Streptomyces parvus lysinopolymerus subsp. 11011A-1 were suspended in tris-maleic acid buffer (pH 6.0). NTG was added to the suspension to 1.5 mg/ml, and the spores and NTG were contacted at 37°C for 60 minutes. The resulting spores were collected by centrifugation, washed with phosphate buffer (0.05M, pH7.0), and cultured in a 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: 0.003%, pH6.8, where % means g/ dl%) overnight. Then the cells were collected by centrifugation, washed with phosphate buffer (0.05M, pH7.0), and cultured on a basic agar medium (glucose: 5%, ammonium sulfate: 1%) containing 20mg/ml AEC and 1mg/ml L-threonine. , 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%, pH6.8 , Agar: 1.5%, where % represents g/dl%), plated in 30° C. for 3 to 4 days. The colonies formed were collected. The resulting strains resistant to high concentrations of AEC were evaluated for epsilon PL yield. As a result, the strain with the highest yield was Streptomyces parvus lysinopolymerus subspecies B21021 (Institute of Biotechnology, Institute of Industrial Technology, MITI (1-3, Higashi 1-chome, Tsukuba-shi, Ibaraki-ken, 305, Japan ), international deposit number: FERM BP-5926, which was converted from the original deposit (FERM P-15193, date of original deposit: September 20, 1995) to international deposit on April 18, 1997 according to the Budapest Treaty), followed by Strains B22107 and B22201.

The resistance of the improved strain resistant to high concentration of AEC to AEC was evaluated as follows. Inoculate each resistant strain into the basic agar medium (as above), the concentration of AEC added to the medium is shown in Table 1 below, the concentration of L-threonine is 1mg/ml, and incubated at 30°C for 2 to After 7 days, the growth was observed with naked eyes. The results are shown in Table 1. Growth was observed in the parental strain 11011A-1 at an AEC concentration of 5 mg/ml, but no growth was observed at a concentration of 10 mg/ml, while growth was observed even at an AEC concentration of 40 mg/ml for the highly resistant strain B21021 . The improved strains of the present invention are resistant to high concentrations of AEC and in this respect they can be clearly distinguished from the parental strains.

Table 1

Test strain AEC concentration Incubation time (days)

(mg/ml) 2 3 3 4 5 6 7

B21021 5   + + + + + + +

+ + +

+ +

                                                                                  

B22107 5   + + + + + + +

+ + +

+ +

                                                                                  

B22201 5   + + + + + + +

+ + +

+ +

                                       +

+

Parental strains 10 - - - - ± ± +

20 - - - - - - - - - -

40 - - - - - - - - - -

Notes: + growth ± little growth - no growth

The mycological characteristics of strain B21021 are described below.

1). Morphological characteristics

i) Branching mode and form of spore hyphae: simple branching, closed spiral

ii) Number of spores: dozens

iii) Spore surface structure and size:

The spores are each round or oval in size from 1.2 to 1.5 μm and have a spiny surface structure.

iv) No pili, sclerotia and sporangia not seen

v) Spore tip adhesion site: aerial hyphae

2). Culture characteristics in culture medium

The culture characteristics in various media shown in Table 2 are the results observed after incubation at 30°C for 10 to 14 days.

Table 2 culture medium Vegetative mycelia aerial hyphae water soluble pigment Sucrose·Nitrate Agar Medium not formed not formed none Glucose Aspartic Acid Agar Medium white to light yellow brownish yellow none Glycerol-Aspartic Acid Agar Medium light yellow white, powdery none Lysine agar medium Light Brown White brown Nutrient agar medium yellow small amount of white none Yeast and malt agar medium brownish yellow brown gray, powdery none Oatmeal Malt Agar Medium brownish yellow white, large none

3). Physiological characteristics

i) Growth temperature range: about 15 to 40°C, optimum growth temperature: about 30°C.

ii) Gelatin liquefaction, starch hydrolysis and skim milk peptonization were all positive.

iii) Coagulation of skim milk: Negative.

iv) Melanin-like pigment formation: Brown pigment was formed on lysine agar medium.

v) Cell wall composition

According to the method of Becker et al. (Applied Microbiology, Vol. 13, p. 236 (1965)), the form of diaminopimelic acid, a component of the cell wall, is L, L.

4). Carbon source assimilation (Pridham-Gottlieb agar medium)

L-Arabinose -

D-xylose -

D-glucose +

D-fructose +

L-rhamnose -

D-Galactose +

Sucrose -

Raffinose -

D-Mannitol +

Inositol +

Salicin +

+: assimilable, -: different

It can be seen from the above that the improved strain 21021 of the present invention is different from the parent strain 11011A-1 in some mycological characteristics. For example, in terms of carbon source assimilation, strain 11011A-1 could not assimilate salicin, but strain B21021 could. In terms of culture characteristics, the 11011A-1 strain formed brown-gray aerial hyphae on the sucrose·nitrate agar medium, but the strain B21021 did not. The 11011A-1 strain formed a large number of white aerial hyphae on the nutrient agar medium, while the strain B21021 only formed a small amount of aerial hyphae. The 11011A-1 strain formed water-soluble pigments in both glucose·aspartic acid agar medium and glycerol·aspartic acid agar medium, while the B21021 strain did not form pigments in both media. Therefore, the strain B21021 can be clearly distinguished from the parent strain, namely the strain 11011A-1, in terms of mycological characteristics.

In order to produce εPL with the improved strain, the improved strain is inoculated to a medium and cultured, and then formed and accumulated εPL is isolated and purified from the medium. Any medium can be used as long as it contains appropriate amounts of carbon sources, nitrogen sources, inorganic substances and other nutrients. As the carbon source, carbon sources that can be assimilated by the improved strain such as glucose, fructose, glycerol and starch can be used without any limitation. The amount added is preferably 1-5% (% means g/dl%). Among nitrogen sources, peptone, casein hydrolyzate, amino acid, inorganic ammonium salt, etc. can be used, and ammonium sulfate is preferred. The nitrogen source is preferably added in an amount of 0.2 to 2% (% means g/dl%). During cultivation, carbon source and nitrogen source can be added continuously. Examples of inorganic substances include phosphate ions, potassium ions, sodium ions, magnesium ions, zinc ions, iron ions, manganese ions, nickel ions, and sulfate ions. Adding 0.1 to 0.5% (% represents g/dl%) yeast extract can promote the growth of microorganisms and is beneficial to the production of εPL.

Under aerobic conditions, shaking culture, stirring culture or other methods of culture. The culture temperature is preferably 25 to 35°C. The pH of the medium is preferably close to neutral (pH 6-8), but the pH will decrease after starting the culture. When the pH dropped to 4, base was added to maintain the pH at 4. The added base is preferably ammonia, but sodium hydroxide, potassium hydroxide or other bases can also be used. Usually εPL accumulates in the medium after 1 to 7 days. After removing cells by centrifugation or filtration, the remaining solution is purified, decolorized and concentrated. εPL can be obtained by crystallizing the concentrate in an organic solvent such as acetone or ethanol.

Best Mode for Carrying Out the Invention

The present invention will be described more specifically below.

The amount of εPL produced in the medium was measured according to the method of Itzhaki et al. (Analytical Biochemistry, 50, 569 (1972)). That is, the amount of εPL in the culture medium was determined as follows: the culture was centrifuged to remove cells, 2 ml of supernatant (εPL: 0 to 200 μg) was mixed with 2 ml of 1 mM methyl orange aqueous solution, and the resulting mixture was left at room temperature for 30 minutes, centrifuged The generated εPL-methyl orange complex was removed and the absorbance of the supernatant was measured at 465 nm.

In Examples and Comparative Examples, % means weight (g)/volume (dl)% unless otherwise specified.

Example 1

In a container with a volume of 3 liters, fill 2 liters of medium consisting of: glucose: 5%, ammonium sulfate: 1%, _yeast extract: 0.5%, _K2HPO4 : 0.08%, _{KH2PO 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 6.8. Inoculate 100 milliliters of Streptomyces albicans lysinopolymerus subspecies B21021 bacterial strain, namely the preculture of the improved bacterial strain of the present invention, into the culture medium, cultivate aerobically at 30° C. at 700 rpm for 72 hours, and the air flow rate is 3 L/min. The pH was maintained at 4 with 10% aqueous ammonia. When the concentration of residual glucose and ammonium sulfate decreases, add glucose and ammonium sulfate continuously.

Table 3 shows the yield of εPL and the conversion rate of glucose after 72 hours of cultivation. "Glucose conversion (%)" is expressed herein as (εPL production/glucose consumption) x 100.

Example 2

Except for using Streptomyces albicans subsp. lysinopolymerus B22107 strain instead of Streptomyces albicans subsp. lysinopolymerus B21021 strain, culture was carried out as in Example 1, and the amount of εPL in the culture was measured. Table 3 shows the production of εPL and the conversion rate of glucose after 72 hours of cultivation.

Example 3

Except for using Streptomyces albicans subspecies lysinopolymerus B22201 strain instead of Streptomyces albicans subsp. lysinopolymerus B21021 strain, culture was carried out as in Example 1, and the amount of εPL in the culture was measured. Table 3 shows the production of εPL and the conversion rate of glucose after 72 hours of cultivation.

Comparative Example 1

Cultivation was carried out in the same manner as in Example 1, except that the Streptomyces albicans subsp. lysinopolymerus subsp. 11011A-1 strain was used instead of the Streptomyces albicans subsp. lysinopolymerus subspecies B21021 strain. Table 3 shows the production of εPL and the conversion rate of glucose after 72 hours of cultivation.

table 3

Strain εPL yield (g/l) Glucose conversion rate (%)

B21021 (Example 1) 16.2 12.1

B22107 (Example 2) 15.6 11.1

B22201 (Example 3) 14.3 13.0

11011A-1 (Comparative Example 1) 9.2 7.7

Example 4

In a container with a volume of 3 liters, fill 2 liters of medium consisting of: glucose: 5%, ammonium sulfate: 1%, _yeast extract: 0.5%, _K2HPO4 : 0.08%, _{KH2PO 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 6.8. Inoculate 100 milliliters of Streptomyces albicans lysinopolymerus subspecies B21021 strain, the preculture of the improved bacterial strain of the present invention, into the culture medium, cultivate aerobically at 30° C. and 700 rpm for 168 hours, and the air flow rate is 3 L/min. The pH was maintained at 4 with 10% aqueous ammonia. When the concentration of residual glucose and ammonium sulfate decreases, add glucose and ammonium sulfate continuously.

Table 3 shows the yield of εPL and the conversion rate of glucose after culturing for 168 hours. .

Example 5

Except for using Streptomyces albicans subsp. lysinopolymerus B22107 strain instead of Streptomyces albicans subsp. lysinopolymerus B21021 strain, culture was carried out as in Example 4, and the amount of εPL in the culture was measured. Table 4 shows the production of εPL and the conversion rate of glucose after 168 hours of cultivation.

Example 6

Except for using Streptomyces albicans subsp. lysinopolymerus subsp. B22201 strain instead of Streptomyces albicans subsp. lysinopolymerus subsp. B21021 strain, culture was carried out as in Example 4, and the amount of εPL in the culture was measured. Table 4 shows the production of εPL and the conversion rate of glucose after 168 hours of cultivation.

Comparative Example 2

The cultivation was carried out in the same manner as in Example 4, except that the Streptomyces albicans subsp. lysinopolymerus subsp. 11011A-1 strain was used instead of the Streptomyces albicans subsp. lysinopolymerus subspecies B21021 strain. Table 4 shows the production of εPL and the conversion rate of glucose after 168 hours of cultivation.

Table 4

Strain εPL yield (g/l) Glucose conversion rate (%)

B21021 (Example 4) 31.0 12.4

B22107 (Example 5) 28.6 12.6

B22201 (Example 6) 25.0 11.4

11011A-1 (comparative example 2) 19.1 7.8

Example 7

The culture obtained in Example 4 was cultured for 168 hours to remove cells by centrifugation, and then the pH was adjusted to 7.5. The residue was separated, purified using IRC-50 (cation exchange resin), IRA-402 (anion exchange resin), and XT-1006 (cation exchange resin), and then concentrated through a reverse osmosis membrane (RO) to obtain εPL. The yield of εPL is shown in Table 5.

Comparative Example 3

The culture obtained by culturing for 168 hours in Comparative Example 2 was centrifuged to remove cells as in Example 7, and then the pH was adjusted to 7.5. The residue was separated, purified using IRC-50 (cation exchange resin), IRA-402 (anion exchange resin), and XT-1006 (cation exchange resin), and then concentrated through a reverse osmosis membrane (RO) to obtain εPL. The yield of εPL is shown in Table 5.

table 5

Strain εPL yield (g)

B21021 (Example 7) 39

11011A-1 (Comparative Example 3) 24

It can be seen from Tables 3, 4 and 5 that the improved strains were significantly higher than strain 11011A-1 in terms of εPL production and glucose conversion rate.

Industrial Applicability

The improved strain resistant to high-concentration AEC of the present invention has the ability to efficiently produce εPL in large quantities. Using this εPL-producing strain, εPL can be produced with high yield and high yield.

Claims (2)

1. B21021 strain B21021 (preservation number FERM BP-5926) that produces a large amount of ε-poly-L-lysine, which is obtained by mutagenizing Streptomyces parvus lysinopolymerus subspecies 11011A-1 strain (FERM BP-1109) obtained, which is resistant to S-(2-aminoethyl)-L-cysteine at a concentration of 10 mg/ml or higher. 2. A method for producing ε-poly-L-lysine, the method comprising aerobically culturing the B21021 strain of claim 1 in a culture medium, and collecting ε-poly-L-cysteine formed and accumulated in the culture solution.