JPH0411196B2 - - Google Patents

Description

[Detailed description of the invention]

(Industrial Application Field) The present invention relates to a method for producing L-lysine by fermentation. More specifically, the present invention relates to a method for producing L-lysine by a fermentation method using L-lysine producing bacteria of the genus Brevibacterium or Corynebacterium having enhanced superoxide dismutase activity. (Prior art) Conventionally, as a method for producing L-lysine by fermentation, there has been a method using S-(2-aminoethyl)-L-cysteine (hereinafter referred to as AEC)-resistant bacteria (Tokuko Showa).
48-28078), AEC resistant and L-leucine,
Method using L-homoserine, L-proline, L-arginine, or L-alanine auxotrophic (hereinafter referred to as Ala ^- ) mutant strain (JP-A-49-36888
No., JP-A-49-80289, JP-A No. 51-21078),
A method using a mutant strain resistant to AEC and leucine analogs such as β-hydroxylleucine (hereinafter referred to as HL) (Japanese Patent Publication No. 53-1833), a method using a mutant strain resistant to α-chlorocaprolactam (hereinafter referred to as CCL) (Japanese Patent Publication No. 53-1833); Publication No. 53-43591), Method using γ-methyllysine (hereinafter referred to as ML) resistant mutant strain (Japanese Patent Publication No. 56-19235), Fluoropyruvate (hereinafter referred to as ML)
A method using a susceptible mutant strain (referred to as FP) (Japanese Patent Application Laid-open No. 55-9783), a method using a mutant strain with decreased pyruvate kinase activity (Japanese Patent Application Laid-Open No. 58-170487)
No.) are known. (Problems to be Solved by the Present Invention) The problems to be solved by the present invention are to establish a method for producing L-lysine by a fermentation method at a lower cost. (Means for Solving the Problems) In order to solve the above problems, the present inventors
- As a result of various studies aimed at increasing the fermentation yield by improving the strain of lysine-producing bacteria, the L-lysine-producing ability of this bacteria was significantly increased by increasing the superoxide dismutase activity of the L-lysine-producing bacteria. Based on this finding, the present invention has been completed. That is, the present invention relates to L-
The present invention relates to a method for producing L-lysine using a fermentation method, which comprises culturing a mutant strain capable of producing lysine, producing and accumulating L-lysine in a culture solution, and collecting the L-lysine. The microorganisms used in the present invention belong to the genus Brevibacterium or Corynebacterium and have the ability to produce L-lysine, and are selected from among the resulting mutant strains as superoxide production agents and superoxide dismutase induction inhibitors. This can be obtained by selecting strains that are resistant to agents, superoxide radical reaction promoters, or oxidizing agents that supply oxygen to form superoxide. The mutant strain thus obtained has enhanced superonicide dismutase activity. As the parent strain of the mutant strain induced in the present invention, any strain of microorganisms belonging to the genus Brevibacterium or Corynebacterium may be used, regardless of the species or strain. - Bacteria known as glutamic acid producing bacteria are preferred. Brevibacterium devaricatum
ATCC14020 Brevibacterium flavum ATCC14067 Brevibacterium lactofamentum
ATCC13869 Brevibacterium roseum ATCC13825 Corynebacterium acetoacidophyllum
ATCC13870 Corynebacterium Lilium ATCC15990 In addition to these wild strains, strains that have properties advantageous for lysine production, such as AEC resistance, CCL resistance,
ML resistance, homoserine auxotrophy, alanine auxotrophy,
Fluoropyruvate sensitivity, threonine sensitivity,
Mutant strains with properties such as methionine sensitivity can also be used. In order to obtain the mutant strain of the present invention using a strain belonging to the Brevibacterium genus or Corynebacterium genus and having properties advantageous for lysine production, microorganisms such as ultraviolet irradiation, X-ray irradiation, mutagenic agent treatment, etc. are generally used. Any conventional method for inducing mutations may be used. An example of treatment with a mutagen is treatment with 250 μg/ml of N-nitro-N'-methyl-N-nitrosoguanidine at 30° C. for 20 minutes. In the present invention, from the mutant strains thus obtained, strains resistant to superoxide production enhancers, superoxide dismutase induction inhibitors, superoxide radical reaction promoters, or oxidizing agents that supply oxygen to form superoxide are selected. get. All of these drugs facilitate the formation of lipid peroxides through the reaction between superoxide (O ₂ ⁻ ) and unsaturated fatty acids. In other words, these drug-resistant strains should be strains with strong cell activity that proliferate in environments where cell activity tends to decrease, and the present inventors believe that such strains are extremely suitable as strains for L-lysine production. They found that Superoxide production enhancers are substances that have the effect of increasing superoxide in vivo.
For example, methyl viologen, nitrofurantoin,
vitamin _K1 , morphine, streptonigrin,
Adriamycin, mitomycin C, daunomycin, bleomycin, β-lapachone, cis-
Such as platinum()diaminodichloride. A superoxide dismutase induction inhibitor is one that inhibits the induction of superoxide dismutase, which is an enzyme that converts superoxide into hydrogen peroxide, in a living body, and Pieromycin can be mentioned as an example. A superoxide radical reaction accelerator is one that promotes a reaction in which superoxide reacts with an unsaturated fatty acid to produce lipid peroxide, and phenylhydrazine can be mentioned as an example. The oxidizing agent is one that supplies oxygen that forms superoxide in vivo, such as benzoyl peroxide and ammonium persulfate. Obtaining strains resistant to these drugs can be carried out in accordance with the usual method for obtaining drug-resistant strains, for example, by spreading and culturing the mutant strain on an agar medium containing one of the above drugs at a concentration that does not allow the growth of the parent strain. ,
All you have to do is collect the grown colonies. The concentration of the drug in the medium varies depending on the type of drug; for example, methyl viologen is about 1.5 μg/ml, pieuromycin is about 0.5 μg/ml, and phenylhydrazine is about 20 μg/ml. ml, and in the case of benzoyl peroxide, about 20 μg/ml is appropriate. Needless to say, when using other drugs, a culture test may be conducted in advance to determine the appropriate concentration. In addition, wild strains belonging to the genus Brevibacterium or Corynebacterium are subjected to mutation treatment, and superoxide enhancers, superoxide dismutase induction inhibitors, superoxide radical reaction accelerators, or superoxide are added to the mutant strains obtained. After selecting strains that are resistant to the oxidizing agent that supplies oxygen to form, these resistant strains are endowed with properties useful for lysine production, such as AEC resistance, homoserine auxotrophy, CCL resistance, ML resistance, and fluoropyruvate sensitivity. However, it is possible to obtain a strain with significantly increased lysine production ability. Examples of microorganisms that can be used in the present invention include Brevibacterium lactofermentum AJ12220 (FERM-P8248, FERM
BP-996), and for superoxide dismutase induction inhibitors, Corynebacterium acetoglutamicum, which is resistant to Pieuromycin.
AJ12223 (FERM-P8251, FERM BP-998),
In addition, regarding the superoxide radical reaction accelerator, Brevibacterium flavum AJ12222 (FERM-
P8250, FERM BP-997) and Brevibacterium lactofermentum AJ12221, which is resistant to benzoyl peroxide, for the oxidizing agent that supplies oxygen to form superoxide.
(FERM-P8249, FERM BP-2329). In order to produce and accumulate L-lysine using such a microorganism, a conventional method used for L-lysine fermentation may be used. That is, the medium to be used is a conventional medium containing a conventional carbon source, nitrogen source, inorganic ions, and other nutrients. As the carbon source, for example, sugar raw materials such as sugar juice or blackstrap molasses from sugar cane or sugar beet, starch hydrolyzate, or organic acids such as acetic acid may be used. As the nitrogen source, ammonium salts, aqueous ammonia, urea, etc. used in ordinary L-lysine fermentation may be used, and organic ions such as phosphate ions, magnesium ions, and vitamins such as thiamine may be used as appropriate. When using strains that require specific substances for growth, such as auxotrophic strains, add these substances themselves to the culture medium, or add protein hydrolysates containing these substances, peptone, cornstap liquor, meat extract, yeast extract, etc. The added medium may be used. Regarding culture conditions, temperature is 30-40℃, pH 6-8.
It may be carried out by a conventional method such as carrying out under aerobic conditions within the range of . Furthermore, it goes without saying that L-lysine can be obtained from the fermentation liquid by a conventional method. The present invention relates to the completely new finding that L-lysine production ability is increased by enhancing the superoxide dismutase activity of L-glutamic acid producing bacteria, and the present invention relates to the completely new finding that L-lysine production ability is increased by enhancing the superoxide dismutase activity of L-glutamic acid producing bacteria.
-Lysine can be obtained easily and inexpensively. Next, an example of obtaining microorganisms used in the method of the present invention will be shown. The microorganism shown below was used as a parent strain. Brevibacterium lactofermentum
AJ3990, FERM-P3387 (AEC ^r , ML ^r , Ala ^- ) Brevibacterium flavum FAEC1-30,
FERM−P282 (AEC ^r ) Corynebacterium acetoglutamicum
AJ3792, FERM-P2650 (AEC ^r , β-HL ^r ) These strains were cultured in bouillon slant for 24 hours, and each strain was treated with 250 μg/ml of N-
Mutants were obtained by treatment with nitro-N'-methyl-N-nitrosoguanidine at 30°C for 20 minutes. Yeast extract 1g/dl, peptone 1g/dl,
In a medium consisting of 0.5 g/dl of salt and 2 g/dl of agar, 1.5 μg/ml of methyl viologen, 0.5 g/ml of pieuromycin, 20 μg/ml of phenylhydrazine,
Added 10μg/ml of benzoyl peroxide, both
After adjusting the pH to 7.0, the mixture was sterilized at 120°C for 15 minutes to prepare an agar medium. The mutant strain was spread on this agar medium and incubated at 31.5℃ for 48 hours.
After culturing for a period of time, the resulting colonies were collected to obtain each drug-resistant strain, and each AJ number listed in Table 1 was assigned. The results of growth tests conducted on each of the drug-resistant strains and the parent strain thus obtained by varying the concentration of each drug are shown below. The test method involved culturing all strains in bouillon slant for 24 hours, and adding 1 g of yeast extract/
dl, peptone 1 g/dl, salt 0.5 g/dl, and agar 2 g/dl containing a pH 7.0 medium in an 8 cm diameter shear dish.The number of bacteria per plate was ¹⁰⁵ to ^106. I sprinkled it so that it looked like this.
Then, a paper disk containing each drug at the concentration shown in Table 1 was placed thereon and cultured for 16 to 48 hours, after which the degree of growth was determined based on the presence or absence of a growth inhibition circle formed. Table 1 shows the results obtained for each drug-resistant strain and its parent strain. In the table, + indicates the degree of bacterial growth, and - indicates that no growth was observed.

[Table] The results of measuring the superoxide dismutase activity of each drug-resistant strain and its parent strain described above are shown in the second table.
Shown in the table. The measurements were carried out as follows. First, 20 ml of the culture solution of each strain was centrifuged at 10,000 rpm for 10 minutes to collect the precipitate, which was washed twice with 0.1 M phosphate buffer, pH 7. Add 20 ml of the above phosphate buffer to the washed precipitate.
Add, suspend, and perform ultrasonic treatment for 5 minutes.
The mixture was centrifuged at 10,000 rpm for 10 minutes, and the resulting supernatant was used as a sample. 0.05M sodium carbonate buffer with PH10.2 in a test tube
Take 2.4ml and add 3mM xanthine, 3mM EDTA,
0.1 ml each of 0.15% bovine albumin and 0.75 mM nitroblue tetrazolium was added. Add 0.1ml of the above sample to this and leave it at 25℃ for 10 minutes.
0.1 ml of the xanthine oxidase solution described below was added, quickly stirred, and incubated at 25°C.
After 20 minutes, 0.1 ml of 6mM CuCl ₂ was added to stop the reaction, and the absorbance at 560 nm was measured. on the other hand,
The same operation was performed using distilled water instead of the sample, and the obtained absorbance was used as a blank. One unit of activity was defined as superoxide dismutase activity that inhibits xanthine oxidase reaction by half under such measurement conditions. The xanthine oxidase solution was obtained by diluting xanthine oxidase with 2M (NH ₄ ) ₂ SO ₄ so that the absorbance in the blank test was around 0.23, and the xanthine oxidase concentration was about 2.1 × 10 ^-7 M. Ta.

[Table] Mutase Examples are shown below. Example 1 Glucose 36mg/ml, ammonium chloride 20mg/ml
ml, KH ₂ PO ₄ 1mg/ml, MgSO ₄・7aq0.4mg/ml,
FeSO ₄・7aq10μg/ml, MnSO ₄・4aq8μg/ml,
Soy protein acid hydrolyzate (as nitrogen) 1μg/ml,
Thiamine hydrochloride 0.1μg/ml and biotin
A medium containing 0.3 μg/ml was prepared, 30 ml of each was placed in 500 ml shaking flasks, and sterilized by heating at 115° C. for 10 minutes, and 1 g of calcium carbonate, which had been previously dry heat sterilized, was added. This medium was inoculated with the strains shown in Table 2, and cultured at 31.5°C using a reciprocating shaker. Fermentation was completed in 48 hours, and L-lysine accumulated in the fermentation liquid was measured. As a result, as shown in Table 3, all drug-resistant strains accumulated a good amount of L-lysine (in terms of hydrochloride).

[Table] Example 2 Cane molasses as sugar 80mg/ml, KH ₂ PO ₄ 1mg/
ml, MgSO ₄・7aq 1mg/ml, soybean hydrolyzate 1mg/ml nitrogen equivalent and ammonium sulfate
Prepare a medium with a composition of 500 μg/ml (PH7.0),
20 ml of the mixture was dispensed into 500 ml shaking flasks, sterilized by heating, and 1 g of calcium carbonate, which had been separately sterilized in advance, was added. Inoculate the following bacterial strains into this medium,
Culture was carried out at 31.5°C using a reciprocating shaker. Fermentation ended in 72 hours and L accumulated in the fermentation liquid.
- Lysine was measured. As a result, as shown in Table 4, all drug-resistant strains accumulated L-lysine (in terms of hydrochloride) well.

【table】

Claims (1)

[Scope of Claims] 1. A mutant strain belonging to the genus Brevibacterium or Corynebacterium and having enhanced superoxide dismutase activity and L-lysine producing ability is cultured, and L-lysine is added to the culture solution. A method for producing L-lysine by a fermentation method, which comprises producing, accumulating, and collecting lysine. 2. Claim 1, wherein the mutant strain is resistant to a superoxide production enhancer, a superoxide dismutase induction inhibitor, a superoxide radical reaction promoter, or an oxidizing agent that supplies oxygen to form superoxide. manufacturing method.