JPH0441991B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for culturing isoleucine-producing microorganisms, and more specifically, to a method for culturing isoleucine-producing microorganisms, which improves the productivity of isoleucine by culturing the isoleucine-producing microorganisms under irradiation with light in a specific wavelength range. Regarding culture methods. The present inventor happened to discover how light conditions affect the growth of various useful plants, the control of the growth of pathogenic fungi on useful plants, the cultivation of algal plants, etc. The present inventors have discovered that isoleucine productivity is greatly improved when isoleucine-producing microorganisms are cultured under active irradiation with a specific light beam containing at least 280 nm or more, leading to the completion of the present invention. Thus, according to the present invention, in the method of culturing an isoleucine-producing microorganism while irradiating it with light, the microorganism is a microorganism selected from bacteria, actinomycetes, and fungi that have the ability to produce isoleucine; The irradiation start time of the light beam is after the logarithmic growth phase of the microorganism in the culture system, and the light beam substantially contains light in a wavelength range of at least 280 nm or more. A method for culturing isoleucine-producing microorganisms is provided. As used herein, the term "isoleucine-producing microorganism" refers to a microorganism that has the ability to produce isoleucine as a metabolic product outside the microbial cell,
Such microorganisms include fungi, actinomycetes, bacteria, and yeasts. Generally speaking, the method of the present invention can be applied to any type of microorganism, as is clear from the experience of the present inventors and the results of the examples described below. Although it can be expected to improve the productivity of isoleucine to a greater or lesser extent, it is particularly effective against microorganisms such as bacteria and actinomycetes, and bacteria are particularly preferred. Examples of typical microorganisms to which the method of the present invention can be applied are as follows. In the following examples of microorganisms, the name of the microorganism is shown in Japanese with the genus and species, followed by the original name in parentheses. 1 Bacteria (1) Corynebacterium
Genus (1) Corynebacterium glutamicum (C.
glutamicum) (2) C.acetocidophilum (3) Corynebacterium equi (C.equi) (2) Brevibacterium
Genus (1) Brevibaclerium flavum (Br.
flavum) (2) Brevibacterium ammoniagenes (Br.ammoniagenes) (3) Brevibacterium lactofamentum (Br.lactojermentum) (4) Brevibacterium herlborum (Br.
helvolum) (3) Genus Serratia (1) Serratia marcietsiens (S.
marcescens) (4) Bacillus genus (1) Bacillus subtilis (2) Bacillus megaterium (B.
megaterium) (5) Micrococcus genus (1) Micrococcus variens (M.
(2) M.luteus (6) Microbacterium
Genus (1) Microbacterium flavum (Mi.
flavum) (7) Genus Pseudomonas (1) Ps.
aeruginosa) (2) Ps. fluorescens (3) Ps. aureofaciens (8) Aerobacter (1) Aerobacter cloacae (A.
cloacae) (2) Aerobacter aerogenes (A.
aerogenes) (9) Escherichia (1) E. coli (10) Arthrobacter genus (1) Arthrobacter citreus (Ar.
citreus) (2) Ar.aminofacience (11) Sarcina spp. (1) Sarcina lutea (12) Methanomonas spp. (1) Methanomonas methylovora (Me.
methylovora (13) Protaminobacter genus (1) Protaminobacter ruber (14) Hafnia genus (1) Hafnia alvei (H.alvei), etc. , among others, Corynebacterium, Brevibacterium, Serratia, Bacillus,
The genera Aerobacter and Arthrobacter are preferred, and the genera Coneribacterium, Brevibacterium, Serratia and Arthrobacter are particularly preferred. 2 Actinomycetes (1) Streptomyces
Genus (1) St. typhimurium (2) St. flaveolus, etc. Conventionally, the industrial production of isoleucine by fermentation has generally been carried out in a dark tank from beginning to end, under conditions that substantially avoid exposure to light. In contrast to the conventional fermentative production method of isoleucine, the present invention is to culture microorganisms while actively controlling the light beam containing the above-mentioned specific light beam, and in this respect, the method of the present invention is substantially different from conventional fermentation methods. Irradiation with the above-mentioned specific light beam can be applied to both the pre-culture and the main culture in the isoleucine production process, but the effect will be greater if it is applied during the main culture. There is no particular restriction on the light beam that can be irradiated, as long as it substantially contains light in the wavelength range of at least 280 nm or more, and as long as it substantially contains light in the above-mentioned specific wavelength range, it can be used not only as artificial light. ,
Natural light can also be used. Therefore, when using artificial light and/or natural light, use an optical filter as necessary to reduce the amount of light irradiated with light that substantially contains light in the wavelength range of at least 280 nm and above.
It is preferable to culture under artificial light irradiation at a level of 100,000 μw/cm ² or less, preferably 30,000 μw/cm ² or less, more preferably 10,000 to 1 μw/cm ² . According to the method of the present invention, the intensity of the light rays irradiated onto the microorganism culture system is not strictly limited, and varies depending on the type of microorganism to be cultured and other culture conditions, and is determined to be optimal in each case. Irradiation conditions can be easily determined by those skilled in the art by conducting small-scale experiments, but in general,
The amount of light in the visible light region with a wavelength in the range of 400 nm to 700 nm is 100,000 μw/cm ² or less, preferably 50,000 to
1 μw/cm ² , more preferably 10000 to 5 μw/cm ² ,
It is particularly advantageous to irradiate with a light beam adjusted to a range of preferably 5000 to 50 μw/cm ² . Also,
It is preferable that the light in the ultraviolet region with a wavelength of 280 nm to 400 nm is not very strong, and the intensity of the ultraviolet light in this wavelength range is generally less than 800000 μw/cm ² , preferably less than 40000 μw/cm 2, and more preferably 10000 μw/cm ² or less.
~1 μw/cm ^{2 ,} particularly preferably 5000 to 1 μw/cm ² . Further, according to the method of the present invention, a specific method of actively irradiating the microorganism culture system with the specific light beam includes, for example, in a system (inside a tank) that is substantially sealed from external light; at least
Light rays substantially containing light in the wavelength range of 280 nm and above, preferably light in the wavelength range of 280 to 600 nm, more preferably light in the wavelength range of 280 to 500 nm, most preferably light in the wavelength range of 280 to 400 nm and/or
Artificial light containing substantially light in the wavelength range of 400 to 500 nm (in this case, the artificial light source itself may emit light with such light quality characteristics, or the artificial light source may be filtered with a suitable filter) A method of irradiating the irradiated light (by covering the irradiated light with the above-mentioned light quality characteristics); under the irradiation of the sun or natural light, the irradiated light is substantially irradiated with light in the wavelength range of at least 280 nm and above. , preferably light in the wavelength range of 280 to 600 nm, more preferably light in the wavelength range of 280 to 500 nm, most preferably,
By a transparent colorless to colored organic or inorganic coating material (for example, a synthetic resin film containing an ultraviolet absorber) that transmits light substantially containing light in the wavelength range of 280 to 400 nm and/or 400 to 500 nm. Possible methods include culturing under coated conditions; and a combination of both of the above methods. According to the method of the present invention, irradiation of the isoleucine-producing microorganism with the light beam substantially containing light in the specific wavelength range is preferably started during the logarithmic growth phase of the microorganism in the microorganism culture system. When the microorganism is inoculated into a culture system, it does not rapidly proliferate immediately, but after passing through a lag phase, it enters a logarithmic growth phase in which it proliferates rapidly. After the logarithmic growth phase, the microorganisms go through a stationary phase as nutrient sources in the system are depleted, and then decrease in number and die. According to the invention,
The irradiation of the light beam substantially containing light in the specific wavelength range starts during the logarithmic growth phase, preferably the middle phase of the logarithmic growth phase. The period of irradiation with the light beam substantially containing light in the specific wavelength range includes, for example, from the start of irradiation until the end of fermentation, or until a certain stage of fermentation is reached, but preferably. is up to the point where fermentation ends. Note that the irradiation format includes a continuous irradiation method in which irradiation is performed continuously, an intermittent irradiation method in which irradiation and darkness are alternately repeated, and a combination method thereof, which can be selected as appropriate. In the present invention, "substantially containing light in the wavelength range of Xnm or more" means not only that ultraviolet rays in the wavelength range of less than Xnm are completely absent from the total amount of light irradiated, but also that the ultraviolet rays in the wavelength range of less than Xnm are completely absent. This means that there is no problem even if it is contained in a small amount within a range that does not adversely affect the culture of the present invention. Furthermore, in the present invention, "substantially containing light in the wavelength range of Y to Znm" means not only a case where the light in the wavelength range of Y to Znm accounts for 100% of the total light dose to be irradiated, but also 60% of the total light dose. The ratio is preferably 80% or more, more preferably 90% or more. The cultivation of isoleucine-producing microorganisms according to the method of the present invention can be carried out under exactly the same conditions as those conventionally used, except for the condition that it is carried out under irradiation with the above-mentioned specific light rays. For example, this can be carried out by culturing isoleucine-producing microorganisms in a suitable nutrient medium in liquid or solid state. Nutrient sources, nitrogen sources, inorganic salts, etc. of the culture medium at that time are appropriately selected depending on the microorganisms and culture means used, but those commonly used for culturing microorganisms are widely used. As a carbon source,
Any assimilable carbon compound may be used, such as glucose, sucrose, lactose, maltose, starch, dextrin, molasses, glycerin, hydrocarbons, ethyl alcohol, methyl alcohol, acetic acid, and the like. The nitrogen source may be any nitrogen compound that can be used, such as corn stew liquor, soybean flour, soybean protein hydrolyzate, cottonseed oil,
Wheat gluten, peptone, meat extract, yeast extract, yeast, casein hydrolyzate, ammonium salts, nitrates, etc. are used. Examples of inorganic salts include phosphate, magnesium, calcium,
Salts such as potassium, sodium, zinc, iron, and manganese are used, and if necessary, various vitamins, amino acid salts, and nucleic acids are also used. The culture temperature and culture time vary somewhat depending on the microorganism used, and can be changed as appropriate within the range that allows the microorganism to grow sufficiently. In the case of actinomycetes, it is preferable to culture at a temperature of about 26 to 32°C. More specific culture conditions vary depending on the individual microorganism, but for example, Patent Publication Nos. 51-6237 and 51-6237.
-7755, 51-21077, 52-4629, 52
-4629, 52-30593, 53-10153, 54
−22513, No. 54-32070, No. 56-3035 and No.
It can be carried out using the culture conditions disclosed in No. 56-29998 and the like. In the present invention, the material used for culturing under the above light quality conditions is not particularly limited as long as it has the above light transmission characteristics, and any type of material may be used. It can also be used as a dressing. Such materials can usually consist of inorganic or organic films, plates, or other molded bodies. For example, as an inorganic film or plate, glass plates are typically mixed with dyes or pigments (e.g. emerald green), glass plates coated with or laminated with synthetic resin films containing ultraviolet absorbers as shown below, and glasses. Examples of the organic film or board include a synthetic resin film or board coated with or containing an ultraviolet absorber. In addition to the thermoplastic resins described below, thermosetting resins such as melamine resins, phenolic resins, epoxy resins, silicone resins, urea resins, alkyd resins, and allyl phthalate resins can also be used as resins that can be used for this molding. be able to. The transparent film or plate that can be used in the present invention can be produced, for example, by blending a suitable ultraviolet absorber with a normal film-forming thermoplastic resin and molding the mixture into a film or plate. Examples of usable film-forming thermoplastic synthetic resins include polyvinyl chloride, polyvinylidene chloride, polyethylene, polypropylene, polystyrene, polyester, polyamide, polycarbonate, polymethyl methacrylate, polyacrylate, polyvinyl acetate, polyvinyl alcohol,
Fluorine-containing resin, cellulose resin, ABS resin, etc.
It also includes copolymers or blends containing these polymers as the main component (preferably 50% by weight or more), particularly polyvinyl chloride, polyethylene, polypropylene,
Polyester, fluorine-containing resin, cellulose resin, and polycarbonate are suitable. As the artificial light source that can be used in the present invention, any light source that emits light in the above-mentioned specific wavelength range can be used. Such light sources include, for example, fluorescent lamps, mercury lamps, sunlight lamps, general light bulbs, flood light bulbs, special light bulbs (from the Toshiba Lamp Catalog, Toshiba Electric Materials Co., Ltd.), and the like. More specifically, fluorescent lamps (product names are shown in parentheses). ] as daylight (FL40SW-E/M, Toshiba), Plantorx (FL40B/NL, Toshiba), Fuitsyulux (FL40SBRF/NL, Toshiba), blue (FL40B/NL, Toshiba), blue-white (FL40BW/NL,
Toshiba), white (FL40SD/NL, Toshiba), daylight (FL40SD/NL, Toshiba), deluxe (FL40SW
-DL-X/NL, Toshiba), warm white (FL40SW/
NL, FL40SW-A/NL, Toshiba), for leaf tobacco
6100〓 (FL40SRD-SDL6100〓, Toshiba), for photography (FL40SD-SDL-CP/NL, Toshiba), blacklight fluorescent lamp (FL40SBL-B Matsushita),
There are fluorescent lamps for health line (FL20S-E Matsushita), fluorescent chemical lamps (FL20S-BL Matsushita), high color rendering fluorescent lamps (FL20SW-EDL-50K, Toshiba), and insect-trapping fluorescent lamps (FL20SBA-37K, Toshiba), etc. ,this house,
Blacklight fluorescent lamps, health line fluorescent lamps, blue and blue-white lamps are preferred. According to the method of the present invention described above, in the production of isoleucine by the fermentation method, by culturing microorganisms under specific light quality conditions, the production of isoleucine is greatly promoted, which contributes to the fields of medicine, food, etc. It is extremely serious. Next, the present invention will be further explained with reference to Examples. Examples 1 to 4, Comparative Examples 1 and 5 Glucose 5g, peptone 10g, yeast extract 10
Dispense 100 ml of a pH 7.0 aqueous solution consisting of 5 g of NaCl, 5 g of distilled water, and 1 ml of distilled water into a 500 ml Erlenmeyer flask, autoclave it, and inoculate the isoleucine-producing microorganism Brevibacterium flavum in advance into a bouillon slant. flavum
One platinum loop of FERM-P6373 (a microorganism described in Japanese Patent Publication No. 56-29998) was inoculated and precultured for 20 hours under dark conditions at a culture temperature of 30° C. and a shaking speed of 200 times/min. Prepare the aqueous solution shown in Table 1 in advance,
Dispense 20 ml of the aqueous solution into a 500 ml plate-necked flask made of Halio glass, sterilize it at 110°C for 10 minutes, inoculate it with 2 ml of the above preculture suspension, and incubate at 30°C, shaking at 200 times/min, and in the dark. The main culture was carried out below. Seven hours after the start of main culture, it was confirmed by the number of viable bacteria that the bacterial growth had reached the middle of the logarithmic growth phase. Five culture flasks were transferred for each light irradiation section to four shaking culture machines (culture temperature 30°C, shaking rotation speed 200 times/min) each equipped with a fluorescent lamp that emits light. Culture was continued. The remaining five culture flasks continued to be cultured in the dark. Culture time is 72 hours from the start of culture.
Continuous irradiation culture was performed in various light quality irradiation sections. Further, from the beginning of the main culture, culture was carried out under the same light irradiation conditions as in Example-2 (Comparative Example-5). The results are shown in Table-3. In addition,
The amount of isoleucine produced is the average value of the values obtained from the culture flasks (5 flasks) in each light irradiation group.

【table】

【table】

【table】

[Table] Examples 5 to 8, Comparative Example 2 Corynebacterium glutamicum was used as an isoleucine-producing microorganism.
glutamicum FERM-P2842, Special Publication Showa 54-32070
Table 4 and Table 5 (microorganisms described in the publication)
Examples 1 to 4 using the pre-culture medium and main culture medium shown in
Preculture and main culture were performed in the same manner as described above. However, the culture time was 69 hours, and the culture results are shown in the table below.
6.

【table】

【table】

【table】

[Table] Examples 9 to 12, Comparative Example 3 Isoleucine-producing microorganisms were isolated from Serratia marcescens.
ATCC21741), and preculture and main culture were performed in the same manner as in Examples 1 to 4 using the preculture medium and main culture medium shown in Tables 7 and 8. However, the culture time was 72 hours, and the culture results are shown in Table 9.

【table】

【table】

[Table] Examples 13 to 16, Comparative Example 4 The isoleucine-producing microorganism was Arthobacter citreus.
ATCC21040), and preculture and main culture were performed in the same manner as in Examples 1 to 4 using the preculture medium and main culture medium shown in Tables 10 and 11. However, the culture time was 72 hours, and the culture results are shown in Table 12.

【table】

【table】

【table】 [Brief explanation of drawings]

FIGS. 1 and 2 are specific energy curves by wavelength of irradiation light used in Examples and Comparative Examples.

Claims (1)

[Claims] 1. A method for producing isoleucine by culturing an isoleucine-producing microorganism while irradiating it with light, wherein the microorganism is selected from bacteria, actinomycetes, and fungi that have the ability to produce isoleucine. is a microorganism, the irradiation time of the light beam is after the logarithmic growth phase of the microorganism in the culture system, and the light beam substantially contains light in a wavelength range of at least 280 nm or more. , A method for culturing isoleucine-producing microorganisms, characterized by: 2. The method according to claim 1, wherein the culturing is carried out under irradiation with light that substantially contains light in the wavelength range of at least 280 to 600 nm. 3. The method according to claim 1, wherein the culturing is carried out under irradiation with light that substantially contains light in the wavelength range of at least 280 to 500 nm. 4. The culture is exposed to at least 280-400 nm and/or
The method according to claim 1, which is carried out under irradiation with a light beam substantially containing light in the wavelength range of 400 to 500 nm. 5. The method according to any one of claims 1 to 4, wherein the irradiation intensity of the light beam is in the range of 100000 to 1 μW/cm ² . 6. The method according to any one of claims 1 to 5, wherein the light beam is an artificial light beam and/or a natural light beam. 7. The method according to any one of claims 1 to 6, wherein the microorganism is a bacteria or an actinomycete. 8. The method according to claim 7, wherein the microorganism is of the genus Serratia, Corynebacterinm, Arthrobacter, and Brevibacteriun.