JPS61274692A - Production of l-histidine - Google Patents

Abstract

PURPOSE:To produce, a large amount of L-histidine inexpensively, by cultivating cultivating a specific Coryneform mold capable of producing histidine in a mold. CONSTITUTION:A bacterium (e.g., Corynebacterium glutamicum GAUPTr-113) capable of producing L-histidine is varied with nitrous acid, to give a variant (e.g., Corynebacterium glutamicum Re-13) which has no ability or extremely reduced ability to grow using D-ribose as the only carbon source. Then, it is aerobically cultivated in a medium consisting of a carbon source (e.g., molasses), a nitrogen source (e.g., urea), an inorganic salt (e.g., NaCl), etc., at 20-40 deg.C at 5-9pH for 2-7 days, and L-histidine is accumulated in the culture mixture. Then, it is isolated by ion exchange resin method, etc.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention has wide applications in food, medicine, and other fields.
A new industrial method for producing histidine is provided.

BACKGROUND ART Regarding methods for producing L-histidine using microorganisms, methods using mutant strains such as histidine analog resistant strains of bacteria such as Corynebacterium, Brevibacterium, and Serratia are known.

Problems to be Solved by the Invention The demand for L-histidine has increased in recent years, and improvements in methods for increasing the productivity of L-histidine are always desired.

Means for Solving the Problems The present inventors have discovered that L.
- Among histidine-producing bacteria, it was discovered that a strain with a significantly reduced ability to grow using D-ribose as the only carbon source has an excellent ability to produce L-histidine, and the present invention was completed.

The present invention will be explained in detail below.

The present invention involves culturing in a medium a microorganism that belongs to coryneform glutamate-producing bacteria, has the ability to produce L-histidine, and has a deleted or significantly reduced ability to grow using D-ribose as the sole carbon source. Provided is a method for producing L-histidine, which comprises accumulating L-histidine in the culture and collecting the accumulated L-histidine from the culture.

The microorganism used in the present invention can be any microorganism that belongs to the coryneform glutamate-producing bacteria and that has been deficient or significantly reduced in its ability to grow using D-ribose as the sole carbon source. That is, the microorganism of the present invention does not grow or grows very slowly on a minimal medium containing D-'J bose as the sole carbon source.

As used herein, coryneform glutamate-producing bacteria are
A group of glutamic acid-producing bacteria belonging to the genus Corynebacterium, Brevibacterium, Microbacterium, or Arthrobacter [see "Fermentation and Industry" Vol. 40, 102, 1982].

The microorganisms used in the present invention are obtained by subjecting microorganisms capable of producing L-histidine to mutation treatment to induce f) mutant strains lacking or significantly reduced in the ability to grow using 9-port as the sole carbon source. Obtainable.

As the mutation treatment, a conventional mutation treatment method such as ultraviolet irradiation or chemical treatment with N-methyl-N'-nitro-N-nitronguanidine (NTG), nitrous acid, etc. is employed. Mutant strains can also be induced by other genetic techniques (eg, genetic recombination, transduction, cell fusion, etc.). Furthermore, in addition to the above-mentioned properties, the microorganism of the present invention also has other properties (e.g., various nutritional auxotrophies, drug resistance, drug sensitivity, drug dependence, changes in the ability to assimilate various sugars or organic acids, etc.). You can.

A specific example of the microorganism used in the present invention is Corynebacterium glutamicum G, which is an L-histidine producing bacterium.
ALIPTr-113 (Microtechnical Research Institute No. 1874) (
Rb derived from Japanese Patent Publication No. 52-18798)
-11 and Re-13.

Next, a specific example of operation for obtaining such microorganisms will be explained.

Operation example> Corynebacterium glutamicum GAIIPTr-1
13 cells in 0.2N Tris-Maleide buffer (pH
6,0) to a concentration of 10" cells/ml,
To this, N-methyl-N'-nitrosoguanidine was added to a final concentration of 0.25 μ/ml, and after standing at room temperature for 30 minutes, the bacterial cells were collected. It was suspended in a buffer solution. This cell suspension was placed on a minimal agar medium containing glucose as the only carbon source [glucose 0.5%, (NH4)].
2SO40, 15%, KH, Po, 0.1%, J
PO40.3%, NaCj20.1%, Mg5O,・
7H,00,1%, CaC1x・2H201g/ml,
Fe5Q, '7HzOI Ox/ml, MnCl2
・4H207011g/ml, Thiamin・HC(l
1g/ml biotin 30g/l, shikimic acid 50x/ml
, 2% agar (adjusted to p 17.8 with NaOH)] so that 100 to 1,000 colonies appeared per plate, and the cells were statically cultured at 30°C for 3 days.

Next, the cells grown in colonies were placed on the above-mentioned minimal agar medium and a minimal agar medium containing D-ribose as the sole carbon source (
After removing glucose from the above-mentioned glucose medium and transferring it to a medium supplemented with 0.5% D-ribose using the replica method and statically culturing at 30°C for 3 days, it was found that no growth was observed in the medium using glucose as the carbon source. However, it does not grow on a medium containing D-ribose as a carbon source, or the parent strain GALI
Many mutant strains with significantly slower growth than PTr-113 were obtained.

100 mutant strains with these properties were subjected to an L-histidine fermentation test (same method as in Example 1), and Rh-11 and Re-1 were identified as strains with superior L-histidine productivity compared to the parent strain.
I chose 3.

Rb-11 was deposited with the Institute of Microbial Technology (Feikokuken) of the Agency of Industrial Science and Technology on May 2, 1985, and its deposit number is FERM P-5'244.

In the method of the present invention, the medium for culturing microorganisms is
Nutrient or synthetic media containing carbon sources, nitrogen sources, inorganic salts, growth factors, etc. are used.

As the carbon source, carbohydrates such as glucose, fructose, chiny sucrose, molasses, starch, starch hydrolyzate, and fruit juice, and alcohols such as ethanol, methanol, and propatool can be used.

Nitrogen sources include ammonium sulfate, ammonium nitrate, ammonium chloride, ammonium phosphorus, ammonium acetate, urea, ammonia, amines, peptone, meat extract, yeast extract, cornstarch liquor, casein hydrolyzate, various fermenting bacteria and their digestion. Things can be used.

Inorganic salts include potassium phosphate, dipotassium phosphate, magnesium phosphate, sodium chloride, ferrous sulfate,
Manganese sulfate, calcium carbonate, etc. can be used.

When the bacteria used have auxotrophic properties, those auxotrophic substances are added as standard products or natural products containing them.

Cultivation is performed under aerobic conditions such as aeration and shaking culture. The culture temperature is 20 to 40°C, and the number of culture days is 2 to 7 days. The pH of the culture solution is maintained in the range of 5-9. To adjust the pH, urea, calcium carbonate, ammonia gas, aqueous ammonia, magnesium phosphate, etc. are used as a neutralizing agent.

As a method for isolating L-histidine from the culture solution after completion of culture, ion exchange resin method, solvent extraction method, and other known methods are used.

The present invention will be specifically illustrated by giving examples below.

Example 1 Mutant strains Corynebacterium glutamicum Rb-11 and R with significantly reduced D-ribose assimilation ability as inoculum
e-13 was used. This mutant strain was seeded in a seed medium [glucose 4
%, peptone 1%, meat extract 0.5%, yeast extract 0.
5%, KH, PO, 0,1%, KJPO4o, t%, u
gso, ・7Lo o, 05%, pesOa4B
20 20mg/j! , MnCj! 2
・4) 120 2 0 mg/1 urea 0.5
%, (NH4) lSO40.5% (pF17.2)
] A 309 ml Erlenmeyer flask containing 220 m was inoculated and cultured at 30°C for 24 hours. Add 1 ml of this seed culture to fermentation medium [glucose 7%, (NH4) zsOa 2.3
%, H2PO40.2%, KJPOn 0.1
%, Mg5O, 7L 0.05%, NaC1O,
25%, urea 0.2%, thiamine/HCl 200n/
l, piotine 500 ttg/l, nicotinamide 500/11 calcium pantothenate 5 Q Og/
l, Fe5L 4LO 10 mg/l.

Mn1Jz・4)1zo l Omg/j! , soybean meal sulfuric acid hydrolyzate 0.5% (solid content equivalent), shikimic acid 0.
3 containing 120 ml of 02%, pH 7,2 (NL mouth H)
The mixture was inoculated into a 00ml Erlenmeyer flask and cultured with shaking at 30°C for 4 days.

The parental strain GAIIPT was simultaneously grown under the same conditions as a control.
r-113 was cultured. The amount of L-histidine accumulated by each strain was as shown in Table 1.

Rh-119,5 Example 2 The same procedure as in Example 1 was carried out except that a fermentation medium containing 7% blackstrap molasses (in terms of glucose) was used instead of glucose. The amount of L-histidine accumulated by each strain is shown in Table 2.

Effects of the Invention According to the method of the present invention, L-histidine is produced! Since production can be significantly improved, L-histidine can be supplied in large quantities at low cost.

Claims (3)

[Claims] (1) A microorganism that belongs to the coryneform glutamate-producing bacteria, has the ability to produce L-histidine, and has lost or significantly reduced the ability to grow using D-ribose as the sole carbon source is cultured in a medium, and the culture is accumulates L-histidine in
A method for producing L-histidine, which comprises collecting accumulated L-histidine from the culture. (2) The method according to claim 1, wherein the microorganism belongs to the genus Corynebacterium, Brevibacterium, Microbacterium, or Arthrobacter. (3) The microorganism is Corynebacterium glutamicum,
Brevibacterium flavum, Microbacterium
The method according to claim 2, characterized in that it belongs to Ammoniaphyllum or Arthrobacter citreus.