JPS5940439B2 - Method for producing L-histidine by fermentation method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing monohistidine by fermentation.

More specifically, the present invention relates to a method for producing 1-histidine using a fermentation method in which a microbial mutant strain resistant to growth inhibition by 2-fluorohistidine is cultured in a nutrient medium and L-histidine is accumulated in the culture solution.

The purpose is to provide a new industrial method for producing histidine, which has a wide range of uses in foods, medicines, and other fields.

Conventionally, various methods are known for producing L-histidine by fermentation, but among these methods, methods using analog-resistant strains include Brevibacterium, Corynebacterium, Arthrobacter, Using L-histidine analog (i.e., 2-thiazole-alanine and 1,2,4-4-lyazole-3-alanine) resistant strains of the genera Microbacterium, Nocardia, Bacillus, or Azotobacter. Method (Japanese Patent Publication No. 48-18829, Japanese Patent Application Publication No. 49-41592) and its improvement method (Japanese Patent Application Publication No. 49-100291, Japanese Patent Application Publication No. 50-4
9490, JP-A-50-49491. Japanese Unexamined Patent Publication 1986-1969
292, JP-A-50-70591), 2-thiazole alanine-resistant strain of Escherichia coli (JP-A-55-165798)
A method using a 2-methylhistidine-resistant strain of Serratia genus and another method using a 2-methylhistidine-resistant strain are known.

There is always a need for an excellent method for producing histidine, and as a result of research into L-histidine-producing bacteria, we have found that
- We discovered that a mutant strain endowed with 2-fluorohistidine resistance, which was completely unknown as a property of histidine-producing bacteria, accumulated a significant amount of L-histidine in the culture solution, and completed the present invention. It's arrived.

The 2-fluorohistidine-resistant mutant strain of the present invention can be obtained by a known method for obtaining analog-resistant mutant strains.

For example, histidine-producing bacteria are treated with various drugs or
It can be obtained by treatment with rays, γ-rays, Co irradiation, etc., or by genetic recombination methods.

The mutant strain of the present invention can be derived from histidine-producing bacteria such as Co11 Nebacterium glutamiculum TCC13761 and Brevibacterium ATCC14902.

In addition to the property of 2-fluorohistidine resistance,
Other drugs (e.g., 2-thiazolealanine, 1,2.
4-triazole-3-alanine, 3-amino-1,2
, 4-) Histidine analogs such as lyazole, other purine analogs, pyrimidine analogs, sulfa drugs, analogs of money-making amino acids, glutamine analogs, glycine analogs, etc.) or various auxotrophic strains (e.g., various amino acid It is expected that histidine can be produced at even higher yields by using mutant strains that have the following requirements:

A specific example of operation for obtaining such a mutant strain will be described below.

Operation example Corynebacterium glutamicum wild strain ATCC1
3761 was cultured on yeast broth agar medium for 16 hours, centrifuged and washed, suspended in 0.1 M Tris-maleic acid buffer to a cell concentration of 103 cells/d, and added to this to a concentration of 300 μi/ml. N-methyl-N'-nitro-N-nitrosoguanidine was added to the mixture, and the mixture was left at room temperature for 30 minutes.

The washed cells were inoculated into an agar medium containing 100 μg/ml of 2-fluorohistidine having the following composition in 107 cells/Petri dish and cultured at 30° C. for 3 days.

30 colonies expressing 2-fluorohistidine resistance
An L-histidine production test is carried out by sampling the bacteria, and strains that accumulate L-histidine of 1 μ/ml or more in the culture solution are preserved as L-histidine producing bacteria.

Among these L-histidine producing bacteria, the mutant strain with the highest production amount was named C3-9.

Composition of agar medium Gelcose 10g, (NH4)2S041g.

KH2PO40.5g, 2HP0.0.5g, KCl
0.2,! li', MgSO4・7H200,2g
.

F e So ・7HOO,1jj, Mn SO
4.2 7H2050"2S', CaCl2 20m?,
11 contains 30μ9 of biotin, 10μ of rutin, 0.1g of 2-fluorohistidine and 20g of agar (PH7
,2).

Similarly, a mutant strain AB 39 having 2-fluorohistidine resistance was obtained from Brevibacterium 5pl ATCC14902.

These bacteria were sent to the Microbial Technology Research Institute as Corynebacterium glutamicum C3-9.
No. 9 and Brevibacterium 3p, B-39, which has been deposited as Microtechnology Research Institute No. 6228.

As for the medium composition used in the present invention, either a synthetic medium or a natural medium can be used as long as it contains a sufficient amount of carbon sources, inorganic substances, and other necessary nutrients that can be utilized by the strain used.

In other words, carbon sources include various carbohydrates such as glucose, fructose sorbitol, glycerol, sucrose, starch, starch hydrolysates, molasses, fruit juice, acetic acid, fumaric acid,
Organic acids such as lactic acid, gluconic acid, and cono-phosphoric acid, as well as alcohols such as ethanol and methanol, can also be used.

Nitrogen sources include ammonium salts of various inorganic acids such as ammonia, ammonium chloride, ammonium sulfate, ammonium acetate, and ammonium phosphate, urea, amines, and their base nitrogen compounds, as well as Beftone, meat extract, yeast extract, and corn steep. - Liquor, casein hydrolyzate, soybean meal acid hydrolyzate, various fermented microbial cells and their digested products, etc. can be used.

Furthermore, as inorganic substances, potassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate,
Sodium chloride, ferrous acid, manganese sulfate, calcium carbonate, etc. are used.

Of course, if the microorganisms used in the present invention require specific nutrients for growth, appropriate amounts of those nutrients must be present in the culture medium, but these substances may be substituted with the natural products exemplified as nitrogen sources. It may be included in and added to.

Cultivation is performed under aerobic conditions such as shaking culture or deep aeration agitation culture.

Cultivation is usually carried out at a temperature of 20 to 40°C, at a pH of 3 to 9, at a temperature of 1 to 5.
It will be held on the day.

A known method such as the ion exchange resin method is used to collect L-histidine from the culture solution after completion of the culture.

Examples are shown below.

Example 1 One type of bacteria is Corynebacterium glutamicum C3.
-9 is used.

Glucose 40S1 polypeptone 20g
,KH2PO41,5,! if', 2HPO40,5
g, MgSO4・7H200.5g, biotin 5.0μ
pH containing I, 3g of urea, and 5y of yeast extract per iz
The mixture was inoculated into a 300-capacity Erlenmeyer flask containing 40 ml of seed medium of No. 7.2, and cultured with shaking at 30°C for 24 hours.

Inoculate 1 ml of this seed culture into a 300 ml Erlenmeyer flask containing 20 ml of fermentation medium with the following composition, and hold at 30°C.
When cultured with shaking for 3 days, the amount of histidine produced was 3.2711Ii/ml.

As a control, the amount of L-histidine in the culture solution of wild strain ATCC13761, which was similarly cultured, was 0.1 to 0.1/ml or less.

The composition of the fermentation medium used is as follows.

Blackstrap molasses (glucose equivalent) 70g, meat extract 5g, (N
H4) 2SO440g.

KH2PO41.5g, 2HPO40.5g.

MgSO4・7H200,! l, 3 g of urea.

Contains 330 g of CaCO per 11 ml (pH 7.4).

Example 2 The procedure was carried out in the same manner as in Example 1 except that Brevibacterium sp, B-39 was used as the inoculum. As a result, 4-2"
? Histidine/ml accumulated.

The content of histidine in the culture solution of the wild strain ATCC14902 cultured as a control was 0.1 m9/rnl or less.

Claims (1)

[Claims] 1 Cultivate a microbial mutant strain belonging to the genus Corynebacterium or Brevibacterium and having resistance to growth inhibition by 2-fluorohistidine in a nutrient medium to accumulate L-histidine in the culture solution; A method for producing perilla-histidine by a fermentation method, which is characterized by collecting mustard-histidine from a liquid.