JPH01120297A - Production of l-tyrosine by fermentation process - Google Patents

Abstract

PURPOSE:To obtain in an industrially extremely efficient manner the titled L-tyrosine without need of using any expensive precursor, by aerobic culture in a liquid medium of microorganisms which belongs to Citrobacter group and capable of producing L-tyrosine. CONSTITUTION:Microorganisms which belongs pref. to Citrobacter group as a mutant resistant to phenylalanine antimetabolite and is capable of producing L-tyrosine [e.g. Citrobacter freundii-OFPR 36-158 (FERM P-9536)] are subjected to culture under aerobic conditions e.g. at 24-37 deg.C and pH5-9, in a liquid medium containing carbon source such as glucose, nitrogen source such as ammonium sulfate, inorganic ions and, if needed, other organic trace component(s) to accumulate the product followed by separation and collection of said product, thus obtaining the objective L-tyrosine.

Description

[Detailed description of the invention] <Industrial application field> The present invention relates to a method for producing L-tyrosine by fermentation.

<Conventional technology> Conventionally, L- using microorganisms belonging to the genus Citrobacter
As a method for producing tyrosine, an enzymatic method using phenol, ammonia, and pyruvic acid as raw materials is known (B
iochen, Biophysic.

Res, Commun, Volume 46, Page 370, 19
1972; Cheaical Abstract 10
Volume 0, 119321d, 1984).

<Problems to be Solved by the Invention> However, the conventional method uses an expensive precursor and is economically extremely disadvantageous.

Means and Effects for Solving the Problems In order to solve the problems, the present inventors conducted intensive research and found that L-tyrosine accumulates in significant amounts through fermentation, and thus arrived at the present invention. Shina.

That is, the present invention belongs to the genus Citrobacter and
A fermentation method characterized by culturing a microorganism capable of producing tyrosine aerobically in a liquid medium to produce and accumulate L-tyrosine in the culture solution, and collecting L-tyrosine from the culture solution. This is the manufacturing method for 1,000 rosin.

It has not been known until now that microorganisms belonging to the genus Citrobacter can produce and accumulate L-tyrosine in significant amounts by fermentation.

Next, the present invention will be explained in detail.

The microorganism used in the present invention is not particularly limited as long as it belongs to the genus Citrobacter and has the ability to produce katsu-tyrosine. Preferably, it belongs to the genus Citrobacter,
A mutant strain that is resistant to phenylalanine antimetabolites is used. Examples of phenylalanine antimetabolites include 0-fluorophenylalanine and β-thienylalanine.

Typical microorganisms used in the present invention include, for example, the following.゛Citrobacter freundii (Citrobacter freundii) OF
PR36-158 (R Koken Bokuyori No. 9536). This seedling strain is Citrobacter freundii FTR66-3
This is a mutant strain derived from No. 6 (Feikoken Bibori No. 9339), which is resistant to 0-fluorophenylalanine. Mutant strains can be induced relatively easily by conventional mutant strain treatment methods. That is, a mutant strain that is resistant to phenylalanine antimetabolites can be created by irradiating the parent strain with ultraviolet rays or by using a mutagenic agent (for example, N-methyl-N-
After treatment with N-nitrosoguanidine, ethyl methanesulfonic acid, etc.), a strain that can grow in a medium containing an amount of phenylalanine antimetabolite that cannot grow the parent strain may be collected. Here, a mutant strain that is resistant to phenylalanine antimetabolites is a strain that has stronger resistance than its parent strain, and preferably a strain that has a phenylalanine antimetabolite that has a relative growth rate of 40% or less of the parent strain after 24 hours. It refers to those that exhibit a relative growth rate of 50% or more when cultured in a medium containing an anti-metabolite.

The liquid medium for producing L-tyrosine in the present invention is a normal liquid medium containing a carbon source, a nitrogen source, inorganic ions, and other organic R components as necessary. Carbon sources include glucose, fructose, starch and cellulose hydrolysates, saccharides such as molasses, all acids such as fumaric acid, citric acid, succinic acid, etc., alcohols such as glycerol, etc. at 2 to 15%, As a nitrogen source, organic ammonium salts such as ammonium acetate, H
Inorganic ammonium salts such as D ammonium, ammonium chloride, ammonium phosphate, ammonium nitrate, ammonia gas, aqueous ammonia, urea, etc.
In addition to these, small amounts of potassium hexadiphosphate, magnesium sulfate, ferrous sulfate heptahydrate, manganese chloride tetrahydrate, etc. are added.

Cultivation is performed under aerobic conditions. During cultivation, the pH of the liquid medium was adjusted to 5 to 9, the temperature was adjusted to 24 to 37 °C, and the temperature was adjusted to 48 to 12 °C.
Favorable results can be obtained by culturing with shaking or aeration for 0 hours.

L-tyrosine can be collected from the culture solution by known methods such as ion, exchange resin method, and activated carbon adsorption method.

<Example> The present invention will be specifically explained below with reference to Examples.

Experimental Example 1 (Obtaining an O-fluorophenylalanine-resistant mutant strain) Cells of Citrobacter freundii FTR66-36 were treated with N-methyl-N-12tro N-nitrosoguanidine (150 μg/ml, 37℃,
After 15 minutes), the cells were treated with O-fluoro-D,L-
Agar plate medium supplemented with phenylalanine 0.2+r/J (glucose 0.2%, ammonium sulfate 0.1%, monopotassium phosphate 0.2%, dipotassium phosphate 0.7%, magnesium VX 7 water) A synthetic medium containing 0.01% of hydrate, 0.05% of sodium citrate, 0.2% of L-tryptophan, and 0.04% of L-tyrosine).

Next, the bacteria were cultured at 37°C for 4 to 6 days, and the resulting large colonies were isolated to isolate the 0-fluorophenylalanine metabolic mutant Citrobacter freundii 0FPR36-1.
58 (Feikoken Bibori No. 9536).

Experimental Example 2 (Raininess of 0-Fluorophenylalanine Resistant/Note Mutant Strains) Each strain shown in Table 1 below was grown in a liquid medium (tryptone 1%
, yeast extract 0.5%, sodium chloride 0.5%, P
H7,5) was cultured with shaking at 30°C for 6 hours, and the grown bacterial cells were collected and thoroughly washed with physiological saline. This bacterial cell suspension was mixed with 0.1.5mM of 0-fluorophenylalanine.
Minimal medium (medium composition: Nigelcose 0.2%, ammonium sulfate 0.1%, monopotassium phosphate 0.2%) with aK of
, potassium phosphate 0.7%, magnesium sulfate heptahydrate 0.01%, sodium citrate 0.05%, L-
Tryptophan 0.2%, L-tyrosine 0.02%)5
ml was inoculated and cultured at 30°C for 14 hours, and the growth rate of each strain was examined. The results are shown in Table 1.

0-fluorophenylalanine-resistant mutant strain Citrobacter freundii 0FPR36 used in the method of the present invention
-158 is the parent strain Citrobacter 70 inday-F
Compared to TR66-36, it is clear that growth is not inhibited by O-fluorophenylalanine and that it has acquired resistance to Q-fluorophenylalanine.

Table 1*) The turbidity of the culture solution at 660 nm was measured, and the turbidity of the culture solution to which O-fluorophenylalanine of each strain was not added was expressed as 100%.

Example 1 40 ml of a fermentation medium having the following composition was placed in an 11-volume Erlenmeyer flask and steam sterilized at 115°C for 15 minutes. Second to this
Each strain shown in the table was inoculated with a platinum loop and cultured with rotational shaking at 30°C for 96 hours.

Fermentation medium composition Glucose 10% (NH4)23
04 2.5%KH2PO40,04% M gS O4・7H200,04% Fe50+ ・7H201opp+i MnCJ 4 ・4H207ppn After culturing, L in the solution from which bacterial cells and calcium carbonate were removed
- The concentration of 1,000 rosine was determined by a microbial assay method using Leuconostoc mesenteroides ATCC 8042, and the results shown in Table 2 were obtained.

Table 2 Also, the liquid after the completion of fermentation was subjected to a strong acid cation exchanger, the resin was washed with water, the eluate was concentrated with 0.5N ammonia water, and the eluate was decolorized by activated carbon treatment, and after cooling, the culture liquid 11 Obtain 0.37 g of L-tyrosine crystals per sample.

<Effects of the Invention> According to the method of the present invention, it is possible to accumulate a significant amount of L-tyrosine without using an expensive precursor, and therefore it is extremely advantageous industrially.

Claims (2)

[Claims] (1) A microorganism belonging to the genus Citrobacter and capable of producing L-tyrosine is aerobically cultured in a liquid medium to produce and accumulate L-tyrosine in the culture solution. 1. A method for producing L-tyrosine by a fermentation method, which comprises collecting L-tyrosine. (2) A method for producing L-tyrosine by a fermentation method according to claim 1, wherein the microorganism is a mutant strain having resistance to a phenylalanine antimetabolite.