JPS5971698A - Preparation of l-phenylalanine by fermentation - Google Patents

Abstract

PURPOSE:To prepare L-phenylalanine, economically, by culturing a specific mutant belonging to Bacillus genus and capable of producing L-phenylalanine aerobically in a liquid medium, and collecting the produced and accumulated L- phenylalanine. CONSTITUTION:A mutant (e.g. Bacillus subtilis FERM-P 6759 and Bacillus subtilis FERM-P 6760) resistant to phenylalanine analogue, demanding L-tyrosine for growth, producing L-phenylalanine, and preferably resistant to sulfa drugs, is prepared by the mutagenic treatment of wild strain of Bacillus subtilis. The mutant is cultured aerobically in a liquid nutrient medium containing L-tyrosine, and after the cultivation, the produced and accumulated phenylalanine is separated from the culture liquid, e.g. by ion exchange resin treatment.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention uses a fermentation method to produce one phenylalanine C, hereinafter simply referred to as phenylalanine. ) production method.

Conventionally, as a method for producing phenylalanine by fermentation, a method using tyrosine-requiring bacteria of the genus Brevibacterium or bacteria of the genus Mycukotsucus (Japanese Patent Publication No. 37-6345
A method using a mutant strain that requires tyrosine for L. growth and is resistant to 5-methyltryptophan (Tokuko Shou 5l).
-21079), a method using mutant strains resistant to phenylalanine analogs (Japanese Patent Publication No. 51-28712)
Furthermore, a method using a definin-sensitive mutant strain (Japanese Patent Publication No. 56-64793) is known.

On the other hand, in the case of No. 1, there is a method using L-tyrosine auxotrophic mutants (Japanese Patent Publication No. 37-6345), or I.
A method using mutant strains resistant to lanin and β-2-cheninoalanine is known (Japanese Patent Application Laid-Open No. 48-67488), but the productivity of phenylalanine is 0.2.
~0.4&'/dl, which is significantly inferior to the phenylalanine-producing bacteria of the genus Brevibacterium or Corynebacterium. For this reason, breeding of bacterial strains for the purpose of industrial production of phenylalanine has hardly been studied, and in fact, strains with high L-phenylalanine productivity are not known.

The present inventors believe that microorganisms of the genus Bacillus are actually used in the fermentation field for nucleic acid fermentation, and that they have potentially excellent fermentation productivity. We bred phenylalanine-producing bacteria for the purpose of As a result, we found that a mutant strain of Bacillus subtilis with 15-tyrosine auxotrophy and phenylalanine analog resistance was added to the wild strain, and among these mutant strains was one that had sulfa drug resistance. Or/del
It was discovered that there are many strains that produce significant amounts of phenylalanine. The present invention has been completed based on this discovery, and provides a novel and effective method for the industrial production of phenylalanine by direct fermentation.

The phenylalanine analog referred to in the present invention refers to a compound that inhibits the growth of microorganisms belonging to the genus Bacillus, but whose growth inhibition is partially or completely canceled by the presence of phenylalanine. - or P-fluorophenylalanine, m-fluorotyrosine, 0-1m-1 or P-amino/phenylalanine, β
- phenylserine, ncrohexylalanine, α-amino-β-phenylethanesulfonic acid, 0-lm- or P-furomophenylalanine, β-2 or β-3-chenylalanine, β-2-pyrrolealanine, ]-7 clopentene-1-alanine, ■-7 clohexene-1-alanine, 2-amino-4-methyl-hexenoic acid, S-(
Examples include 1,2-chlorovinyl)-nostine, β-4-pyrinolalanine, β-2-pyriflualanine, β-4-pyranolalanine, and P-nitrophenylalanine.

Sulfa drugs are drugs that have an antagonistic effect on γ-aminobenzoic acid, such as zulfanilamide, acetosulfamine, sulfapyridine, sulfaguanidine, sulfathiazole, sulfadiazine, sulfamethacin, sulfamethacin, and sulfine. Examples include xazole and sulfinmidine.

The mutant strain used in the present invention belongs to the genus Bacillus, has resistance to phenylalanine analogs, and grows +: L--y-.
It is a mutant strain that requires sulfa drugs, desirably has resistance to sulfa drugs, and has the ability to produce phenylalanine. Specifically, the following mutant strain is mentioned as a representative example.

Bacillus subtilis AJ11971F E RM
-P 6759 (p-F-pher+Tyr-) Bacillus subtilis AJ11972F E RM -P
6760 (p-F-pha-+m-p-Tyrr,
Tyr-) Bacillus subtilis AJ11973F
E RM-P 6761 (p-F-pher+SG
r, Tyr-) Note) pF-pher: p-fluorophenylalanine resistant Tyr-: L-tyrosine requirement m-F-Tyrr: m-fluorof+12 Schiff resistant SGr: sulfaguanidine resistant The above mutant strain of the present invention is A wild strain of the genus Bacillus is used as the parent strain, and this is subjected to conventional mutation induction procedures such as ultraviolet rays, X-ray irradiation, or N-methyl-N+-nitro-N-nitrosoguanidine (
(abbreviated as NG), treated with chemical agents such as nitrous acid, and cultured in an agar plate medium containing an amount of phenylalanine analog such that the mutant parent strain does not grow.
Colonies growing on the plate medium are isolated and phenylalanine analog-resistant bacteria are induced, and this phenylalanine analog-resistant bacteria is used as the parent strain to grow. This is obtained by inducing a mutant strain that requires L-tyrosine. In this case, the order of addition of analog resistance and hypophilicity can be arbitrary as it is not related to the productivity of phenylalanine.

Next, the specific method for inducing the above-mentioned mutant strain used in the present invention and the degree of resistance to analogs will be shown.

Example Bacillus subtilis AJ 11708 Yosu-induced sittyrosine auxotrophic phenylalanine-producing bacterium △J1]Cj6
6 was cultured on yeast bouillon agar slant medium, and the grown cells were collected to yield 1750M! J acid buffer (I) H7,0
) is suspended in 108~10° pieces/m1! (including bacterial cells)
, add NG (NG concentration is 200μf/ml)
, and kept at room temperature for 30 minutes.

like this? After thoroughly washing the strained and NG-treated bacterial cells with the same phosphate buffer, one plate of the minimum agar plate shown in Table 1e containing P-fluorophenylalanine was applied.
The cells were cultured for 4 to 10 days in r.

Glucose 209/ammonium sulfate 5 Urea 2
〃KH2PO41// MgSO4・7H201// F e” + M n++ Io7 2
ppm biotin 50μy
/l thiamine hydrochloride 200//p
-F-ph e 200M
9/ IL-Chiro 7n 100
Among the colonies grown on the agar plate, large ones were collected as P-fluorophenylalanine-resistant strains.

Among the resistant strains thus obtained, many were found to have superior phenylalanine production ability than the parent strain.

Among these, strain AJ11971 with the highest production capacity was selected.

Using the same mutational procedure as the parent strain, A311972 strain with meta-fluorotyrosine (m-F-Tyr) resistance and AJ11973 strain with sulfaguanine resistance were derived, respectively.

Examples A minimal medium (Table 11) containing phenylalanine analog or sulfaguanidine at the concentrations shown in Table 2 was dispensed into test tubes at 4.0 Tne and heat sterilized.

This medium? A fixed amount of the above mutant strain was inoculated into a 31.5 U shaking ring. Dilute each culture solution 26 times with water, and
The degree of growth was determined by measuring the absorbance at 62 nm. The results are shown in Table 2.

Note that Table 2 shows relative growth values, with the growth rate when no chemicals were added being 100.

Table 2 Resistance to drugs The culture medium used in the present invention includes carbon sources, nitrogen sources, and inorganic salts:
A conventional nutrient medium containing L-tyrosine and other organic micronutrients such as amino acids, vitamins, and nucleic acids as necessary is used. The carbon source may be any carbon source that is available for the mutant strain used, such as sugars such as haculcose, fructose, sucrose, maltose, starch decomposition product molasses, and others such as ethanol, propatool, etc. Alcohols, organic acids such as acetic acid, citric acid, etc. are used.

Nitrogen sources include ammonium sulfate, ammonium chloride,
Inorganic or organic nitrogen sources such as ammonium salts such as ammonium phosphate, nitrates, urea, ammonia, meat extracts, etc. are used. As organic micronutrients, amino acids, vitamins, fatty acids, nucleic acids, peptones containing these, casamino acids, yeast extracts, protein decomposition products, etc. are used.

Since the mutant strain of the present invention requires L-tyronone for growth, it is necessary to add about 2 to 50 mb/de of L-tyrosine to the medium.

It is desirable that the culture be carried out under aerobic conditions, with the pH of the medium being between 5 and 9 and the temperature between 201C and 40R during the culture period.
By carrying out shaking culture or aerated agitation culture for 1 to 4 days under controlled conditions, a significant amount of phenylalanine is accumulated in the culture solution. Is there a known method to collect phenylalanine from culture solution? After separating and removing the bacterial cells from the culture solution, the cells are collected by a method of concentration and crystallization or a method of using an ion exchange resin.

Hereinafter, the trowel of Example t will be explained.

Example 1 A phenylalanine production medium shown in Table 3 below was prepared, and dispensed into 20ml 500m/volume 1 cylinder flasks.
Heat sterilized at +201r for 10 minutes. Carbonated powder 1. Added Of.

Table 3 Composition of phenylalanine production medium Glucose
80.0 fNH4C11
0,0// KCl2.0N KH2P0.
1.0 11Mg5O,@7H,00,4〃Fe-1+ 4. n++ 2 ppm each and 0. I51η dog bean protein autolysis solution* 20 me One cup of phenylalanine-producing bacteria shown in Table 4 was inoculated into this medium and cultured with shaking at 30C for 72 hours. The amount of phenylalanine produced in the culture solution was determined, and the results are shown in Table 4.

Table 4 Accumulation amount of phenylalanine AJ+1708 (parent strain)
0AJII966 (Tyr'-)
2.0AJII971 (Tyr, pFp
her) 8.0AJI]972 (T
yr-, pFpher, mFTyrr) 10.5
AJI+93 (Tyr, pFpher, 5Gr)
11.0448-

Claims (1)

[Scope of Claims] A mutant strain belonging to the genus Bacillus that is resistant to phenylalanine analogs, requires L-tyrosine for growth, and has the ability to produce L-phenylalanine is aerobically cultured in a liquid medium. A method for producing phenylalanine according to the Bn method, characterized in that L-phenylalanine is produced and accumulated, and L-phenylalanine is collected. (2) The method for producing mono-phenylalanine according to claim 'AI', wherein the mutant strain is a mono-phenylalanine-producing bacterium that is further resistant to sulfa drugs.