JPS59120091A - Preparation of l-tryptophan by fermentation - Google Patents

Abstract

PURPOSE:To prepare a large amount of L-tryptophan, by cultivating a variant belonging to the genus Bacillus, having tryptophan analog resistance and reduced chorismate mutase, capable of producing L-tryptophan. CONSTITUTION:A bacterium belonging to the genus Bacillus, having tryptophan analog resistance, capable of producing tryptophan, as a parent strain, is subjected to common operation for induction of variation, a strain having low activity of chorismate mutase is selected among a mold solution subjected to variation treatment, to give Bacillus subtilis (FERM-P 6845) or Bacillus subtilis (FERM-P 6846). The strain is cultivated in a common medium at 5-9pH at 20-40 deg.C for 1-4 days aerobically, and a large amount of L-tryptophan is formed and accumulated.

Description

[Detailed Description of the Invention] Tree J6 Ming describes the production of L-1-u butophane (hereinafter referred to as tryptophan) using a fermentation method.

The method for producing K-11 fl-fan is to use anthranilic acid, which is a precursor of tryptophan, indole or
- A method for producing tryptophan from indolepyruvic acid is known.

A direct fermentation method using a mutant strain of Bacillus that is resistant to tributophane analogs and uses sugars as a carbon source instead of using precursors. Method for producing fans (Tokukoku Sho 48-1.8828, Sho 53-39517)
or has been developed.

Therefore, the inventors conducted research to develop a method to inexpensively produce trizo)phane from carbon sources such as sugars by direct fermentation using microorganisms of the genus Bacillus. We have discovered that a mutant strain with weakened activity of chorismate mucus derived from a tryptophan-producing bacterium that is resistant to l-lyptophan analogs can produce a large amount of tryptophan.

The present invention was completed as a result of further research based on this knowledge.

The chorismate mutase referred to in the present invention is an enzyme involved in the biosynthesis of L-thylonnara and L-phenylalanine in microorganisms of the genus Bacillus, and isomers chorismate, which is an intermediate in the biosynthesis of aromatic amino acids, to prephenic acid. It is an enzyme (enzyme number EC5, 4, 99, 5) that causes oxidation. Chorismate mutase activity can be determined using known methods such as R,G
, H., Cotton and F., Gibson.

Biochim, Biophys, AcLa, 1
Volume '00, page 76 7. 1965.

Or 1.5hiio and S, Sugimoto
, J. Biochem, vol. 86, p. 17 7,
It can be measured by the method described in the literature such as 1979.

The tryptophan analog referred to in the present invention refers to a drug that inhibits the growth of microorganisms belonging to the genus Bacillus, and this growth inhibition is partially or completely canceled by the presence of L-tryptophan, such as 5-methyl i-liptophan. 5.6 or 7-lower alkyl tryptophan, 5-
5- or 6-valogenotryptophan, such as fluorotryptophan, tryptophan hydroxamate, or 7
- Azatryptophan The mutant strain used in the present invention belongs to the genus Bacillus, is resistant to the above-mentioned tryptophan analogs, has weakened chorismate mutase activity, and has the ability to produce tryptophan. A typical example is a mutant strain.

Bacillus subtilis AJ11984 FERM-
P 6845 (5-F-Trpr, CMW) Bacillus subtilis AJ11985 FERM-
P 6846 (5-F-Trpr, CMW) (Note) 5FTrpr: 5-fluorotryptophan resistant CMw Chorismate mutase weakened A parent strain is used as a parent strain, and it is subjected to conventional mutagenesis procedures such as ultraviolet irradiation or chemical treatment with N-methyl-N'-nitro-N-nitron guanidino, nitrous acid, etc., and frismic acid is extracted from the mutagenized bacterial solution. Obtained by selecting strains with low mutase activity.

Various methods can be used to select mutant strains with low chorismate mutase activity, including a method of directly measuring chorismate mutase activity, but the present invention is not limited to the method of selecting mutant strains. This method is characterized by the use of a weakened strain of chorismate mutase F.

The above parent strain has properties useful for tryptophan production in addition to tryptophan analog resistance.
! J-i hfan-producing bacteria, such as L-arginine, L-linno, ■,-loin, or L-phenylalanino-requiring tryptophan a4 [(Japanese Patent Publication No. 53-3
9517), tryptophan-producing bacteria resistant to tryptophan analogs and indolemynon (Japanese Unexamined Patent Publication No. 56-92796), etc. can also be used.

In addition, the mutant strain of the present invention can also be induced by using a wild strain of the genus Bacillus as a parent strain, selecting a mutant strain with weakened 7Er properties of furismate mutase, and then imparting tryptophan analog resistance to the mutant strain. It's coming.

The medium used in the present invention is a conventional nutrient medium containing a carbon source, a nitrogen source, inorganic salts, and other organic micronutrients such as amino acids and vitamins as necessary. As the carbon source, glucose, euclose, maltose, starch melting product, molasses, etc. are used, and ethanol, acetic acid, citric acid, etc. are also used alone or in combination with the above-mentioned carbon sources. Nitrogen sources include ammonium salts such as ammonium sulfate, ammonium chloride, ammonium phosphate, nitrates,
Organic or inorganic nitrogen sources such as urea and peptone are used.

Organic micronutrients include amino acids, vitamins, fatty acids,
Nucleic acids, peptones containing these substances, casamino acids, yeast extracts, soybean protein decomposition products, etc. are used to supplement the required nutrients when using auxotrophic mutant strains that require amino acids, etc. for growth. It is necessary to attach the As the inorganic salts, phosphates, magnesium salts, carnoum salts, iron salts, manganese salts, etc. are used.

Cultivation can be carried out under normal culture conditions, and by controlling the pH to 5 to 9 and the temperature at 20 to 40°C and culturing with shaking or aeration for 1 to 4 days, a significant amount of tryptophan can be produced in the culture solution. Accumulated. If the pH decreases during culturing, add the carbonaceous solution after separately sterilizing it, or neutralize it with an alkali such as aqueous ammonia or ammonia gas.

In addition, when an organic acid is used as a carbon source, the pH increase is neutralized with a mineral acid or an organic acid.

Tryptophan can be collected from the culture solution by any known tryptophan recovery method, such as by removing bacterial cells from the culture solution and then concentrating and crystallizing it, or by ion exchange chromatography.

Examples will be described below.

Example 1 A culture medium having the composition shown in Table 1 was dispensed into a 50011e shaking flask and heat sterilized at 10°C for 10 minutes to prepare a culture medium.

Table 1 Composition of bacterial culture medium (pH 7,0)
Minutes Concentration Gurufus
5oy/ ammonium chloride l Q
//K H4F O,l //K CI
'l //MnSO4・4H2010m
9/l FeSO4* 7H2010// Casamino acid 4 2/1M g S
O4・7H200,4//CaCO340// This medium was inoculated with the strains shown in Table 2, and incubated at 30°C for 24 to 30 minutes.
The culture was cultured with shaking for 48 hours, and the culture was stopped when the absorbance at 570 nm of the 26-fold dilution of the culture solution reached 0.3 to 0.4.
After washing twice with 50 mM potassium phosphate buffer (pH 7,0) containing dithiothreitol, the cells were suspended in 10 mM of the same buffer (!), and after the cells were sonicated,
The supernatant obtained by centrifugation at 000 rpm for 20 minutes was used as a crude enzyme solution.

The activity of furismate mutase is 1.5hiio, S,
Sugimoto.

J. Biochem, Volume 86, Page 117,]9
It was measured by the method described in 1979. The reaction solution was 50 mM potassium phosphate buffer (p, H7.0) in 0.4me.
, 2.5mM chorismate and crude enzyme solution, and the reaction was carried out for 3
After running for 20 minutes at 0°C, it was stopped by adding 0.4me of 1N hydrochloric acid.

Then, the prephenic acid was further heated at 37°C for 10 minutes to quantitatively convert it into phenylpyruvic acid, and the resulting volume was 3.2ml! of 1N caustic sorter was added and phenylpyruvate was measured by absorbance at 320 nm. In addition, the amount of protein in the crude enzyme solution was determined by a method such as 0, H, Lowry.
et al., J. Biol, Chem, vol. 193, 2.
It was quantified by the method described in ]951, page 65.

On the other hand, the same strain was cultured with shaking at 30° C. for 96 hours using the above medium, and tryptophan produced in the culture solution was quantified according to a conventional method. Table 2 shows the nophrimate mutase activity and tryptophan accumulation of the mutant strains of the present invention. The results shown in Table 2 indicate that tryptophan productivity is increased by attenuating chorismate mutase activity.

Table 2: Chorismate mucus activity (L-)lyptopha/accumulation amount FT-145 (FERM-P1783) 346
1.9 (5-FTr) AJ 11984 105 4.
3 (5-FTr, CMw) AJ 11985 28 5.
9 (5-FTr, CMW) Patent applicant Ajinomoto Co., Inc.

Claims (1)

[Claims] A monotributophane/producing mutant strain belonging to the genus Bacillus that has tryptophan analog resistance and has weakened chorismate mutase activity is cultured aerobically to obtain L-1 in the culture medium.
- Generate liptophano, store fJ'i, set 8 y L
-1-L-Tryptophan Na method using a fermentation method which is characterized by the extraction of L-ributophane.