JPS6083594A - Production of l-tryptophan by fermentation method - Google Patents

Abstract

PURPOSE:To produce L-tryptophan, by cultivating a microorganism, belonging to the genus Bacillus, and having resistance to sulfaguanidine under aerobic conditions. CONSTITUTION:A microorganism e.g. Bacillus subtilis (FERM-P No.7294) or Bacillus subtilis (FERM-P No.7295), belonging to the genus Bacillus, having the ability to produce L-tryptophan and resistance to sulfaguanidine is subjected to ordinary variation operation. The resultant variant strain is then cultivated under aerobic conditions to produce a remarkable amount of L-tryptophan.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention is directed to a method for preparing L-) lysotophan (hereinafter referred to as tricytophan) using the reaction method.

Conventional methods for producing tricytophan include anthranyl f2, indole or 3, which are tryptophan precursors.
- Methods for producing indolepyruvate and citricytophan are known.

In contrast to previous methods that use small substances, we have developed a direct fermentation method using a mutant strain of Bacillus that is resistant to tryptophan analogs, using a sugar such as a carbon source without using a precursor substance. Yo,? ) Method for producing risotophan (Special Publication No. 48-18828, Special Publication No. 53-39517)
') has been developed.

Therefore, the present inventors conducted research to develop a method to inexpensively produce tryptophan from carbon sources such as 82 species by direct fermentation using microorganisms of the genus Bacillus.
In addition to the above-mentioned resistance to tryptophan analogs in the genus Bacillus, some microorganisms that have been conferred resistance to sulfa guanidine, also known as sulfa drugs, have the ability to produce even larger amounts of L-)liptophan than previously known. We have discovered that there are strains that have this. The present invention was completed as a result of further research based on this knowledge.

In the present invention, a microorganism having the ability to produce tryptophan refers to a microorganism that belongs to the genus Bacillus tj and thrives on tryptophan analogs such as 5-methyltryptophan or 5-fluorotryptophan. In addition to this property, L-phenylalanine, L-
Bacillus with auxotrophic properties such as tyrosine or L-histidine, or resistance to phenylalanine analogs, tyrosine analogs, hisnano/analogs, etc.)
Microorganisms belonging to category 4 are also included in the microorganisms that have the ability to produce tritophane.

The mutant strain used in the present invention belongs to Bacillus linizi and is a microorganism that has the above-mentioned ability to mainly produce tryptophan and is resistant to sulfur drugs.
For example, the following mutant strains are used.

Bacillus subtilis AJ12098%P-'lyu9Φ (5-FTr, 5Gr) 729 left Bacillus subtilis AJ12099 FERM-P
(5-FTr, IMr, 5Gr) 5-FTr: 5-fluorotryptophan 1 Sokawa IMr: Indolmycin resistance SGr: Sulfaguanidine resistance These mutant strains used in the present invention are tryptophan analogs of the Bacillus genus. A resistant L-)litfan-producing strain is used as a parent strain, and it is subjected to conventional mutation induction operations, such as external radiation irradiation or chemical treatment with N-methyl-N'-nitro-N-nitrosoguanidine, nitrous acid, etc. After the application, the parent strain was cultured on an agar plate medium containing sulfur guanidine at a concentration of 1000 μμ so that the parent strain could not grow, and the colonies growing on the agar plate medium were isolated.
4.

The above parent strain is tryptophan analog IIJt
Trit7an-producing bacteria that have useful properties for trithophan production, such as L-argini/, L-lysine, and L-leucine, also have Llj:L-phenylalanine-requiring trithophan-producing bacteria (Tokukosho). 53-39517
In addition, tryptophan-producing bacteria that are resistant to tryptophan analogs and indolmycin (Japanese Patent Application Laid-Open No. 1982-92796) are used. As concrete &1, the following tryptophan analog-resistant tryptophan-producing bacteria are used.

Bacillus subtilis FT-145FERM-P17
83 (5-FTr) Bacillus subtilis AJ11483 FERM-7
P5216 (5-FTr, IMr) 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, imparting resistance to sulfaguanidine to the parent strain, and then imparting resistance to a tryptophan analog.

However, the above-mentioned tricytophan analog inhibits the growth of microorganisms belonging to the genus Bacillus, and this growth inhibition causes L-
Refers to drugs that are partially or completely released by the presence of tryptogua, such as 5-methyl, 5- or 6-lower alkyl tryptophan such as IJ detophyl, 5- or 6-lower alkyl tryptophan such as 5-fluorothrin, etc. - Balognotrinotophane, tryptophan hydroxamate, or 7-azatritofan, etc.

In addition, the sulfur drug used in the present invention refers to a 1'f/J substance that competitively inhibits the metabolism of p-aminobenzoic acid.
Examples include sulfuramide, acetosulfamine, sulfur hyridine, sulfur guanidine, sulfur thiazole, sulfur cyanone, sulfur methacin, and sulfur methacin.

The degree of resistance to sulfaguanidine of the simian guaguanidine-resistant tryptophan-producing bacteria of the present invention will be shown below in an experimental example.

Experimental Example Add sulfur guanidine to the minimal medium with the composition shown in Table 1 to the concentration shown in Table 2,
A liquid medium was prepared by dispensing the mixture in ml portions and sterilizing it by heating.

34. Add 0.1 ml of the culture solution of the Bacillus subtilis mutants shown in Table 2, which were cultured in a drug-free medium with 241 kl of diaphragm, to the above liquid medium, and shake at 30°C for 48 hours. ! The growth rate was determined by culturing and measuring the absorbance at 570 nm. The results are shown in Table 2.

Dashi table 1 Composition of minimal medium (PH7,0) Glucose
5.0 g/l ammonium sulfate 1. O〃 KH2PO48,65// MgSO4・7H200・21/l FcSO4・7H7H2O10/l MnSO4・4H2010Z' Sodium citrate 0.5 g/IIKOH2,18
/I L -7x =/' 77 = 7 100 m9/
IL-cysteine 125 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. Glucose, sucrose, maltose, starch melt J3 honey, etc. are used as carbon sources, and other sources include ethanol, acetic acid,
Citric acid and the like can also be used alone or in combination with the other carbon sources mentioned above. As nitrogen sources, ammonium salts such as ammonium sulfate, ammonium chloride, and ammonium phosphate, nitrates, urea, and organic or non-organic nitrogen sources are used.As organic micronutrients, amino acids, vitamins, fatty acids, and nucleic acids are used. In addition, pet products containing these substances, casamino acids, yeast extracts, soybean protein decomposition products, etc., are used, and nutrients that are stolen when using auxotrophic mutant strains that require amino acids, etc. for growth. It is necessary to supplement dogs ((4 Mori salts include phosphorus salt, magnesium 1
Beans, calcium salts, iron salts, manganese salts, etc. are used.

Cultivation can be carried out under normal culture conditions, and by culturing with shaking or aeration for 1 to 4 days while controlling the Pll to 5 to 9 and the temperature at 20 to 40°C, double the amount in the culture solution can be obtained. Tricytophane accumulates. If the pH decreases during culture, calcium carbonate should be separately sterilized and added, or neutralized with an alkali such as aqueous ammonia or ammonia gas. In addition, when using 43K as a carbon source, the increase in pH is neutralized with a mineral acid or a mineral acid.

Tricytophan can be collected from the Ir culture medium by a known method for collecting tricytophan, such as by removing microbial cells from the culture solution and then performing secondary condensation crystallization, or by ion exchange chromatography.

Examples will be described below.

Example 1 Tryptophan production medium 2 having the composition shown in Table 3 below
After dispensing Qnrl into a 500 ml flask and heating it at 110°C for 10 minutes, 1 of each of the microorganisms shown in Table 4 was added.
/3 slant amount and cultured with shaking at 30°C for 96 hours. The amount of tritophane produced in each culture solution was as shown in Table 4.

Table 3 Composition of medium for producing triodoane (Pll7.Q
) Component Concentration Glucose 80 9/11 Ammonium chloride 10 〃 KH2PO41// KC721/ 81nso4'4H2010', ry/lFcSO4・
7H2010 Kazaminoba 4 g/l MgSO4・7H20o, 4 ft CaCO540# Table 4 Tributophan production amount

Claims (1)

[Claims] microorganisms of the genus Bacillus that are resistant to sulfaguanidine and have the ability to produce L-tricytophane are aerobically cultured in a liquid medium to produce L-)IJnotophane in the medium; L- characterized by collecting this
) Method for producing liptophan.