JPH0525476B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing L-tryptophan, and more particularly to a method for producing L-tryptophan by an enzymatic method using specific microbial cells.

The following formula (): L-tryptophan, represented by
It plays an important role in growth, weight retention, body fat retention, blood cell component formation, milk secretion, etc., and is essential for life support, and is widely used as an additive in medicines, nutritional sources, and animal feed. It is an important amino acid.

Various chemical synthesis methods and biochemical production methods are known as methods for producing L-tryptophan.

Although the chemical synthesis method is suitable for mass production, it has the disadvantage that the tryptophan obtained is in the DL-form and optical resolution must be carried out.

Among biochemical production methods, methods using microorganisms include direct fermentation methods to produce and accumulate L-tryptophan using carbohydrates, and methods in which microorganisms are cultured by adding indole or anthranilic acid to the culture solution. There is a precursor fermentation method that produces and accumulates L-tryptophan.

On the other hand, as an enzymatic method, indole and serine;
Alternatively, there is a method for producing L-tryptophan from indole, pyruvic acid, and ammonium ion. This enzymatic method uses inexpensive raw materials produced by chemical synthesis, and is expected to be an industrially powerful method, but there is a desire to develop an inexpensive method for producing enzymes.

Conventionally, methods for producing L-tryptophan by enzymatic action include
Escherichia), Genus Proteus,
A method using microorganisms belonging to the genus Pseudomonas, Genus Aerobacter, or Genus Erwinia (Special Publication No. 46917/1987); Neurospora), Genus Saccharomyces, Genus Bacillus, Achromobacter (Genus
A method using microorganisms belonging to the genus Achromobacter or Genus Alcaligenes (French Patent No. 1207437, Japanese Patent Application Laid-Open No. 1983-1998)
39693, Special Publication No. 53-1836), etc. are known.

However, Brevibacterium (Genus
There is no known method to utilize microorganisms belonging to the genus Brevibacterium. Conventionally, a substance converted from indole using a microorganism belonging to the genus Brevibacterium is L-methoxytryptophan (Japanese Patent Publication No. 52-8400) obtained when Brevibacterium ammoniagenes is used.
(No.) is only known.

The inventors have the following formula (): Indole denoted by and the following formula (): As a result of a wide search for microorganisms that have the ability to produce L-tryptophan represented by the above formula () from serine represented by By utilizing this, we have completed the present invention.

That is, in the method for producing L-tryptophan of the present invention, Brevibacterium spp.
This is a method for producing L-tryptophan by an enzymatic method by using Flavum MJ-233 cells or an enzyme obtained therefrom.

The microorganism used in the present invention belongs to the genus Brevibacterium and has the ability to produce L-tryptophan from indole and serine.
flavum) MJ-233 (Microtechnical Research Institute No. 1479; JP-A-Sho
51-130592). Its mycological properties are shown below.

Mycological properties: Short rods measuring 0.6-0.8 x 1.0 x 2.4μ. They usually form a V-shaped pair, and fence-like patterns are also recognized. Gram-positive, non-sporulating, non-motile, no capsule, non-acid fast.

Cultural properties 1. Juicy agar colony: round, the surface is smooth, the ridges of the colony are flat or slightly ridged, the periphery of the colony is slightly wavy, yellow, opaque, and slightly glossy.

2 Gravy agar slope Medium growth, filamentous, yellow.

3 Gravy Medium growth, sediment, no turbidity.

4 Meat juice agar puncture It grows well on the surface, and the internal growth is sparse and filamentous.

Growing conditions 1 Growth temperature: Suitable temperature 30-37℃, range 15-40℃ 2 Growth PH: Optimal PH7-8, range 5-10 3 Oxygen requirement: aerobic 4 Utilization of ammonium salt: Yes, 5 Usability of urea: Yes 6 Availability of nitrate: Yes Physiological properties Other 1 Do not liquefy gelatin.

2 Litmus milk does not change.

3 Reducibility of nitrate: Positive 4 Indole production: Negative 5 Hydrogen sulfide production: Negative 6 Starch hydrolysis: Negative 7 Ammonia production from peptone: Positive 8 Vougesproskauer reaction: Negative 9 Methylred test: Positive (weak) ) 10 Catalase: Positive 11 Urease: Positive 12 Production of acid from carbohydrates Acid is produced from glucose, sutucarose, trehalose, maltose, and salicin, but no gas is produced. Arabinose, xylose, lactose, rhamnose, raffinose, mannite, zulcitrate, inosito,
Adonis does not produce acids or gases.

13 Vitamin requirement: Biotin requirement.

The medium used for culturing this strain uses ethanol as the main carbon source, and commonly used nitrogen sources and inorganic salts may be used. That is, ammonia as a nitrogen source,
Ammonium sulfate, ammonium chloride, urea, etc. are used alone or in combination. As the inorganic salt, potassium monohydrogen phosphate, potassium dihydrogen phosphate, magnesium sulfate, etc. are used. In addition, nutrients such as peptone, meat extract, yeast extract, cornstarch liquor, casamino acids, and various vitamins are also used in amounts necessary for the growth of the bacteria.

Cultivation is performed under aerobic conditions such as aerated stirring culture or shaking culture, and the culture temperature is 20 to 40°C, preferably 25°C.
~30℃. The pH during culturing is maintained at 5 to 10, preferably around neutral. The pH during culture can be adjusted by adding acid or alkali as appropriate. The ethanol concentration at the start of culture is 1 to 5% by volume, preferably 3 to 4% by volume. In this way, by using ethanol at a relatively high concentration, so-called contamination by various germs can be efficiently prevented.

The culture period is 1 to 8 days, preferably 1 to 3 days.

When producing L-tryptophan using this strain, the culture obtained by culturing as described above (culture product), the bacterial cells obtained by separating the culture by centrifugation, or the bacterial cells processed materials, such as washed bacterial cells,
Various forms of bacterial cells are used, such as dried bacterial cells, destroyed bacterial cells, and immobilized bacterial cells. Furthermore, enzymes obtained by extracting and purifying the various forms of bacterial cells described above or immobilized enzymes obtained by immobilizing the enzymes on a vinyl polymer system may also be used.

The concentration of the substrate indole in the reaction solution is not particularly limited, but is preferably 0.1 to 15 w/v%, and the concentration of serine in the reaction solution is similarly not limited, but is preferably 0.1 to 15 w/v%. /v%.

In addition to the substrates indole and serine, pyridoxal phosphoric acid is preferably added to the reaction solution in order to increase the production rate of L-tryptophan.

Water is generally used as a solvent, and if necessary, Triton It is preferable to add a surfactant such as ionic surfactant).

The pH in the reaction solution is usually 5 to 12, and the reaction temperature is preferably 15 to 60°C.

By processing according to the above conditions, the target L-tryptophan can be obtained efficiently and at low cost. The obtained L-tryptophan can be separated and purified by combining the usual ion exchange resin method and other known methods.
It can be done easily.

EXAMPLES Hereinafter, the present invention will be explained in more detail with reference to Examples, but these Examples are not intended to limit the scope of the present invention in any way.

Example 1 Medium shown in the table below (excluding ethanol)10
Dispense ml into test tubes with a diameter of 24 mm, sterilize at 120°C for 15 minutes, then add 2 v/v% ethanol under aseptic conditions to remove Brevibacterium flavum MJ.
-233 was inoculated with one platinum loop and cultured with shaking at 30°C for 20 hours to obtain a preculture solution. Next, 50ml of medium prepared in the same way
was placed in a 500 ml Erlenmeyer flask and sterilized at 120°C for 15 minutes, then 2.5 ml of the preculture was inoculated into a medium containing 2v/v% ethanol, and incubated at 30°C.
It was cultured with shaking for 20 hours.

After culturing, collect the bacteria by centrifugation and dilute to 100mM.
Wash with phosphate buffer and add indole 1.0
g, L-serine 1.2 g, pyridoxal phosphate 10
mg, 5 ml of Triton X-100, and 95 ml of water (PH8.5).
The reaction was carried out at 30°C for 17 hours. The reaction solution was immediately centrifuged, and L-tryptophan was added to the supernatant obtained.
Production of 16g/was observed.

Example 2 Washed bacterial cells obtained in the same manner as in Example 1 were mixed with 1.0 g of indole, 1.2 g of L-serine, 10 mg of pyridoxal phosphate, 5 ml of Tween 20, and 95 ml of water (PH8.5).
Suspend in 10 ml of a reaction solution with the composition of
The reaction was carried out with stirring for hours. The reaction solution was immediately centrifuged, and 15 g/L-tryptophan was observed to be produced in the supernatant obtained.

Table medium composition Urea 2.0g Ammonium sulfate 7.0g KH ₂ PO ₄ K ₂ HPO ₄ MgSO ₄・7H ₂ O 0.5g each FeSO ₄・7H ₂ O MnSO ₄・4～6H ₂ O CaCl ₂・2H ₂ O NaCl ZnSO ₄・7H ₂ O 2 mg each Yeast extract 0.5g Casamino acids 0.5g Water 1

Claims (1)

[Claims] 1. In an aqueous solution containing indole, L-serine and pyridoxal phosphate, Brevibacterium
A method for producing L-tryptophan by an enzymatic method by reacting Flavum MJ-233 cells or an enzyme obtained therefrom.