JPH02234687A - Production of n-acetyl-l-tryptophan by fermentation method - Google Patents

Abstract

PURPOSE:To obtain acetyltryptophan at a low cost in good yield by culturing a specific bacterium of the genus Micrococcus in a culture medium and collecting N-acetyl-L-tryptophan from the cultured product. CONSTITUTION:A bacterium belonging to the genus Micrococcus and having N-acetyl-L-tryptophan producing ability is cultured in a culture medium and N-acetyl-L-tryptophan is produced and accumulated in a cultured product. Then the aimed N-acetyl-L-tryptophan is collected from the cultured product. As the bacterium used, tryptophan analog resistant bacterium is preferably used. Specific example of the bacterium includes Micrococcus varians, preferably Micrococcus varians 80-171-95. Either one of synthetic culture medium and natural culture medium may be used as the culture medium, when the medium is a medium moderately containing carbon source, nitrogen source, inorganic source, growth factor, etc.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention provides N-acetyl-L-}! The present invention relates to a method for producing Jptophan (hereinafter referred to as acetyltryptophan).

Acetyltryptophan is useful as a feed additive, infusion, etc.

Conventional technology Acetyltryptophan is a method of acetylating tryptophan [Journal of Biological Chemistry]
Chemistry) 144, 193 (194
2)], a method for decarboxylating mono-ruboxy-DL-acetyltryptophan, and a method for reacting acetamido T-formyl acetic acid with phenylhydrazine (Japanese Patent Application Laid-open No. 1973
-14661) etc.
- Obtained by racemic resolution of tryptophan.

The conventional method is a synthetic method, and there is no known method for obtaining acetyltryptophan by fermentation.

Problems to be Solved by the Invention There is a need for a method for obtaining acetyltryptophan at low cost and in good yield by fermentation.

Means for Solving the Problems The present invention involves culturing a microorganism belonging to the genus Micrococcus and having the ability to produce acetyltryptophan in a medium, producing and accumulating acetyltryptophan in the culture, and collecting acetyltryptophan from the culture. A method for producing acetyltryptophan by a fermentation method is provided.

The microbial inhibitor used in the present invention may be any microorganism that belongs to the genus Micrococcus and has the ability to produce acetyltributophane. Preferably, tryptophan analog resistant microorganisms are used.

A specific example is Micrococcus variens (Micro
coccus varians)^TCC 399
, preferably Micrococcus variens 80-17
1-95 (5-methyltryptophan resistance) (F[
! RM BP-2255) (hereinafter referred to as FBRM BP-
2255), etc.

Next, a specific example of obtaining FORM BP-2255 will be explained.

Micrococcus variens ATCC 399 cells were added to trisumarate buffer (pH 6.0) at 1011 cells/
ml, and then add N-methyl-N to the suspension.
'-N-nitronic acid at a final concentration of 2 m
g/mI! The mixture was added to the solution and treated at room temperature for 20 minutes.

The treated bacterial cells were washed three times with physiological saline, suspended again in physiological saline, and then the suspension or the solution diluted with physiological saline was diluted with 0.
．． 1 ml of 5-methyltributophane in 2 portions
Minimal medium containing g/l (glucose 10 g/I!, gazamino acid 5 g/β, ammonium phosphate 1 g/1, potassium chloride 0.2 g/R, magnesium sulfate 0.2 g/It,
Piotin 50 r/Il, vitamin B+10mg/1
, trace metal liquid 1rdl/1, agar 20g/1, pH
7. 0).

After culturing at 30°C for 4 days, the emerging colonies were obtained as 5-methyltributophane-resistant strains, and fermentation medium (glucose 100g/j!, monopotassium phosphate 1glll,
Dipotassium phosphate Ig/j! , Magnesium sulfate 7
Water salt 0.5g/j', ammonium sulfate 20g/j'! ，
Cohn Stieble force - 30g/Cp H 7. O)
80-171-95, a mutant strain with improved ability to produce lyacetyltryptophan compared to the parent strain was cultured in test tubes and jars.
selected as. This strain was submitted as FORM OP-22 to the Institute of Microbial Technology, Agency of Industrial Science and Technology on January 24, 1989.
It has been deposited as No. 55.

The medium used in the present invention may be either a synthetic medium or a natural medium as long as it contains a suitable amount of carbon sources, nitrogen sources, inorganic salts, growth factors, etc.

As the carbon source, carbohydrates such as glucose, maltose, sucrose, blackstrap molasses, starch, and cellulose decomposition products, organic acids such as acetic acid, fumaric acid, and malic acid, n-paraffin, and the like are used.

As the nitrogen source, ammonia, ammonium sulfate, ammonium nitrate, ammonium chloride, ammonium phosphate, urea, peptone, meat extract, yeast extract, casamino acid, Cohn-Steepley force, etc. are used.

As the inorganic salt, monopotassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, ferrous sulfate, manganese sulfate, sodium chloride, etc. are used.

As the growth factor, vitamins (eg, biotin, thiamine, nicotinic acid, pantothenic acid), etc. are used.

Furthermore, by adding indole to the culture medium, the production amount of lyacetyltributophane can be significantly improved. The amount of indole added to the culture solution is preferably 1% or less.

Cultivation is performed under aerobic conditions such as shaking culture and aerated agitation culture. The culture temperature is 20-40°C, preferably 25-35°C. The pH of the medium is maintained at 5 to 9, preferably around neutrality. The pH of the medium is adjusted using calcium carbonate, inorganic or organic acids, alkaline solutions, ammonia, pH buffers, and the like.

The culture period is usually 24 to 150 hours, and acetyltributophane is produced and accumulated in the culture.

The isolation of acetyltryptophan produced in the culture medium is
After culturing, acetyltryptophan can be recovered from the supernatant by removing precipitates such as bacterial cells from the culture solution by centrifugation, etc., and then using methods such as ion exchange, concentration, adsorption, and extraction. .

Examples are shown below. In the examples, acetyltributophane was quantified by high performance liquid chromatography.

Example 1 Glucose 30g/Il, peptone 10g/C yeast extract 5g/Il and sodium chloride 2.5g/j! A liquid medium (pH 7.0) consisting of the following composition: 300mi! FBRM BP-2255 was inoculated into a 2 liter flask containing the FBRM BP-2255, and cultured with shaking at 30°C for 16 hours. Thereafter, the entire amount of the culture solution was transferred to fermentation medium 2. Place in a 5l jar jar containing 7 A, lv, v, m, 6 0 0
Aeration and agitation culture was carried out at r, p, m. The culture temperature was 30℃.
The pH was adjusted with 14% aqueous ammonia.

Fermentation medium composition Glucose 100g/J! Monopotassium phosphate - 18/1 Dipotassium phosphate 1g/1 Magnesium sulfate heptahydrate 0.5g/It Ammonium sulfate
20g/β Corn Stieble force - 30g/I
! pH 7. 0 After 50 hours from the start of culture, glucose was sequentially added to the culture solution so that the concentration ranged from 10 to 30 g/1 when glucose was consumed, and after 122 hours of culture, the acetyl concentration in the culture solution was It was 1.7g/A.

Example 2 A seed culture solution obtained in the same manner as in Example 1 was placed in a 5β jar fermenter containing the same fermentation medium composition as in Example 1. After 28 hours from the start of culture, the indole concentration in the culture solution is 0.
, and cultured for 122 hours while sequentially adding a 50% ethanol solution of indole to within 1%. After the completion of the culture, the concentration of acetyl}IJbutophane in the culture solution was 4,
3 g/jl! Met.

Centrifuge the culture solution to remove bacterial cells and reduce the volume to one third.
and hydrophobic adsorption resin [Diaion SP207,
The solution was passed through a column packed with Mitsubishi Kasei ■ and eluted with 30% methanol. The eluate was passed through a cation exchange resin [DOWEX 50W, manufactured by Mitsubishi Kasei@], and both components corresponding to acetyltryptophan were collected and concentrated to obtain acetyl}U-butophane crystals 1. 5 g was obtained.

Effects of the Invention Acetyltributophane can be obtained at low cost and in high yield by the method of the present invention.

Patent applicant (102) Representative of Kyowa Hakko Kogyo Co., Ltd.
Mikio Kato

Claims (1)

[Claims] Cultivating a microorganism belonging to the genus Micrococcus and having the ability to produce N-acetyl-L-tryptophan in a medium, producing and accumulating N-acetyl-L-tryptophan in the culture,
N-acetyl-L- by a fermentation method characterized by collecting N-acetyl-L-tryptophan from the culture.
Method for producing tryptophan.