JPH04320677A - Culture of bacterium belonging to genus pseudomonas - Google Patents

Abstract

PURPOSE:To obtain cells of a bacterium belonging to the genus Pseudomonas having high ASP decarboxylase activity in high yield by regulating concentration of fumaric acid of a main carbon source in culturing a bacterium belonging to the genus Pseudomonas, containing L-ASP decarboxylase. CONSTITUTION:In culturing a bacterium belonging to the genus Pseudomonas, containing L-aspartic acid beta-decarboxylase, fumaric acid, a main carbon source, is maintained at <=1wt.%. For example, Pseudomonas fluorescens IFO 3,081 may be cited as the bacterium. The culture of the bacterium is preferably carried out under an aerobic condition such as aerated stirring at 28-32 deg.C at pH7-8 for 10 hours to 2 days.

Description

Detailed Description of the Invention [0001] The present invention relates to a method for culturing a microorganism belonging to the genus Pseudomonas and containing L-aspartate β-decarboxylase. The present invention relates to a method for simply culturing and obtaining microbial cells with high enzyme activity. [Prior Art] L-aspartate β-decarboxylase (E
C 4.1.1.12) is an enzyme that decarboxylates the carboxyl group at the 4-position of L-aspartic acid to produce L-alanine, and is industrially useful for the production of L-alanine. It is. Methods for culturing microorganisms containing the enzyme include a method of adding lactic acid and pyruvic acid to the medium (Japanese Patent Publication No. 19997-1982), and a method of adding L-glutamic acid to the medium (Japanese Patent Publication No. 27355-1982). Public bulletins) etc. have been proposed. Furthermore, the present inventors also proposed a method for producing L-alanine using bacterial cells cultured in a medium containing fumaric acid as the main carbon source (Japanese Patent Application Laid-Open No. 2-242690).
Publication No.). [0003] However, these methods have drawbacks such as the organic acids and amino acids added are expensive and the yield of the obtained bacterial cells is not sufficient, making it difficult to use them industrially. The method was unsatisfactory. The present invention solves the above-mentioned problems and achieves high aspartate β-
The purpose of this invention is to provide a method for culturing and obtaining a high yield of microorganisms belonging to the genus Pseudomonas containing decarboxylase. [Means for Solving the Problems] As a result of intensive studies to achieve the above object, the present inventors have found that the microorganisms are cultured using fumaric acid, which is the main carbon source, at a concentration of 1% by weight or less. The inventors have discovered that the problem can be solved by maintaining the same conditions, and have completed the present invention. Thus, according to the present invention, a method for culturing a microorganism belonging to the genus Pseudomonas and containing L-aspartate β-decarboxylase is characterized in that fumaric acid as a main carbon source is maintained at a concentration of 1% by weight or less. A method for culturing a Pseudomonas microorganism is provided. The microorganism used in the present invention is not particularly limited as long as it belongs to the genus Pseudomonas and contains L-aspartate β-decarboxylase, but for example,
Pseudomonas dacne
IAM 1152, ATCC 21
192, Pseudomonas putida
as putida) IAM 1506, same ATCC
21812, Pseudomonas fluorescens (Ps
eudomonas fluorescens) IFO
3081, Pseudomonas aeruginosa (Pse
udomonasaeruginosa) IAM 10
54, Pseudomonas syringae
nas syringae) IFO 3310 and the like. [0006] Fumaric acid is used as the main carbon source in the culture medium used to prepare microbial cells containing L-aspartate β-decarboxylase, and its concentration is 1% by weight or less.
It is important to maintain the content preferably at 0.3% by weight or less. Fumaric acid may be added to the medium continuously or intermittently as long as the concentration can be maintained within the above range. As a nitrogen source for the culture medium, inorganic salts such as ammonia, ammonium chloride, ammonium sulfate, ammonium nitrate, urea, etc. can be used, and organic nutritional sources such as peptone, meat extract, yeast extract, corn steep liquor, casamino acids, etc. can be used. be able to. As the inorganic salt of the medium, potassium monohydrogen phosphate, potassium dihydrogen phosphate, magnesium sulfate, etc. are used. [0007] The microbial cells containing L-aspartate β-decarboxylase are cultured under aerobic conditions such as aeration and shaking, and the culture temperature is 20 to 40°C, preferably 20°C.
The temperature is 8-32°C. The pH during culturing is around 5-10, preferably around 7-8. The culture time is 8 hours to 4 days, preferably 10 hours to 2 days. After completion of the culture, the bacteria were collected by centrifugation, and the L-aspartate β-decarboxylase activity of the bacteria was measured by the following method. The total amount of bacterial cells collected from 100 ml of the culture solution was added to the reaction solution (aspartic acid 1500 mM, pyridoxal 5'-phosphate 0.04 mM, sodium pyruvate 5 m
M, polyoxyethylene sorbitan monolaurate 0.
Contains 1% by volume and 0.6M ammonia; pH 4.8)2
The amount of alanine produced was determined by suspending the suspension in 00 ml and shaking at 30°C for 1 hour using silica gel thin layer chromatography or high performance liquid chromatography. Note that the activity was expressed as the amount of L-alanine (μmole) produced per 1 ml of broth in 1 hour. The present invention will be explained in more detail below with reference to Examples. [Example] [Example] Medium (ammonium fumarate 0.
5%, yeast extract 0.5%, potassium dihydrogen phosphate 0.
05%, MgSO4・7H2O containing 0.05%; pH 7
．． 0) Dispense 100ml into a 500ml Erlenmeyer flask, sterilize it at 120°C for 20 minutes, and then add Pseudomonas dacunhae.
One platinum loop of ATCC 21192 was inoculated and incubated at 30°C.
Shaking culture was performed for days (preculture). Next, fumaric acid (neutralized to pH 7 with ammonia) was added to the concentrations shown in Table 1, and main culture was performed. Namely, yeast extract 0.5%, potassium dihydrogen phosphate 0.05%, MgSO4.7H2O
1 liter of medium containing 0.05% (pH 7.0) to 2 liters
After sterilization at 120°C for 20 minutes, fumaric acid was added to 20 ml of the preculture solution and fumaric acid to the concentration shown in Table 1, and the mixture was heated at 1000 rpm and 1 aeration volume.
Culture was performed for 1 day at vvm, temperature 30° C., and pH 7.3. Incidentally, fumaric acid was added so as not to exceed the concentration shown in Table 1, so that the final total amount added was about 4%. As a comparative example, fumaric acid was first added to
．． An experiment was conducted in which fumaric acid was added at 5 w/v% and then twice at 1.2 w/v% after consumption of fumaric acid (3.9% in total). . After culturing, the collected cells were centrifuged from 100 ml and washed with 0.9 w/v% saline, and then the whole amount of cells was used to
L-aspartate decarboxylase activity was measured by the method described above. The results are shown in Table 1. [Table 1]
Table 1
Fumaric acid addition method L-aspartate decarboxylase activity

(μmole/ml broth/hr)
1% x 4 times
1200
0.5% x 8 times
1250 0.2
5% x 16 times
1300 Comparative example (1.5%+1.2%+1.2%)
1000
Effects of the Invention According to the present invention, L-aspartic acid β
- Microorganisms belonging to the genus Pseudomonas having decarboxylase activity can be obtained in high yield.

Claims (1)

[Claims] Claim 1: A method for culturing a microorganism belonging to the genus Pseudomonas and containing L-aspartate β-decarboxylase, characterized in that fumaric acid, the main carbon source, is maintained at a concentration of 1% by weight or less. A method for culturing Pseudomonas microorganisms.