JPH04299979A - Culture of microorganism of genus pseudomonas - Google Patents

Abstract

PURPOSE:To provide a process for the easy cultivation and production of a microbial strain belonging to genus Pseudomonas and rich in aspartic acid beta-decarboxylase. CONSTITUTION:A microbial strain belonging to genus Pseudomonas and containing aspartic acid beta-decarboxylase, e.g. Pseudomonas dacunhae IAM 1152 is cultured in a medium while controlling the pH of the medium with fumaric acid or its salt.

Description

[Detailed description of the invention]

0001

[Field of Industrial Application] The present invention relates to a method for culturing a microorganism belonging to the genus Pseudomonas and containing aspartate β-decarboxylase, and more specifically, it relates to a method for easily culturing and obtaining microorganisms with high enzyme activity. It's about how to do it.

0002

[Prior art] Aspartate β-decarboxylase (EC
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. be. As a method for culturing microorganisms containing the enzyme,
Method of adding lactic acid and pyruvic acid to the culture medium (Special Publication Act 1986)
19997), a method of adding L-glutamic acid to the culture medium (Japanese Patent Publication No. 53-27355), 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).

0003

[Problems to be Solved by the Invention] However, these methods have drawbacks such as the organic acids and amino acids added are expensive and the yield of bacterial cells obtained is not sufficient, so they are not suitable for industrial use. It 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 easily culturing and obtaining microorganisms belonging to the genus Pseudomonas that contain decarboxylase.

0004

[Means for Solving the Problems] As a result of intensive studies to achieve the above object, the present inventors have determined that fumaric acid or a salt thereof can be used to control at least a part of the pH of the medium during culturing of the microorganism. The inventors have found that the problem can be solved by using the method, and have completed the present invention. Thus, according to the present invention, in a method for culturing a microorganism belonging to the genus Pseudomonas and containing aspartate β-decarboxylase, at least part of the pH adjustment of the medium during culturing is performed using fumaric acid or a salt thereof. 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 aspartate β-decarboxylase, but for example, Pseudomonas da
cunhae) IAM 1152, ATCC 2119
2. Pseudomonas putida
putida) IAM 1506, same ATCC 21
812, Pseudomonas fluorescens (Pseu
domonas fluorescens) IFO 3
081, Pseudomonas aeruginosa (Pseud)
omonas aeruginosa) IAM 105
4th grade is mentioned.

[0006] Carbon sources for the culture medium used for preparing microbial cells containing aspartate β-decarboxylase include:
Commonly used substances may be used, such as fumaric acid,
Examples include succinic acid and aspartic acid, among which fumaric acid is particularly preferred. As a nitrogen source for the medium, inorganic salts such as ammonia, ammonium chloride, ammonium sulfate, ammonium nitrate, urea, etc., organic nutrient sources such as peptone, yeast extract, corn steep liquor, casamino acids, etc. can be used. Inorganic salts in the medium include potassium monohydrogen phosphate, potassium dihydrogen phosphate,
Magnesium sulfate and the like are used.

[0007] The microbial cells containing aspartate β-decarboxylase are cultured under aerobic conditions such as aeration and shaking, and the culture temperature is 20-40°C, preferably 28-40°C.
The temperature is 32°C. pH during culture is 5-10, preferably 7
It is around 8. It is important to adjust the pH using fumaric acid alone or fumaric acid and an alkali. As the alkali, one or more alkalis such as ammonia, sodium hydroxide, potassium hydroxide, etc. may be used to neutralize the fumaric acid-containing liquid. In this case, the fumarate-containing solution becomes less effective as an acid as it approaches neutrality (pH 7), so it is preferable to use the solution at a pH of 4 or less. Since fumaric acid has low solubility, it is virtually impossible to completely dissolve it with a free acid or a salt with a pH of 4 or less, but in this case, there is no problem even if it is in the form of a slurry. The concentration of fumaric acid or its salt (in the case of slurry, the weight of fumaric acid or its salt contained in a certain volume of slurry) is not particularly limited, but
Usually 5 to 50% (w/v) is used. Culture time is 8
hours to 4 days, preferably 10 hours to 2 days.

[0008] The present invention will be explained in more detail below with reference to Examples.

【Example】

[Example 1] Medium 1 (sodium fumarate 0.5%, ammonium fumarate 1.0%, yeast extract 0.5%, potassium dihydrogen phosphate 0.1%, MgSO4.7H2O0
．． 05%; pH 7.0) was dispensed into a 500 ml Erlenmeyer flask and sterilized at 120°C for 20 minutes.
One platinum loop of IAM1152 was inoculated and cultured with shaking at 30°C for 12 hours. Pour 1.5 liters of medium 2 (same as medium 1 except that the sodium fumarate and ammonium fumarate in medium 1 were changed to 0.17% and 0.33%, respectively, and 6 g of ammonium chloride was added) to a 3 liter jar fermenter. , sterilized at 120°C for 20 minutes, inoculated with 30ml of the Erlenmeyer flask culture solution, and rotated at 6 rotations.
Culture was performed at 30° C., 00 rpm, 1 vvm ventilation.

Next, fumaric acid 10 was used as a pH adjusting acid.
0g of NaOH and 20g of NaOH were mixed, deionized water was added to make the volume to about 800ml, 52ml of ammonia water was added, and then deionized water was added to make the volume to 1L. This was sterilized at 120° C. for 20 minutes, and after cooling, the pH was measured and found to be approximately 3.6. Use this solution to adjust the pH of the culture solution to 7.3.
After 11 hours, the culture was continued, and the cells were collected by centrifugation to obtain about 40 g of wet cells. The obtained bacterial cells were suspended in 1 liter of 0.9% NaCl, and after washing, the bacterial cells were recovered by centrifugation twice.
Washed bacterial cells were obtained.

[0010] The aspartate β-decarboxylase activity of the obtained bacterial cells was measured by the following method. Reaction solution (L-
Aspartic acid 1500mM, pyridoxal 5'-phosphate 0.04mM, Triton X-100 0.1% (v
/v), containing 0.4M ammonia; pH 4.7) 20m
0.2 g of bacterial cells were suspended in 1 liter of water and shaken at 42° C. for 1 hour, and the amount of L-alanine produced was measured and quantified by thin layer chromatography or high performance liquid chromatography. The activity was expressed as the amount of enzyme that can produce 1 μmol of alanine per hour at 42° C. as 1 U (unit), and the specific activity was expressed as the amount of enzyme per 1 g of wet bacterial cells (U/g cell). The specific activity of this bacterial cell was approximately 37,000 U/g cell.

[Example 2] Pseudomonas dacne (Ps
eudomonas dacunhae) IAM 11
52 to adjust the pH using a fumaric acid suspension (fumaric acid 55
The culture was carried out in the same manner as in Example 1 in an Erlenmeyer flask and then in a jar fermenter, except that the culture was carried out in an Erlenmeyer flask and then in a jar fermenter, and the wet bacterial mass was 45 g/l. obtained at a concentration of Centrifugal washing was performed in the same manner as in Example 1, and the specific activity of the washed bacterial cells was determined to be approximately 40,000 U/g cell.

[Comparative Example] Pseudomonas dacne (Pse
udomonas dacunhae) IAM 115
An Erlenmeyer flask, an Erlenmeyer flask, The cells were then cultured in a jar fermenter to obtain wet bacterial cells at a concentration of 43 g/l. Centrifugal washing was performed in the same manner as in Example 1, and the specific activity of the washed bacterial cells was determined to be approximately 29,000 U/g cell.

[0013]

[Effects of the Invention] According to the present invention, bacterial cells with high aspartate β-decarboxylase activity can be obtained by adjusting the pH of the culture medium of microorganisms belonging to the genus Pseudomonas using fumaric acid or its salt. Can be done.

Claims (1)

[Claims] Claim 1: A method for culturing a microorganism belonging to the genus Pseudomonas and containing aspartate β-decarboxylase, in which at least part of the pH adjustment of the culture medium is performed using fumaric acid or a salt thereof. A method for culturing Pseudomonas microorganisms characterized by: