JPH04248988A - Production of l-proline by fermentation method - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention]

0001

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for producing L-proline by fermentation.

0002

[Prior Art] Conventionally, as a method for producing L-proline by fermentation, a method using microorganisms belonging to the genus Corynebacterium, Curthia, etc. is known (
Amino acid fermentation, edited by Aida et al., pp. 218-242, Society Publishing Center (1986)).

0003

[Problems to be Solved by the Invention] However, in the conventional method, only a limited number of microorganisms have been found that have the ability to produce L-proline by fermentation.

0004

[Means for Solving the Problems] The present inventors have extensively searched and studied microorganisms that are different from conventional microorganisms and have the ability to produce L-proline. It has been found and previously proposed that L-proline can be accumulated in a significant amount in a nutrient medium containing a common carbon source.

However, the accumulated concentration of L-proline produced by these methods or the yield of L-proline produced from raw materials such as sugars were not fully satisfactory.

[0006] As a result of intensive research into a highly productive method for producing L-proline, the present inventors have found that microorganisms belonging to the genus Providencia and capable of producing L-proline are given resistance to aspartate antimetabolites. The present invention was achieved by discovering that the L-proline accumulation concentration and production yield were significantly improved.

That is, the present invention provides Providencia (Pr
A microorganism belonging to the genus Ovidencia, resistant to aspartate antimetabolites, and capable of producing L-proline is cultured, L-proline is accumulated in the culture solution, and L-proline is extracted from the culture solution. This is a method for producing L-proline by a fermentation method, which is characterized by collecting L-proline.

[0008] Here, the aspartate antimetabolite is
Substances that (1) inhibit the growth of microorganisms belonging to the genus Providencia and whose growth inhibition is recovered by the addition of L-aspartic acid, or (2) exhibit suppressive and inhibitory effects on enzymes involved in the L-aspartic acid biosynthesis system. , is a substance whose suppression or inhibition can be restored by adding L-aspartic acid.

Examples of aspartate antimetabolites include aspartate hydroxamate, α-methylaspartic acid, β-methylaspartic acid, cysteine sulfinic acid, difluorosuccinic acid, and hadacidin. The microorganism used in the present invention belongs to the genus Providencia (according to Virsey's Manual of Systematic Bacteriology, Vol. 1 (1984), pages 495 and 496) and is a mutant strain that is resistant to aspartate antimetabolites. . As long as they have such properties, those with other nutritional requirements and drug resistance are also included within the scope of the present invention. In addition, auxotrophy for L-isoleucine and resistance to proline antimetabolites such as 3,4-dehydroproline effectively affect L-proline production ability, so some or all of these characteristics may be combined. Microorganisms are preferably used as parent strains. Moreover, these characteristics can be imparted by ordinary mutagenesis operations. Auxotrophy here has a broad meaning, and incomplete deletion type (so-called lea
ky type). Furthermore, it also includes cases where the requirement is satisfied by the biosynthetic precursor of the required substance.

[0010] Typical mutant strains used in the present invention include, for example, the following. Providencia
Retogeri PX8.5-13 (FERM P-1193
6).

[0011] This mutant strain is Providencia retogeri A
It was obtained by a normal mutation treatment method using Providencia retogeri PP2-704 derived from TCC21118 (L-isoleucine auxotrophy) as the parent strain,
This is a mutant strain that is resistant to aspartate hydroxamate.

[0012] Mutant strains can be induced by irradiating the parent strain with ultraviolet light or
Alternatively, after treatment with a mutagen (e.g., N-methyl-N'-nitro-N-nitrosoguanidine, ethylmethanesulfonic acid, etc.), the parent strain is grown on a solid medium containing an aspartate antimetabolite at a concentration such that it cannot grow. All you have to do is collect the possible strains.

[0013] The aspartate antimetabolite-resistant mutant strain is a strain that has stronger resistance to aspartate antimetabolites than the parent strain, and preferably has a relative growth rate of 3.
This is a mutant strain that exhibits a relative growth rate of 60% or more in a concentration range of aspartate antimetabolite that is 0% or less. The relative growth rate here is expressed as a relative value when the absorbance of the culture solution at 660 nm is measured and the absorbance of the culture solution to which the aspartate antimetabolite of each strain is not added is taken as 100%. A commercially available aspartate antimetabolite may be used for testing resistance.

[0014] The medium for L-proline production in the present invention is a conventional medium containing a carbon source, a nitrogen source, inorganic ions, and other organic trace components as necessary.

Carbon sources include glucose, fructose, starch and cellulose hydrolysates, sugars such as molasses, organic acids such as fumaric acid, citric acid, and succinic acid, alcohols such as glycerol, and the like. As a nitrogen source, organic ammonium salts such as ammonium acetate, inorganic ammonium salts such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium nitrate,
Ammonia gas, ammonia water, urea, etc. from 0.5 to 4
．． 0%, organic micronutrients include 0.001 to 0.4% of required substances such as L-isoleucine, or 0 to 4% of corn steep liquor, peptone, yeast extract, etc. as required. A medium is preferably used. In addition to these, potassium phosphate, magnesium sulfate, ferrous sulfate heptahydrate, manganese sulfate tetra- to hexahydrate, and the like are usually added in small amounts as trace components to such a medium.

[0016] Cultivation is carried out under aerobic conditions. During cultivation, the pH of the medium was adjusted to 5 to 9, the temperature was adjusted to 24 to 37°C, and
Favorable results can be obtained by culturing with shaking or aeration for 8 to 120 hours.

[0017] To collect L-proline from the culture solution,
This can be done in a conventional manner. For example, a culture filtrate from which bacterial cells have been removed is adjusted to pH 2 with hydrochloric acid, and then passed through a strongly acidic cation exchange resin for adsorption, the adsorbed components are eluted with dilute aqueous ammonia, deammoniated, and concentrated. L-proline can be obtained by adding alcohol to this and collecting the generated crystals under refrigerated storage.

[0018]

[Examples] The present invention will be specifically explained below with reference to Examples.

[0019]

[Example 1] Isolation of DL-aspartic acid hydroxamate resistant strain Providencia retgeli PP2-704 (3,4
-dehydroproline resistant, thiazolidine-4-carboxylic acid resistant, L-isoleucine auxotrophic) cells were treated with N-methyl-N'-nitro-N-nitrosoguanidine (300 mg/l, 10 minutes at 30°C) using a conventional method. ), the cells were treated with 0.5 g/DL-aspartate hydroxamate.
Agar medium (glucose 0.5%, ammonium sulfate 0.1%) supplemented with
1%, potassium phosphate 0.3%, potassium phosphate 0.7%, magnesium sulfate heptahydrate 0.01%, L
- minimal medium containing 0.005% isoleucine). Next, it was cultured at 30°C for 6 to 8 days, and the resulting large colonies were isolated by fishing, and a DL-aspartate hydroxamate-resistant strain (Providencia retogeri PX8.5-
13) was obtained.

[0020]

[Example 2] Resistance of DL-aspartate hydroxamate resistant mutant strains Each strain shown in Table 1 was cultured with shaking at 30°C for 16 hours using a liquid broth medium, and the grown cells were collected and saline Washed with water. A minimal medium (glucose 0.5%, ammonium sulfate 0.1
%, potassium phosphate 0.3%, potassium phosphate 0.7%, magnesium sulfate heptahydrate 0.01%, L-
Inoculate 5 ml of isoleucine (0.005%) and incubate at 30°C.
The growth rate of each strain was examined after 48 hours. The results are shown in Table 1. D used in the present invention
Mutant strain resistant to L-aspartate hydroxamate (
Compared to the parent strain Providencia retgeli PP2-704, the growth of Probdensia retgeli PX8.5-13 was not inhibited by high concentrations of DL-aspartate hydroxamate, and it showed strong DL-aspartate hydroxamate resistance. It shows that you have earned it.

[0021]

[Table 1]

[0022]

[Example 3] Culture of L-proline producing bacteria and production of L-proline Each strain shown in Table 2 was grown in 50 ml of liquid broth medium.
After pre-culturing at 30°C for 16 hours with shaking, transfer to a 2-liter mini-jar fermenter containing 950 ml of main fermentation medium with the following composition, which had been previously steam sterilized at 120°C for 20 minutes at 30°C, 800 rpm, aeration rate 1vvm. Aerated agitation culture was started. pH adjustment and nitrogen source supply were performed using 25% ammonia water, and the pH was 6.5 to 8.
It was maintained at 0. Glucose, KH2 PO4, MgS
When cultured for 65 hours while appropriately adding O4.7H2O, L-isoleucine, the results shown in Table 2 were obtained.

Fermentation medium glucose (separately sterilized) 4% (NH4
)2 SO4 0.5%KH2
PO4 0.1%M
gSO4 ・7H2 O 0.04%F
e++ 2p
pmMn++
2ppmL-isoleucine
0.015% yeast extract
0.05% pH 7.0 (neutralized with KOH).

[0024] Sequential addition medium (amount added per 1 liter of culture) Glucose 180g (separately sterilized) KH2 PO4
1.125gMgSO4 ・7H2O
0.45gL-isoleucine
0.675g tap water total 240ml
.

[0025]

[Table 2]

[0026] Providencia letgeri PX8.5-1
Bacterial cells were removed from the culture solution in step 3, and 500 ml thereof was passed through a column of strong cation exchange resin Diaion SK.1B (H type). After washing the column with water, the adsorbed components of the column were eluted with 2N aqueous ammonia, decolorized, and concentrated under reduced pressure. Isopropyl alcohol was added to this, the resulting crystals were collected and dried, resulting in 32 L-proline crystals with a purity of 98%.
g was obtained.

[0027]

Effects of the Invention The method of the present invention makes it possible to produce L-proline at a high yield and at a high accumulated concentration, making it possible to produce L-proline at a lower cost.

Claims (2)

[Claims] Claim 1: Providencia
ia) Cultivate a microorganism belonging to the genus genus that is resistant to aspartate antimetabolites and capable of producing L-proline, accumulate L-proline in the culture solution, and extract L-proline from the culture solution. A method for producing L-proline by a fermentation method, which comprises collecting L-proline. 2. The method for producing L-proline by a fermentation method according to claim 1, wherein the aspartate antimetabolite is aspartate hydroxamate.