JPH0159876B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing L-proline by fermentation.

L-proline is an amino acid useful as a medicine, feed additive, and drug. Conventionally, methods for producing L-proline using fermentation methods include L-proline belonging to the genus Previbacterium, Micrococcus, Kurtia, Satucharomyces, Bacillus, Eschieria, Microbacterium, Corynebacterium, and Arthrobacter. A method is known in which proline-producing bacteria are cultured to produce L-proline in the medium (Japanese Patent Publications No. 11751/1976, No. 44-1198, No. 44-
6631, 44-26911, 46-38557, 48-38876,
51-33190, Japanese Patent Application Publication No. 55-148096, etc.). However, there have been no reports of producing and accumulating L-proline using microorganisms belonging to the genus Serratia.

As a result of various studies on the production method of L-proline by fermentation, the present inventors found that microorganisms belonging to the genus Serratia have strong L-proline degrading enzyme ability, and L-proline biosynthesis is subject to metabolic regulation. Although the microorganism does not normally produce and accumulate L-proline, it produces and accumulates a significant amount of L-proline by mutating the microorganism and imparting L-proline degrading enzyme deficiency and resistance to proline analogs and/or purine analogs. They discovered this and completed the present invention.

That is, the present invention provides a method for producing L-proline by a fermentation method, which comprises culturing a microorganism belonging to the genus Serratia and capable of producing L-proline in a medium, producing and accumulating L-proline in the medium, and collecting the L-proline. It is.

The microorganisms used in carrying out the present invention belong to the genus Serratia, are deficient in L-proline degrading enzymes (e.g., L-proline oxidase), and are deficient in proline analogues (e.g., 3,4-dehydro-DL-proline). , L-thiazoline-4-carboxylic acid, L-azetidine-2-carboxylic acid) and/or purine analogs (for example, 2,6-dibromprine). Preferred examples of such strains include Serratia marsetuscens that is L-prolinease deficient and resistant to proline analogs, Serratia marsetuscens that is L-prolinease deficient and resistant to proline analogs and purine analogs, and the like. Can be mentioned. More specifically,
For example, Serratia marsetuscens deficient in L-proline oxidase and resistant to 3,4-dehydro-DL-proline, Serratia marsetuscens deficient in L-proline oxidase and resistant to 3,4-dehydro-DL-proline and L-thiazolidine-4-carvone. Acid resistant Serratia marsetuscens, L-proline oxidase deficient and 3,4-dehydro-DL-
Preferred examples include Serratia marsetuscens, which is resistant to proline and 2,6-dibromopurine.

The above mutant strain can be obtained as follows. For example, a mutant strain deficient in L-proline degrading enzyme and resistant to proline analogs can be obtained by subjecting the original strain (e.g. Serratia marsetuscens Sr41) to mutagenic treatment, e.g. UV irradiation, or by mutagenic agents (e.g. N-methyl -N'-Nitro-N
- nitrosoguanidine, ethyl methanesulfonate, etc.) to induce mutations, and then plated at 30°C in a plate medium containing L-proline as the main carbon or nitrogen source (e.g. Davis's minimal medium). After culturing for 3 to 5 days and separating small colonies from the resulting colonies, an L-proline degrading enzyme-deficient strain was obtained, and then mutations were induced in the deficient strain in the same manner as above. , cultured at 30°C for 1 to 3 days in a plate medium (e.g., Davis's minimal medium) supplemented with 0.2 mg/ml of proline analog (e.g., 3,4-dehydro-DL-proline), and harvest the resulting colonies. It can be obtained by separating bacteria. A typical example of a mutant strain obtained in this way is Serratia marsetuscens Dr-9 (Feikoken Bacterial Serial No. 6159).
No.) (L-proline oxidizer deficient and 3,
Mutant strain resistant to 4-dehydro-DL-proline)
can be mentioned. In addition, after further inducing mutations in the mutant strain obtained above in the same manner as above, L-
Plate culture medium containing 1.0 mg of thiazolidine-4-carboxylic acid (e.g., 0.5 mg of sodium succinate as carbon source)
3-5% Davis' minimal medium) at 30°C.
By culturing for days and separating the resulting colonies, a mutant strain deficient in L-proline oxidase and resistant to 3,4-dehydro-DL-proline and L-thiazolidine-4-carboxylic acid can be obtained. A typical example of the mutant strain thus obtained is Serratia marsetuscens DTr-12 (Feikoken Joyori No. 171). In addition, a mutant strain deficient in L-proline degrading enzyme and resistant to proline analogs and purine analogs can be obtained from the L-prolyl degrading enzyme obtained above.
- After inducing mutations in a mutant strain that is prolinease deficient and resistant to proline analogues, purine analogues (e.g., 2,6-dibromopurine)
It can be obtained by culturing in a plate medium containing 2 mg/ml at 30° C. for 1 to 3 days, and separating the resulting colonies. A typical example of a mutant strain obtained in this way is Serratia marsetuscens DBr-51 (Feikoken Bibori No. 6158) (L-proline oxidase deficient and 3,4-dehydro-
mutant strains resistant to DL-proline and 2,6-dibromprine).

The method for obtaining a mutant strain capable of producing L-proline is not limited to the above method, but may be obtained by obtaining a mutant strain having different properties such as L-proline degrading enzyme deficiency, proline analog resistance, purine analog resistance, etc. By applying genetic recombination methods such as transduction after obtaining mutant strains with each property in sequence or separately,
A mutant strain capable of producing L-proline can be obtained.

The L-proline production medium used in the method of the present invention contains sugars such as glucose, sucrose, and molasses, organic acids such as succinic acid, citric acid, and fumaric acid, and sugar alcohols such as glycerol as carbon sources. ~20%, organic ammonium salts such as ammonium fumarate, ammonium succinate, inorganic ammonium salts such as ammonium sulfate, ammonium chloride, urea, etc. as a nitrogen source.
5%, 0 to 0 organic nutrients such as cornstarch liquor, peptone, yeast extract, fish extract, etc.
A medium containing appropriate amounts of each in the range of 1% and a small amount of potassium phosphate magnesium sulfate as an inorganic substance can be suitably used.

In addition to these, calcium carbonate, aqueous ammonia, citric acid, etc. may be added as necessary to maintain the pH of the medium at 6 to 8. Furthermore, such a medium contains L, which is a precursor for L-proline biosynthesis.
- A medium to which glutamic acid, L-aspartic acid, etc. are appropriately added can also be suitably used.

According to the present invention, the L-proline producing mutant strain described above is inoculated into the above medium and cultured for 2 to 7 days under aerobic conditions such as shaking culture or aeration and stirring at 25 to 40°C. As a result, a significant amount of L-proline can be accumulated in the culture medium. The generated L-
After completion of the culture, proline can be easily collected by normal separation and purification operations using an ion exchange resin, as shown in the Examples, after removing bacterial cells and other insoluble matter.

The method of the present invention will be described below with reference to Examples and Reference Examples. In the Examples, L-proline was confirmed by ninhydrin reaction and isatin reaction in paper chromatograms. The quantitative determination was carried out by bioassay using Leuconostoc mesenteroides P-60.

Example 1 10% sucrose, 1% urea, dibasic potassium phosphate
0.1%, magnesium sulfate 0.05%, cornstarch liquor 0.1%, and calcium carbonate 1% (PH 7.0) (15 ml) was poured into a 500 ml shaking Kolben and sterilized by heat. However, sucrose must be sterilized separately.
Added. Add 30% to this fermentation medium with a broth slant.
Serratia marsetuscens cultured overnight at °C.
One platinum loop was inoculated with Dr-9 strain (Feikoken Bibori No. 6159). When cultured for 72 hours at 30°C, 140 revolutions/min, and an amplitude of 7 cm, 9.5 mg/ml of L-proline was produced and accumulated in the medium.

Example 2 One platinum loop of Serratia marsetuscens strain DBr-51 (Feikoken Bacterial Serial No. 6158), which had been cultured overnight at 30°C in a bouillon slant medium, was inoculated into a fermentation medium having the same composition as in Example 1. When cultured for 72 hours at 30°C, 140 revolutions/min, and an amplitude of 7 cm, 12.6 mg/ml of L-proline was produced and accumulated in the medium.

Example 3 15% sucrose, 2% urea, dibasic potassium phosphate
0.1%, magnesium sulfate 0.05%, cornstarch liquor 0.6%, and calcium carbonate 1% (PH 7.0) (15 ml) was poured into a 500 ml shaking Kolben and sterilized by heat. However, sucrose must be sterilized separately.
Added. Add 30% to this fermentation medium with a broth slant.
Serratia marsetuscens cultured overnight at °C.
One platinum loop was inoculated with DTr-12 strain (Feikoken Bibori No. 6160). When cultured for 72 hours at 30°C, 140 revolutions/min, and an amplitude of 7 cm, 25.6 mg/ml of L-proline was produced and accumulated in the medium. The culture solution 1 was collected, heat-treated, and then filtered to remove bacterial cells and other insoluble matter. The filtrate was passed through a column filled with cation exchange resin Amberlite IR-120B (H ⁺ type). After washing with water, the adsorbed L-proline was eluted with 5% aqueous ammonia, and the eluate was concentrated under reduced pressure. The concentrated solution was cooled and allowed to stand, yielding 18.0 g of L-proline crystals.

Reference Example 1 (Method for obtaining Serratia marcetuscens Dr-9 strain) A small amount of Serratia marcetuscens strain Sr41 was inoculated into a bouillon medium and cultured at 30°C. When the bacteria in the culture solution reached approximately 10 ⁹ cells/ml,
A 1 mg/ml aqueous solution of N-methyl-N'-nitro-N-nitrosoguanidine was added to a concentration of 0.25 mg/ml, and after culturing at 30°C for 20 minutes, the centrifuged cells were washed with physiological saline. After washing, fresh bouillon medium was added and cultured at 30°C for 6 hours. This culture solution was centrifuged, the supernatant was discarded, and physiological saline was added to suspend the cells. This cell suspension was diluted with physiological saline to approximately 10 ³ cells/ml. 0.1 ml of this suspension was added to an agar plate medium containing L-proline as the main nitrogen source (glucose 0.5%,
L-proline 0.2%, ammonium sulfate 0.0002%,
Potassium phosphate 0.7%, Potassium phosphate 0.7%
0.3%, magnesium sulfate heptahydrate 0.01%, agar 1.5%) and cultured at 30°C for 3 days.
Isolate small colonies from the resulting colonies,
L-proline oxidase activity was determined by the method of Denzinger et al. [Journal of Bacteriology,
103, pp. 144-152, 1970], and a strain lacking this enzyme activity was obtained.
The thus obtained L-proline degrading enzyme-deficient strain was treated with N-methyl-N'-nitro-N-nitrosoguanidine in the same manner as above, and approximately
A cell suspension of 10 ⁹ cells/ml was prepared. Add 0.1 ml of this suspension to 0.2 ml of 3,4-dehydro-DL-proline.
Agar plates containing mg/ml (glucose 0.5%,
Ammonium sulfate 0.1%, potassium phosphate 0.7
%, potassium phosphate 0.3%, magnesium sulfate heptahydrate 0.01%, agar 1.5%).
This was cultured at 30°C for 2 days, the resulting colonies were isolated, and the L-proline productivity was examined as described in Example 1.
Dr-9 stock was acquired.

Reference Example 2 (Method for obtaining Serratia marsetuscens strain DBr-51) Reference example is a mutant strain Dr-9 derived from Serratia marsetuscens Sr41 that is deficient in L-proline degrading enzyme and resistant to 3,4-dehydro-DL-proline. Obtained according to 1. This Dr-9 strain was treated with N-methyl-N'-nitro-N in the same manner as in Reference Example 1.
- Mutation induction treatment was performed with nitrosoguanidine, and a cell suspension was prepared. Add 0.1 ml of this suspension to 2,6-
Agar plate medium containing dibromprine 2 mg/ml (glycerol 0.5%, ammonium sulfate 0.1%, dibasic potassium phosphate 0.7%, dibasic potassium phosphate 0.3
%, magnesium sulfate heptahydrate 0.01%, agar
1.5%). This was cultured at 30° C. for 3 days, the resulting colony was isolated, and the L-proline productivity was examined as described in Example 1 to obtain Serratia marsetuscens DBr-51 strain.

Reference Example 3 (Method for obtaining Serratia marsetuscens strain DTr-12) Reference example is a mutant strain Dr-9 derived from Serratia marsetuscens Sr41 that is deficient in L-proline degrading enzyme and resistant to 3,4-dehydro-DL-proline. Obtained according to 1. This Dr-9 strain was treated in the same manner as in Reference Example 1 to obtain N-methyl-N'-nitro-N-
Mutation induction treatment was performed with nitrosoguanidine, and a cell suspension was prepared. 0.1 ml of this suspension was added to an agar plate medium containing 1.0 mg/ml of L-thiazolidine-4-carboxylic acid (sodium succinate 0.5%, ammonium sulfate 0.1%, monobasic potassium phosphate 0.3%, dibasic potassium phosphate 0.3%). %, magnesium sulfate 0.01%,
Agar 1.5%). This was cultured at 30°C for 4 days, the resulting colonies were isolated, and L-proline productivity was examined as described in Example 1.
Acquired Serratia marsetuscens DTr-12 stock.

Claims (1)

[Scope of Claims] 1. L-proline produced by a fermentation method characterized by culturing a microorganism belonging to the genus Serratia and capable of producing L-proline, producing and accumulating L-proline in a medium, and collecting the L-proline. manufacturing method. 2. The method according to claim 1, wherein the microorganism belongs to the genus Serratia and is an L-proline-producing bacterium that is deficient in L-prolinease and is resistant to proline analogs and/or purine analogs. 3 The microorganism belongs to the genus Serratia and is L-proline degrading enzyme deficient and proline analog resistant.
- The production method according to claim 1, which is a proline-producing bacterium. 4. The production method according to claim 1, wherein the microorganism is an L-proline producing bacterium belonging to the genus Serratia, deficient in L-proline degrading enzyme, and resistant to proline analogs and purine analogs. 5 Claim 1 in which the microorganism is Serratia marsetuscens having the ability to produce L-proline
Manufacturing method described in section. 6. The production method according to claim 5, wherein the Serratia marsetuscens is L-proline degrading enzyme deficient and has an ability to produce L-proline that is resistant to proline analogs and/or purine analogs. 7 Serratia marsetuscens is L-prolinease deficient and proline analog resistant.
- The production method according to claim 5, which is Serratia marsetuscens having proline producing ability. 8. The production method according to claim 5, wherein the Serratia marsetuscens is L-prolinease deficient and has L-purine producing ability that is resistant to proline analogs and purine analogs. 9 Serratia marsetuscens is L-proline oxidase deficient and 3,4-dehydro-
6. The production method according to claim 5, which is Serratia marsetuscens having the ability to produce L-proline that is resistant to DL-proline. 10 Serratia marsetuscens is L-proline oxidase deficient and 3,4-dehydro-
The production method according to claim 5, which is Serratia marsetuscens having the ability to produce L-proline that is resistant to DL-proline and 2,6-dibromopurine. 11 Serratia marsetuscens is L-proline oxidase deficient and 3,4-dehydro-
Claim 5, which is Serratia marsetuscens, which has the ability to produce L-proline that is resistant to DL-proline and L-thiazolidine-4-carboxylic acid.
Manufacturing method described in section.