JPS5860995A - Preparation of l-proline by fermentation - Google Patents

Abstract

PURPOSE:To form and to accumulate the titled substance and to collect it, by cultivating a bacterium capable of producing L-proline, lacking in L-proline degrading enzyme, having resistance to proline analogue and/or purine analogue. CONSTITUTION:A bacterium such as Serratia marcescens, etc. belonging to the genus Serratia, capable of producing L-proline, lacking in L-proline degrading enzyme (e.g., lacking in proline oxidase), having a resistance to proline analogue and/or purine analogue, is cultivated. The medium consists of 10-20wt% carbon source, 1-5wt% nitrogen source, 0-1wt% organic nutrient substance, etc. and kept at a pH of 6-8. The strain is cultivated in the medium under aerobic conditions for 2-7 days, and accumulated L-proline is collected by a common separating and purifying operations using an ion exchange resin etc.

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 that is used as a medicine and feed additive. Conventional methods for producing L-proline using nine fermentation methods include Brevibacterium, Micrococcus, Kurtia, Satucharomyces, Bacillus, Nijieria spp., Microbacterium spp., Corynebacterium spp.
A method for producing KL-proline in a culture medium by culturing L-proline-producing bacteria belonging to Arthrobafter sea bream is known, 38557, 48-38876, 51-3319.
0, JP-A-55-148096, etc.). However, there is no report on the production and accumulation of L-proline using a Δ microorganism 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 are L-proline.
- Because it has a strong proline degrading enzyme ability and L-proline biosynthesis is subject to the chloride Mg1 clause, it does not normally produce and accumulate L-proline.
It was discovered that a significant amount of L-proline can be produced and accumulated by imparting phospholase deficiency and resistance to proline analogs and/or purine analogs, and the present invention has been completed.

Specifically, 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 having L-proline-producing ability in a medium, producing and accumulating L-proline in the medium, and collecting the L-proline. It is.

The microorganisms and organisms used in carrying out the present invention belong to the genus Serraci Lr, are deficient in L-proline degrading enzymes (e.g., L-proline oxidase), and are deficient in proline analogues (e.g., 3,4-dehydrogenase). -DL-proline,
L-thiazoline-4-carboxylic acid, L-azetidine-2
-carboxylic acid-) and/or purine analogs (e.g.
Examples include L-proline producing bacteria that are resistant to 2,6-dibromopurine). Preferred examples of such strains include Serratia marcescens that is L-prolinease deficient and resistant to zoroline analogs, Serratia marcescens that is L-prolinease deficient and resistant to proline analogs and purine analogs, etc. Preferred examples include: More specifically.

6- For example, L-proline oxidase deficient gag3.4-
Serratia marcescens resistant to dehydro-DL-proline, L-proline oxidase deficient and 3.4
-dehydro-DL-7'loline and L-thiazolidine-
4-Carboxylic acid resistant Serratia marcescens, L
Preferred examples include Serratia marcescens which is proline oxidase deficient and resistant to 3,4-dehydro-DL-proline and 2,6-dibromprine.

The above-mentioned alveolar strains can be obtained as follows. For example, a mutant strain deficient in L-prolyl degrading enzyme and resistant to proline analogs can be treated with a mutant strain (e.g., Serratia marcescens sr 41), subjected to a treatment for induction of JllI (eg, Serratia marcescens sr 41), irradiated with ultraviolet light, or irradiated with a mutagenic agent (e.g. ,
N-methyl-N'-nitro N-nitroguanidine,
After treatment with ethyl methanesulfonate, etc.) to induce oxidation.

3 on a plate medium containing L-proline as the main carbon X source or nitrogen source (e.g. Davis's minimal medium).
Cultivate at 0°C for 3 to 5 days, isolate small colonies from the resulting colonies to obtain an L-proline degrading enzyme-deficient strain, and then induce mutations in the deficient strain in the same manner as above. After that, a proline analog (e.g. 3.
4-dehydro-DL-proline) α2 tq/d at 30°C in a culture medium (e.g., Davis's minimal medium).
It can be obtained by culturing for 1 to 3 days and separating the resulting colonies in 4 tubes. (A typical example of a mutant strain obtained by this method is Serratia marcescens Dr-9C Microtechnical Research Institute No. 1t/C9) (L-proline oxidizer deficient and 3゜4-dehydro-DL -,
Mutant strains resistant to proline). In addition, after further inducing mutations in the above-obtained black variant strain in the same manner as above, a plate medium containing 1.0 to 1.0% of L-thiazolidine-4-carboxylic acid (for example, the carbon source is amber) Davis's minimal medium (containing 0.5% sodium hydroxide) at 30°C
By culturing for 5 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 was obtained. It will be done. A typical example of the mutant strain thus obtained is Serratia marcescens DTr-12 (Feikoken Bibori No. 6760). Furthermore, a mutant strain deficient in L-proline degrading enzyme and resistant to proline analogs and purine analogs can be obtained by inducing mutations in the mutant strain deficient in L-prolyl degrading enzyme and resistant to proline analogs obtained above. .

Purine analogs (e.g. 2,6-dibu p mubulin) 2
A typical example of a mutant strain obtained in this way is Serratia marcescens, which can be obtained by culturing it at 30°C for 1 to 3 days in a plate medium containing ~- and separating the resulting colonies. Examples include DBr-51 (Feikoken Mayyori No. bt<9) (a mutant strain deficient in L-proline oxidase and resistant to 3,4-dehydro-DL-proline and 2,6-dibromprine).

Note that the method for obtaining a mutant strain capable of producing L-proline is not limited to the above-mentioned method; Is it given in a different order than the above? Alternatively, by obtaining mutant strains having each property separately and then employing genetic recombination methods such as transduction.

A mutant strain capable of producing L-proline can be obtained.

The L-proline production medium used in the method of the present invention includes glucose as a carbon source and stain 1 axis.

Sugars such as molasses, organic acids such as succinic acid, citric acid, fumaric acid, sugar alcohols such as glycerol, etc.
-20%, organic ammonium salts such as ammonium fumarate, ammonium succinate, inorganic ammonium salts such as ammonium sulfate, ammonium chloride, urea, etc. as nitrogen sources, 1-5%, corn stap liquor peptone, yeast extract as organic nutrients. , fish meat extract, etc. 0-1
%, and a medium containing 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 concentration of the medium at 6 to 8. In a medium like Saraban here, L
- L-glutamic acid, which is a precursor for proline biosynthesis;
A medium to which L-aspartic acid or the like is appropriately added can also be suitably used.

According to the present invention, the above-mentioned L-proline-producing mutant strain is inoculated into the above-mentioned medium, and cultured at 25-40°C under aerobic conditions such as shaking culture or aerobic stirring for 2-7 days. It is possible to accumulate a significant amount of L-proline in it. After the culture is completed, the L-proline produced can be collected in the valley by a normal separation and purification operation using an ion exchange resin, as shown in the examples, after removing bacterial cells and other insoluble substances.

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. Its quantification is Leuconostoc mesenteroides P.
-60 bioassay.

Example 1 Sucrose 10%, urea 1%, dibasic potassium phosphate 0.1%
Fermentation medium (pH
7,0) 15- was injected into a 500-capacity shaker button and sterilized by heat. However, the sea stems were added after being sterilized separately. This fermentation medium was inoculated with a platinum loop of Serratia marcescens Dr-9 strain (No. 2 Tsujihito) cultured at 30°C on a broth slant. 30°C, 140 revolutions/min, amplitude When cultured for 72 hours under 7C1l conditions, 9.
5-q/d of L-proline was produced and accumulated in the medium.

Example 2 A fermentation medium with the same composition as in Example 1 was added with a bouillon slant medium.
Serratia marcescens DBr cultured overnight at 0°C
- 1 platinum loop transplant of 51 strains (Feikoken Bacteria No. BTL, S No.) -
did. 30℃, 140 revolutions/min.

When cultured for 72 hours under the condition of shaking axis 7cx, I2.
6.rg/d of L-proline was produced and accumulated in the medium.

Example 3 Citrus sugar 15%, urine cumulative 2%, dibasic potassium phosphate 0.1%
Fermentation medium (pH
7,0)15/ was injected into a 500 m shaking Kolben and sterilized by heat. However, sucrose was added after separate sterilization. This fermentation medium was inoculated with a platinum loop of Serratia marcefuscens strain DTr-12 (Imitation Koken Bacteria WE 6169), which had been cultured overnight at 30°C in a bouillon slant medium. 30'C, 1
When cultured for 72 hours under the conditions of 40 revolutions, 7 minutes, and 7 amplitudes, 25.6·4 d of L-proline was produced and accumulated in the medium. The culture solution 11 was collected, heat-treated, and then filtered to remove bacterial cells and other insoluble matter. The filtrate was treated with cation exchange resin Amberlite IR-1208 (H+ type,
) was passed through a column packed with After washing with water.

The adsorbed L-proline was eluted with 5% aqueous ammonia, and the eluate was concentrated under reduced pressure. When the concentrated solution was cooled and allowed to stand, crystals of L-proline 1&O/ were obtained.

Reference Example 1 13-f (Method for obtaining Serratia marcescens Dr-9 strain) A small amount of Serratia marcescens 5r41 strain was inoculated into a bouillon medium and cultured at 30°C. When the bacteria in the culture reached approximately 10 cells/d.

Add a 1-width aqueous solution of N-methyl-N'-nitro-N-nitronguanidine to α254d, and heat at 30°C.
After incubation for 20 minutes, centrifugation, washing the octopus cells with physiological saline, adding fresh bouillon medium, and incubating for 30 minutes.
The cells were incubated at ℃ for 6 hours. This culture solution was centrifuged, the upper channel was discarded, and physiological saline was added to suspend the cells. This cell suspension? 11! The cells were diluted with physiological saline and adjusted to about 10 cells. 0.1 m of this suspension was transferred to an agar plate medium containing L-proline as the main source (glucose 0.1 m).
5%.

L-proline 0.2%, sulfuric acid, ammonium o, o
o.

2%, dipotassium phosphate α7%, dipotassium phosphate 0.3%, magnesium sulfate heptahydrate 0.01%, agar 1.5%) and cultured at 30°C for 3 days. did. Small colonies produced were isolated and L-proline oxidase activity was determined by the method of Den Singer et al. [Journal of Bacteriology, Vol. 103, pp. 144-152].

1970], and a strain lacking two enzyme activities was obtained. The thus obtained L-proline degrading enzyme-deficient strain was treated in the same manner as above to obtain N-1+L-N'
- treated with ditro N-ditroguanidine, approx.
A cell suspension of 09 cells/- was prepared. This suspension 0.
1 m of 3,4-dehydro-DL-proline α2FIW
- Agar plate medium containing (glucose α5%, ammonium sulfate 0.1%, dipotassium phosphate α7%, dipotassium phosphate 0.3%, magnesium sulfate heptahydrate 0.
01%, agar 1.5%・)k was applied. 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 to obtain Serratia φ marcescens Dr-9 strain.

Reference Example 2 (Method for obtaining Serratia marcescens DBr-51 strain) L-proline degrading enzyme deficient and 3.4-dehydro-DL- derived from Serratia marcescens 5r41
A proline-resistant mutant strain Dr-9 was obtained according to Reference Example 1. This Dr-9 strain was subjected to mutation induction with N-methyl-N'-nitro-N-nitroguanidine in the same manner as in Example 1, and cells were isolated. The i4 suspension was loaded. This suspension 0.
1d on an agar plate medium containing 2,6-dibromopurine 2 eq; 0.3%.

Magnesium sulfate heptahydrate 0601%, agar 1.5%
) was applied. This was cultured at 30°C for 3 days, the resulting colony was isolated, and L=-proline fertility was determined in Example 1.
I investigated as described in , and collected Serratia marcescens DBr-51 strain. It's 41.

Reference Example 3 (Method for obtaining Serratia marcescens DTr-12 strain) L-proline degrading enzyme deficient and 3.4-dehydro-DL- extracted from Serratia marcescens 5r41
A proline-resistant mutant strain Dr-9 was obtained according to Reference Example 1. This Dr-9 strain was mutagenicly treated with N-methyl-N'-nitro-N-nitronganidine in the same manner as in Reference Example 1 to prepare a cell suspension. This suspension 0.1
d, L-thiazolidine-4-carboxylic acid 1.0, a
y/d! Agar plate medium containing (sodium succinate 05
%, ammonium sulfate 0.1%, monobasic potassium phosphate 0
, 3%, 9J2 potassium phosphate 0.3%, magnesium sulfate 0.01%, agar 1.5%). This is 3
After culturing at 0°C for 4 days, the resulting colonies were isolated,
L-proline productivity was determined as described in Example 1.

Acquired Serratia marcescens DTr-12 stock.

Claims (1)

[Scope of Claims] 1. 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. L
-Production method of proline. 2. The production method according to claim 1, wherein the microorganism is an L-zoroline producing bacterium that belongs to Serratia and is deficient in L-10 phospholase and is resistant to proline analogs and/or purine analogs. 3. The production method according to claim 9isI, wherein the microorganism is an L-zoroline producing bacterium that belongs to the genus Serratia and is deficient in L-proline degrading enzyme and resistant to proline analogs. 2. The production method according to claim 1, wherein the microorganism is an L-proline live drum that belongs to the genus Serratia and is deficient in L-proline degrading enzyme and is resistant to proline analogs and purine analogs. & The microorganism is Serratia spp., which has the ability to produce L-proline.
The method according to claim 8, which is Marcescens. The production method according to claim 5, wherein the Serratia marcescens is L-prolinease deficient and has an L-proline producing ability that is resistant to proline analogs and/or purine analogs. 7. The production method according to claim 5, wherein the Serratia marcescens is a Serratia marcescens that is capable of producing L-proline with L-proline decomposition #depletion properties and resistance to proline analogs. The method according to claim 5, wherein the Serratia marcescens is L-prolinease deficient and has L-purine producing ability that is resistant to proline analogs and purine analogs. Claims: Serratia marcescens is L-proline oxidase-deficient and has an ability to produce L-proline that is resistant to 3,4-dehydro-D-3-L-proline. The manufacturing method described in Section 5. 10 Claim No. 1, which is Serratia marcescens or Serratia marcescens that is deficient in L-proline oxidase and has the ability to produce L-proline that is resistant to 3,4-nahydroDL-proline and 2,6-dibromopurine. Serratia marcescens according to item 5 is L-proline oxidase deficient and 3.
4-dehydro-DL-proline and L-thiazolidine-
The manufacturing method according to claim 5, which is Serratia marcescens having an ability to produce L-proline that is resistant to 4-carboxylic acid.