JPH0425799B2 - - Google Patents

Description

[Detailed description of the invention]

[Industrial Application Field] The present invention relates to microbial industry, and more particularly to L.
- A method for producing L-proline by fermentation using proline-producing microorganisms. L-proline is one of the most important amino acids. L-proline, in the form of an amino acid mixture, is mainly used in medicine as a parenteral nutritional supplement and as a nervous system activator. Furthermore, L-
Proline has found use in the food industry as a food supplement. [Prior art] As a producer of L-proline, the amino acid: L-
isoleucine or L-arginine auxotrophic (see U.S. Pat. No. 3,329,577), and L-isoleucine auxotrophic and sulfaguanidine resistant (see U.S. Pat. No. 3,819,483) and similar compound tolerant of nucleosides and purine or pyrimidine bases (see U.S. Pat. No. 3,819,483). by fermentation using mutants of microorganisms belonging to the genus Brevibacterium and Corynbacterium (European Patent No. 0098122A2/1984).
- Methods for producing proline are known to those skilled in the art. These methods guarantee a relatively low yield of L-proline (15-35 g/; 35 g/
is achieved only when the fermentation is carried out in the presence of L-glutamic acid), which increases the cost of producing the desired product. The production of L-proline by fermentation using mutants of microorganisms of the genus Serratia that are resistant to analogues of L-proline and deficient in proline oxidase is also known to the person skilled in the art (European Patent No. 0076516 reference). Although it is possible to obtain L-proline with a yield of 62.5 g/l by this method, the fermentation requires a considerable amount of sugar (20%).
and the fermentation time is 96 hours. A method for producing L-proline by fermentation using mutants of microorganisms belonging to the genera Brevibacterium, Corynebacterium, and Microbacterium that are resistant to 3,4-dehydro-D,L-proline. are also known to those skilled in the art (see US Pat. No. 4,224,409).
A maximum yield of 36.2 g/l of L-proline at an initial sugar content of 10% was achieved in this method by L-isoleucine auxotrophic, sulfaguanidine-resistant and 3,4-dehydro-D,L-proline resistant microorganisms. This is achieved using mutants of Brevibacterium flavum. This method is applicable to the conventional targets of microbial industry,
Although the advantages present when using glutamate-producing bacteria and the possibility of obtaining relatively high yields of L-proline without the use of proline precursors, this method is L-proline is still not guaranteed. [Problems to be Solved by the Invention] An object of the present invention is to provide a method for producing L-proline that can increase the yield of the desired product. [Means for Solving the Problems] The object of the present invention is to cultivate microorganisms of the genus Brevibacterium in a nutrient medium containing sources of carbon and nitrogen, inorganic salts and growth stimulants, and subsequently to incubate the culture medium. This is accomplished by isolating the desired product from. In this method, according to the invention, a mutant strain of a microorganism of the genus Brevibacterium is simultaneously resistant to both 3,4-dehydroproline and the increased osmotic pressure of the medium and/or incapable of catabolizing L-proline. A mutant strain of . According to the present invention, the yield of L-proline is 90-100
g/, during which L-
72 without introducing any precursors of proline
Over an hour of fermentation 15% of the carbon source is consumed. According to the invention, the All-Union Research Institute of Genetics
and Selection of Industrial Microorganisms (All-Union
Research Institute of Genetics and Selection
of Industria Microorganisms)
Union Collection (All-Union
It has been deposited in the Vsesojuzny Nauchino-Issredbatersky Institut Geneteiki (Vsesojuzny Nauchino Collection).
Nauchno-Issledovatelsky Institut Genetiki;
It is recommended to use the following mutant strains of microorganisms of the genus Brevibacterium, which have been internationally deposited under the Budapest Treaty under the following registration numbers (abbreviated as VNIIGeretica): Brevibacterium flavum strain AP108,
Registration number B3320; Brevibacterium flavum AP109 strain,
Registration number B3319; Brevibacterium flavum AP110 strain,
Registration number B3318; Brevibacterium flavum AP111 strain,
Registration number B3258; Breviibacterium flavum AP112 strain,
Registration number B3259; and Brevibacterium flavum AP113 strain,
Registration number B3260. One aspect of the invention provides high yields of 70-80 g/L using mutant strains of Brevibacterium microorganisms that are simultaneously resistant to both 3,4-dehydroproline and increased osmolarity of the medium. - It consists in making it possible to obtain proline. Particular examples of such mutant strains are the Brevibacterium flavum strain AP111 or the Brevibacterium flavum strain AP112, or the Brevibacterium flavum strain AP109. Another aspect of the invention consists in making it possible to obtain L-proline in yields of up to 30 g/ml using microorganisms of the genus Brevibacterium which are unable to catabolize L-proline. The identification of this mutant strain is an example of Brevibacterium flavum.
It is AP108 strain. Yet another aspect of the invention consists in using a microorganism of the genus Brevibacterium that is simultaneously resistant to both 3,4-dehydroproline and increased osmotic pressure and is unable to catabolize L-proline. Examples of such mutant strains include Brevibacterium flavum AP110 strain and Brevibacterium flavum AP113 strain. Further objects and advantages of the invention are as follows:
It will become more fully clear from the detailed description of the process for the production of proline and a specific example of the implementation of the process. In order to carry out the process for the production of L-proline, it is recommended to use mutant strains of microorganisms of the genus Brevibacterium that are resistant both to 3,4-dehydroproline and to the increased osmotic pressure of the medium at the same time. Additionally, it cannot catabolize L-proline (i.e. cannot assimilate L-proline as the sole carbon source or the sole nitrogen source or simultaneously the sole source of carbon and nitrogen for growth) It is recommended to use mutant strains of microorganisms of the genus Brevibacterium. It is further recommended to use mutant strains of the microorganism Brevibacterium that simultaneously have both of the above-mentioned properties. Any strain of a microorganism belonging to the genus Brevibacterium can be used as a parent strain for selection of mutant strains according to the present invention. The positive effect of the mutations according to the invention on the production of L-proline is shown in combination with other known mutations that contribute to the oversynthesis of L-proline. An example of such a known mutation is the isoleucine deficient mutation (see US Pat. No. 3,329,577). All under the All-Union Research Institute of Genetics and Selection of Industrial Microorganisms (USSR, Moscow), producers of L-proline with the above-mentioned combination of properties. - Examples of strains deposited with the corresponding accession numbers in the Union Collection of Industrial Microorganisms are the following variants of microorganisms of the species Brevibacterium flavum: strain AP108 (B3320); strain AP111 (B3258), strain AP109 (B3319), strain AP110 (B3318), strain AP112 (B3259) and strain AP113 (B3260). These new strains were selected from Brevibacterium flavum ATCC 14067 strain (B-42). Strains AP109, AP111 and AP112 are mutants that are simultaneously resistant to 3,4-dehydro-D,L-proline (DP) and increased osmolarity of the medium, and strain AP108 is L-proline. Strains AP110 and AP113 are mutants that are simultaneously resistant to DP and increased osmotic pressure and are unable to catabolize L-proline. In addition, AP108
The strains AP111, AP112 and AP113 are also mutant strains that require L-isoleucine for their growth. AP108 strain, AP109 strain, AP110 strain, AP111
strain, AP112 strain and AP113 strain, culture characteristics,
The morphological and physiological characteristics are similar to Brevibacterium flavum ATCC14067 strain, except for the above-mentioned properties and ability to produce proline. Mutations of DP and increased osmotolerance, L-proline catabolism inability and L-isoleucine auxotrophy can be achieved by a stepwise approach in any order, using any conventional method (e.g., N-methyl-N′- It can be obtained by mutagenizing bacteria using nitro-N-nitrosoguanidine or treatment with UV irradiation) and subsequently identifying the corresponding mutant strains. These mutations can also be obtained in separate strains and integrated into one genome using any genetic exchange method, such as protoplast fusion. As a compound that increases the osmotic pressure of the medium, sucrose or an alkali metal chloride such as sodium chloride, potassium chloride, etc. can be used. The specific method used to prepare the L-proline producing strain according to the present invention is described below. Preparation of Brevibacterium flavum AP109 strain Brevibacterium flavum
ATCC14067 strain was added to meat-peptone broth (hereinafter referred to as
MPB) at a temperature of 30° C. under aerated conditions to obtain a titer of 10 ⁹ cells/ml. Wash cells and remove MPB by centrifugation, pH5.5
of citrate buffer, precipitated again by centrifugation and resuspended in citrate buffer of pH 5.5. N-methyl-N'-
Nitro-N-nitrosoguanidine (hereinafter referred to as NG) is added to a final concentration of 300 μg/ml and this is incubated for 20 minutes at a temperature of 30° C. under ventilation. Cells were then washed with cold MPB to remove mutagens, and then
Transfer to a test tube with 10 ml of MPB and incubate for 18 hours at a temperature of 30° C. under ventilation. The resulting NG mutagenic culture was pelleted by centrifugation and No. 1 with the following composition (mg/ml) was prepared:
1 medium: Satucarose-250, Na ₂ SO ₄ -2,
K ₂ HPO ₄ −3, KH ₂ PO ₄ −1, MgSO ₄・7H ₂ O−
0.1, NH ₄ Cl−5, NH ₄ NO ₃ −1, MnSO ₄・
5H ₂ O−1×10 ⁻⁴ , FeSO ₄・7H ₂ O−1×10 ⁻⁴ , bition−2×10 ⁻⁵ , thiamine chloride−1×10 ⁻⁴ and DP−1.5, PH=7.4. Transfer into test tubes to bring the initial titer of the culture equal to 10 ⁵ cells/ml. This in vitro culture was grown under aeration at a temperature of 30°C until turbidity, then inoculated onto the surface of No. 1 medium containing an additional 2% agar, and grown for 48 hours at a temperature of 30°C. Time incubation. Proliferated colonies were picked and their ability to produce proline was tested under the conditions of Example 1 described below. One of the L-proline producing mutant strains selected in this way was designated as Brevibacterium flavum AP109. Preparation of Brevibacterium flavum strain AP111 A mutagenized culture of strain ATCC14067 (conditions for mutagenesis are the same as described above for the preparation of strain AP109) was grown in No. 1 with the following composition (mg/ml):
2 medium: glucose _- 20, _Na2SO4-2 ,
_K2HPO4-3 , _KH2PO4-1 , _NH4Cl - _{3 ,}
NH ₄ NO ₃ −1, MgSO ₄・7H ₂ O−0.1, MnSO ₄・
_5H2O -1× ^10-4 , _FeSO4・_7H2O × ^10-4 , bithione-5× ^10-5 , thiamine chloride-1× ^10-4 , L-isoleucine-0.5 and agar-20, PH= Inoculated on 7.4. Among the colonies grown on this medium, 30
After incubation for 48 hours at a temperature of 0.degree. C., mutants were picked that were unable to grow on No. 2 medium without L-isoleucine. The resulting L-isoleucine auxotroph strain was mutagenized again using NG, and 20 mg/ml glucose and 28 mg/ml were added to the No. 1 medium instead of 250 mg/ml sucrose.
Mutants resistant to DP and increased osmolality of the medium were selected following the same procedure described above for the preparation of strain AP109, with the exception that NaCl and 0.5 mg/ml L-isoleucine were added. One of the abundant L-proline producing mutant strains selected in this manner is designated as Previbacterium flavum AP111. Preparation of Brevibacterium flavum AP112 strain The ATCC14067 L-isoleucine auxotroph strain, which had been induced with NG in the same manner as described above for the preparation of AP111 strain, was treated with NG again, and No.
Mutants resistant to DP and increased concentrations of sucrose in medium No. 1 were prepared as described above for the preparation of strain AP109, with the difference that medium No. 1 contained 0.5 mg/ml L-isoleucine. Select. One of the abundant L-proline producing mutant strains selected in this manner is designated as Brevibacterium flavum AP112. Preparation of Brevibacterium flavum AP108 Strain The ATCC14067 isoleucine auxotroph strain induced with NG in the same manner as described above for the preparation of AP111 strain was mutagenized again using NG, and then mutagenized with 20 mg/ml agar. Inoculate into No. 2 medium containing. For each individual colony grown on this medium after 48 hours of incubation at a temperature of 30°C, its Examine proliferation ability. Composition of No.3 medium (mg/ml)
is: L-proline-10, Na ₂ SO ₄
-2, K ₂ HPO ₄ -3, KH ₂ PO ₄ -1, MgSO ₄・
7H ₂ O−0.1, MnSO ₄・5H ₂ O−1×10 ⁻⁴ , FeSO ₄
-1 x 10 ^-4 , biotin - 5 x 10 ^-5 , thiamine chloride - 1 x 10 ^-4 , L-isoleucine - 0.5 and agar-20, pH = 7.4. Incubation for 96 hours at a temperature of 30°C. After that, one of the mutant strains that could not be grown on medium No. 3 was transformed into Brevibacterium flavum.
Shown as AP108. Preparation of Brevibacterium flavum strain AP110 As a first step, mutants are selected that are simultaneously resistant to DP and increased concentrations of sucrose using the method described above for the preparation of strain AP109. Next, using the obtained mutant strain as a parent strain, a mutant strain incapable of catabolizing L-proline is selected by the method described above for the preparation of the AP108 strain. One of the selected abundant L-proline producing mutant strains is designated as Brevibacterium flavum AP110. Preparation of Brevibacterium flavum strain AP113 A mutant strain incapable of catabolizing L-proline is selected from the ATCC14067 strain using the method described above for the preparation of strain AP108. As the next step, from the obtained L-proline catabolism incapable strain,
Using the method previously described for the preparation of strain AP109
Select mutants that are resistant to DP and increased concentrations of sucrose. In the resulting double mutant strain that is incapable of L-proline catabolism and resistant to the DP and increased osmolarity of the medium, L
-Select isoleucine auxotrophic mutants. One of the abundant L-proline producing mutants thus prepared is designated as Brevibacterium flavum AP113. The salient features of the mutant strains used in the present invention compared to each other and to the ATCC14067 strain are shown in Table 1 below.

〔Example〕

Example 1 Under sterile conditions, in a 250 ml fermentation flask, the following composition (g/): Sucrose - 150, (NH ₄ ) ₂ SO ₄
−55, KH ₂ PO ₄ −1, MgSO ₄・7H ₂ O−10,
CaCO ₃ −50, FeSO ₄・7H ₂ O−0.01, MnSO ₄・
5H ₂ O−0.01, ZnSO ₄・7H ₂ O−0.01, biotin−
0.0005, thiamine chloride - 0.0005 and a PH of 8.0. Each flask is inoculated with one of the following strains: Brevibacterium flavum AP109, Brevibacterium flavum AP111 and Brevibacterium flavum AP112. AP111 strain and
Add 0.15 g/L-isoleucine to the fermentation medium of AP112 strain. The flask is incubated for 72 hours at a temperature of 30° C. with shaking. The content of L-proline in the culture solution obtained after completion of fermentation is shown in Table 2 below. Under the same fermentation conditions, the content of L-proline in the culture solution of Brevibacterium flavum strain ATCC 14067 does not exceed 4 g/, while the content of L-proline in the culture solution of the isoleucine auxotrophic strain of ATCC 14067 is 20 g/ / was not exceeded.

[Table] Obtained culture solution of Brevibacterium flavum AP112 containing 38.2g of L-proline
Centrifuge 500 ml to remove bacteria and other insoluble matter. The resulting supernatant is passed through a column with an ion exchange resin in H ⁺ form for the adsorption of L-proline. The column is washed with 2 volumes of water and the adsorbed L-proline is eluted with 3% aqueous ammonia solution. The resulting eluate is concentrated under reduced pressure, cooled and left to crystallize L-proline therefrom. The yield of L-proline is 20.8 g. Example 2 Brevibacterium flavum strain AP108 is cultured under the conditions described in Example 1. The L-proline content in the culture solution obtained from fermentation is
Equal to 28.2g/. After purifying L-proline from culture broth 1 using the method described in Example 1, 15.4 g of L-proline are obtained. Example 3 Each flask is inoculated with the microorganisms Brevibacterium flavum AP110 strain and Brevibacterium flavum AP113 strain. The composition of the fermentation medium and the culture conditions are the same as those described in Example 1, except that the culture medium for AP113 strain is 0.15
Contains L-isoleucine/g/. The content of L-proline in the culture solution of Brevibacterium flavum AP110 is equal to 40.5 g/
The content of L-proline in the culture solution of Brevibacterium flavum AP113 strain is equal to 97.5 g/. L-proline was purified from 500 ml of the culture solution of Brevibacterium flavum AP113 obtained using the method described in Example 1 to obtain crystalline L-proline.
- Obtain 27.2 g/proline.

Claims (1)

[Scope of Claims] 1. A property selected from the group consisting of the property of a microorganism that is simultaneously resistant to both 3,4-dehydroproline and increased osmotic pressure, and the property of a microorganism that is unable to catabolize L-proline. using a mutant strain of a microorganism of the genus Brevibacterium having at least one of the following and having the ability to produce L-proline, in a nutrient medium containing a carbon and nitrogen source, an inorganic salt, and a stimulant for the growth of the microorganism. The mutant strain of the Brevibacterium microorganism is grown until the desired product L-proline is accumulated in the culture solution, and the desired product is recovered from the culture solution. A method for producing L-proline. 2 Vsesojuzny Nauchno-Issledovatelsky Institute
Mutant strains of the following Brevibacterium flavum species deposited at the Institut Genetiki; Brevibacterium flavum AP 108 strain;
Registration number B3320; Breviibacterium flavum AP 109 strain,
Registration number B3319; Brevibacterium flavum AP 110 strain,
Registration number B3318; Breviibacterium flavum AP 111 strain,
Registration number B3258; Breviibacterium flavum AP 112 strain,
Registration number B3259; or Brevibacterium flavum AP 113 strain,
Registration number B3260; The method for producing L-proline according to claim 1. 3 Brevibacterium flavum strain AP 111, Brevibacterium flavum strain AP 112 or Brevibacterium flavum AP that is simultaneously resistant to both 3,4-dehydroproline and the increased osmotic pressure of the medium The method for producing L-proline according to claim 2, using strain 109. 4. The method for producing L-proline according to claim 2, which uses Brevibacterium flavum AP 108 strain that is unable to catabolize L-proline. 5 Brevibacterium flavum AP 110 strain or Brevibacterium flavum AP 113 strain that is simultaneously resistant to both 3,4-dehydroproline and increased osmotic pressure and is unable to catabolize L-proline The method for producing L-proline according to claim 2, which uses the following.