FR2601692A1 - Process for preparing bacitracin by fermentation using modified bacillus strains - Google Patents

Abstract

Process for preparing bacitracin by aerobic fermentation using modified bacillus strains in submerged culture. There is used as producing strain a defective mutant or a strain of bacillus resistant to arsenate or a strain of bacillus which is a good bacitracin producer but which is resistant to virulent phages and which is prepared by fusion of the protoplasts of various bacillus strains, the culture is carried out at 37-45 DEG C, the production at 35-37 DEG C, a nutrient medium is used whose starch value is 60 to 85 per litre, with a ratio of 0.30 to 0.95 : 1 between the carbon source and the organic nitrogen source, and at an NH<+>4 ion concentration of not more than 16 mM and a phosphate ion concentration of not more than 5mM before inoculation.

Description

The invention relates to a process for the preparation of bacitracin by fermentation of modified strains of Bacillus.

Among the strains belonging to the species
Bacillus subtilis and Bacillus licheniformis, several are known to produce bacitracin in abundant quantities. In United States Patent No. 2,813,061 a strain is described which produces 323 U / cm3 (units per cm3 of culture medium) (throughout the present application, the units of bacitracin are those used in the United States patent. USA No. 2,498,165 i.e. 1 U = 23.8 micrograms). The strain described in Czechoslovakian Patent No. 206,910 produces 580
U / cm, the strain described in the Hungarian patent
3 No. 173,315 produces 570 to 590 U / cm3 and the strain described in Japanese Patent No. 74,46,079,500 U / cm3. Czechoslovak Patent No. 173,450 describes a strain of Bacillus licheniformis which even at the virulent phage contaminated state, still produces 240-280 U / cm3.

Some patents also give indications on the production of bacitracin by defective mutants or strains resistant to antimetabolites and strains with impaired metabolism.

Thus, for example, the strain of Japanese Patent No. 74. 46,079 is auxotrophic to methionine, the strain of Hungarian Patent No. 173,315 is resistant to isoleucine hydroxamate and the strain of Polish Patent No. 120,267 is unable to value isoleucine.

A comparison of published production figures shows that strains resistant to virulent phages and therefore well suited to fermentation do not have a particularly high production, while cultures of good producing strains are always exposed to the risk of contamination by phages. Until now, strains producing bacitracin with high activity and simultaneously resistant to phages have never been described.

The Applicant has sought to prepare Bacillus strains whose production capacity reaches or exceeds the production capacities indicated in the scientific literature but whose fermentation would not be disturbed by virulent phages. The Applicant: has also studied novel parameters which can be used in the fermentation of the strains sought.

It is known that the addition of ornithine to chemically defined nutrient media leads to an increase in the production of bacitracin (FEMS Microbiol. Letters 10 (2), 111-114 / 1981 /). It is also known that the good producing strains have a common characteristic a catabolism of ornithine which is inhibited (Acta Biochim. Pol. 21 (2), 213-214 / 1974 /). In some cases, arsenate resistance, produced artificially, decreases the transport of phosphate within the cell (J. Bacteriol. 137 (1), 69-72 / 1979 /). Finally, it is known that protoplast fusion is suitable for the transfer of oxygen properties (Proc. Natl. Acad. Sci.

USA 73, 2147-50 and 73, 2151-55, / 1976 /) and also that recombination is possible between Bacillus substilis and Bacillus licheniformis (Arch. Microbiol. 134 (4), 303-308 / 1983 /).

It has now been found that the inhibited catabolism of ornithine, characteristic of good producing strains, can also occur in defective mutants. It has furthermore been found that the reduced transport of phosphates also allows a high yield of antibiotic when the concentration of inorganic phosphate is greater than the optimum concentration. Finally, it has been found that it is possible to prepare good producing strains resistant to virulent phages by interspecific fusion of the protoplasts of strains which are bad producing or even non-producing bacitracin but resistant to virulent phages.

Upon study of the fermentation conditions, it was found that, for multiplication of the producing strain, the optimum temperature was different from that which is optimum for the biosynthesis of bacitracin. It was further found that the ammonium ions formed as the breakdown products of proteins inhibited, at a concentration of 70 to 80 mM, the biosynthesis of bacitracin and, therefore, the ratio between the sources of protein had to be adjusted accordingly. organic nitrogen and mineral nitrogen sources, as well as the ratio of carbon sources to nitrogen sources. Finally, it was found that soybean meal could be advantageously replaced by pea or pea meal. alfalfa flour when the starch value of the nutrient medium was considered a constant and the concentration of natural components of the nutrient medium was chosen taking into account this constant and the carbon / nitrogen ratio (the starch number is calculated according to the procedure of the Hungarian standard MSZ 6830-53).

The subject of the invention is therefore a process for the preparation of bacitracin by fermentation of modified strains of Bacillus, a process in which the fermentation is carried out under aerobic conditions, with temperature control, in culture submerged in a nutrient medium containing sources of carbon, organic and mineral sources of nitrogen, trace elements and, as a buffer, calcium carbonate.The process according to the invention is characterized in that it is used as a producing strain
- a defective mutant, or
- a strain of Bacillus resistant to arse-
niate, or
- a strain of Bacillus which produces
bacitracin, resistant to virulent phages,
prepared by fusion of the protoplasts of sou
different from Bacillus, the cultivation is carried out at 37-45 "C, the production is carried out at 35-37" C and a nutrient medium with a starch number of 60 to 85 per liter is used, which contains the carbon source and the organic nitrogen source in a ratio of 0.30 to 0.95: 1 and which, before seeding, has an NH4 ion concentration of up to 16 mM and a phosphate ion concentration 5 mM maximum.

The preparation of some preferred Bacillus strains is described below. The properties of the starting strains are reported in the table below.

Table I
S ouches property
MNG 00110 MNG 101012 MNG 101013
Phylaxia ATCC 9800 NCTC 6816 bacitracin production good bad none
Resistance to virulent phages susceptible resistant resistant
Antibiotic resistance
oxytetracycline resistant susceptible resistant
erythromycin resistant susceptible resistant
bacitracin resistant susceptible resistant
Preparation of mutants using
N-methyl-N'-nitro-N-nitroso-guanadine
Bacillus strain MNG 00110 is grown overnight in a nutrient medium of the following composition
yeast extract 5 g
tryptone 10 g
NaC1 5g
MgSO4 10-4 4 M
-5
CaCl2 5 x 10 M
water 1 liter (hereinafter abbreviated: nutrient solution YT).

The bacteria suspension is diluted 50 times and cultured for a further 2 h. The cells are then centrifuged and suspended in 1/15 M phosphate buffer (pH 6.0). 20 micrograms of N-methyl-N'-nitro-N-nitroso-guanadine per ml are added to the suspension and the mixture is allowed to stand for 1 hour. The cells are centrifuged, washed with the buffer, centrifuged again and then suspended in the nutrient medium of the above composition. The treated cells are aerated for 2 h at 30 ° C. and then isolated to obtain monocolonies (description of the procedure: cf. Sherrat and Collins,
J. Gen. Microbiol. 76, 217-230 / 1973 /).

After isolation, a strain is obtained in which the production of bacitracin is 20% greater than that of the starting strain. The new strain was called PP-188.

By UV irradiation
The spore suspension of the strain is washed
PP-188 twice with sterile distilled water then it is irradiated with a UV lamp (Philips Germicid, 30 W) from a distance of 30 cm for 1.5 min then it is isolated on YTA nutrient medium ( YTA nutrient medium has the same composition as YT nutrient solution but with the addition of 1.8% agar).

After the UV treatment, a strain of mutants defective in ornithine / arginine is isolated. This strain was called AH 22 and was deposited under number MNG 00343 in the Hungarian collection of microorganisms.

The strain grows well on minimum nutrient medium at 50 micrograms / ml of ornithine, it grows poorly on minimum nutrient medium at 50 micrograms / ml of arginine and its antibiotic production is approximately 25% higher than that of the strain initial.

Isolation of arsenate resistant mutants
Cells of strain PP-188 are washed with physiological saline and suspended in Spizizen minimum nutrient medium containing, instead of phosphates, 200 millimoles of arsenate.

The culture is aerated overnight at 30 ° C. The surviving cells are centrifuged, washed with YT nutrient solution and then isolated (procedure
Alfesi, Friedberg and Friedberg, J. Bacteriol. 137, (1), 69-72 / 1979 /).

The strain PP-188 has 25 descendants resistant to arsenate whose bacitracin productions are higher by 10% for 5 of them, by about 25% for two of them and about 50% for two of them. 'between them. The best-producing strain was named AS 19 and deposited in the Hungarian microorganism collection under No. 00344. To produce the optimum amount of bacitracin, this strain requires supplementation of phosphate (which is not necessary for the other strains) .

Protoplast fusion
The preparation of the protoplasts, the fusion and the regeneration were carried out according to the procedure of Gabor and Hotchkiss (J. Bacteriol. 137 (3), 1346-1353 / 1979 /). Of the strains used for fusion, one was phage resistant and antibiotic insensitive and the other was phage sensitive and antibiotic resistant. The PP-188 strain was fused in one case with the MNG strain 101012 and in the other case with the MNG strain 101013.

For the preparation of the protoplasts, the cells in the exponential phase were washed with a solution of the following composition
0.1 mM CaCl2
MgC12 0.15 mM
0.05 mM K2SO4
gelatin 1 g
sucrose 68.5 g
0.05 M Tris-HCl buffer
(pu 7.4) supplement to 1 liter - (hereinafter abbreviated: BP solution). The cells were then taken up in a BP solution containing 0.1 mg / ml of lysozyme and subjected to a 45 min incubation at 37 ° C. The protoplasts formed were washed twice with the solution. BP. Equal volumes of the protoplasts to be fused were mixed together. To 0.2 ml of the mixture of protoplasts, 1.8 ml of PEG solution (PEG solution: 4 g of polyethylene glycol were carefully mixed). 4000 in 6 ml of BP solution). The mixture was left to stand for 1 min and then diluted with BP solution, isolated on YTA nutrient medium supplemented with 68.5 g / liter of sucrose and then regenerated.

Recombinants were selected, after isolation, in the presence of virulent phages and antibiotic in order to exclude the starting strains.

The selection was carried out in detail as follows: the colonies developed after fusion and regeneration of strains PP-188 and 101013 were removed by scraping and suspended in YT nutrient solution containing 10 virulent phages per ml. The mixture was stirred for 16 h at 37 ° C; during this shaking most of the phage sensitive cells died. Supernatant cells were collected by centrifugation and treated with phage antiserum for inactivation of virulent phages. Cells were then isolated on YTA nutrient medium, and developed colonies were transferred using a sample loop to nutrient medium.
YTA and respectively on YTA nutrient medium containing, per cm3, about 106 cells of Micrococcus flavus ATCC 10240. The plates were incubated for 16 h at 37 ° C. On colonies which show an inhibition ring on nutrient medium containing Micrococcus flavus, bacitracin production and phage resistance were investigated, resulting in good phage-producing and phage-resistant fused recombinant strains, the best of which, even in the presence of virulent phages, gave about 80 % of the production of strain PP-188 This strain was named HS-124.

The fusion of strains PP-188 and 101012 was carried out in an analogous manner but the YT solution contained virulent phages and 20 U / ml of bacitracin, which eliminated the two starting strains. Numerous strains that are good producers and resistant to phages have been formed. The best of these had an identical production to that of strain PP-188 even in the presence of phages, and in the absence of phages, its production was 50% higher. This strain was called HS-13.

The preparation of the strains is illustrated schematically in the single figure of the accompanying drawings.

The examples which follow illustrate the invention without, however, limiting its scope.

Example 1
Production capacity of individual strains
1. Preparation of seed material
(general operating mode)
YTA nutrient medium was seeded with the relevant strain of Bacillus licheniformis and incubated for 120 h at 37 ° C. The culture bearing the spores at + 4 ° C was stored. From the culture stored on agar slant, the size of a pinhead was removed, transferred to 100 ml YT nutrient solution and shaken for 120 h. At the end of this period, the culture is 80% spore carrier. In each case, 2 ml of the spore suspension are introduced into a test tube with a sterile cap and stored at + 40 ° C. until use. With 2 ml of the spore suspension, 100 ml are inoculated each time. of nutrient solution YT and shaking for 16 hours at 30 ° C. These cultures constitute the seed material for the following stage of operation.

2. Productive culture
10% of the seed material is added to 50 ml of BTK nutrient solution, the composition of which is as follows (in% w / v)
soy flour extracted 6.0% w / v
corn flour 2.4 "
(NH4) 2SO4 0.2
CaCO3 0.4
MgSO4.7H2O 0.02
MnSO4, 4H2O 0.0015
Antihabzin A / 3 1.0 in tap water. (Antihabzin A / 3 is described in Hungarian Patent No. 160,474).

50 ml of nutrient solution are introduced into a 500 ml Erlenmeyer flask and sterilized over 20 minutes at 1200C <pH 7-7.2). The cultures are shaken at 30 "C on
-1 a table to shake at a frequency of 220 min -1 for 48 h. The different strains give the following productions of bacitracin
U / ml
MNG 00110 320
PP-199 384
MNG 00 343 480
MNG 576
HS-124 310
HS-13 480
Example 2
Efficiency of phosphate addition
MNG 00344 strain is grown as described in ex. 1 but supplementing the nutrient solution with 0.02% w / v monopotassium phosphate.

The culture liquid reaches a titre of 640 U / ml whereas one arrives at 576 U / ml in the absence of phosphate (ex. 1).

Example 3
Industrial fermentation with variation of
temperature
1. Preparation of seed material
100 ml of a 16-hour old YT culture is used 3 to inoculate 4 m of the K-3/72 culture medium, the composition of which is as follows
soy flour extracted 4.5% w / v
mals flour 2.25
(NH4) 2SO4 0.2
CaCO3 0.1
Antihabzin A / 3 0.2% by volume in tap water. The seed material is cultivated at 30 ° C. under an aeration of 0.25 liters of air per liter of liquid and per minute for 12 hours. Before inoculation in the fermenter, the culture medium is sterilized over 45 minutes. at 125 ° C and then has a pH of 6.8 to 7.2.

2. Productive culture
3 19 m of nutrient solution K-6 is inoculated
3 per 1 m of inoculate. Composition of the nutrient solution
in weight / volume
soy flour extracted 6.74
corn flour 3.47
<NH4) 2SO4 0.13
CaCO3 0.39
MgSO4, 7H20 0.021
MnSO4, 4H 20 0.0016
Antihabzin A / 3 0.2 in tap water. The fermenter is sterilized over 60 minutes at 125 "C at the end of which the pH is 6.8 to 7.2. Fermentation is carried out at 300C for the first eight hours then at 37" C from the thirtieth hour until the end. In the first stage of operation, the aeration rate is 0.25 liters of air per liter of liquid and culture and per minute and at the second stage of operation, it is 0.5 liters of air. The fermentation time is 30 to 35 hours; at the end of fermentation, the pH is 8.0 to 8.3. The following activities in bacitracin are determined:
PP-188 530 U / ml
HS-13 570 U / ml.

Example 4
Substituting soy flour
We operate as described in ex. 3 but the extracted soy flour is replaced by 10% w / v alfalfa flour (or 10.53% w / v pea flour) and 2.1% w / v corn flour. The results obtained for the synthesis of bacitracin are the same.

Example 5
Optimization of the temperature profile
We operate as described in ex. 3 but with a fermentation temperature of 39 C in the first nine hours which is then lowered by 1 C per hour until the twelfth hour. From the twelfth hour until the end, the temperature is 36 C. The aeration rate is constant, at 0.5 liters of air per liter of culture liquid and per minute. The fermentation time falls to 25-28 h and the bacitracin yield is the same.

Example 6
Optimal nutrient medium 3
4 n of inoculate nutrient medium is inoculated
AGI per 100 ml of a 16 h old YT culture. The composition of the AGI medium is as follows
soy flour extracted 4.0% w / v
corn flour 1.0
CaCO3 0.3 I '
MgSO4, H2O 0.02 'I
Antihabzin A / 3 0.2% by volume
The nutrient medium is sterilized over 5 min at 125 ° C. After inoculation, the pH is 6.8 to 7.2.

Cultivation is carried out at 37 ° C. with stirring, with aeration of 0.25 liters of air per liter of medium and per minute for
3 24 h, until spores form. With 1 m of the inoculate, 19 m of AG nutrient medium is inoculated to the following composition
soybean flour extracted 6.74% w / v
corn flour 5.7S
(NE) 2S 4 0.05
CaCO3 0.53
MgSO4.7H2O 0.021
kinSOS, 4H2O 0.0016
Antinabzin A / 3 0.2% by volume
The fermenter was sterilized over 60 minutes at 125 ° C. at the end of which the pH was 6.8 to 7.2. The aeration and the temperature regime are the same as in Example 5. The fermentation time is 28 to 30 h and at the end of fermentation the pH is 7.4 to 7.8.

Bacitracin titer
PP-188 630 U / ml
HS-13 817 U / ml

Claims (5)

1. Process for the preparation of bacitracin by fermentation of modified Bacillus strains, in which the fermentation is carried out under aerobic conditions with temperature control, in culture submerged in a nutrient medium containing carbon sources, organic and mineral sources nitrogen, trace elements and calcium carbonate as a buffer, the process is characterized in that it is used as producing strains - a defective mutant, or - a strain of Bacillus resistant to arsenic niate, or - a strain of Bacillus which produces bacitracin, resistant to virulent phages, prepared by fusion of aif protoplasts All strains of Bacillus are grown at a temperature of 37 to 45 "C, produced at a temperature of 35 to 37" C and a nutrient medium with a starch number of 60 to 85 per liter is used. , which contains the carbon source and the organic nitrogen source at a ratio of 0.30 to 0.95: 1 and which, before seeding, has a maximum NH4 ion concentration of 16 mM and a phosphate ion concentration 5 mM maximum. 2. Method according to rev. 1, characterized in that the mutants defective in ornithine / arginine deposited under n " MNG 00343 to the Hungarian Microorganism Collection. 3. Method according to rev. 1, characterized in that the strain of Bacillus licheniformis resistant to arsenate deposited under the number MNG 00 3a4 in the Hungarian microorganism collection is used as the producing strain and in that the nutrient medium is completed by phosphate. 4. Method according to rev. 1, characterized in that one uses as naked producer strain a strain prepared by interspecific fusion of a good producer strain but sensitive to phages and of a strain poor producer or non-producer but resistant to phages, preferably the strain obtained by fusion of strains PP-188 and 101013 or PP-188 and 101012. 5. Method according to one of rev. 1 to 4, characterized in that on passing from the culture temperature to the fermentation temperature the temperature is reduced by about 1 ° C. per hour.