DE2300056A1 - Pyridine-2,6-dicarboxylic acid prepn - by culture of bacillus subtilis N26 atcc 21855 in nutrient soln. and recovery of prod from sepd spores - Google Patents

Abstract

Pyridine-2, 6-dicarboxylic acid (I) is prepd. by (i) submerged cultivation of Bacillus substilis N 26 ATCC 21855 (A) in a nutrient soln. until growth is completed and cells are changed to spores, and (ii) recovering (I) from spores septd. from the nutrient soln. Fermentation takes place with airing at 30-40 (pref. 35-38 degrees C) at pH 6-9 (pref. 7-8), esp. with starch as C-source and casein or casein hydrolysate as N-source. An amino acid, esp. valine, leucine or lysine is added when using an N-source other tahn casein or casein hydrolysate. Salt base pref. contains 27-137 ppm Ca2+. Culture takes place continuously and partic. in 2 steps. Fresh nutrient soln. is continuously added to growing culture in 1st fermentor. Bacteria-contg. culture is drawn off from 1st to 2nd fermentor and from 2nd to 3rd. Spores are formed in 2nd and following fermentors, and recovered from overflow of last fermentor.

Description

Process for the production of pyridine-2,6-dioarboxylic acid on fermentative Ways It has long been known that pyridine-2,6-dicarboxylic acid - hereinafter PdC called - is part of the wall of bacterial spores (Biochem. J. 54, 210), chne that so far attempts would have been made to use this deposit for the technical extraction of Exploiting PdC. The considerable expenditure of time and equipment at a relatively low cost Yield made a process by fermentation uneconomical.

It has now been found that an economical production of pyridine-2,6-dicarboxylic acid is possible by fermentation, if as Bacillus Bacillus subtilis N 26 ATCC 21355 is used for cultivation. The method according to the invention for nnung of pyridine-2,6-dicarboxylic acid by fermentation by submerged cultivation of Bscillus strains until growth stops and the cells become spores is characterized in that Bacillus subtilis N 26 ATCC 21855 in a Nutrient solution cultured submerged and from the spores separated from the nutrient solution the pyridinedicarboxylic acid is isolated.

In numerous experiments with different Bacillus strains showed that Bacillus subtilis N 26 ATCC under winter conditions a gives a very favorable yield of PdC. Table 1 shows the results with a small one Selection of different Bacillus strains, some of which iterated as good PdC producers are described in comparison with Bacillus subtilis N 26 ATCC 21855.

Tab. 1 Suitability of various Bacillus strains for the fermentative production of pyridine-2,6-dicarboxylic acid Bac. subtilis Bac. subtilis Bac. pumilis Bac. megater. Bac. megater. ATCC 13956 N 26 ATCC 2 @ 855 ATCC 19546 ATCC 7051 DP I. PdC µg / ml 40 202.6 41.7 170.3 1 @ 7.8 TRGW * mg / ml 0.54 2.24 1.15 1.75 1.71 % PdC / TRGW 7.4 9.06 3.98 9.73 9.82 Modium: 1% maize starch - 1% yeast extract - salt base - Cultivation: 96 hours at 37 ° C in a shaking incubator "TRGW: dry weight ATCC 21855 Bacillus subtilis N 26 / is an amino acid auxotrophic mutant which, by treating the parent strain with N -Methyl-N'-nitroso-N'-nitroguranidine was prepared by conventional methods The parent strain was determined to belong to Bacillus subtilis according to Bergey's Manual of Determinative Bacteriology (7th edition, 1957 Baltimore).

The procedure is carried out by referring to Bacillus subtilis ATCC 21855 N 26 / submersed in a suitable nutrient solution with aeration and stirring in one cultivated in a suitable fermenter until the PdC content of the Nänr "solution is at its maximum has reached. The temperature can be between 30 and 450 C, preferably at 35 to 380 C. The pH of the nutrient solution can be between 6 and 9, preferably at PH7 to 9. After the formation of the spores, they are absorbed by the nutrient solution separated and worked up in a known manner on PdC. This is preferred in the DOS 2 222 597 described method for the isolation of the PdC used, the PdC by heating the anhydrous spore mass with alcohol in the presence of acid is brought into solution as pyridine-2,6-dicarboxylic acid ester, the resulting diester isolated after filtering off the spore mass and then hydrolyzed in a known manner will.

For the analytical determination of the PdC, based on the distant volume, an aliquot of the culture solution is centrifuged and a certain amount Water (PH 7) added. This suspension is in an autoclave at 121 ° C. for one hour treated, whereby the PdC goes into solution; then it is centrifuged again. The PdC can then quantitatively in the supernatant using the Jansser method. et al. (Science 127, 26) or can be determined by thin layer chromatography, by doing 1 to 10 liters of solution are applied to a silica gel plate, with methanol - 5 N NH3 (80 + 20), the PdC stain eluted with water and the absorbance of the Eluate measures at 273 nm. The amount of PdC corresponding to the measured value becomes a calibration curve remove. The values given in the following tables were obtained using this method certainly. You just pose. Comparative values, because in later work was found that when the suspension of dry matter is autoclaved at pH 12 about 20 to 50 5S more PdC will go into solution.

Possible sources of carbon in the nutrient solution include: monosaccharides such as glucose, galactose, xylose; Disaccharides such as sucrose, lactose, maltose; Tri- and polysaccharides such as raffinose, dextrin, starch.

In addition, all other carbon sources that allow growth are suitable and allow spores of Bacillus subtilis N 26 itTCC'21855.

Experiments with different nutrient media have shown that starch both the cheapest carbon source in terms of both yield and economy as shown in Table 2.

Influence of the C source on PdC formation by Bacillus subtilis N 26 ATCC 21855. C source PdC TRGW% PdC / TRGW 1% µg / ml mg / ml Thickness 179.5 2.20 8.16 Maltose 117.0 1.88 6.23 Glueose 105.2 1.67 6.30 Dextrin 113.6 2.37 4.88 Whey 74.3 1.66 4.47 Laetose 92.7 1.66 5.59 Galactose 75.2 1.64 4.58 Sa @ charos @ 131.9 2.85 4.63 Raffinose 110.2 1.59 6.95 Xylose 74.8 1.44 5.20 Medi @ m: 1% C source + 1% peptone + salt base.

Cultivation: 96 hours at 37 ° C in a shaking incubator as Nitrogen sources include casein peptone, casein, casein hydrolyzate, meat extract, Yeast extract, soy powder, soy peptone, corn steep liquor and all other sticky ingredients Substances that promote the growth and prominence of Bacillus subtilis N26 ATCC 21855 enable As Table 3 shows, casein and casein derivatives are the cheapest Nitrogen sources.

When using a nitrogen source other than casein, it is recommended that nor casein hydrolyzate or the amino acids contained therein, preferably lysine and / or leucine and / or valine to add to the nutrient solution to reduce the PdC content the spore is increased as shown in Tables 4 and 5.

The influence of the N source on PdC formation by Bacillus subtilis N. 26. ATCC 21855. N source PdC TRGW% PdC / TRGW 1% µg / ml mg / ml Peptone 179.5 2.20 8.15 Corn steep water 74.3 2.16 3.44 @ efeextra @ t 129.4 2.37 5.55 Soy powder * 148.7 Meat extract 145.2 1.96 7.42 Casein * 263.9 Cascin hydrolyzate 237.2 2.30 10.3 Soy peptone 214.6 2.12 10.1 Medium: 1% starch + 1% N-source + salt base Cultivation: 96 hours at 37 ° C in a shaking incubator * Since these @ -quells do not dissolve clearly in water, no dry weight determinations were carried out.

Tab. 4 The influence of amino acids on PdC formation by Baeillus subtilis N 26 ATCC 21855. Addition PdC TRGW% PdC / TRGW µg / ml mg / ml without 153.7 2.02 7.60 Valine 187.9 2.19 8.57 Lysine 187.0 2.05 9.12 Glutamic acid 168.7 2.26 7.45 Leucine 182.9 2.26 8.10 Medium: 1% starch + 1% peptone + 0.1% additive + salt base Cultivation: 96 hours at 37 ° C im. Shaking incubator Tab. 5 The influence of increasing amounts of Bacto-Casamino Acid on PdC formation by Bac llus subtilis N 26 ATCC 21855 PdC TRGW N source% PdC / TRGW µg / ml mg / ml Yeast extract 1% 98.6 1.83 5.28 Yeast extract 1% + 0.1% Casamino Acids 106.0 1.68 6.30 Yeast extract 1% + 0.5% Casamino Acids 137.7 1.71 8.05 Yeast extract 1% @@@@@ @@@ @@@ + 1% Casamino Acids 180.4 2.22 8.1 1% Casamino Acids 295.6 2.64 11.2 Medium: 1% starch + N source + salt base Cultivation: 48 hours at 37 ° C in a shaking incubator Casein hydrolyzate from Difco Laboratories Inc., Detroit, USA The following inorganic nutrient salts have proven themselves in the experiments.

Na2HP04 2 H20 0; 3% of the nutrient solution (weight / volume) KH2PO4 0.3% NaCl 0.3% 1: MgCl2. 6 H20 0.01% ii II Na2SO4 0.011% "MnCl2. 4 H20 0.001% CaCl2 . 2 H20 0.02 '"" This mixture is called salt base in the following; she has proved to be suitable in the tests. It is of course possible to their qualitative and to change quantitative composition.

In order to determine the optimal amount of calcium ions, the The above salt mixture varies the CaCl2 content and the influence on the PdC formation certainly. The results are shown in Table 6. From this it can be seen that for optimal growth and optimal PdC-Saynthese about 0.01 to 0.05% Calcium chloride corresponding to 27 to 137 ppm Ca2 + are required.

For maximum yield, the concentration of the carbon and nitrogen source of importance. With increasing concentration of the carbon source increases with the same concentration of the N source, the growth of the bacteria is true significantly, but not the relative PdC content of the spores, which only increases with the The concentration of the nitrogen source increases. This illustrates the importance of Nitrogen source for the PdC content of the spores.

Tab. 6 The influence of calcium ions on PdC formation by Bacillus subtilis N 26 ATCC 2/855 % CaCl2.2@2O 24 hours 48 hours 72 hours 96 hours in the nutrient medium Cell number PdC Cell number PdC Cell number PdC Cell number PdC x 108 / ml µg / ml x 108 / ml µg / ml x 108 / ml µg / ml x 108 / ml µg / ml without 20.4 - 12.5 108.5 10.3 87.7 10.4 78.5 0.001 22.3 - 14.9 121.9 15.3 101.0 11.6 93.6 0.01 28.9-24.1 237.2 22.8 192.9 14.9 160.4 0.05 19.2 - 18.3 194.6 19.4 150.3 15.1 162.0 0.1 17.5 - 13.6 140.3 17.8 128.6 18.4 112.8 Medium: 1% starch + 1% peptone + salt base Cultivation: at 37 ° C in a shaking incubator.

The time in which the maximum of PdC is formed also depends on the composition of the nutrient solution depends on the culture vessel used. she lies on average between 24 and 48 hours, usually after 24 hours an optimal yield is achieved. From Table 7 it can be seen that in the fermenter significantly higher yields can be achieved than in the shake flask.

The foregoing test results show that Ver-ATCC 21855 application of Bacillus subtilis N 26 / spores in such a relatively short time Number and are formed with such a high content of PdC that de fermentative Tin plating of PdC becomes economically interesting. In addition, the inventive Method has the advantage that the PdC is absolutely isomer-free without further purification actions accrues, which is not the case with synthetic processes.

A particular advantage of the method according to the invention, however, is that it can be carried out continuously.

In the continuous cultivation of. Bacillus subtilis TCC 285 N 26 is continuously fed fresh nutrient solution into the first fermenter, and the cell-containing culture solution is in the same amount as' the inflow into a second Fermenter, in which the cells then form and spor PdC. To the temporal Differences between the generation time of the vegetative cells and the sporulation time To balance out, the second fermenter should be larger, at least twice as large like the first. It is even cheaper to ferment in three stages by using the overflow of the second fermenter is passed into a third fermenter.

Tab. 7 Dependence of the PdC formation on the culture vessel shake flask Fermenter after hours

100 ml 8 1 PdC µg / ml 164.5 248.8 24 TRGW mg / ml 4.07 3.76 So PdC / TRGW 4.04 6.63 PdC µg / ml 189.5 48 TRGW mg / ml- 2.68 unchanged% PdC / TRGW 7.07 medium : 1 Vo starch + 1% peptone + salt base, pH 7.5 Cultivation: at 370 ° C To the Obtaining the spores can overflow the last fermenter stage into a closed one self-discharging separator. The supernatant can be used to prepare fresher Nutrient solution can be used. In our ATCC 21855 experiments with Bacillus subtilis N 26 We could not see any significant decrease in growth, and in particular in Determine sporulation if two parts supernatant with one part fresh iCasser mixed and fresh C and N sources and calcium were added.

The comparative tests compiled in Table 8 clearly show the higher yields using continuous culture.

Tab. 8 Yields with different types of fermentation a) continuous fermentation Outlet 2nd stage Outlet 3rd stage Yield 1) 2) Pass PdC TRGW PdC TRGW PdC h-1 µg / ml mg / ml µg / ml mg / ml mg / STD / 1 0.200 334 6.36 - - 66.8 0.280 258.2 6.46 300.4 5.92 84.11 b) Batch fermentation after 24 hours 248.8 3.76 1.05 Continuous fermentation: Volume ratio of 1st: 2nd: 3rd stage = 1: 2: 2 Medium: 1% corn starch + 1% yeast extract + salt base 1) based on one liter of fresh nutrient solution = one liter of outflow = total yield 2) throughput = inflow per fermenter volume per time Example 1 200 ml of standard broth medium (Oxoid) in 2 Erlènmeyer flasks are mixed with Bacillus subtilis N 26 SeimpfQ and the cultures for 16 hours in a horizontal shaker at 37? C incubated. With these cultures 8 1 medium (2, G corn starch + 1% casein + salt base, pH 7.5) are inoculated in the fermenter. The fermenter is vented, stirred and kept at 370.degree. After 48 hours, the culture contained 434.2 µg PdC / ml. 51.8 g of spore-containing dry substance can be obtained from 8 l of culture by centrifugation.

The anhydrous spore mass is concentrated with 230 ml of ethanol and 20 ml Sulfuric acid 'and heated to reflux for 1 hour with stirring. Afterward is filtered and the filtrate is concentrated to about 100 ml. With an aqueous slurry of calcium carbonate is neutralized. Then it is extracted with benzene, the organic Phase washed with water, clarified with sodium sulfate and the benzene in a rotary evaporator distilled off.

The remaining crystallizing residue is by Kugelrohr distillation cleaned at reduced pressure.

4.43 g of pyridine-2,6-dicarboxylic acid diethyl ester are obtained from that by saponification with potassium hydroxide solution 3.32 g of pyridine-2,6-dicarboxylic acid in pure form be won.

Example 2 tin 1 liter glass fermenter and a 2 liter glass fermenter are connected to a 2-stage fermenter unit in such a way that the outlet of the first fermenter enters the second fermenter.

The first fermenter is filled with nutrient solution (1% starch + 1% yeast extract + Salt base, pH 7.5) and inoculated with Bacillus subtilis N 26. During the logarithmic growth phase is started with the help of a fresh pump sterile nutrient solution from a storage tank continuously into the first Fercenter to pump at a flow rate of 0.2 h @. The culture is in both Fermenters in equilibrium can spout one liter from the second in 5 hours Stage 6.36 g of dry matter are obtained, from which, as described in Example 1, 334 mg of pyridine-2,6-dicarboxylic acid can be isolated.

Example 3 One 1 liter glass fermenter and two 2 liter glass fermenters are connected to a 3-stage fermenter unit. The 1st fermenter is with Nutrient solution filled (1 c starch + 1 5'o yeast extract + salt base, pH 7.5) and with Bacillus subtilis N 26 inoculated. During the logarithmic growth phase, it begins to with the help of a pump, fresh sterile nutrient solution from a storage tank continuously to pump into the first fermenter at a flow rate of 0.28 h-1. Located If the culture in all three fermenters is in equilibrium, it can be finished in 3.57 hours 5.92 g dry matter are obtained from one liter of the discharge from the third fermenter, from which, as described in Example 1, 300.4 mg of pyridine-2,6-dicarboxylic acid were isolated will.

Claims (8)

Claims 1. Process for the production of pyridine-2,6-dicarboxylic acid by submerse Cultivate Bacillus strains until growth has stopped and the cells close Spores have become from which the pyridine-2,6-dicarboxylic acid is obtained, thereby characterized that Bacillus subtilis N 26 ATCC 21855 in a nutrient solution summers cultivated and from the spores separated from the nutrient solution the pyridine-2,6-dicarboxylic acid is isolated. 2. The method according to claim 1, characterized in that the nutrient solution Calcium ions, preferably 27 to 137 ppm, can be added. 3. The method according to claim 1 and 2, characterized in that the Fermentation at 30 to 400 C, preferably at 35 to 380 C, and at a pH value from 6 to 9, preferably at pH 7 to 8, is carried out. 4. The method according to claims 1 to 3, characterized in that that starch is used as the C source and casein or casein hydrolyzate as the N source will. 5. The method according to claim 4, characterized in that when used another nitrogen source than casein or casein hydrolyzate in addition one Amino acid, preferably valine, leucine or lysine, is added. 6. Process according to claims 1 to 5, characterized in that the cultivation is carried out continuously by adding in the growing Bacillus culture continuously fed fresh nutrient solution and bacteria-containing to the same extent Culture is derived. 7. The method according to claim 6, characterized in that the continuous Cultivation is done in several, preferably in 2 stages, adding in the first Fermenter continuously fed fresh nutrient solution to the growing Bacillus culture and to the same extent bacteria-containing culture from the first to the second fermenter and is derived from the second to the third and so on, where in the second and in the following fermenters the spores are formed and from the overflow of the last one Fermenters are harvested from which the pyridine-2,6-dicarboxylic acid is isolated. 8. Process according to claims 1 to 7, characterized in that the fermenter broth after harvesting the spores to prepare fresh nutrient solution is used.