Microbiological process for the preparation of heteroaromatic carboxylic acids using Alcaligenes faecalis

A microbiological process for the preparation of a heteroaromatic carboxylic acid or one of its physiologically tolerated salts of the formulae I or II: ##STR1## wherein R.sub.1 and R.sub.2 are identical or different and each denotes a hydrogen or halogen atom, and X denotes a nitrogen atom or CH--, from a heteroaromatic nitrile of the formulae III or IV, respectively: ##STR2## First, a microorganism, Alcaligenes faecalis (DSM 6335), is cultured in the presence of an inducer, 2-cyanopyridine, and a carbon source such as a dicarboxylic acid, a tricarboxylic acid or a carbohydrate. Substrate III or IV is then reacted with the cultured microorganism. The biotransformation is carried out under anaerobic conditions for compounds of the formula I, and under aerobic conditions for compounds of the formula II.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
The invention relates to a novel microbiological process for the 
preparation of heteroaromatic carboxylic acids or their physiologically 
tolerated salts of the general formulae: 
##STR3## 
wherein R.sup.1 and R.sup.2 are identical or different and each denotes a 
hydrogen or halogen atom, and X denotes a nitrogen atom or --CH--. 
Heteroaromatic carboxylic acids, such as, for example, 6-hydroxypicolinic 
acid, are important intermediate products for the preparation of 
pharmaceuticals, such as, for the preparation of 2-oxypyrimidine Berichte 
der Deutschen Chemischen Gesellschaft, (1912), 45, pages 2456-2467! or for 
the preparation of herbicides (European Published Patent Application No. 
0,447,004). 
It is known in general that microorganisms containing nitrile hydratases 
and amidases or nitrilases convert nitriles into the corresponding acids. 
For example, European Published Patent Application No. 0,187,680 describes 
a microbiological process for the preparation of organic acids, such as, 
nicotinic acid using microorganisms of the genus Corynebacterium, 
Nocardia, Bacillus, Bacteridium, Micrococcus and Brevibacterium. It is 
obligatory to carry out this reaction in the presence of light energy. 
European Published Patent Application No. 0,444,640 discloses a 
microbiological process for the preparation of organic acids, such as, 
nicotinic acid using microorganisms of the genus Rhodococcus. It is 
obligatory to carry out this reaction in the presence of a lactam. 
It is furthermore known that microorganisms of the species Rhodococcus 
rhodochrous J1 convert, for example, 2-cyanopyrazine to pyrazinecarboxylic 
acid Kobayashi et al., J. of Antibiotics, Vol. 43, No. 10, (1990), pages 
1316-1320!. However, these microorganisms are unable to convert 
2-cyanopyridine into picolinic acid Mathew et al., Appl. Environmental 
Microbiology, Vol. 54, No. 4, (1988), pages 1030-1032!. 
It is also known that 2-cyanopyridine-utilizing microorganisms of the genus 
Alcaligenes convert 2-cyanopyridine into 6-hydroxypicolinic acid (European 
Published Patent Application No. 0,504,818). It is a disadvantage of this 
process that the 6-hydroxypicolinic acid is formed only in moderate yield. 
BROAD DESCRIPTION OF THE INVENTION 
It is an object of the present invention to provide a more economic 
microbiological process for the preparation of heteroaromatic carboxylic 
acids or their physiologically tolerated salts, such as, 
pyrazinecarboxylic acid, picolinic acid or chromium picolinate using 
microorganisms of the genus Alcaligenes, wherein the formed carboxylic 
acids or their physiologically tolerated salts are formed in good yield. 
Other objects and advantages of the invention are set out herein or are 
obvious herefrom to one skilled in the art. 
The objects and advantages of the invention are achieved by the process of 
the invention. 
The invention involves a microbiological process for the preparation of 
heteroaromatic carboxylic acids or their physiologically tolerated salts 
of the general formulae: 
##STR4## 
wherein R.sup.1 and R.sup.2 are identical or different and each denotes a 
hydrogen or halogen atom, and X denotes a nitrogen atom or --CH--. The 
invention process includes converting heteroaromatic nitriles of the 
general formulae: 
##STR5## 
wherein R.sup.1 and R.sup.2 and X have the above-stated meanings, as 
substrate, using 2-cyanopyridine-utilizing microorganisms of the genus 
Alcaligenes which have been cultured before the biotransformation in the 
presence of a dicarboxylic acid, a tricarboxylic acid or a carbohydrate, 
into the corresponding carboxylic acid. The latter is converted where 
appropriate into physiologically tolerated salts.

DETAILED DESCRIPTION OF THE INVENTION 
The process is carried out according to the invention in such a way that a 
heteroaromatic nitrile of the general formulae: 
##STR6## 
wherein X, R.sup.1 and R.sup.2 have the above-stated meanings, is 
converted as substrate, using 2-cyanopyridine-utilizing microorganisms of 
the genus Alcaligenes which have been cultured before the 
biotransformation in the presence of a dicarboxylic acid, a tricarboxylic 
acid or a carbohydrate, into heteroaromatic carboxylic acids according to 
Formula I or II. The heteroaromatic carboxylic acids are then converted 
where appropriate into physiologically tolerated salts. Physiologically 
tolerated salts of these carboxylic acids mean hereinafter, for example, 
chromium, calcium or ammonium salts. 
Before the actual biotransformation, the microorganisms of the genus 
Alcaligenes used for the process are normally cultivated (cultured) and 
their effective enzymes expediently induced with 2-cyanopyridine. 
2-Cyanopyridine can be used for culture and induction in a concentration 
of 0.1 to 20 percent by weight, preferably in a concentration of 0.1 to 1 
percent by weight. A dicarboxylic acid means hereinafter fumaric acid, 
succinic acid, maleic acid, glutaric acid and malonic acid, and their 
salts and derivatives such as esters. A tricarboxylic acid means 
hereinafter citric acid and isocitric acid and their salts and derivatives 
such as esters. Salts and derivatives of these dicarboxylic acids and 
tricarboxylic acids which can be used are fumarate, malate, malonate, 
oxalacetate, citrate, aconitate, isocitrate, 2-oxoglutarate, succinate and 
succinyl-CoA. Fumarate, malonate or succinate is preferably used. 
Carbohydrates mean hereinafter monosaccharides such as glucose, 
disaccharides such as sucrose, trehalose or maltose, trisaccharides such 
as raftnose, and sugar alcohols such as glycerol. Glycerol is preferably 
used as the carbohydrate. The dicarboxylic acid, tricarboxylic acid or the 
carbohydrate is expediently used in a concentration of 0.1 to 20 percent 
by weight, preferably in a concentration of 0.5 to 5 percent by weight. 
It is possible to use as culture medium the media customary among those 
skilled in the art, such as, the mineral salt medium of Kulla et al. 
Arch. Microbiol., 135, 1-7, (1983)!, low molarity phosphate buffer or as 
shown in Table 1. The mineral salt medium described in Table I is 
preferably used. 
After the culture phase and before the actual addition of the substrate, 
either the microorganisms are harvested by conventional separation 
processes, or the substrate is added directly to the microorganisms. 
The substrates used for the biotransformation, the heteroaromatic nitriles 
of the formulae III and IV, such as, 2-cyanopyridine, are purchasable 
compounds. 
X in the general formulae I to IV denotes a nitrogen atom or --CH--, 
preferably --CH--. The radicals R.sup.1 and R.sup.2 are identical or 
different and denote hydrogen or halogen, such as, fluorine, chlorine, 
bromine or iodine. Possible substrates are, accordingly, 2-cyanopyridine, 
6-chloro-2-cyanopyridine, 5,6-dichloro-2-cyanopyridine, 2-cyanopyrazine, 
6-chloro-2-cyanopyrazine and 5-bromo-6-chloro-2-cyanopyrazine. 
2-cyanopyridine, 2-cyanopyrazine or 6-chloro-2-cyanopyridine is 
expediently used as the substrate. 
The substrate can be added all at once or continuously for the 
biotransformation. The substrate is expediently added in such a way that 
the substrate concentration in the medium does not exceed 20 percent by 
weight, preferably in such a way that the substrate concentration does not 
exceed 10 percent by weight. 
The biotransformation, which is normally carried out with stationary cells, 
is expediently carried out using the 2-cyanopyridine-utilizing 
microorganisms of the species Alcaligenes faecalis which are disclosed in 
European Published Patent Application No. 0,504,818 and are designated DSM 
6335, and using their functionally equivalent variants and mutants. These 
microorganisms were deposited on Jan. 3, 1991, (03/01/1991) at the 
Deutsche Sammlung yon Mikroorganismen und Zellkulturen GmbH, Mascheroder 
Weg 1b, D-38124 Braunschweig, Germany, in accordance with the Budapest 
treaty. 
"Functionally equivalent variants and mutants" mean microorganisms which 
have essentially the same properties and functions as the original 
microorganisms. Variants and mutants of this type can be formed 
adventitiously, for example by UV irradiation. 
The same media can be used for the biotransformation as for the culturing 
of the microorganisms. The biotransformation can also take place in the 
presence or absence of the dicarboxylic acids, tricarboxylic acids or 
carbohydrates described above. 
The pH is expediently in a range from 4 to 10, preferably in a range from 5 
to 9. The biotransformation can be carried out at a temperature from 
10.degree. to 50.degree. C., preferably at a temperature from 20.degree. 
to 40.degree. C. 
After a usual conversion time of 6 to 100 hours, the appropriate carboxylic 
acids according to formula I or II can then be obtained by customary 
working up methods, such as, by acidification. The carboxylic acids can 
also be isolated in the form of salts, such as, ammonium or chromium salt. 
If the prepared heteroaromatic carboxylic acids are heteroaromatic 
carboxylic acids hydroxylated in position 6 (general formula II), the 
biotransformation is expediently carried out under aerobic conditions. If, 
however, a non-hydroxylated heteroaromatic carboxylic acid, such as, 
picolinic acid is prepared, the biotransformation is expediently carried 
out under anaerobic conditions. 
EXAMPLE 1 
Preparation of 6-hydroxypicolinic Acid 
The conditions chosen for the preparation of 6-hydroxypicolinic acid using 
the strain Alcaligenes faecalis DSM 6335 were as follows. A 7.5 l 
fermenter with an operating volume of 5 l was used. Alcaligenes faecalis 
DSM 6335 was cultured in a mineral salt medium (Table 1 ) with sodium 
fumarate as the sole source of carbon and energy, and 2-cyanopyridine as 
inducer at 30.degree. C., 600 rpm and a pH of 7.0. The aeration rate was 
about 3 l/min during this operation. Addition of the sodium fumarate took 
place under pO.sub.2 control when the pO.sub.2 was &gt;30 percent. A 20 
percent strength stock solution of sodium fumarate to which 0.5 percent, 
2-cyanopyridine was added was used. The cells were cultured until the 
optical density, measured at 650 nm (OD.sub.650), was 16 over the course 
of 23 hours before the biotransformation was started. For the growth 
phase, about 160 g of sodium fumarate in the form of a 20 percent strength 
solution (about 800 ml) was used. 
No source of carbon and energy was added during the aerobic 
biotransformation of 2-cyanopyridine to 6-hydroxypicolinic acid. The 
biotransformation took place with stationary cells. 
The addition of the 2-cyanopyridine took place by means of a pump with 
limitation. The pump rate was monitored "online" by means of HPLC. The 
concentration of the intermediate picolinic acid, whose rate of formation 
is about 2.5 times greater than the rate of conversion of picolinic acid 
into 6 -hydroxypicolinic acid (10 g/l.multidot.h to 4 g/l.multidot.h), was 
limited to values &lt;2 g/l, since otherwise the conversion of picolinic acid 
into 6-hydroxypicolinic acid was inhibited. 
Since 2-cyanopyridine is a solid at room temperature, it was necessary to 
heat the reservoir containing 2-cyanopyridine to 50.degree. C., making it 
possible to add 2-cyanopyridine in liquid form. It was possible with this 
process to prepare 75 g/l 6-hydroxypicolinic acid within 31 hours. The 
intermediate picolinic acid was no longer detectable at the end of the 
biotransformation. 
To isolate the 6-hydroxypicolinic acid, the cells were removed by 
filtration. The cell-free solution was then heated to 60.degree. C. and 
acidified with concentrated sulfuric acid to a pH of 2 to 2.5. At this pH, 
the 6-hydroxypicolinic acid was precipitated from the solution. 
It was then slowly cooled, with stirring, to 4.degree. C. and filtered, and 
the residue was washed with demineralized water and dried (100 mbar, 
55.degree. C.). About 2 g/l 6-hydroxypicolinic acid remained in the mother 
liquor from this washing. The yield was 87 percent based on 
2-cyanopyridine used. 
TABLE 1 
______________________________________ 
Composition Concentration, (g/l) 
______________________________________ 
Disodium fumarate 10 
Yeast extract 1 
MgCl.sub.2.6H.sub.2 O 
0.8 
Na.sub.2 SO.sub.4 0.25 
(NH.sub.4).sub.2 SO.sub.4 
1.0 
NH.sub.4 Cl 2.33 
NaCl 0.2 
CaCl.sub.2.2H.sub.2 O 
0.16 
MnSO.sub.4 1.8 .multidot. 10.sup.-2 
H.sub.3 BO.sub.3 3 .multidot. 10.sup.-2 
NiCl.sub.2 2 .multidot. 10.sup.-3 
NaMoO.sub.4 3 .multidot. 10.sup.-3 
FeSO.sub.4.7H.sub.2 O 
0.3 
Na.sub.2 EDTA.2H.sub.2 O 
0.75 
2-Cyanopyridine 1 
KH.sub.2 PO.sub.4 0.4 
Na.sub.2 HPO.sub.4 0.96 
______________________________________ 
EXAMPLE 2 
Preparation of Picolinic Acid 
The biomass was cultured as described in Example 1. The formation of 
picolinic acid took place under strictly anaerobic conditions. A 500 ml 
glass bottle with rubber septum charged with 400 ml of biomass of 
OD.sub.650 =20 was used for the biotransformation. Incubation took place 
at 30.degree. C. Before the biotransformation was started, the mixture was 
made anaerobic with pure nitrogen. For this purpose, nitrogen (50 mbar 
gauge pressure) was passed through a needle into the stirred mixture for 
about 30 minutes in order to drive out the oxygen quantitatively. In order 
to prevent oxygen from entering during the biotransformation or on 
addition of the 2-cyanopyridine, the gas introduction was maintained 
during the biotransformation (about 10 mbar gauge pressure). 
2-cyanopyridine was added in 12 steps each of 10 g/l, in each case after 
one hour had elapsed. The addition can, however, also take place 
continuously. HPLC was used to check whether 2-cyanopyridine had been 
completely converted into picolinic acid before adding another portion. 
During the biotransformation there was no detectable formation of 
picolinamide. It was possible with this process to prepare about 150 g/l 
picolinic acid within 26 hours. 6-Hydroxypicolinic acid was not formed 
during this. 
For isolation, the cell-free picolinic acid solution was precipitated with 
CaCl.sub.2 /H.sub.2 SO.sub.4. For this purpose, the cell-free picolinic 
acid solution from Example 2 was diluted 3-fold and, while stirring, 0.5 
equivalent of CaCl.sub.2 per equivalent of picolinic acid was added after 
the cell-free fermentation solution had been heated to 90.degree. C. The 
resulting calcium/picolinic acid complex precipitated immediately. The 
resulting complex was cooled to 4.degree. C. with stirring, filtered off 
on a glass flit (porosity 3) and washed with demineralized water. The 
filter cake was suspended in demineralized water and acidified to a pH of 
2.5 with concentrated sulfuric acid. During this operation, the picolinic 
acid was dissolved out of the complex and, at the same time, insoluble 
calcium sulfate was formed. Since the free picolinic acid is very soluble 
in water it was possible to remove calcium sulfate by filtration. The 
picolinic acid solution was evaporated to dryness and analyzed. The crude 
yield was about 70 percent with a purity of 86 percent by titration. The 
water content was 0.7 percent measured by the Karl-Fischer method. 
EXAMPLE 3 
Preparation of Chromium(III) Picolinate 
Aqueous chromium trichloride hexahydrate solution (23.95 g, 0.09 mol of Cr 
in 63 ml of water/was added dropwise over a period of 3.5 hours to an 
ammonium picolinate solution (271.4 g; 0.325 mol; 16.8%), pH 7.1, 
73.degree. C. in a 500 ml flask. The resulting violet solution was stirred 
for a further 1 hour and then slowly cooled to 3.degree. C. After the red 
solid which had formed had settled out, the upper blue phase was decanted 
off. The solid was suspended in 100 ml of water for 30 min., and 
decantation was repeated. After a second suspension in 50 ml of water (30 
min.), the solid was filtered off with suction and dried at 50.degree. C. 
in vacuo. 33.64 g of dark red crystals was obtained (90 percent yield). 
EXAMPLE 4 
Culturing of Alcaligenes faecalis DSM 6335 with Various Sources of Carbon 
300 ml conical flasks containing 100 ml of A+N medium (Table 1 without 
disodium fumarate) were used for culturing Alcaligenes faecalis (DSM 
6335), and in addition, 2 gl.sup.-1 2-cyanopyridine and 10 gl.sup.-1 of 
one of the following sources of carbon were added to the medium: 
disodium fumarate 
glycerol 
disodium malonate 
disodium succinate 
Incubation took place in a shaker at 30.degree. C. After growth for 16 
hours, the cells were spun down and resuspended in fresh A+N medium 
(without source of carbon) containing 10 gl.sup.-1 2-cyanopyridine. The 
optical density of the cell suspension, measured at 650 nm (OD.sub.650), 
was 10. The cell suspensions (total volume 10 to 20 ml) were then 
incubated again at 30.degree. C. The formation of 6-hydroxypicolinic acid 
was followed by spectrophotometry, measuring the absorption of the 
cell-free solution at 308 nm. The following average productivities were 
determined for the formation of 6-hydroxypicolinic acid: 
______________________________________ 
Carbon Productivity (in gl.sup.-1 h.sup.-1) 
______________________________________ 
Disodium fumarate 
2.4 
Glycerol 2.0 
Disodium malonate 
4.2 
Disodium succinate 
0.14 
______________________________________ 
EXAMPLE 5 
Preparation of 6-hydroxypyrazinecarboxylic Acid 
Alcaligenes faecalis (DSM 6335) was cultured as in Example 4 with fumaric 
acid as the source of carbon. The washed cells were resuspended in A+N 
medium containing 10 gl.sup.-1 2-cyanopyrazine (OD.sub.650 =10) and 
incubated at 30.degree. C. The formation of 6-hydroxypyrazinecarboxylic 
acid was followed by spectrophotometry, measuring the absorption of the 
cell-free solution at 320 nm. It was possible to determine the decrease in 
the concentration of 2-cyanopyrazine (substrate) by measuring the 
absorption at 270 nm. The amount of 2-cyanopyrazine used had been 
converted into 6-hydroxypyrazinecarboxylic acid after 7 hours. 
EXAMPLE 6 
Preparation of 6-chloropicolinic Acid and Pyrazinecarboxylic Acid 
Alcaligenes faecalis (DSM 6335) was cultured as in Example 4 with fumaric 
acid as source of carbon. The washed cells were resuspended in A+N medium 
in glass vessels (OD.sub.650 =10) which could be closed with rubber 
stoppers, and nitrogen was introduced through needles in order to remove 
dissolved oxygen. Then, 2-cyanopyrazine or 6-chloro-2-cyanopyridine was 
added as substrate to the cell suspensions, until the final concentration 
was 10 gl.sup.-1, and incubated at 30.degree. C. After 3 hours, the 
starting substances had been converted quantitatively into the 
corresponding acids detection by thin-layer chromatography; silica gel 60 
with fluorescence indicator, mobile phase: chloroform 30/ethanol 
55/NH.sub.4 OH (25%) 10/H.sub.2 O 5!.