The way microbiological oxidation of methyl groups in aromatic heterocycle before carboxylic acid

 

(57) Abstract:

The use of biotechnology. The inventive methylated derivative of thiophene, furan, pyrrole, thiazole, pyridine, pyrimidine or pyrazine having no substituent at the neighboring oxidizable methyl group carbon atom, incubated with assimilative xylene strain of the microorganism Pseudomonas putida was ATSS 33015 or assimilative zymol strain of microorganisms Pseudomonas putida DSM 5709. Oxidizing microorganism cultures pre-treated for induction of microbial enzymes. Incubation is carried out at pH 4 to 11 and a temperature of 15°C to 50°C. the Substrate-heterocycle is administered once, or continuously until the concentration in the culture medium is not more than 5 wt.%/ volume. Receive the appropriate carboxylic acid with the release of 40 - 90%. 3 C. p. F.-ly, 2 ill., table 2.

The invention relates to a new method of microbiological oxidation of methyl groups in aromatic 5 - or 6-cyclic heterocyclic compounds to the corresponding carboxylic acid, with heterocycle at the carbon atom adjacent to oxidize the methyl group has no substituent. This heterocycle is a substrate for the reaction, performed using toluene, KS the heterocyclic carboxylic acid present, for example, an important intermediate products in the production of pharmaceuticals. Thus, nicotine (3-piridinkarbonovaya) acid is an important intermediate product in the production of the amide of nicotinic acid, representing a vitamin complex of vitamin b and of great importance in the diet of humans and animals (Ullmann, T. 19, 1980, S. 603).

Next, 2-pyrazinecarboxamide acid, for example, is an important intermediate product in the production of tuberculostatic means pyrazinamide (amide 2-pyrazinecarboxamide acid) (Romps Chemie Lexikon, T. 5, 1987). 4-Diazocarbonyl acid is used to retrieve thiabendazole highly effective sedative, which in turn serves as the starting material in the production of other new antihelminthic means, for example, cambendazole (Ullmann, T. 23, 1980, S. 46). 2-Thiencarbazone acid has anti-allergic effect (Ullmann, T. 23, 1980, S. 219).

Detailed studies of the oxidation of methyl groups still were conducted only with aromatic hydrocarbons.

Obtaining carboxylic acids by microbiological oxidation of methylated aromatic hydrocarbons is described in detail in the works of Raymond and his zatrudnionych groups in aromatic hydrocarbons using a gram-positive strain of microorganism of the genus Nocardia.

The disadvantage of this method is that, for example, in the oxidation of methyl groups in aromatic hydrocarbons is disclosed benzene ring of the appropriate acid.

For Pseudomonas putida was ATSS 33015 known that the methyl group of toluene in the three-stage biochemical oxidation is converted into benzoic acid. When this methyl group under the influence of the toluene monoxygenase first converted into benzyl alcohol, and then in two subsequent stages under the catalytic action of alcohol and aldehyde dehydrogenase is converted to the acid.

In this strain as KSIL-genes encoding enzymes for the degradation of xylene, and genes regulating KSIL-genes located on plasmid pWWO. This archetypal tol plasmid has already been thoroughly investigated molecular-biological methods (Harayama with TCS. J. Bacteriol. 171, 1989, S. 5048-5055; Burlage with TCS. Appl. Environ Environ. 55. 1989, S. 1323-1328).

From the literature also known microbiological methods oxidation of the methyl groups of N-heterocycle. According to the patent of the USSR N 417468 2-methylpyridin using the gram-positive strain of microorganism of the genus Nocardia can oxidize to the corresponding acid.

In the patent of the USSR N 228688 described Microbiology of the genus Mycobacterium. From the patent of the USSR N 302341 known microbiological method for the production of nicotinic acid using gram-positive bacteria of the genus Nocardia.

Disadvantages of oxidation of the methyl groups of N-heterocycles using gram-positive bacteria are that in the case of these bacteria using alkanes, alkane ratio with oxidizable substance must be strictly coordinated to ensure biotransformation, and that it is necessary to prevent biotransformation of the substrate in the absence of alkane, i.e., that used for the induction of alkane must be present and the reaction of interaction of the substrate with microorganisms. Comparative experiments with gram-positive bacteria Nocardia, on the one hand, and with the proposed gram-negative bacteria Pseudomonas, on the other, clearly showed that in the case of bacteria Nocardia even in the presence of an alkane, for example, dodecane, 3-methylpyridin not oxidized to nicotinic acid.

Further, in U.S. patent N 4859592 describes a method for pikolinos acid using Pseudomonas putida, according to which of the alkyl substituted aromatic hydrocarbon in the presence of molecular oxygen in the first stage under the influence of the primary amine is translated in 2-Pikalyovo acid.

The disadvantage of this method is that the corresponding picolina acid is formed only at the second stage in the reaction of semialdehyde 2-oxymoronical acid with ammonia.

Therefore the task of the invention is to eliminate the above drawbacks in the development of a simple, single-stage method of microbiological oxidation of methyl groups, for which it is possible to select the appropriate acid in good yield and purity without the disclosure aromatic heterocycle.

Another challenge is to develop a method, according to which connection is necessary for the induction of enzymes, after the last within of the oxidation reaction of the substrate may not be present, and thus the degree of oxidation is independent of the number present enzyme inducer.

Thus, the subject invention is a microbiological method for the oxidation of methyl groups in aromatic 5 - or 6-cyclic heterocyclic compounds to the corresponding carboxylic acid, with heterocycle at the carbon atom adjacent to oxidize the methyl group has no substituent, and the methylated heterocycle is a substrate for oxidation reactions, assests the work of the enzymes of the latter.

It is advisable to carry out the oxidation reaction using using toluene, xylene or zymol microorganisms of the species Pseudomonas putida.

Preferably used using xylene microorganism of the strain Pseudomonas putida with the designation of ATS 33015 or effective mutant of this organism, or use zymol microorganism strain Pseydomonas putida with the designation of DMSD 5709, or effective mutant of this organism. The oxidation reaction, in particular, is carried out using a strain of Pseudomonas putida ATCC 33105.

A strain of Pseudomonas putida (DSM 5709) was deposited in the German collection of microorganisms and cell cultures (Deutsche Sammlung von Mikroorganismen (DSM) und Zellkulturen GmbH) Macerative 16, 3300, Braunschweig, Germany, 22.12.89 for N DSM 5709.

A strain of Pseudomonas putida ATCC 33105 was deposited in the American type culture collection (American Type Culture Collection 12301 Parklawn Drip. Raquel, PCs Maryland 20852, USA, for N ATS 33015.

The induction of the enzyme can be realized by using compounds that serve as the microorganism source of carbon and energy, such as p-xylene, m-xylene, p-zimola, m-zimola or toluene, and through connections, not serve as a source of carbon and energy, for example mono - and disubstituted methyl-, ethyl - and chlortoluron, benzyl spydertv already described (M. Abril-A. with TCS. J. Bacteriol. so 171, 1989, 6782-6789).

The induction of the enzyme is preferably carried out by using p-xylene, m-xylene, 2-chlorotoluene, or 2-bromthymol.

Used for induction connection or present during the oxidation reaction of the substrate, or their supply is suspended until the beginning of this reaction.

Select the concentration of the inductor should be less than the minimum concentration at which inhibited responsible for the reaction of enzymes.

Depending on the variant of the process flow used for the induction of enzyme compounds during the reaction preferably cease or suspend supply of the compounds, or by removing cells by centrifugation.

These strains usually grown with p-xylene, m-xylene, p-timolol, m-timolol or toluene as the sole source of carbon and energy in mineral medium (Kulla with TCS. Arch. Environ. 135, 1983, S. 1-70 or on a complex medium (Nutrient Broth Nr 2, Oxoid Ltd. UK) or on minimal medium, the composition of which is specified below. The growth substrate according to Claus and Walker (J. Gen. Environ. 36, 1964, S. 107-122) was filed on Wednesday in a gaseous form from the gas transmission rate of 0.5 volume/min

Prior to entering the substrate nm.

It is advisable to carry out the reaction with single or continuous input substrate so that its concentration in the culture medium did not exceed 20%, preferably 5% (wt./volume). Depending on the implementation method, the substrate can be introduced simultaneously with the enzyme inducer, for example, using a mixture of the inductor and the substrate.

Reaction it is advisable to carry out at pH 4-11, preferably 6-10.

It is advisable to carry out the reaction at a temperature of 15-50aboutWith, preferably 25-40aboutC.

The reaction is carried out for 1-24 h

As substrate for the reaction, it is advisable to use a methylated aromatic 5-cyclic heterocycles containing one or more heteroatoms selected from the group comprising oxygen, nitrogen and sulfur, such as methylated tifany, furans, pyrrole, thiazole, pyrazoles or imidazoles having no substituent at the carbon atom adjacent to oxidize methyl group. Preferably used furans, tifany, pyrrole and thiazole, in particular 3,5-dimethylpyrazol, 5-methylthiazole, 4-methylthiazole, 2.5-dimethylthiophene, 2-methylthiophene, 3-methylthiophene, 2.5-dimethylfuran, 2.5-dimethylpyrrole.

The rationale for the multiple nitrogen atoms as heteroatoms, such as methylated pyridine, pyrimidines, pyrazine or pyridazine having no substituent at the carbon atom adjacent to oxidize methyl group. Preferably used pyridine, pyrazine and pyrimidines, for example, 2-methylpyridine, 3-methylpyridine, 4-methylpyridine, 2.5-dimethylpyridine, 2,4-dimethylpyridine, 6-chloro-3-methylpyridine, 2-chloro-3-ethyl-5-methylpyridin, 4,6-dimethylpyrimidine, 2-methylpyrazine, 2.5-dimethylpyrazine, 2,6-dimethylpyrazine, 2,3,5-trimethylpyrazine and 2-chloro-3,6-dimethylpyrazine.

In Fig.1 shows a preferred embodiment of the method.

Under this option, perform the inducer of the enzyme and/or the methylated heterocycle as a substrate is introduced into the bioreactor 2, and the quantity of substances is regulated through the concentration of the inducer in the exhaust air 3 bioreactor 2. The concentration of the inducer of the enzyme in the exhaust air 3 is preferably measured by measuring device 4. As the measuring device 4 can be used, for example, a light meter for ultraviolet light, gaschromatograph or gaschromatograph, coupled with a mass spectrometer. It is recommended to connect the probe 4 through the system 5 control that regulates the amount of inducer of the enzyme methylated heterocycle in bioreactor 2. Feed enzyme inducer and/or methylated heterocycle is expedient to regulate with the help of the above system of interconnected devices. In the preferred case, using this system, the concentration of the inducer in the exhaust air 3 is maintained at a constant level.

It is expedient to regulate the concentration of the inducer in the exhaust air 3 so that it ranged from 0.001 to 10 mmol/l exhaust air, preferably 0.1 to 3 mmol/l exhaust air.

The inductor and/or the methylated heterocycle, serving as a substrate introduced into the bioreactor 2 is preferably in the form of a mixture.

A suitable ratio of the inductor and the substrate is 5:1-3:1.

According to this variant of the process it is expedient to introduce the inductor and/or the methylated heterocycle in bioreactor 2 through the supply air.

In Fig. 2 shows the results of biotransformation according to a preferred variant of the process.

After the reaction produce the corresponding acid by known methods, for example, extraction with organic solvents or in case of formation of salts of heterocyclic carboxylic acids with alkaline m the ing the pH of the culture medium, for example, by adding a hydroxide of an alkali metal.

According to the invention offers a simple, single-stage microbiological method for the oxidation of methyl groups in aromatic 5 - or 6-cyclic heterocyclic compounds, which are allocated to the corresponding acid with high yield and purity. Another advantage of this method lies in the fact that the aromatic heterocycle is not disclosed, and the degree of conversion does not depend on the amount of an enzyme.

P R I m e R 1. 5-Methyl-2-pyrazinecarboxamide acid.

A strain of Pseudomonas putida ATCC 33015 grown on complex medium (100 ml) "Nutrient Broth Nr.2" (firm Oxoid Ltd. England) in the fermenter at pH 7.0 and temperature 30aboutC, and enter the inducer of the enzyme p-xylene in a gaseous form in accordance with these authors Claus and Walker (J. Gen. Environ. 36, 1964, S. 107-122) to achieve a concentration of 1 mmol/L.

The cells are then washed twice with mineral medium (Kulla with TCS. Arch. Environ. 135, 1983, S. 1-7) and grown to an optical density of 10 at 650 nm in 100 ml of mineral medium. To the obtained cell suspension was added 1 mmol of 2,5-dimethylpyrazine, which corresponds to the substrate concentration to 0.108% (wt. /vol) in 100 ml After incubation for 4 h at 30

P R I m m e R 2. According to example 1 as an inducer of the enzyme also use the following connection, which together with the corresponding concentrations are given in table.1.

P R I m e R 3. 5-Methyl-2-pyrazinecarboxamide acid.

A strain of Pseudomonas putida ATCC 33015 grown in accordance with example 1, but with the difference that the mineral medium (Kulla with TCS. Arch. Environ. 135, 1983, S. 1-7) contains p-xylene as the sole source of carbon and energy. Then to the resulting suspension of cells (100 ml) with an optical density of 10 add 10 mmol of 2,5-dimethylpyrazine, which corresponds to the concentration to 0.108% (wt./volume). During the oxidation reaction of the substrate to suspend the supply of p-xylene. Under these conditions, 1 mmol of 2,5-dimethylpyrazine for 4 h becomes 0.9 mmol 5-methyl-2-pyrazinecarboxamide acid, which corresponds to a yield of 90% counting on the original 2.5-dimethylpyrazine.

P R I m e R 4. The strain Pseudomonas putida DSM 5709 grown as in example 3, but with p-timolol as the sole source of carbon and energy. After 16 h are 0.5 mmol of 5-methyl-2-pyrazinecarboxamide acid, which corresponds to a yield of 50% counting on the original 2.5-dimethylpyrazine.

P R I m e R s 5-28 conducted in accordance with example 3 with use of the Sabbath.

P R I m e R 5. Getting 5-methyl-2-pyrazinecarboxamide acid.

Bacteria strain Pseudomonas putida ATCC 33015 grown on minimal medium, the composition of which is specified below, in the bioreactor 2 with a capacity of 20 l with a working volume of 15 liters

The temperature is 30aboutC. the pH Value by the introduction of potassium hydroxide is maintained at the level of 7.0. The gas flow rate is 20 l/min. as a substrate a mixture of 4 hours (volume/volume) of p-xylene and 1 h 2,5-dimethylpyrazine. This mixture is introduced into the bioreactor 2 pump 1. Supply system of the substrate through the meter 4 for controlling the supplied amount of xylene in the exhaust air 3 bioreactor connected to the system 5 control. Using the latest maintain the concentration of xylene in the exhaust air 3 0.2 mmol/l (Fig.1).

Biotransformation cease only upon completion of the growth of bacteria. In Fig. 2 shows the results of this kind of biotransformation.

Curve a in Fig.2 means the optical density at 650 nm.

Curve In Fig.2 indicates the concentration of 2,5-dimethylpyrazine, g/l

Curve C in Fig.2 indicates the concentration of the potassium salt of 5-methyl-2-pyrazinecarboxamide acid, g/l

According pickup>6H2O 0,8

CaCl20,16

(NH4)SO42

NH4Cl 5

Na2SO40,25

KH2SO40,4

Na2HPO40,9

Trace elements, ml/l 1

FeEDTA, ml/l 15

The composition of trace element solution, g/l:

KOH 15

EDTA-Na22H2O 10

ZnSO47H2O 9

MnCl24H2O 4

H3BO32,7

CoCl26H2O 1,8

CuCl22H2O 1,5

NiCl26H2O 0,18

Na2MoO42H2O 0,2

The composition of FeEDTA, g/l:

EDTA-Na22H2O 5

FeSO47H2O 2

Comparative examples

P R I m e R 6. Bacteria strain Pseudomobas putida ATCC 33015.

The biotransformation of 3-methylpyridine with bacteria strain Pseudomonas putida ATCC 33015 carried out according to example 3 using 1 mmol of 3-methylpyridine. After an incubation period of 8 h are 0.25 mmol nicotinic acid, which corresponds to a yield of 25% considering the source 3-methylpyridin.

P R I m e R 7. Bacteria strain Phodoccocus rhodochrous and Nocardia.

Bacteria strain Phodoccocus rhodochrous DSM 43002 (ATCC 19149) grown on mineral medium according to example 3 with the use of 0.4% dodecane as a source of carbon and energy, and then they are washed in the same sreach add the following connections: a) 1 mmol of 3-methylpyridine, b) 1 mmol of 3-methylpyridine and by 0.055 mmol dodecane, 1 mmol of 3-methylpyridine and 5.5 mmol dodecane.

After an incubation period of 8 h at 30aboutWith none of the mixtures is not detected nicotinic acid.

1. The WAY MICROBIOLOGICAL OXIDATION of METHYL GROUPS in AROMATIC HETEROCYCLE TO a CARBOXYLIC ACID, comprising the incubation of the primary substrate heterocycle having no substituent at the neighboring oxidizable methyl group carbon atom with an oxidizing culture of the microorganism, characterized in that the incubation is carried out at pH 4 to 11 and a temperature of 15 - 50oWith as oxidizing cultures use assimilative xylene - microorganism strain Pseudomonas putida was ATSS 33015 or assimilative timol - microorganism strain Pseudomonas putida DSM 5709 after prior induction of microbial enzymes, substrate use methylated derivative of thiophene, furan, pyrrole, thiazole, pyridine, pyrimidine or pyrazine, while the substrate is injected once or continuously during incubation to its concentration in the culture medium is not more than 5 wt./about.

2. The method according to p. 1, characterized in that the induction of microbial enzymes perform pomai.

3. The method according to PP.1 and 2, characterized in that the inductor microbial enzyme and/or substrate is introduced into the bioreactor, and the number of input connections is measured by the meter through the concentration of inducer microbial enzyme in the exhaust air and regulate management system.

4. The method according to p. 3, characterized in that the concentration of inducer microbial enzyme in the exhaust air support in the limit of 0.001 to 10 mmol/l exhaust air.

 

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