Microbiological method for the terminal oxidation of the ethyl groups in the carboxyl group

 

(57) Abstract:

Usage: microbiological method for the terminal oxidation of alkyl groups to carboxylic acid. The invention is described microbiological method for the terminal oxidation of alkyl groups to carboxylic acid 5 - or 6-membered heterocycles, cyclically substituted by at least one alkyl group containing more than two carbon atoms; the transformation is carried out using microorganisms processing alkane and/or alkanol. 6 C. p. F.-ly, 3 tables.

The invention relates to a new microbiological method for the terminal oxidation of the alkyl groups in the carboxyl group.

The obtained derivatives of carboxylic acids may be used, for example, as intermediates for pharmaceuticals, for example, thiophene-2-acetic acid for the synthesis of the antibiotic penicillin or a cephalosporin (Ullmann, 1983, Band 23, p. 219).

Thorough research on the microbiological oxidation of alkyl groups in aliphatic operatorto were, in particular, carried out with strains of microorganisms Pseudomonas oleovorans.

Relatively Pseudomonas oleovorans ATCC 8062 and ATCC 29437 it is known that biochemical okelani the basics first formed corresponding alcohol, then in two subsequent steps by the action of dehydrogenase-alcohol dehydrogenase-aldehyde turn into acid.

In the above strains have genes responsible for enzymes that oxidation on the OCT plasmid (Witholt et.al. TIBTECH, Vol.8, 1990, S. 46-52).

Using the specified strain was described oxidation only compounds with linear saturated C6-C12alkyl residues and ethylbenzene.

Oxidation of substituted cyclic alkyl, aromatic, or unsaturated hydrocarbons to the corresponding carboxylic acid processing alkane microorganisms type Rhodococcus, Mycobakterium and Pseudomonas described in Raymond, R. L., Process Biochemistry, 1979, S. 71-74.

The disadvantage of this method is that the reaction is non-specific for alkyl groups containing more than two carbon atoms. In this case, can be cleaved cycles, and methyl groups can be oxidized in an aromatic hydrocarbon compounds.

The objective of the invention is to develop a simple and single-step method of microbiological oxidation of alkyl groups in the compounds with which the corresponding acids may be selected with great output, visocica was unexpectedly solved in accordance with the microbiological method for the terminal oxidation of the ethyl groups in the carboxyl group, including the interaction of the substrate with microorganisms, in which the substrate using 5 - or 6-membered heterocycle, substituted by at least one alkyl group with at least two carbon atoms, and from microorganisms are used such that turn alkane and/or alkanol to the corresponding carboxylic acid. However, the imposition of the substrate is carried out one time or continuously until the concentration in the culture medium does not exceed 20% (weight/volume) at pH 4 to 11 and a temperature of 15-50oC, and the obtained carboxylic acid does not undergo further catabolism.

Possibly in addition to induce enzymes of the microorganism compounds, which serve as the microorganism source of carbon or energy, or compounds that do not serve the microorganism source of carbon and energy.

Of organisms typically used bacteria strains of Pseudomonas oleovorans ATCC 8062 or Pseudomonas oleovorans ATCC 29347.

It is also possible from microorganisms to use the yeast strain Candida tropicalis ATCC 32113 or bacteria strain Rhodococcus rhodochrous ATCC 19607.

As the substrate used aromatic 5 - or 6-membered heterocycle, substituted with at least one C2-C6alteltysese 5 - or 6-membered watercoil, substituted ethyl group which contains one or more heteroatoms from the series oxygen, nitrogen or sulphur.

Group 6-membered heterocycles are preferred heterocycles with nitrogen atoms.

Acceptable representatives of the 5-membered heterocycles are tofani, furans, pyrrole, thiazole, pyrazoles or imidazoles. Particularly preferred are 5-membered heterocycles 2-ethylthiophen or 2-ethylfuran.

Acceptable representative 6-membered getarticles are pyridine, pyrimidines, pyrazine or pyridazine. As a 6-membered heterocycle is particularly desirable 3-ethylpyridine, 2-ethylpyrazine or 5-ethyl-2-methylpyridin.

It is reasonable to induction of enzymes of microorganisms processing alkane and/or alkanol. The concept of "alkanes" or "the alkanols" also includes substituted alkanes or alkanols (alkylated cyclic compounds), for example, ethylbenzene.

The induction of the enzyme can be carried out with compounds that serve the organism as a source of carbon and energy (alkanes, alkanols, alkylated cyclic compounds, for example, octane, octanol, dodecan, dodecanol, hexane, hexanol, ethylbenzene), and connect ylmethanol or diethoxyethane), described Grund et.al, J. Bacteriol. 123, S. 546-556, (1975).

Preferably the induction of the enzyme produced for Pseudomonas oleovorans n-octane, Candida tropicalis n-hexadecanol and Rhodococcus rhodochrous n-decane.

Usually the introduction used for inducing compounds during the conversion of the substrate is stopped. However, the conversion of the substrate can also be carried out in the presence of inducer of the enzyme.

Typically, the flow used for inducing compounds during the conversion of substrate to cease or suspend supply or ottsentrirovat cells.

The transformation can be carried out using microorganisms processing alkane and/or alkanol, for example, microorganisms of the species Pseudomonas, yeasts of the species Candida or microorganisms of the species Pseudomonas.

For the implementation of the proposed method is also suitable mutants of these organisms, as well as other microorganisms, either by conjugation or by using genetic engineering techniques impose necessary for the conversion of genetic information, forming effective enzymes for the conversion.

As the microorganisms can be used processing alkane strains microorg the oleovorans ATCC 29347.

Strains of microorganisms Pseudomonas oleovorans ATCC 8062 or ATCC 29347 and Rhodococcus rhodochrous ATCC 19067, as well as the yeast of the species Candida tropicalis ATCC 32113 are deposited in the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852).

The above-mentioned strains grow with alkanes, alkanols or alkylated cyclic compounds in the mineral salts medium (Kulla et. al. Arch. Environ 135, 1983, S. 1-7) or in a complex environment ("Nutrient Broth Nr.2", Oxoid Ltd. England).

Before the introduction of the substrate cells grown in the usual way, followed by the conversion of the substrate when the optical density 1-200 at 650 nm in a nutrient medium, preferably in optical density 5-100 at 650 nm.

The transformation can be carried out either in single or continuous introduction of the substrate so that the substrate concentration in the nutrient medium does not exceed 20% (weight/volume).

The introduction of the substrate is recommended so that the substrate concentration in the nutrient medium does not exceed 5% (weight/volume), while the preferred concentration of the substrate in the nutrient medium does not exceed 1% (weight/volume).

The transformation is carried out at a pH value of 4 to 11, preferably at pH 6 to 10.

The transformation is usually carried out at a temperature of 15-canie period of time lasting from 1 hour to several days, preferably the conversion is conducted in a continuous way in a few days.

After conversion to the corresponding acid can be isolated in a known manner.

Example 1. Getting 2-methylpyridin-5-acetic acid. Pseudomonas oliovorans ATCC 29347 was incubated in a mineral salts medium (Kulla et. al. Arch. Environ. 135, 1983, S. 1-7) with n-octane as the sole carbon and energy source at 30oC and a pH value of 7.

After that, the cells twice washed with the same medium mineral salts and installed an optical density of 10 at 650 nm in 100 ml of mineral salts medium. To this cell suspension was added 1 mmol of 5-ethyl-2-methylpyridine, which corresponds to the substrate concentration, 0.12% (weight/volume).

After 16-hour incubation period at 30oIn the absence of n-octane was obtained 0.5 mmole of 2-methylpyridine-5-acetic acid, with output equal to 50% (based on the original 5-ethyl-2-methylpyridin. Under these conditions, there was no oxidation of the methyl group.

Examples 2-5 were carried out in accordance with example 1 with the number of substrate equal to 1 mmol per 100 ml of cell suspension, and United in table 1.

Examples 2 and 6.

Examples 7-11.

Transformation by microorganisms Pseudomonas oleovorans ATCC 8062 (PL.3).

1. Microbiological method for the terminal oxidation of the ethyl groups in the carboxyl group, which includes the interaction of the substrate with microorganisms, characterized in that the substrate using 5 - or 6-membered heterocycle, substituted by at least one alkyl group with at least two carbon atoms, and from microorganisms are used such that turn alkane and/or alkanol to the corresponding carboxylic acid, the introduction of the substrate is carried out one time or continuously until the concentration in the culture medium does not exceed 20% (weight/volume) at pH 4 to 11 and a temperature of 15 to 50oS, and the obtained carboxylic acid does not undergo further catabolism.

2. The method according to p. 1, characterized in that it additionally induce enzymes of the microorganism with compounds that serve as the microorganism source of carbon or energy, or with compounds that do not serve the microorganism source of carbon or energy.

3. The method according to p. 1 or 2, characterized in that the organisms use bacteria strains of Pseudomonas oleororans ADS 8062 or Pseudomonas oleororans ADS 29347.

5. The method according to p. 1 or 2, characterized in that the microorganisms use bacteria strain Rhodococcus rhodochrous ADS 14067.

6. The method according to PP. 1-5, characterized in that as the substrate used aromatic 5 - and 6-membered heterocycle, substituted by at least one2-C6alkyl group containing several heteroatoms from the series oxygen, nitrogen or sulphur.

7. The method according to p. 6, characterized in that as the substrate used aromatic 5 - or 6-membered heterocycle, substituted ethyl group which contains one or more heteroatoms from the series oxygen, nitrogen or sulfur.

 

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