The way microbiological oxidation of 2-methylinosine (options)

 

The invention relates to the field of biotechnology and can be used to produce 2-khinoksalinona acid. The method of obtaining the latter involves contacting 2-methylinosine with the microorganism, providing its oxidation to the acid. The eleven strains of eight species of microorganisms (fungi, bacteria). The resulting mixture was incubated in the medium for cultivation of microorganisms placed in the flask or fermenter. After incubation of the target product, if necessary, allocate by extraction and purified. The method allows us to simplify technology for acid with the release of 81-90%, depending on the time of cultivation. 3 S. and 26 C.p. f-crystals, 5 PL.

The technical field This invention relates to new methods of producing 2-khinoksalinona acid, and more particularly to microbiological oxidation of 2-methylinosine to 2-khinoksalinona acid.

The level of technology In the existing prior art methods of microbiological oxidation of certain aromatic heterocycles, in particular the methods of microbiological oxidation of methyl groups to certain aroma of the Oxidation of Both Toluene and Benzyl Alcohol by the incorporated Product," S. Harayama et al., J. Bacteriol., 167(2): 455-461 (1986) and "Enzymatic Oxidation of Methyl Groups on Aromatic Heterocycles: A Versatile Method for the Preparation of Heteroaromatic Carboxylic Acids," A. Any, Angew. Chem. Int. Ed. Engl., 31(6):774-775 (1992).

In U.S. patent 4859592 described microbiological method of obtaining pikolinos acid, which can then be converted into products of pyridine chemical methods.

In U.S. patent 5104798; 5213973 and 5236832 described microbiological methods for the oxidation of methyl groups to certain aromatic heterocycles with 5 - or 6-membered ring to the corresponding carboxylic acids, which are carried out by bacteria of the species Pseudomonas when using toluene, xylene or zimola as an inductor. As noted herein, in the existing prior art it is known that the oxidation of the methyl group of toluene to benzoic acid by using a strain of Pseudomonas putida ATCC 33015 involves three steps catalyzed by colormanagement, spindegenerate and aldehyddehydrogenase respectively.

As described earlier in the above article Harayama et al., TOL plasmid pWWO P. putida mt-2 is a sexually extrachromosomal element that encodes all types of enzymes required for oxidative catabolism of certain aromatic hydrocarbons, including aromaticheskie hydrocarbons into the corresponding aromatic carboxylic acids such as l operon which encodes the enzyme decomposition of xylene, and the genes responsible for regulating l gene, located in the TOL plasmid pWWO. Genes located in the TOL plasmid pWWO that encode the enzymes required for the above oxidation, should be induced to produce such enzymes. Therefore, description of such induction has in the above U.S. patents 5104798; 5213973 and 5236832.

As described in Gaucher et al., in Dev. Ind. Environ., 22:219-232 (1981), the fungus Penicillium griseofulvum contain enzymes for the conversion of m-cresol m-hydroxybenzoic acid m-cresol metalhydroxide, (m-hydroxybenzoyl alcohol dehydrogenase and m-hydroxybenzaldehyde.

Repeating said, in the existing prior art it is known that certain fungi and bacteria contain enzymes for the oxidation of methyl groups to certain aromatic rings to the corresponding carboxylic acids. Although it is known that the methyl group heteroaromatic rings can be oxidized to the corresponding carboxylic acids using microorganisms, specialists in this field will be clear that the output of chemical and optical oxidation products using microorganisms typically varies substantially in zawiya so on.

At the present time discovered that a number of microorganisms, including fungi and bacteria, substantially oxidize 2-methylphenoxy to 2-khinoksalinona acid. In addition, this method allows you a convenient way to distinguish the 2-khinoksalinona acid.

In the application for U.S. patent 60/073801 ("application 801") dated 5 February 1998, at the present time, which PCT application PCT/IB99/00067 from January 18, 1999, describes the use of 2-khinoksalinona acid as intermediate compounds in the synthesis of new dihydroxypentanoic acids, which are suitable for the treatment of, for example, inflammation and other immune disorders. 2-Khinoksalinona acid obtained by the method of this invention may be used for the synthesis of such dihydroxypentanoic acids.

All documents referred to herein, including the above mentioned herein for information in their entirety.

Description of the invention This invention relates to a microbiological method for producing a 2-khinoksalinona acid from 2-methylinosine.

More specifically this invention relates to a microbiological process for the production of compounds of formula Ithrough prevedanastasia methyl group of compounds of formula II to the carboxyl group of the compounds of formula I, and incubating the mixture in suitable conditions, with a number of the compounds of formula I.

Accordingly, this invention provides methods for the microbiological oxidation of compounds of formula II, 2-methylinosine, which include the interaction of the compounds of formula II with a microorganism or its mutant, which is known or can be obtained otherwise by experts in the relevant field of technology and which is capable, despite the mutation, to perform this oxidation ("it (microorganism) suitable mutant"), and incubating the mixture under conditions sufficient to obtain a certain amount of the compounds of formula I, i.e., 2-khinoksalinona acid,
where the specified microorganism selected from the group including Absidia glauca ATCC 22752, Absidia glauca ATCC 74480, Absidia pseudocylindrospora ATCC 24169, Absidia repens ATCC 14849, Absidia repens ATCC 74481, Actinomucor elegans ATCC 6476, Alternaria solani ATCC 11078, Aspergillus tamarii ATCC 16865, Coniophora puteana ATCC 12675, Cunninghamella echinulata ATCC 8688a, Cunninghamella echinulata ATCC 8688b, Cunninghamella echinulata ATCC 8983, Cunninghaniella echinulata ATCC 9244, Cunninghamella echinulata ATCC 9245, Cunninghamella echinulata ATCC 10028b, Cunninghamella echinulata ATCC 26269, Cunninghamella echinulata ATCC 36190, Cunninghamella echinulata ATCC 36112, Cunninghamella homothallica ATCC 16161, Cylindrocarpon destructans ATCC 66963, Diplodia gossypina ATCC 20575, Epicoccum neglectum ATCC 12723, Glomerellamonas putida was ATSS 202190, Rhodococcus rhodochrous ATCC 19067 and Thamnostylum piriforme ATCC 8686 and suitable mutants, provided that if the specified microorganism is a Pseudomonas putida ATCC 33015 or Pseudomonas putida ATCC 202190, the Pseudomonas putida ATCC 33015 or specified Pseudomonas putida ATCC 202190 must be induced by the interaction with the inductor to bring these Pseudomonas putida ATCC 33015 or Pseudomonas putida ATCC 202190 in contact with 2-methylinosine.

In addition, the methods include optional selection of the desired product, 2-khinoksalinona acid, in whatever way is appropriate. For example, the reaction mixture may be extracted with an organic solvent, preferably ethyl acetate, and then extracted material may be subjected to chromatography. Alternative 2-khinoksalinona acid can be adsorbed from the reaction mixture at a resin, preferably a polymeric adsorption resin, then eleirovania organic solvent, preferably ethyl acetate and crystallized from the eluate using organic solvent or combination of organic solvents, preferably ethyl acetate and methanol. Next, 2-khinoksalinona acid obtained by the method of this invention may be the 2-khinoksalinona acid. Alkaline salt of 2-khinoksalinona acid can then be isolated from bioconversion environment by removing the cells from the medium by filtration or centrifugation, followed by concentration of the medium not containing cells, for example, by evaporation.

Consider the microorganism is preferably the intact organism.

In the preferred embodiment of this invention, the microorganism is a fungus.

In a preferred embodiment of the present invention, in which the microorganism is a fungus, the fungus is chosen from the group comprising the genera Absidia, Aspergillus, Alternaria, Penicillium, Diplodia and Cunninghamella.

In a particularly preferred embodiment of the present invention, in which the microorganism is a fungus, the fungus is selected from the genus Absidia. In the most preferred embodiment of the present invention, in which the microorganism is a fungus of the genus Absidia, this microorganism is a A. glauca ATCC 22752 or A. glauca ATCC 74480, or a suitable mutant, or, alternatively, any Deposit A. glauca ATCC 22752 or a suitable mutant, obtained under the terms of the Budapest Agreement.

In another most preferred embodiment of the present invention, in which the microorganism is a fungus kind of AOM, any Deposit A. repens ATCC 14849 or a suitable mutant, obtained under the terms of the Budapest Agreement.

The preferred density of cells for fungal cultures of the present invention is from about 10 to about 30 g dry weight of cells per liter.

In another preferred embodiment of the present invention the microorganism is a bacterium.

In a preferred embodiment of the present invention, in which the microorganism is a bacterium found a bacterium selected from the group comprising the genera Pseudomonas and Rhodococcus.

In a particularly preferred embodiment of the present invention, in which the microorganism is a bacterium, the bacterium is a bacterium of the genus Pseudomonas.

In the most preferred embodiment of the present invention, in which the microorganism is a bacterium of the genus Pseudomonas, the microorganism is a P. putida was ATSS 33015 or P. putida ATCC 202190, or a suitable mutant, or, alternatively, any Deposit of P. putida was ATSS 33015 or a suitable mutant, obtained under the terms of the Budapest Agreement.

The preferred density of cells for bacterial cultures of the present invention is of such density that gives an optical density of from about 10 to about 30 at 650 nm.

In a preferred embodiment of the present invention, in which the microorganism is a P. putida ATCC 33015 or P. putida ATCC 202190 or a suitable mutant, and the microorganism is cultivated in a culture medium in vitro, the inductor add to this the growing medium to bring the organism into contact with 2 methylinosine and incubated in this medium for cultivation in a period of time sufficient for practical completion of the specified induction. Cells induced microorganism is collected by centrifuging the contents of the tubes by removing, for example decanter used environment for growing (and, thereby, the inductor), washing the precipitate cells in the test tube after centrifugation and re-suspendirovanie precipitate in the test tube in a water environment, such as SRD (Biowhittaker), before bringing into contact the specified 2-metilfenolom microorganism represents R. putida ATCC 33015 or P. putida ATCC 202190 or a suitable mutant, and the microorganism is cultivated in a culture medium in a fermenter, the inductor continuously or continually add to this the growing medium prior to bringing into contact of the microorganism with 2 methylinosine, and incubated in this medium for cultivation during the time required for almost complete induction, and then stop adding before bringing 2-methylinosine in contact with the specified microorganism.

In yet another preferred implementation of the present invention are considering bringing into contact is carried Appendix 2-methylinosine in the culture medium containing the microorganism, where the microorganism is a fungus. In a preferred embodiment of the present invention, in which the bringing into contact is carried Appendix 2-methylinosine in the culture medium containing the indicated fungus, growing medium is a medium containing corn steep. Especially preferred medium containing corn extract comprises from about 20 to about 40 g/l corn extract and 20 g/l dextrose, having a pH of about 4,85. Another preferred tein) and about 20 g/l dextrose, having a pH of about 7.2.

In yet another preferred embodiment of this invention, the bringing into contact is carried out by adding the compounds of formula II, adsorbed on the resin. Cm. for example, the article by J. T. Vicenzi et al., "Large-scale stereoselective enzymatic ketone reduction with In situ product removal via polymeric adsorbent resins". Enzyme and Microbial Technology, 20:494-499 (1997).

In another preferred embodiment of this invention, the bringing into contact is carried out by adding 2-methylinosine in the aqueous medium containing the washed cells of the microorganism.

In another preferred embodiment of the present invention the microorganism is washed before bringing a microorganism in contact with 2-methylinosine. In a preferred embodiment of the present invention, in which the microorganism is washed before bringing a microorganism in contact with 2-methylinosine, the washed microorganism immobilized before making contact.

In yet another preferred embodiment of the present invention the microorganism is grown in a medium containing corn steep, within about 24 hours to about 72 hours before bringing into contact, which is performed by the addition of 2-methylinosine to the environment.

The methods of the present invention, furthermore, does not necessarily include Wydm solvent, adsorption on the resin, crystallization or, as described previously, when using alkali metal salts of 2-khinoksalinona acid concentration by evaporation of the medium not containing cells, or similar methods.

The invention also includes the use of 2-khinoksalinona acid in the synthesis of new dihydroxypentanoic acids described in the above-identified application 801 according to any one of the methods described in the application 801 or any other suitable method.

The person skilled in the art will understand terms used to describe this invention; however, some of the terms used are described below.

The term "intact microorganism" means that the cells of the microorganism, in essence, are in their innate (and/or induced, depending on the circumstances) mechanical, physical and biochemical integrity.

The term "microbial oxidation" refers to the process of oxidation according to this invention is performed using the intact microorganism or any drug based on it, etc.,

The term "microorganism" includes any intact microorganism or a suitable composition, including, for example, washed Mick is.p., in some cases, where the microorganism is immobilized, for example, in the column attached to the granules, etc.,

Detailed description of the invention
Unless otherwise specified, in this specification and the claims, the following symbols and abbreviations:
oC - degrees Celsius;
% - percentage;
Atsn - acetonitrile;
DMSO - dimethyl sulfoxide;
SPRD - phosphate buffered saline for Dulbecco;
EtOAc is ethyl acetate;
EtOH - ethanol;
g - grams;
HPLC - high performance liquid chromatography liquid chromatography high resolution);
l - liter;
Meon - methanol;
mg - milligram;
min - minute or minutes;
mm - millimeter;
mmol - millimoles;
ml - milliliter;
m-xylene meta - xylene;
N. - normal (concentration);
nm - nanomoles (concentration);
SFR - phosphate buffered saline;
p-xylene - para-xylene;
rpm - revolutions per minute;
TPA - triperoxonane acid;
μl - μl;
about/about - the volume on the volume;
American National Canlocated in Menasha, Wisconsin, USA;
Becton DickinsonLabware is located in Franklin Lakes, New Jersey, USA;
Becton Dickinsonlocated in Walkersville, Maryland, USA;
Column Engineering,, Inc. located in Ontario, California, USA;
IECCentrifuge is located in Needham Heights, Massachusetts, USA;
Rohm and Haaslocated in Philadelphia, Pennsylvania, USA
Traders Proteinlocated in Memphis, Tennessee, USA.

ATCC means American Type Culture Collection (American type culture collection), which is located at 10801 University Boulevard, Manassas, Virginia, 20110-2209, USA. Table 1 lists the microorganisms described in this invention and the depositor(s) (see www.ATCC.com).

As previously described, this invention relates to a microbiological process for the production of compounds of formula I

by bringing into contact of the compounds of formula II

with a microorganism capable of implementing the oxidation of the methyl group of the compounds of formula II, 2-methylinosine, to the carboxyl group of the compounds of formula I, 2-khinoksalinona acid, and incubation of the mixture in suitable conditions to obtain 2-khinoksalinona acid.

The methods of this from microorganism represents R. putida was ATSS 33015 or P. putida ATCC 202190, or a suitable mutant, and then brought into contact with 2 methylinosine to oxidize the methyl group of 2-methylinosine to-COOH group 2-khinoksalinona acid. 2-Khinoksalinona acid can then be subjected to, for example, further processed according to the methods described in the above-mentioned application 801", with the receipt of a new dihydroxypentanoic acids described in the application 801", which is used in the treatment of inflammation and other immune disorders. Activity, methods of evaluation activity, dosages, dosage forms, routes of administration, and other information regarding new dihydroxypentanoic acids disclosed in the application 801", presented in this application (the"application 801").

As described above, in the methods of this invention can be used in any suitable microorganisms or their appropriate mutants. Based on this description, a person skilled in the art will understand that the terms of these methods can be selected depending on, for example, on the type of microorganism and its particular drug. For example, pH, temperature, concentration of components, and so forth, for example, the fermentation medium and of an organic solvent, as well as the specific desired result using the selected microorganism.

Preferred fungi include representatives of the genera Absidia, Actlnomucor, Alternaria, Aspergillus, Coniophora, Cunninghamella, Cylindrocarpon, Diplodia, Epicoccum, Fusarium, Glomerella, Penicillium, Pseudocochilobolus, Thamnostylum and Verticillum, and their species is not limited, provided that the microorganisms or their mutants, able to carry out this oxidation.

Especially preferred fungi are fungi of the genera Absidiar Alternaria, Aspergillus, Cunninghamella, Diplodia and Penicillium.

The most preferred fungi belonging to the genus Absidia.

More specifically, the preferred fungi include A. glauca was ATSS 22752, A. glauca ATCC 74480, A. pseudocylindrospora ADS 24169, A. repens was ATSS 14849, A. repens was ATSS 74481, A. elegans was ATSS 6476, A. solani was ATSS 11078, A. tamarii ADS 16865, C. puteana ADS 12675, C. echinulata ADS a, C. echinulata ADS 8688b, C. echinulata ADS 8983, C. echinulata ADS 9244, C. echinulata ADS 9245, C. echinulata ADS 10028b, C. echinulata ADS 26269, C. echinulata ADS 36190, C. echinulata ADS 36112, S. homothallica ADS 16161, C. destructans was ATSS 66963, D. gossypina ADS 20575, E. neglectum ADS 12723, G. lagenaria ADS 14724, R. clavlforme ADS 10426, P. duclauxii ADS 10440, P. glabrum ADS 11080, P. lunatus ADS 24155, and So piriforme ADS 8686 and suitable mutants.

Preferred fungi include A. glauca was ATSS 22752, A. glauca was ATSS 74480, A. repens was ATSS 14849, A. repens was ATSS 74481, A. solani was ATSS 11078, A. tamarii ADS 16865, C. echinulata ADS 8983, D. gossypina ATCC 20575 and P. glabrum ATCC 11080 and suitable mutants.

Especially the.

The most preferred fungi include A. repens ATCC 14849 and A. repens ATCC 74481 and suitable mutants.

Preferred bacteria include bacteria belonging to the genera Bacillus, Brevibacterium, Micrus, Pseudomonas and Rhodococcus, and their species is not limited, provided that the microorganisms or their mutants are able to carry out this oxidation.

Particularly preferred bacteria include bacteria belonging to the genera Pseudomonas and Rhodococcus.

Preferred bacteria include bacteria of the genus Pseudomonas.

More specifically, preferred bacteria include P. putida was ATSS 33015, P. putida was ATSS 202190 and R. rhodochrous ADS 19067 and suitable mutants.

Particularly preferred bacteria are P. putida was ATSS 33015 and P. putida was ATSS 202190 and suitable mutants.

As previously described, this invention includes the use of any suitable mutants of any suitable microorganisms. In addition, a group of mutants with more desirable properties, for example, is able to oxidize a greater amount of substrate in comparison with the parent strain, can also be used in these ways, and these new strains can be obtained by known techniques, including, for example, standard mutagenesis and breeding techniques, and recom is ucaut chemical mutagenesis using N-methyl-N'-nitrosoguanidine (Delic et al. (1970), Mutat. Res., 9: 167), nitrous acid (Crueger and Crueger (1984), Biotechnology: A Textbook of Industrial Microbiology, p.16, Sinauer Associates, Inc., Sunderland, MA, USA) and exposed to ultraviolet rays (Thrum (1984), in Biotechnology of Industrial Antibiotics (vandamne together, ed.), Marcel Dekker, New York, pp.373-374).

Methods of selection include simple reisolation strain through breeding separated colonies, breeding colonies with defined morphology and breeding colonies resistant analogues of components that are present or suspected present in the biosynthetic pathway of metabolism of compounds of formula I (Crueger and Crueger (1984), Biotechnology: A Textbook of Industrial Microbiology, p.24-25, Sinauer Associates, Inc., Sunderland, MA, USA).

These new strains used in the methods of the present invention, since they, for example, have improved properties compared to the parent strains, for example, they give a greater number 2-khinoksalinona acid, show less unwanted innate destructive effect on 2-methylphenoxy and/or 2-khinoksalinona acid, and/or intermediate compounds that can be obtained in the method of the present invention depending on, for example, selected from a particular organism. In addition, if the mutant is used because of its ability to give the Betwa 2-khinoksalinona acid according to the method of the present invention, you need to grow fewer crops, resulting in significant cost savings.

As described previously, in the method of the present invention may be used any suitable composition of the microorganism, such as, for example, the microorganism in the culture medium, washed microorganism that does not contain, for example, a fermentation medium, the culture broth, and so forth, or immobilized microorganism, for example, in the column attached to the granules, and so on.

From this description, the person skilled in the art will understand the method of obtaining a suitable immobilized intact microorganism, such as described, for example, A. Bauer et al., in the article "Polyvinyl alcohol-immobilized whole-cell preparations for biotransformation of nitriles", published in Biotechnology Letters, 18(3):343-348 (1996).

Preferred intact microorganisms include those that are, essentially, oxidizes 2-methylphenoxy to product, specifically 2-khinoksalinona acid, while leaving the product, essentially unchanged, i.e. free from congenital activity that may destroy or otherwise adversely affect the desired product at any stage of these ways.

Microorganisms suitable for the application of the m, well-known specialist in this field. An example of a suitable method of producing microorganism from commercially available starting sample below. Based on this description, containing the following examples, the person skilled in the art will understand the modifications of any part of these methods, such as method of producing microorganism, free or immobilized; method of bringing into contact 2-khinoksalinona acid with a microorganism; components and environmental conditions for cultivation, such as temperature, pH, and so forth; the respective concentrations of 2-methylinosine, inductor (if used) or incubation conditions to achieve the desired result using any suitable microorganism.

In embodiments of the present invention, in which the microorganism is a fungus, the preferred concentration of 2-methylinosine is from about 0.01 to about 2.5 g/l, especially preferred concentration is from about 0.1 to about 2.0 g/L. In embodiments of the present invention, in which the microorganism is a fungus selected from the group comprising A. repens ATCC 14849, A. repens ATCC 74481, A. glauca ATCC 22752, A. glauca ATCC 74480 and suitable mutants, prepost etenia, in which the microorganism is a bacterium, the preferred concentration of 2-methylinosine is from about 0.01 to about 1.5 g/l and especially preferred concentration is from about 0.1 to about 1.0 g/L. In embodiments of the present invention, in which the bacteria are selected from the group including P. putida ATCC 33015, P. putida ATCC 202190 and suitable mutants, the preferred concentration of 2-methylinosine is from about 0.1 to about 1.0 g/L.

In addition, as described previously, the bacteria carrying the TOL plasmid, such as P. putida was ATSS 33015 or P. putida ATCC 202190 required for this oxidation must be induced. In embodiments of the present invention, in which P. putida was ATSS 33015 or P. putida was ATSS 202190 cultured in the medium in the fermenter, the inductor, preferably p-xylene, add with the preferred speed of adding from about 4.5 mmol/lhour to about 6.5 mmol/lan hour and a particularly preferred speed of about 4.9 mmol/lhour to about 6.1 mmol/lhour.

In embodiments of the present invention, in which P. putida was ATSS 33015 or P. putida was ATSS 202190 are in the medium in the flask, the inductor, preferably p-xylene, is added to the environment cont the above articles and patents (for example, U.S. patent 5236832), the concentration of inducer is usually chosen such that it was less than the minimum inhibitory concentration of the enzymes responsible for the oxidation. Cm. also Claus and Walker, J. Gen. Environ., 36:107-122 (1964).

In this invention may be used any suitable method of bringing into contact of the substrate, 2-methylinosine, with the microorganism. The substrate may be contacted with the microorganism in any suitable manner. For example, 2-methylphenoxy can be added to the environment, such as culture broth containing the microorganism, free or immobilized, or some combination thereof, or the environment may contain 2-methylphenoxy, and the microorganism can then be added in such an environment, or 2-methylphenoxy and the microorganism may be added together in such an environment, or 2-methylpenicillin, or the microorganism may be added in a suitable solvent containing a different reagent, or 2-methylphenoxy can be adsorbed on the resin, and so forth. From this description, the person skilled in the art will understand how you can, if desired, to modify any part of these methods to suit your needs.

As previously described, in this invention it is preferable that a MIC is about the conditions of the Budapest Treaty on 13 January 1999. This newly deposited culture got a new Deposit number ATCC 74480. Therefore, the present invention also preferably, the microorganism was an A. glauca ATCC 74480. All access restrictions the General public to deposited thus the culture of the microorganism will be permanently removed after publication of a patent on this application.

Also, as previously described, the present invention is especially preferably, the microorganism was an A. repens ATCC 14849. Dried sample A. repens ATCC 14849 was deposited in the ATCC under the terms of the Budapest Treaty on 13 January 1999. This newly deposited culture got a new Deposit number ATSS 74481. Therefore, the present invention also preferably, the microorganism was an A. repens was ATSS 174481. All access restrictions the General public to deposited thus the culture of the microorganism will be permanently removed after publication of a patent on this application.

Culture of fungi A. repens was ATSS 14849 (or A. repens was ATSS 74481, A. glauca was ATSS 22752 or A. glauca was ATSS 74480) can be obtained from ATS, and an example of a suitable method of obtaining of such strains is presented below. The original culture is brown rice and about 20 ml of distilled water, placed in an autoclave at a temperature of about 121oWith approximately 30 min, the suspension of vegetative cells or spores of A. repens was ATSS 14849 (or A. repens was ATSS 74481, A. glauca was ATSS 22752 or A. glauca was ATSS 74480) is produced by adding either an aliquot of the liquid culture or smear grown on agar medium culture on the sloped agar sterile distilled water. Each flask with rice seeded with 5 ml of a suspension of spores or cells and incubated for about 10 days at a temperature of approximately 28oWith, then the original solution with spores get irrigating rice culture with 0.5% solution of Tween 80 in distilled water, while the spore suspension with rice and adding from about 10% to about 20% glycerol. The original solution with spores stored at a temperature of about -70oC.

As will be clear to the person skilled in the art, for any of the selected fungi and, as specifically described in the examples are preferred for A. glauca was ATSS 22752 or ATSS 74480 and especially preferred A. repens was ATSS 14849 or ATSS 74481, a suitable method of obtaining the selected fungus is the following way: the fungus is plated from frozen original culture of vegetative cells or spores, such as described above, in a flask or glass tube with a metal cap, sodergran water), the composition of which is described in more detail below. The fermentation is carried out at temperatures in the range of from about 22 to about 32oC, preferably at a temperature of about 29oSince, under suitable agitation speeds of preferably from about 200 to about 220 rpm and more preferably at about 210 rpm If desired pH for cultivation can be supported through the use of appropriate buffers, put into fermentation medium and/or as necessary to periodically reported to the desired level by the addition of either base or acid. The preferred pH range is from pH 6 to pH 7.

In this invention can be used for any suitable duration of cultivation of the microorganism (fungi or bacteria), bringing into contact of the microorganism with 2 methylinosine and incubation 2-methylinosine with the microorganism. Suitable growth of the microorganism can be achieved, for example, within 24 hours, after which the culture can be added to either (a) 2-methylphenoxy in pure form, or (b) a suitable aliquot of the solution of 2-methylinosine in a suitable, i.e. no undesirable influence on the growth or function of the organism, the solvent, Uchenie, for example, from about 2 to about 24 days, depending on, for example, from the vessel in which the bioconversion, medium and incubation conditions, such as temperature, pH, and mixing conditions. The incubation broth can then be extracted using any suitable methods of extraction, such as (a) a method in which a suitable solvent, such as EtOAc, methyl isobutyl ketone, methyl ethyl ketone, dichloromethane, etc., preferably EtOAc, removes organic components of the incubation broth, or (b) adsorption of the product, 2-khinoksalinona acid to a suitable resin, preferably a polymeric adsorption resin, preferably a resin selected from resins Amberlite(Rohm and Haas), most preferably XAD4 (from among the resins Amberlite). After extraction of the incubation broth with a suitable organic solvent and separation of organic and aqueous phase compounds comprising the organic residue may be determined using any suitable technique, such as chromatography. Alternative after extraction 2-khinoksalinona acid from the incubation broth with resin 2-hinoksolinov, and then crystallized, for example, from EtOAc with, for example, EtOAc and Meon.

In the method of this invention can be used in any suitable environment for growing, and a suitable growing medium contains a source or sources of assimilable carbon, available nitrogen and inorganic salts containing minerals. Usually many carbohydrates, such as glucose, maltose, mannose, sucrose, starch, glycerin, millet jelly, syrup, soy, and so forth, can be used as sources of assimilable carbon. Sources of digestible nitrogen include, for example, materials such as hydrolysates and yeast casein, primary yeast, yeast extracts, cottonseed flour, dry soybean residue, wheat germ, meat extracts, peptone, corn extract, dried corn residue and salts of ammonia. Suitable inorganic salts, which are nutrients for use in the environment for the cultivation of this invention include, for example, regular salt contains sodium, iron, magnesium, potassium, cobalt, phosphate, and so forth.

More specifically, the components of the environment for cultivation, suitable for use in this invention, when �/img.russianpatents.com/chr/174.gif" align="BASELINE">and malt extract. Environment based on corn extract contains 40 g/l corn extract and 20 g/l dextrose, and its pH adjusted to 4,85 before sterilization. Environment on the basis of dry corn residue contains from about 20 to about 40 g/l of dry corn residue and about 20 g/l dextrose, and its pH adjusted to 4,85 before sterilization. Other suitable environment for use in the methods of the present invention contains about 20 g/l Pharmamediaand about 20 g/l dextrose, and its pH was adjusted to 7.2 before sterilization. Environment based on malt extract contains about 10 g/l malt extract, 10 g/l of dextrose, about 5 g/l peptone and about 2 g/l yeast extract, and its pH was adjusted to 7 before sterilization. Other suitable environment for use in the methods of the present invention contains about 20 g/l dextrose, 5 g/l nutrient soy flour, about 5 g/l yeast extract, 5 g/l NaCl and 5 g/l To2NRA4its pH was adjusted to 7.0 using H2SO4before sterilization. Especially preferred medium for cultivation of fungi suitable for this method represents the above-mentioned environment on the basis of dry kukuruznaya R. putida ATCC 33015. Lyophilized sample of P. putida ATCC 33015 was deposited in the ATCC under the terms of the Budapest Treaty on 13 January 1999. This newly deposited culture got a new Deposit number ATCC 202190. Therefore, the present invention also preferably, the microorganism was represented by P. putida ATCC 202190. All access restrictions the General public to deposited thus the culture of the microorganism will be permanently removed after publication of a patent on this application.

In addition, the growing medium suitable for use in this invention, when the microorganism is a bacterium, includes any suitable known environment, such as Nutrient Broth (about 32 g/l, Becton Dickinson Microbiology Systems) and glycerin (about 5 g/l). As will be clear to the person skilled in the art, for any selected bacteria, as specifically described in the examples for P. putida ATCC 33015, a suitable method of obtaining the selected bacteria is the following: bacteria are plated from frozen original culture obtained by known in the art methods (approximately 17% glycerol original solution), in a flask or glass tube with a metal lid or a fermenter containing a growing medium (soderi described below. The fermentation is carried out at temperatures in the range of from about 20 to about 40oC, preferably at temperatures of from about 25 to about 32oSince, under suitable agitation, with a speed of preferably from about 200 to about 220 rpm, and most preferably about 210 rpm If desired pH for cultivation can be supported through the use of appropriate buffers, put into fermentation medium and/or
as necessary to periodically reported to the desired level by the addition of either base or acid. Preferably, the inoculum is from about 1 to about 20% V/V (inoculum/environment). The preferred range of pH is from pH 6 to pH 8.

It should be noted that reference to particular buffers, media, reagents, conditions bring into contact or cultivation, the amount of the substrate, the number of inductor when it is used, and so on, in any part of this description are not considered as limiting and include all such related materials that are obvious to the person skilled in the art and which may be of interest or value in the particular context of this description. For example, you can often substitute one buffer system for the e purposes seeks to achieve using the proposed method, material and composition. Moreover, it should be clear that this invention includes the application of this method on an industrial scale.

Consider microbiological oxidation further includes, optionally, the selection of the desired product, 2-khinoksalinona acid. 2-Khinoksalinona acid may be selected, as described below, from the environment where the new process microbiological oxidation and, more specifically, from any intermediate compounds which can be obtained, but not completely transformed into 2-khinoksalinona acid, depending on, for example, from the selected microorganism and conditions of incubation.

In this invention can be used in any suitable methods of separation and/or purification of any intermediates or the desired product of this method, including filtration, extraction, crystallization, chromatography on a column, thin-layer chromatography, preparative liquid chromatography low-pressure HPLC, adsorption on the resin, or any suitable combination of such methods.

Detailed examples, assumptions khinoksalinona acid, which then can be separated from undesirable unreacted 2-methylpenicillin or any intermediate compounds and then subjected to interact well known in the art methods of obtaining, for example, compounds described in the application 801".

Although this description primarily refers to the use in the methods of intact microorganisms, the person skilled in the art will understand that the microbiological processes can be performed with suitable drugs considered microorganisms, such as drugs destroyed and digidrirovanny cells extracted materials containing microbial enzymes capable of this oxidation, or by enzymes, together with any necessary cofactors, and so on. The invention is illustrated by the following examples. Assumes different ways in the previous and subsequent description of the present invention do not limit the scope of the present invention, and only illustrate it. Therefore, it should be clear that this invention is not limited to the specific details of these examples.

EXAMPLE 1.

of the fungus A. repens ATCC 14849.

Three subjects of culture (T1, T2 and T3) are obtained as follows. 2.5 ml sterile environment for growing (about 20 g/l dextrose, 5 g/l nutrient soy flour, about 5 g/l yeast extract, 5 g/l NaCl and 5 g/l To2NRA4with pH, increased to around 7.0 using H2SO4before sterilization) is added to each of the three 16 x 125 mm glass test tubes, each of which has a metal lid (T1, T2 and T3), followed by the addition of spores (about 1% V/V of the original culture of the dispute) A. repens ATCC 14849 in T1, T2 and T3.

Three test tubes with culture is incubated at a temperature of about 29oC, shaking speed of 210 rpm at 48 hours (T1), 72 hours (T2) or 96 hours (T3) in tubes with culture add 0.05 ml of the original solution (50 mg/ml in 100% EtOH to a final concentration of about 1 mg/ml) 2-methylinosine.

After further incubation at a temperature of about 29o(See table 2) fermentation broth contained in test tubes with culture, adjusted to pH 2 with 4 N. Hcl. The contents of each tube is extracted with equal volumes of EtOAc (net) add EtOAc, test tube culture is shaken and then centrifuged with a speed of 2000 rpm (IEC Centrifuge). The EtOAc layer removed and the aqueous layer was extracted with VIR>oC.

C. the Yield of 2-khinoksalinona acid according to HPLC with reversed phase.

Each extract obtained as described above, re-suspended in about 1 ml of a mixture of atsn:water (1:9 V/V) and 20 ml of each re-suspended extract, analyze, injecting into the column for HPLC (column Inertsil HPLCC8 C8, a 4.6 x 250 mm, Column Engineering, Inc.). Compounds contained in each injected resuspending extract, separate isocrate at a rate of about 1.0 ml / minute mobile phase (atsn: of 0.05% aqueous TFA, 1: 4, V/V). Under these conditions, 2-khinoksalinona acid eluted in approximately 8,6 min, and 2-methylphenoxy eluted after about 15 minutes the Yield of 2-khinoksalinona acid is determined according to the HPLC analysis for multiple sets of experimental conditions (i.e. T1, T2 and T3), the results are presented in table 2.

As can be seen from the data for T1, T 2 and TK, are presented in table 2, analysis by HPLC showed that the microbiological method gives a yield of the desired 2-khinoksalinona acid 56, 76, and 79%, respectively.

Therefore, the introduction of the intact microorganism, for example A. repens ATCC 14949 causes oxidation of 2-matildis unaffected.

EXAMPLE 2.

OXIDATION of 2-METHYLINOSINE IN VITRO CULTURE WITH the USE of A. repens ATCC 14849 IN FOUR DIFFERENT ENVIRONMENTS FOR GROWING
A. Obtaining four different environments for growing.

Wednesday 1 is prepared with 40 g/l corn extract and 20 g/l dextrose and bring the pH up to around 4.85 before sterilization.

Wednesday 2 is prepared with 40 g/l of dry corn residue and 20 g/l dextrose and bring the pH up to around 4.85 before sterilization.

Wednesday 3 is prepared with 20 g/l Pharmamediaand 20 g/l dextrose and bring the pH to 7.2 before sterilization.

Wednesday 4 is prepared from 10 g/l malt extract, 10 g/l of dextrose, 5 g/l peptone and 2 g/l yeast extract and bring the pH to 7 before sterilization.

Century Bioconversion using fungus A. repens ATCC 14849.

Eight of the tested cultures (T1a, T1b, T2A, 2b, TA, 3b, TA and 4b) was obtained as follows. 2.5 ml sterile environment for growing (Medium 1, Medium 2, Medium 3 and Wednesday 4 respectively) is added to each of the eight 16 x 125 mm glass test tubes, each of which has a metal cover (T1a, T1b, T2A, 2b, TA, 3b, TA and 4b), followed by the addition of spores (1% V/V of the original culture of the dispute) A. repens ATCC 14849 in each tube with culture.

Eight tubes with culture inqulab 72 hours (T1b, T2b, 3b and 4b) in tubes with culture add about 0.05 ml of the original solution (50 mg/ml in DMSO, final concentration of about 1 mg/ml) 2-methylinosine.

After further incubation at a temperature of about 29oWith over 12 days fermentation broth contained in test tubes with culture, is extracted and the combined organic extracts dried as described in example 1.

C. the Yield of 2-khinoksalinona acid according to HPLC with reversed phase.

Each extract, obtained as described above, then treated and analyzed by HPLC with reversed phase, as described in example 1. The yield of 2-khinoksalinona acid is determined according to the HPLC analysis for multiple sets of experimental conditions (i.e., T1a, T1b, T2A, 2b, TA, 3b, TA and 4b), the results are presented in table 3.

As can be seen from the data presented in table 3, analysis by HPLC showed that the microbiological method, in which the microorganism is a A. repens ATCC 14849, gives the yield of 2-khinoksalinona acid in all tested environments. The data presented in table 3 also show that of the four test environments environment 2 gives the highest % yield of the desired product, 2-henok repens ATCC 14849 OR A. glauca ATCC 22752 A. Bioconversion using fungus A. repens ATCC 14849 or fungus A. glauca ATCC 22752.

Four subjects of culture (T1a, T1b, T2a and 2b) was obtained as follows. 25 ml of sterile medium for cultivation (20 g/l dextrose, 5 g/l nutrient soy flour, 5 g/l yeast extract, 5 g/l NaCl and 5 g/l To2NRA4with pH, increased to around 7.0 using H2SO4before sterilization) is added to each of the four conical flasks (300 ml) followed by addition of dispute (1% V/V of the original culture of the dispute) or A. repens ATCC 14849 (T1a, T1b) or A. glauca ATCC 22752 (T2a, T2b).

Four flasks with cultures incubated at a temperature of about 29oC, shaking speed of 210 rpm Immediately after sowing (T2a) or 24 hours (T2b) in flasks with culture A. glauca ATCC 22752 add about 0.5 ml of the original solution (50 mg/ml in 100% EtOH to a final concentration of about 1 mg/ml) 2-methylinosine, and 48 hours (T1a) or 72 hours (T1b) in flasks with culture of A. repens ATCC 14849 add about 0.5 ml of the original solution (50 mg/ml in 100% EtOH, the final concentration of about 1 mg/ml) 2-methylinosine.

After further incubation at a temperature of about 29oWith over 24 (T1a), 16 (T1b), 25 (T2a) or 24 (T2b) days of fermentation broth contained in flasks with culture, adjusted to pH 2 with p the combined EtOAc extracts under reduced pressure to get crude product.

C. the Yield of 2-khinoksalinona acid according to HPLC with reversed phase.

Each extract, obtained as described above, re-suspended in about 5 ml of the Meon:atsn (3:2 V/V) and diluted 1:19 with water for analysis by HPLC. Analysis by HPLC were carried out as described in example 1. The yield of 2-khinoksalinona acid is determined according to the HPLC analysis for multiple sets of experimental conditions (i.e., T1a, T1b, T2a and 2b), the results are presented in table 4.

As can be seen from the data presented in table 4, the introduction of the intact microorganism, for example, A. glauca ATCC 22752 or A. repens ATCC 14849 causes oxidation of 2-methylinosine to 2-khinoksalinona acid. The percentage of the source material, i.e., 2-methylinosine, which remains in T1a, T1b, T2a and T2b, is 7, 7, 6 and 6%, respectively.

EXAMPLE 4.

SCREENING of MICROBIAL TRANSFORMATION of 2-METHYLINOSINE 2-KHINOKSALINONA ACID
Cells of various microorganisms are grown in test tubes containing 2.5 ml of medium on the basis of dextrose and nutritious soy flour, described in example 1. Each tube is seeded with spores to vegetative cells (1% V/V of spores to vegetative cells in the original culture) various up>With under stirring (210 rpm) on a rotary shaker. After 48 hours in each tube add 0.05 ml of 10 mg/ml solution of 2-methylinosine in DMSO. After 4 days of incubation the contents of each tube are extracted and separate extracts analyzed by HPLC as described in example 1. Outputs 2-khinoksalinona acid was determined by HPLC, the results of the analysis are presented in table 5.

EXAMPLE 5.

OXIDATION of 2-METHYLINOSINE IN FLASKS WITH CULTURE USING P. putida ATCC 33015
Cells of P. putida ATCC 33015 grow in the Environment 5 (Nutrient Broth (about 32 g/l) and glycerol (about 5 g/l)). Six conical flasks (300 ml) containing about 30 ml of medium, seeded with 0.10 ml of a suspension of cells of P. putida ATCC 33015 in glycerol, previously stored at -70oC. After adding about 2 ml of p-xylene contained in a 15 ml conical polypropylene centrifuge tube (Falcon, Becton Dickinson Labware), bulb seal Parafilm(American National Can) and stirred (speed 225 rpm) on a rotary shaker for about 18 hours at a temperature of about 29oC. Data culture in flasks, have an optical density of about 1.9, serpentinous in 20 ml SPR (Biowhittaker) in 300 ml conical flask.

The bioconversion start by adding about 0.1 ml 100 mg/ml solution of 2-methylinosine in DMSO, which corresponds to an initial concentration of about 0.5 g/l Incubation continued for 4 days at a temperature of about 29oWith under stirring with a speed of 225 rpm Samples of the broth in which occurred bioconversion, taken at different points in time and, after removal of cells by centrifugation and dilution Meon to the extent necessary, analyzed by HPLC. About 20 µl of each of these samples analyzed by injection into the column for HPLC InertsilC8 (a 4.6 x 250 mm). Each column elute at a speed of 1.0 ml/min mobile phase consisting of atsn:of 0.05% aqueous TFA (1: 4, V/V). The yield of 2-khinoksalinona acid is 86, 90 and 94% after 1, 2 and 4 days of incubation, respectively.

EXAMPLE 6.

OXIDATION of 2-METHYLINOSINE IN FERMENTER CULTURE WITH the USE of P. putida ATCC 33015
P. putida ATCC 33015 grown in a fermenter containing 10 l of medium 5. The fermentor inoculated six cultures of P. putida, each of which is grown in Erlenmeyer flask (300 ml) containing 50 ml of medium 5. Each flask culture seeded with 175 μl of the original solution of the dispute P. putida ATCC 33015, paste 15 ml r/174.gif" align="BASELINE">. Flasks with culture is incubated at a temperature of about 29oC for 17 hours while stirring with a speed of 210 rpm After sowing fermentor 6 cultures contained in the flasks in the fermenter add p-xylene in a 2 ml aliquot every 20 min for 2 hours. Then in the fermenter add 2.5 ml aliquot of p-xylene every 20 min for 3.5 hours. Then adding xylene stop and add 2-methylphenoxy through the 5.25 hours (1,95 g) and 7.75 hours (7,76 g) after sowing. Incubation continued for 22 hours after the last addition of 2-methylinosine. Sample incubation medium centrifuged to remove cells, dilute the Meon and analyzed by HPLC according to the method of example V. This analysis showed an 81% yield of 2-khinoksalinona acid.


Claims

1. The way microbiological oxidation of 2-methylinosine to 2-khinoksalinona acid, characterized in that 2-methylphenoxy brought into contact with the microorganism and incubated the mixture at conditions that provide 2-khinoksalinona acid, while the microorganism is chosen from the group including Absidia glauca ATCC 22752, Absidia glauca ATCC 74480, Absidia repens ATCC 14849, Absidia ATCC 33015 and Pseudomonas putida ATCC 202190, provided that if the microorganism is Pseudomonas putida ATCC 33015 or Pseudomonas putida ATCC 202190, the microorganism before putting in pin 2-methylinosine induce the addition of inducer.

2. The method according to p. 1, characterized in that the microorganism is chosen from the group including Absidia glauca ATCC 22752, Absidia glauca ATCC 74480, Absidia repens ATCC 14849, Absidia repens ATCC 74481, Alternaria solani ATCC 11078, Aspergillus tamarii ATCC 16865, Cunninghamella echinulata ATCC 8983 and Diplodia gossypina ATCC 20575.

3. The method according to p. 2, characterized in that the microorganism is chosen from the group including Absidia glauca ATCC 22752, Absidia glauca ATCC 74480, Absidia repens ATCC 14849, Absidia repens ATCC 74481, Alternaria solani ATCC 11078 and Aspergillus tamarii ATCC 16865.

4. The method according to p. 3, characterized in that the microorganism is chosen from the group including Absidia glauca ATCC 22752, Absidia glauca ATCC 74480, Absidia repens ATCC 14849 and Absidia repens ATCC 74481.

5. The method according to p. 4, characterized in that the microorganism is chosen from the group including Absidia repens ATCC 14849 or Absidia repens ATCC 74481.

6. The method according to p. 1, characterized in that the microorganism is chosen from the group comprising Pseudomonas putida ATCC 33015 or Pseudomonas putida ATCC 202190.

7. The method according to any of paragraphs.1-6, characterized in that the inductor is p-xylene.

8. The method according to p. 7, wherein the Pseudomonas putida ATCC 33015 or Pseudomonas putida ATCC 202190 is in the growing medium.

9. The method according to p. 8, otlichie cultivation is in the flask.

11. The method according to p. 9, wherein the growing medium is in the fermenter.

12. The method according to p. 9, characterized in that it further includes a step of collecting the cells of the microorganism after induction.

13. The method according to p. 12, characterized in that the collecting is carried out by centrifuging the contents of the flask, desantirovaniya liquid, followed by washing the precipitate cells after centrifugation and re-suspendirovanie specified sediment cells in the buffer.

14. The method according to p. 13, characterized in that 2-methylphenoxy brought into contact with the microorganism by adding it to the buffer after re-suspension.

15. The method according to p. 9, characterized in that the addition of p-xylene on Wednesday to stop growing after induction.

16. The method according to p. 15, characterized in that the addition to the culture medium 2-methylinosine carried out after termination of the addition of p-xylene.

17. The method according to p. 1, characterized in that it further includes a step of allocating 2-khinoksalinona acid.

18. The method according to p. 17, wherein the selection is carried out by extracting the mixture with an organic solvent.

19. The method according to p. 18, characterized in that the organic restout.

21. The method according to p. 17, characterized in that the separation of the 2-khinoksalinona acid carried out by its adsorption of the mixture to the resin, followed by elution of the adsorbate with an organic solvent.

22. The method according to p. 21, wherein the adsorption is carried out on polymeric adsorbent resin.

23. The method according to p. 21, wherein the organic solvent is ethyl acetate or methanol.

24. The method according to any of paragraphs.1, 22 and 23, characterized in that it further includes a step of crystallization elyuirovaniya 2-khinoksalinona acid from ethyl acetate.

25. The way microbiological oxidation of 2-methylinosine to 2-khinoksalinona acid, characterized in that 2-methylphenoxy brought into contact with a microorganism selected from the group including Absidia repens ATCC 14849 or Absidia repens ATCC 74481, and incubated the mixture at conditions that provide 2-khinoksalinona acid.

26. The method according to p. 25, characterized in that the microorganism is in the growing medium.

27. The method according to p. 25, characterized in that 2-methylphenoxy brought into contact with the microorganism by adding it to the growing medium.

28. The way microbiological akisu contact with the microorganism, selected from the group including Pseudomonas putida ATCC 33015 or Pseudomonas putida ATCC 202190, after the induction of enzymes of the microorganism interaction with the inductor, and incubated the mixture at conditions that provide 2-khinoksalinona acid.

29. The method according to p. 28, characterized in that the inductor is p-xylene or m-xylene.

 

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