DE2054785A1 - Process for the preparation of cytidine diphosphatcholine - Google Patents

Description

PATENT Attorney E. 2054785 Dipi. formerly Dr. D. Thomsen Dipi.-mg. H. Tiedtke
Dipl.-Chem. G. BÜhlmg Dipl.-Ing. R. ΚΪΠΠΘ MUNICH 15
KAISER-LUDWIG-PLATZ β
TEL. 0811/530211
530212
CABLES: THOPATENT
TELEX: TO FOLLOW Dipi.-mg. W. We Inka uff FRANKFURT (MAIN) 50
FUCHSHOHL 71
TEL. 0611/514666

Reply requested to: Please reply to:

8000 Munich 15 ^ 6, NOV. ! 970

T 3889 / case MFP-IOl J \ sahi

Asahi Kasei Kogyo Kabushiki Kaisha Osaka (Japan)

Process for the production of cytidine diphosphate

oholin

The invention relates to a method of manufacture of cytidine-diphosphate-choline (hereinafter referred to as CDP-choline) with the use of an enzyme of a Microorganism. GDP-choline plays an important role in the metabolism of living tissue and is important as a medicine or biochemical reagent. It is said that CDP-choline is present in living tissue and particularly in the tissue of the Brain works.

Hericönimlicherweiaö vdrd CDP-Choline prepares, .Inder ;, oue:;: iom \ k; 7 / jbe oxtrnr: iert und dor, ::: xür: .lci r ^ i.kt, '- '■>! -). c; ri ciu li _v : null.; _; i ^ ;. y; iuci.fti, '-. ■. ·;: ..,.'. ;; ■.

ORIGINAL

2034785

However, these processes are inferior in terms of yield and are not advantageous as a commercial process.

An economical process for the production of CDP-choline has now been found and developed. According to the invention, cells of cultivated microorganisms, with or without physical or chemical treatment, to a liquid added which S'-cytidylic acid or cytidylic acid, their Salts or one of their precursors, a choline, its salts or phosphatydilcholine, sources of carbohydrates, phosphate compounds and Contains magnesium salts.

The invention includes a novel microbiological process for the production of cytidine-diphosphate-oholin, which is effective for the metabolism of living tissue. This procedure consists in that one dried cells of Microorganisms applied to a medium, welohes 5'-cytidylic acid, its salts or its precursors, choline, its salts or phosphatydil-oholin (lecithin), sources of carbohydrates, Contains phosphate compounds and magnesium salts.

The invention uses microorganisms that are efficient in producing CDP Chclis. Characteristics of the microorganisms, weloh® have the ability to produce CDP-choline

ORIGINAL INSPECTED

the production of fructose 1,6-diphosphate from carbohydrates. Microorganisms that have a high ability to produce fructose-1,6-diphosphate Produced from carbohydrates are preferred. Expressed more concretely, the ability is related to consumption inorganic phosphate in relation. You use those that are at least 20 inches, preferably 40 inches or have higher consumption of inorganic phosphate. For example, the yeasts mentioned in the examples have an inorganic phosphate consumption of 40 to 80 pounds.

Measurement of the ability to make fructoee 1.6-diphosphate is done as follows:

1 g of dried cells, 1.5 com of water and 0.7 oom of toluene are sufficiently mixed with one another. The mixture is left with intermittent,. Stir for about 30 minutes at room temperature. To this mixture is added 50om of a 0.54 m phosphate buffer solution with a pH of 7.0 (prepared by dissolving 120 mg of KH ₂ PO. And 650 mg of Ua ₂ HPO .. 12HgO in water, so that 5 ecm Solution) and add 1 g of glucose (or sucrose). The thus obtained mixture is sufficiently mixed and then leaves it at 30 to 37 ⁰ C are. The portions of the reaction mixture are gradually discharged and the content of inorganic phosphate radical is analyzed quantitatively. The reaction mixture (1 to 2 com) is brought into

1 0

2 3 / / / 1

• into a narrow hole and put a drop of dodeoyl alcohol added. The mixture is heated for 3 minutes in a boiling water bath. The heated Gemisoh is used to precipitate cells centrifuged. The liquid above (0.5 oom) is taken and 2.0 oom 6% HClO is used. added. The proteins are filtered and the inorganic phosphate content is measured for part of the nitrate. The amount of inorganic phosphate consumed in 5 to 6 hours is considered to be the ability to produce fructose-1,6-diphosphate. The microorganisms include yeasts, Fungi and bacteria. The yeasts are those to which Brettanomyoes, Saooharomyoes, Candida, Torulopsis, Zygosaooharomyoes, Debaryomyoes, Endomyoopsia, Lipomyoes, Endomyoes, Sporoboloayoes, Piohia, Hansenula, Trigonopsis, Cryptooooous, Shizosaooharomyoes and Bhodotorula. Preferred examples are"

Brettanomyoes petrophilum n. Ep ATCO 20224 » Saooharomyoes rouxii AMS 1024 » Sacoharomyoes oarlsbergensis IAM 4206, Candida utilis IAM 4220, Candida petrophilum n. Ep ATCC 20226, Tolulopsis petrophilum n. Sp ATOC 20225, Torulopsis oandida IFO 0768, Zygosaooharomyoes soy IFO 0495,

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Debaryomyoes handenii IFO 0023 » Endomyoopsis ohodati (N) WICKERMAN and BUHIOH OUT 6270,

Lipomyces lipoferue IPO 0673 » Endomyoes hordei IFO 0104, Sporobolomyoes roseus IFO 1037 » Piohia miso IFO 0193, Hansenula Jadini IFO 0987, Trigonopsis variabilia IFO 0671, Cryptoooo0U8 albidus IAH 4830, Shizosaooharomyoes pombe AUS 1022 and HiLodot ο mil a glutinis IFO 0389

Brettanomyoes petrophilum, Saooharomyoes rouxli, Saooharomyoes oarlsbergensiB, Candida petrophilum, Torulopsis oandida, Zygosaooharomyoes soja, Debaryomyoes handenii, etc. are on most preferred.

The bacteria are for example} Miarobaoterium ammoniapnilum ATCC 15354, Corynebaoterium petrophiIum ATCC 19080 and Brevibaoterium ammoniagenes ATOC 6871

Particularly suitable strains of the fungi are the following »

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Aepergillua fumigatus IAH 4040, Penioillium ohrysogenum IPO 4626, Rhizppue nlgrioans IFO 478 and Muoor javanious IAM 6087.

The microorganisms are first cultured to prepare cells. Cultivating is done in a way durohführung, which is familiar to the expert. A liquid one Culture medium, which sources of carbon, nitrogen sources, containing inorganic substances and, if necessary, a trace of organic nutrients, is most brewable.

The carbon sources include: starch, glucose, suorose, Maltose, glycerine, waste molasses, hydrocarbons, organiaohθ acids, alcohols, etc. The organic acids include Acetic acid, citric acid, fumaric acid, etc. The alcohols are saturated lower primary alcohols with 1 to 3 carbon atoms. Illustrative examples of sources of nitrogen are ammonia, nitrates, urea, ammonium salts, etc. Nitrates include sodium nitrate, potassium nitrate, magnesium nitrate, Ammonium nitrate, etc. As inorganic substances, salts such as potassium phosphate, magnesium sulfate and other metal salts can be used be mentioned. Organic nutrients include: Grain Preserving Lye, NZ-Amine, Fisohmeal, Fepton, Meat Extract, Yeast Extract, etc. An example of a culture medium

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(11) is the following

Glucose 6 96 ( urea 0.4 ( Ar ⁰ InOTiI iwffui ^ r * " 0.2 ( phosphate 0.2 ( Magnesium sulfate 0.1 ( Calcium chloride ( 0.01 *

■)

Grain Soaking Lye 0.2 # ( ^N )

Yeast ExtraJrt 0.059 ^ (") Water rest

The optimal conditions for cultivation depend on the microorganisms used. The pH value of the culture medium, one of the parameters, should be kept at the desired value depending on the microorganisms. In general, the pH for yeasts and fungi is 3 to 7 and for bacteria 6 to 8. A pH controller is used to maintain the pH. Such agents include ammonia, sodium hydroxide, hydrochloric acid, sulfuric acid, etc. The cultivation temperature is 25 to 35 ⁰ C, though the microorganisms have optimum temperatures within this range Respective. The cultivation is carried out for 1 to 3 days under aerobic conditions.

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For this purpose, air is introduced into the culture medium.

As indicated above, the formation reaction of CDP-choline carried out by using the thus obtained Cells added to the solution, which, in addition to gytosine compounds and choline compounds as substrates, carbohydrates, Contains inorganic phosphates and magnesium ions.

The cells of the microorganisms are used as a source of enzyme. The culture medium obtained above can, as it is, be used. Cells which have been separated from this culture medium with or without washing are preferred. That Washing is done with water, a physiological saline solution, a buffer solution, etc.! The washed cells can be used in the form of a suspension. Further can the cells at low! temperature with acetone, ether, Toluene, ethanol, a surface active agent, etc. can be treated. The cells can also be ground. Naturally you can use dried cells. The 5'-cytidylic acid can be in free form or in the form of their water-soluble salts such as sodium or alkali metal salts. Insoluble Salts like their! Alkaline earth salts are not preferred. Instead of 5'-ytidylic acid, its precursors such as Cytosine or cytidine can be used. Their optimal concentration (1 each) is in the range from 5 to 50 m-mol.

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The choline can for example by such salts as Gholin chloride, choline phosphate, or lecithin (phosphatidylcholine) be replaced. A preferred amount is 20 to 200 m-mol / l.

The carbohydrates include glucose, sucrose, pructose and maltose. These zuokers can be brewed to phosphate the cytosine compounds. They can be contained in an amount of 2 to 15 i> (weight AoI.).

The inorganic phosphates include sodium phosphate, primary potassium phosphate, secondary potassium phosphate, etc. These Phosphates act as a source of the phosphoric acid part, which is a component of CDP-choline. Your concentration ranges from 0.1 to 0.5 mol / l.

The magnesium ion can be in the form of magnesium sulfate, Magnesium chloride, etc. are present. Ss plays a very important role Bolle in biological reactions and takes part in the formation of CDP choline. The addition of magnesium ion supports the handling of the present reaction. The suitable concentration is in the range from 0.01 to 0.05 mol / l. Furthermore, the presence of a substance such as surface-active agents, sodium fluoride, borates, copper salts, sodium bicarbonate, Acetaldehyde, toluene, ether, etc. the enzymatic ones

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- ίο -

Reaction. These substances suppress the de-amination effect of the cytosine compounds. It is a surfactant preferably non-ionic. These include polyoxyethylene sorbitan monoalkylate, sorbitan monoalkylate, polyoxyethylene orbit alkylate, etc. The borates include sodium borate, Potassium borate, magnesium borate, etc. Any other metal salts can be used as long as they are not used in the reaction Form medium-insoluble phosphates by binding to the phosphoric acid part.

An example of a composition (11) of the above-mentioned solution is as follows:

5'-cytidylic acid (or

their precursor) 10 - 20 mM

Choline (or choline derivative) 50-100 mM Phosphate buffer (pH 7.5) 150-250 mM Glucose 4 - 8? 6 (w / v) Magnesium sulfate 5-15 mM Cells 3 - 10 56 (w / v) Toluene 1- $ 5 (v / v)

The pH of the solution should be kept in the range from 6 to 9, preferably from 7 to 8. The reaction is carried out at a temperature of 20 to 45 ⁰ C under stirring leiohtem S0Mitteln or duroh. The reaction continues,

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until the maximum amount of CDP-choline is produced. The implementation is usually completed within about 15 to 20 hours.

When the reaction is complete, the ODP-choline is removed from the Reaction mixture in a conventional manner, for example by sediment! eren, isolated. But you can also isolate with Ion exchange resin, active charcoal or solvents carry out. Any combination of these operations can be used.

In the following examples, the percentages are expressed as weight / volume (g substance in 100 com mixture).

example 1

A strain of Brettanomyces petrophilum n. Sp ATCG 20224 40 hours at a pH value of 5 to 6 and at 3O ⁰ C in a culture medium cultured aerobically which # 6 sucrose, 0.4 # urea, 0.2 i »ammonium sulfate , 0.2 ^ primary potassium phosphate, 0.05 5 * magnesium sulfate, 0.01 i> calcium hydroxide and 0.2 $> cereal steeping liquor. This culture medium is centrifuged and 144 g of cells are obtained. The cells obtained in this way are treated with acetone and then dried. The cells (120 g) are added to 2000 com of an aqueous medium which contains 15 g of sodium 5'-oytidylate, 145 g of glucose,

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Contains 6 g of magnesium sulfate ι 20 g of primary potassium phosphate, 30 g of secondary sodium phosphate and 5 to 9 g of choline chloride. This mixture is allowed under shaking at 32 ⁰ C for 12 hours to react, wherein g / dl 0.725 form CDP-choline.

The reaction mixture is heat-treated ^{at 90 ° C. for 3 minutes.} Solids are removed by filtration and washed with water. The wash water is added to the filtrate.

The filtrate, adjusted to pH 3.5, is passed through a column of active charcoal. The column it is washed with water and eluted with 50 # aqueous ethanol solution which contains ammonia. The eluted solution concentrated so far that it has a pH of 8. The solution concentrated in this way is passed through a column Go Dowex 1x2 resin (Cl type). Then the column with Washed water and fractionally eluted by a solvent of the HCl-NaCl system. The CDP-choline fractions are collected. The fractions collected in this way are in turn desalinated by passing them through a column of active charcoal lets go through. Barium chloride is added to the desalted solution to produce the CDP-choline in the form of the barium salt to fail. Sodium sulfate is added to remove the barium in the form of barium sulfate, while the CDP-choline is isolated as the sodium salt. The yield is

1 09823/22 1 A

carries 10.2 g as free CDP-choline. In addition, 1.8 g of cytidine-5'-triphosphate (CfDP) and 0.7 g of cytidine-5'-diphosphate (CDP) formed.

Example 2

A strain of Torulopsis petrophilum n. Sp ATCC 20225 is cultivated in the same manner as in Example 1. The culture medium obtained in this way is centrifuged off and cells are obtained. The cells are freeze-dried. The freeze-dried cells (80 g) are added to 1000 oom of an aqueous medium containing 7.35 g of 5'-cytidylic acid, 5'-01 g of choline phosphate, 72 g of glucose, 4 g of magnesium sulfate, 9.8 g of primary potassium phosphate, 15 , 2 g secondary sodium phosphate, 18% toluene and 22 g sodium bicarbonate. This Gemisoh allowed to 8 hours at 37 ⁰ O to react, with 7.44 g / l CDP-Oholin form. The CDP-choline thus obtained is processed in the same way as in Example 1 and 5.21 g of free CDP-choline are obtained.

Example 3

A strain of Candida petrophilum n. Sp. ATCC 20226 is 24 hours in the same culture medium (2 1) as that of Example 1 is cultivated.

The entirety of the culture medium thus obtained becomes

1 0 9 8 2 3/2 2 1 A

500 oom of a 0.18 M phosphate buffer solution (pH 8) was added, which contains 2.2 g of cytidine, 32 g of Suoroae, 1.5 g of magnesium sulfate, 25 oom of toluene and 2.1 g of choline phosphate. This mixture is left to 18 hours at 30 ⁰ O react to form SiOH 0.928 g CDP-choline form. The Gemisoh is worked up in the same way as in Example 1 and 650 mg of pure CDP-choline are obtained.

Example 4

A strain of Sacoharomyces rouxii AMS 1024 is, with stirring for 22 hours at 3O ⁰ C aerobically in a culture medium (pH to 7) cultured containing 3 $> waste molasses (as glucose concentration), 0.2 i> urea, ammonium sulfate 0.1 # containing 0.1 # potassium primary phosphate and 0.05 i ° magnesium sulfate. The end. Cells are collected from the culture medium obtained in this way by separating them in the centrifuge and these are washed once with water. _{3 tons of} 7 g of sodium S'-oytidylate, 2.1 g of choline chloride, 72 g of glucose, 1.8 g of magnesium sulfate, 4.9 g of primary potassium phosphate, 7.6 g of secondary sodium phosphate are added to 500 ecm of the cell suspension in city water and add 25 oom of ethanol. Sas Gemisoh allowed to 16 hours at 37 ⁰ C to react, with 3.84 g of CDP-choline form. The gel is worked up in the same way as in Example 1 and 2.69 g of CDP-choline are obtained.

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Example 5

A strain of Miorobacterium ammoniaphilum ATCC 15354, is cultivated under the same conditions as those of Example 3. Duroh separation in the centrifuge cells are obtained from the culture medium and left under the same conditions as those of Example 3 react. 1.08 g of CDP-choline are formed in the reaction medium. The reaction medium is worked up in the same way as in Example 1 and 760 mg of CDP-choline are obtained.

Example 6

A strain of Corynebacterium petrophilum ATCC-19080 is cultivated in waste molasses in the same manner as in Example 4. The culture medium thus obtained is processed in the same manner as in Example 4 to obtain dried cells. The dried cells (10 g) are added to a mixture of 360 mg cytosine, 15 g glucose, 300 mg magnesium sulfate, 501 mg choline phosphate, 20 mg sodium fluoride and 100 ecm of a 0.2 molar phosphate buffer solution (pH 7.5). The mixture is allowed to ^{react for 24 hours at 30 °} C. and 270 mg of CDP-choline, 240 mg of CTP and 140 mg of CDP are formed. The mixture is processed in the same way as in Example 1 and 190 mg of free CDP choline are obtained,

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- 16 Example 7

A strain of Zygosacoharomyces soja IPO 0495 is cultivated in the same manner as in Example 1. This culture medium is treated with acetone and 50 g of dried cells are obtained. The dried cells obtained in this way (50 g), together with 4 g of 5'-cytidylic acid, 4.2 g of choline phosphate, 40 g of glucose, 2.0 g of magnesium sulfate, 10 com of toluene and 5 ecm of acetaldehyde, with 600 com of a 0.2 molar phosphate buffer solution (pH 7.5) mixed "This mixture is left to 16 hours at 32 ⁰ C to react while shaking, with 3.68 g GDP-choline form. The mixture is worked up in the same way as in Example 1 and 2.58 g of free CDP-choline are obtained.

Example 8

A strain of Muoor javanious IAM 6087 is sohüttelkultiviert 72 hours at 30 ⁰ C in a culture medium (pH 6.2) containing 5 $> glucose, 0.5 # peptone, 0.5 9 ^ meat extract, 0.5 $> soybean powder containing 0.05 fi magnesium sulphate, 0.01 calcium chloride and 0.2 #? 6 potassium nitrate. Cells are separated from the culture medium by a centrifugal separation device and then freeze-dried. The cells are then treated with acetone and 5 g of dried cells are obtained. The cells (5g), together with 380 mg of sodium 5'-oytidylate, 147 mg of choline, 4.2 g of glucose,

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160 mg of magnesium sulfate and 20 mg of sodium bicarbonate, added to 50 μm of a 0.2 molar phosphate buffer solution (pH 7.0). This mixture is allowed to ^{react for 8 hours at 37 °} C. and 118 mg of CDP-choline are formed. The mixture is worked up in the same way as in Example 1 and 83 mg of free CDP-choline are obtained.

Example 9

A strain of Piohia miso IPO 0193 is cultivated under the same conditions as those in Example 1. This culture medium is treated with acetone and 25 g of dried cells are obtained. The dried cells (25 g), together with 2.0 g of 5'-cytidylic acid, 1.4 g of ololine phosphate, 20 g of glucose, 0.9 g of magnesium sulfate, 6 com of toluene and 2 ocm of acetaldehyde, with 300 com of 0, 2-molar phosphate buffer solution (pH 7.0) · vermisoht This mixture is allowed to 6 hours at 35 ⁰ O with shaking react, forming 1.43 g of CDP-choline, and CTP 0.43 g 0.11 g CDP. The mixture is worked up in the same way as in Example 1 and 1.0 g of free CDP-choline is obtained.

Example 10

A strain of Brevibaoterium ammoniagenes ATCC 6871, is cultivated in a waste molasses culture medium given in Example 4. Using a centrifuge separator

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cells are separated from the culture medium and these are freeze-dried, giving 10 g of dried cells. The dried cells (10 g), together with 720 mg of 5-cytiylic acid, 9.0 g of glucose, 250 mg of magnesium sulfate and 501 mg of choline phosphate, are added to 100 oom of a 0.2 molar phosphate buffer solution (pH 7.0). This mixture is allowed to ^{react for 8 hours at 28 °} C., 680 mg of CDP-choline, 120 mg of CTP and 150 mg of CDF being formed. The mixture is worked up in the same way as in Example 1 and 480 mg of free CDP-choline are obtained.

Example 11

A strain of Aspergillus fumigatus IAM 4040 is found under cultured under the same conditions as those of Example 8, and cells are obtained therefrom. The cells become Treated with acetone to achieve dried cells. The cells so obtained are used as an enzyme and cytosine as a substrate used instead of 5'-cytidylic acid »The reaction leads in the same manner as in Example 8 to give 78 mg of CDP-choline. The mixture works in the same way As in Example 1arf and obtain 55 mg CDP-choline.

Example 12

The reaction is carried out using 12 g of lecithin (phosphatidyl-oholin) instead of choline chloride,

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under exactly the same conditions as those in Example 1. 0.418 g / dl CDP-choline is obtained. The cfemish it is worked up in the same way as in Example 1 and 5.8 g of CDP-choline are obtained in 2 liters of reaction mixtures

Example 13

The cultivation, treatment of the cells and the enzymatic reaction are carried out in the same manner as in Example 4 with the exception that a strain of Debarycmyces handenii IFO 0023 is used as the microorganism instead of Saccharomyces rouxii, and 500 mg of polyoxyä-fchylene-sorbitan monoaikylat used instead of ethanol. 3.64 g of CDP-choline were formed in the reaction mixture. The mixture is worked up in the same way as in Example 1 and 2.55 ε choline is obtained.

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Claims (11)

Claims Process for the production of cytidine-diphosphatcholine, characterized in that an enzyme which is contained in cells of microorganisms that has the ability of Produced by fructose "! .6-diphosphate" on a medium applies, the latter being a pyrimidine compound S'-cytidylic acid, its salts, cytidine and / or cytosine, as Choline compound choline, its salts and / or phosphatidylcholine, also a phosphoric acid donor, carbohydrates and contains a magnesium salt. 2. The method according to claim 1, daduroh characterized in that 1 1 medium, the pyrimidine compound in an amount of 5 to 50 m-mol § the choline compound in an amount of 20 up to 200 m-moles; the phosphoric acid donors in an amount of 0.1 to 0.5 mol; the carbohydrates in an amount from 2 to 15 #! and the magnesium salt in an amount from 0.01 to 0.05 Contains moles. 3 · The method according to claim 1, characterized in that the phosphoric acid donor is anorganic before phosphate 109823 / 22U Coils used, namely sodium phosphate, primary potassium phosphate and / or secondary potassium phosphate. 4. The method according to claim 1, characterized in that the carbohydrates are glucose, sucrose, fructose and / or Maltose used. 5. The method according to Anspruoh 1, characterized by that the magnesium salt is magnesium sulfate and magnesium chloride used. 6. The method according to claim 1, characterized in that the enzyme in the form of a culture medium of microorganisms, their cells, dried cells, extracts and ground substances are used. 7. The method according to claim 1, characterized in that microorganisms are used whose ability to produce pructose-1,6-diphosphate, 20 $> or higher. 8. The method according to claim 1, characterized in that the microorganisms used are ι (1) yeasts from the group Brettanomyoes, Torulopsis, Candida, Saccharomyces, Zygosaooharomycea, Debarylomyoes and Piohia, (2) bacteria 109823/221 the group Miorobacterium, Gorynebacterium and Brevibaoterium, and (3) pungi from the group Mucor and Aspergillua. 9. The method according to claim 1, characterized in that that one uses as microorganisms: Brettanomyces petrophilum n. Sp ATCC 20224t Saocharomyces rouxii AMS 1024 "Saocharomyoes carlsbergensie IAM 4206, Candida utilis IAM 4220, Candida petrophilum n. Sp ATCC 20226, Torulopeis petrophilum n. Ep ATCC 20225, Torulopsis candida Ii ¹ O 0768, Zygosaccharomyces soja IPO 0495, Debaryomyces handenii IFO 0023, Endomyoopsis chodati (N) WICKERMAN and BURTON OUT 6270, Lipomyoes lipoferus Ii ¹ O 0673, Endomyces hordei IPO 0104, Sporobolomyoes roseus IPO 1037, Pichia miso IPO 0193, Hanaenula jadabilis IPO 0671, Crypto-docopsis IPO 0671, Trididis IPO 0987 IAM 4830, Shizosaocharomyoes pombe AMS 1022, Rhodotorula glutinia IPO 0389, Miarobaoterium ammoniaphilum ATCC 15354, 109823 / 22U Corynebacterium petrophilum ATCO I9O8O, Brevibacterium ammoniagenes ATOC 6871, Aspergillus fumigatus IAM 4040,
Penicillium chrysogenum IFO 4626, Rhizopus nigricans IPO 478 and
Mucor javanicus IAM 6087. 10. Procedure according to. Claim. 1, characterized in that the reaction at 20 to 45 ⁰ C at a pH of bit 9 performs. 11. The method according to claim 1, characterized in that that one uses an enzymatic reaction mixture which Toluene, ethanol, acetaldehyde, sodium bicarbonate, sodium fluoride, Contains ethers, borates and / or surfactants. 109823/221 U