Microbiological production method of amides

BACKGROUND OF THE INVENTION The present invention relates to a process for obtaining microbiological anhydrides by the action of a nitrihydrase enzyme from a microorganism on a nitrile substrate.

A process for obtaining microbiological amides obtained by cultivating light microorganisms such as Rhodococcus, Arthrobacter or Microbacterium on a liquid culture medium is known, after which the cells are washed, acrylonitrile solution is added intermittently for 3 hours and stirred for several hours. Finally, the cells are removed.

The aim of the present invention is to broaden the range of possibilities for microbiological production of amines.

The problem solved by the invention is to determine the optimal parameters for the biological process.

The process according to the invention consists in that it provides for the cultivation of a microorganism from the Rhodococcus rhodochrous spaces, preferably R. rhodochrous FERM 1478, registered in the International Depository of Microorganisms, Japan, in a culture medium containing 5 ... 15 mg / 1 cobalt ions in the form of cobalt chloride and 2 ... 6 g / 1 inductor of enzyme represented by crotonamide, at a temperature of 15 ... 50 ° C, of ​​7; ., 4 preferably 2O ... 45 ° C, pH == 7 ... 9, preferably - «rv * rinse 8, for 30 ... 120h, when a 7 * 7 ^ hydration reaction takes place of the substrate selected from the aromatic nitrile framework, such as. 4-, 3- and 2-cyanopyridines, benzonitrile, o-, m- and para-chlorobenzonitrile, o- m- and pf luoro- <benzonitrile, o- and m- nitro benzonitrile, paminobenzonitrile, o-, m- and p -tolunitrile 4-cyanophenol, anisonitrile, phthalonitrile, isophthalonitrile, terephthalahitrile, 2,6-dichlorobenzonitrile, 2,4-dichlorobenzonitrile, 2,6-difluorobenzonitrile, alpha- and aafa-naphthonitrile, 2-thiophenecarbonitrile, 2-thiophenecarbonitrile, , its cyanopyranazine of aliphatic nitriles having 2.): 6 carbon atoms, preferably acrylonitrile and the transformation of this substrate into a suitable amide, this when the substrate is present in a concentration of 0.5 ... 12 moles / 1 growing medium, and in the variant that the substrate is added to the culture in concentration 0.5, 12 moles / 1 culture medium, and in the culture there is the accumulated nitrilhydrate enzyme or the substrate is contacted with the cells of said microorganism isolated from the culture, where they have been accumulated, with the enzyme extracted from the cells or with the immobilized cells or their enzymatic extract, the culture is carried out at a temperature of 10 ... 30 ° C and pH = 7 ... 9, after which, finally, the obtained amide is isolated by the known method .

The following are 15 examples of embodiments of the invention.

The microorganism used in the process according to the invention is of the species of Rhodo coccus rhodochrous. Preferably the strain. FERM 1478 registered in the International Imorganization Deposit of Microorganisms, Japan. , · Among the most important characteristics5 of the microorganism are mentioned:

(a) Nymphology.

(1) Form and size of cells: 0,91,0 ux 3—10 u.

(2) Presence of cell polymorphism: 10 cells have a rod of ahingite form.

get the initial stage of culture, increase with interruptions along the curvature and then sedivide in small bacilli.

(3) Motility: lack.

(4) Presence of spores: missing.

(5) Gram's positive leg.

(6) Acidity properties: negative, (7) Heterophilic granulocyte: detected.

(b) Growth stage in different culture media 20 (30 ° C).

(1) Broth culture on agar plate ^ circle with a diameter of 1 mm (48 h), irregular and smooth, the surface being drier_rflat, opaque, orange-pink, (2) Broth culture on agar blade?

filament with a smooth surface, being faster dry, overhanging section with a slight orange-pink color, dry.

(3) Liquid broth culture medium: rich growth 30 forming cell membrane â.

bacteria and moderate turbidity and sediment formed along with the growth.

(4) Perforated gelatin culture: final growth on the surface, in the form of an elongate along the perforated part, but not in the lower layer; liquefaction is not observed in gelatin.

(5) Sunflower milk: no changes, (c) Physiological properties:

(1) Nitrate reduction: positive.

(2) Denitrification: negative ..

(3) MR test: negative.

(4) VP test: negative.

(5) Indole generation: positive.

(6) Generation of hydrogen sulphide: positive.

(7) Starch hydrolysis: negative.

(8) Use of citric acid: culture medium Kocur: negative; culture medium

Christensen: Positive.

(9) Use of inorganic nitrogen source: nitrates; positive, ammonium salts :: positive.

(10) Generation of coloring matter: no 55 cake.

. (11) Urease: positive.

(12) Oxidase: negative.

(13) Catâlaza: positive.

(14) Cellulose hydrolysis: negative.

(15) Growth range: pH = 5 ... 10-.

temperature 1O ... 41 ° C.

(16) Attitude towards oxygen: aerobic, (17) Tyrosine decomposition: positive.

(18) Adenine decomposition: positive, (19) Phosphatase: positive.

The reaction solution is diluted with water and subjected to centrifugation for bacterial cell removal. The supernatant is concentrated to dryness (on a rotary evaporator), dissolved in methanol and then recrystallized from methanol to obtain acetamide crystals.

Example 7. The suspension of bacterial cells (corresponding) to 4.66 mg dry cells, obtained, according to example 2, 1 'is added to 4 ml of reaction solution containing 10 mM potassium phosphate buffer (pH = 8.0) and 3 M of 3-hydroxypropionitrile. The reaction is carried out at 25 ° C with the addition of portions of .3 M 3-hydroxypro-; 1 · pionitrile (4 times in the total 1 hour interval from the initiation of the reaction). After 5 hours from the start of the reaction, 15 moles of 3-hydroxypropionamide are obtained with 100% conversion. When the additional 3 M portions of 3-hydroxypropionitrile are added, 18 moles of 3-hydroxypropionamide are obtained with 100% conversion after 11 hours from the initiation of the reaction. This means 1600 g of 3-hydroxypropionamide obtained and accumulated^2!width per liter dfe reaction solution.

Example 8. In a flask with a capacity of 1 1, 400 mg culture medium is introduced as in Example 1, which contains cobalt chloride and crOtonamide, added respec-x to me. ^3<ed in the proportions of O, OÎ g and 2 g / l of the environment./.-'/ The mixture is grown on a shaker at 28 ° C. Cultivation is continued with the addition of 0.2% (w / v) crotonamide (800 mg / 400 ml) at 26 and 56 h after the initiation of cultivation and stops at 76 h after the initiation of the culture.

Bacterial cells are collected by centrifuging the culture medium at 10,000 rpm for 2Q.niin, with a separator ^;centrifugally, wash. then with 0.85% solution. . Sodium chloride, centrifuged again.⁴ 'Then a solution described above is suspended in 40 ml. A small portion of the suspension is 4? uses to measure the weight of cells in the dry state of the suspension._s

The suspension containing the cells (corresponding to 2.96 'mg dry cells) was added to 4 ml of reaction solution containing 10 mM 5 (buffer, potassium phosphate (pH = 8.0), and 3cyanopyridine in different concentrations. The reaction was is carried out at 25 ° C. Eight moles of 3-Cyanopyridine are converted into nicotinamide with 100% conversion after 9 h of reaction and 9 mol of 3-cyanppyridine after .22 h of reaction. On the other hand, when a suspension containing bacterial cells in the amount corresponding to 5.92 mg Dried cells, instead of 2.96 mg dried cells priced above, is added to the simi -4 ml reaction 9 moles of 3-cia no.pyridine are converted into nicotinamide eu> 0% conversion after 5 h of reaction and 12 M of 3-cyanopyridine after 9 h of reaction; gi

Obtaining of nicotinic acid was not reported. .

The concentration of 12 M corresponds to 1,765 g of nicotinamide produced and accumulated 5 per 1 1 reaction solution. :

Nicotinamide is recrystallized from the solution that produced it. The crystals are collected and recrystallized from methanol.

Example .9. The bacterial cell suspension (corresponding to 5.92 mg of dried cells), obtained according to Example 8, was added to 4 ml of reaction solution containing 10 mM potassium phosphate buffer (pH-8.0) and 1 M benzonitrile. The reaction is carried out at a temperature of 25 ° C with the addition of IM benzonitrile to the reaction solution, skein 1 ,. 2, 3, 4, 5 and 7 hours after the reaction started. After 24 hours of initiation of the reaction, 7 M benzamide was obtained with a conversion of00%, 3 corresponding to 848 g / l reaction-solution.

: Exampler lO. A bacterial cell suspension (corresponding to 5.92 mg dry cells), obtained according to Example 8, was⁵ add to 4 ml of reaction solution containing 10 mM potassium phosphate buffer (pH = 8.0) and 1 M of 2,6-difluorobenzonitrile. The reaction is carried out at 25 ° G with the addition of 1 M 2.6 difluorobenzonitrile to the reaction solution. After 7 2, 4, 6 and <Ș> h - from / upon initiation of the reaction. ' After}; '? · 22 h from: initiation of the reaction, 2,5 moles (393 g / l) 2,6-difluoro'benzamide are obtained.

. . Example 11 Suspension of bacterial cells (yielding 5.92 mg of dry cells) obtained in accordance with Example .8 was added to 4 ml of reaction solution containing 10 mM potassium phosphate buffer (pill = 8 , 0) and 2-thiophenecarbonitrile IM. The reaction is carried out at a temperature of 25 ° C with the addition of 1 M '2-thiophenecarbonitrile to the reaction solution after ilh since the reaction was initiated. After 5 hours ld 'ΰΐΐίίΐί'ίΐ Μ (254 g / l | 2-ii0fe'n ^' rFdMa; hÎidli 100% cojuivation girls_; Example 12. Suspension of bacterial cells 'retina' (fluorescent at 5.92 mg dry cells), obtained according to example 8, is added to 4 ml of reaction solution containing 10 mM potassium phosphate buffer (pyl - 8) ₍ and 1 M 2-furonitrile. The reaction is carried out at a temperature of 25 "C with the addition of-M 2-furonitrile to the reaction solution after 1, 2 / 4,6, 8, 11 and 23 li since the initiation of the reaction! After 30 hours of initiating the reaction, 8 M (88Ș g / l) 2-furancarboxamide is obtained with a 100% conversion. = · /. · - '

Example 13. Ό bacterial cell suspension (corresponding to 5.92 mg dry cells), obtained; As in Example 8, add the l / 4'mr reaction solution containing 10 mM potassium phosphate buffer (pH, = 8) and 4 M indolacetonitrile. The reaction is carried out at 25 ° C. After 24 hours. upon initiation of the reaction, 4 M (69.7 .g / l) 3-indoacetamide is obtained with • 0 '100% conversion. /

Example 14. The cells of the following stem: R. rhodochrous pines, obtained by cultivation at 30 ° C for 42 ha of the strain in the culture medium described above, were contacted with 3-cyanopyridine as substrate. The reaction was carried out on cells obtained from 1 ml of culture and 1 ml of reaction mixture of 50 mM phosphate buffer (pH 7.2) and 10 mM substrate at 0 ° C for 5 marbles and 0.1 ml acid. hydrochloric acid IN (added to stop the reaction). The amount of nicotinamide produced was. termed by HPLC analysis and the activity of the nitrile hydrase strain was calculated. For comparison, the same experience was performed, except that CoCl was not used₂.

The results obtained are given in table 2.

(II)

Characteristics for this are 4-, 3- and 2-cyanopyridines.

· - / CN z \ ..

R + a + b₂ \ z which R₄ and R₂, respectively, represents II, CH₃, OH, OCHs, C1, CN; NH₂ or NO.,. For these, characteristics are benzonitrile. o- and m- nitrobehzonitrile-p-amiiitF · benzonitrile / m- and -p-tolunitriliij ^ caryofciiole, anisonitrilub phthalonitrile, isophtanyitrile, terfeftalonitrile, 2,6-dichlorobenzo15¹ nitrile, 2,4-dichlorobenzonitrile and 2,6-difluorobenzonitrile.

Z \ Z \ | O | O + CN \ z \ z

Characteristics for this are alpha and ie / c-naphthonitriles:

(ΠΙ)

Table 2

<td rowspan="2">Stem</td><td colspan="2">Specific activity (U / mg cells)</td>

<td>Without CoCl₂</td><td>With CoCl₂</td>

<td>ATCC 33278</td><td> 0,248</td><td> 2,42</td>

Example · 15. The process is similar to that of example 14, except that another culture medium has been used which has the following composition: 2g yeast extract, 0.5 g KH₂P0₄, 0.5 g K₃HPO₄, 0.5 g MgSO₄-7₂O, 5g glucose, 5g polypeptone, 2 g isobutyramide and 2 g isobutironitrile.

The results obtained are given in table 3.

. /^X\ '| O + CN (IV) where X is S or O.

Characteristic for these are 2-thiophenitrile and 2-furonitrile.

z \ z^N\ lo II \ X- (V) 'CN

Characteristic for these is 5-cyanoindole.

, Table 3

<td rowspan="2">Stem</td><td colspan="2">"Specific activity (U / mg cells)</td>

<td>Without CoCl₂</td><td>Cii CoCl₂</td>

<td>ATCC</td><td> 0,05</td><td> 0,64</td>

For a better understanding of the invention, the following are also shown:

Nitriles which may be used, as a substrate of the nitrile hydrate produced by the microorganism described above, may be aromatic and aliphatic mononitriles or dinitriles, in particular mononitriles.

Preferred nitriles, according to the features of the present invention, are aromatic nitriles, particularly those having 4 to 10 carbon atoms forming an aromatic ring. Some types of examples of aromatic nitriles are the compounds represented! of the formulas, general

I ... VI below.

Z^N\ .

IO | -CN \ Z (VI)

ZX • ZS O + CN

Characteristic for these is cyanopyrazine.

Another group of nitriles is formed by the group of aliphatic nitriles, especially mono- or dinitriles having 2 to 6 carbon atoms, preferably mononitriles. Depending on the usefulness of the amides produced, acrylonitrile is characteristic and has a good productivity. The amides corresponding to these nitriles are those obtained by converting the CN grouping of the latter into the CONH group₂. in the case of dinitriles, it should be considered as the corresponding amides obtained by converting at least some of the CN groups into CONH groups₂.

Cultivation of the microorganism of the species Rhodococcus rhodochrous can be conducted under any of the appropriate conditions, provided that the cobalt ion is present in the sulphide medium. It may be a common practice to pinch a cart enzyme inducer will be described in detail below, (I)

J (20) Hydrolysis of Tween 80: positive.

(21) OF test: negative.

(22) Temperature resistance (in 10% whipped milk at 72 ° C, 15 min): none: change.

<td>(23) Acid generation and so on</td><td colspan="2">i gas from sugar</td>

<td>- Arabinose</td><td>acid</td><td>gas</td>

<td>- D-xylosis</td><td> ·</td><td> _ </td>

<td>- D-glucose</td><td>rp</td><td> — </td>

<td>- D-mannose</td><td> — '</td><td> — </td>

<td>- D-fructose</td><td></td><td></td>

<td>- maltose</td><td></td><td> —</td>

<td>- sugar</td><td></td><td></td>

<td>- lactose</td><td></td><td></td>

<td>- trehalose</td><td> — &lt;.</td><td> :—</td>

<td>- D-sorbitol</td><td>/ -F:</td><td></td>

<td>D-mannitol</td><td></td><td> —</td>

<td>- glycerin</td><td> . ₊ ··. '</td><td> —</td>

+ ·· positive; -: negative · &lt;(24) Growth in a single carbon source:

- - inositol -.

- maltose -p - D-mannitol + - rhamnose -;:

- D-sorbitol + - m-hydroxybenzoic acid + - sodium adipate + - and sodium benzoate Η- ·· sodium citrate + - sodium lactate - testosterone + - L-tyrosine +. - glycerol (1%) (gr / vol) (+):

- trehalose (+) - p-hydroxybenzoic acid (1%) (gr / vol). + 4- means positive; - it means negative; (-i) weakly positive. . ·.

(25) Fatty acid and cell wall analysis: contains linear saturated and unsaturated fatty acids and thiiberculo-stearic acid. TLC of mucolic acid gives a single stain,

As a result of the classification according to the bacteriological properties mentioned above, the strain is aerobic, gram-positive, resistant in a weak acidic environment, catalase-positive and non-supporting bacillus generator, which does not insert flagella. It also has an elongated bacillus shape like a mycelium in the initial growth stage, grows with branching and then divides into short bacilli. Therefore, it is recognized as belonging to a bacterium of the Nocardia type.

Analysis of the fatty acid composition shows that - the bacterium contains unsaturated and linearly saturated fatty acids, containing tuberculostearic acid. TLC analysis of mycolic acid shows a single stain having the same Rf value as the standard bacterium Rodocoecus rhodochrous (IFO 3338) and thus distinct from the genus.Mycobacterium. It is also distinct from the Nocardia genus by com position (number of carbon atoms) of the mycolic acid. As a result. of the examination of other biochemical properties, the bacterium is recognized as a Rhodococcus rhodochrotis. .

Knowing that microorganisms tend to undergo mutations, any mutant of the respective strain can be used, according to the process of the present invention, as long as the product of its culture provides nitrilhydrate in the presence of cobalt ion.

Example 1. The working strain cells, obtained by culturing the strain in the culture medium containing 2 g / 1 protonamide and 10 mg cobalt chloride, react with a variety of nitriles used as substrate. The reaction is carried out using 2 ml of reaction solution comprising cells obtained from 2 ml of culture, 10 mM potassium phosphate buffer (pH = 8.0) and 200 mM substrate, at 25 ° C, for 76 h.

The reaction is stopped by the addition of 0.2 ml of 1N hydrochloric acid. The activities of the nitrilhydrate on the respective substrates are presented, and the values ​​of the reaction product or the amount consumed from the substrate, measured by HPLC relative to the activity of the nitrilehydrate, measured with 3 cyanopyridine as substrate, are presented as specific activity (%).

The results are presented in table 1. '

Table 1

<td>substrate</td><td>Specific activity: (%)</td>

<td>3-Ciandpiridină</td><td> 100</td>

<td>acrylonitrile</td><td> 106</td>

<td>4-cyanopyridine</td><td> 129</td>

<td>2-cyanopyridine</td><td> 64 ·.·.</td>

<td>5-Gianoindol</td><td> / 9 -</td>

<td>2-Tiofenonitril</td><td> 116</td>

<td>2-FuronitriI</td><td> 71.</td>

<td>benzonitrile</td><td> 80</td>

<td>4-CianofencI</td><td> 24</td>

<td>'p-Aminobenzonitrile</td><td> 16</td>

<td>m-nitrobenzonitrile</td><td> ,·..·.. 7 . .</td>

<td>a-nitrobenzonitrile</td><td> 16</td>

<td>m-chlorobenzonitrile</td><td> .29</td>

<td>p-ToIunitril</td><td> . ·. .. . 5. .</td>

<td>o-Tolunitrile,</td><td> . 46 ·</td>

<td>m-tolunitrile</td><td> 32</td>

<td>Anis-nitrile</td><td> 20</td>

<td>. a-Clorobenzohitril</td><td> ..... 41 </td>

<td>p-chlorobenzonitrile</td><td> 7</td>

<td>2,4-Diclorbenzonitril</td><td> . 2</td>

<td>2,6-DiclorbenzonitriI</td><td>and</td>

<td>Cianopirâzihă</td><td> 80 '</td>

Example 2. In a flask with a capacity of 11, 400 ml of the above-mentioned culture medium (containing cobalt chloride and crotonamide, added respectively in the proportions of 0,01 g and 2 g / 1 of medium). The mixture is grown on a shaker at 28 ° C. Culture and culture get with. further adding to the culture medium an additional 0, 2% (w / v) crotocamide (800 mg / 400 ml) at 30 and 60 h after culture initiation. It was interrupted at 80 h of 1 of the initiation of the culture.

t

J: Bacterial cells have. formerly, collected by centrifugation of the culture medium at 12000: Tot / min, for 15 min, with a centrifugal separator, washed with 0.85% sodium chloride solution. centrifuged again and suspended, in 40 ml solution; described above. A small portion of the suspension was taken as a sample and used for measurement. the dry weight of the bacterial cells in the suspension.

The suspension; containing the appropriate cells. to 2.33 g dry cells, add. to¹ 7 ml of reaction solution containing 10 mM potassium phosphate buffer (pH = 8.0), and 7.59 M 3-cyanopyridine. The reaction proceeds at a temperature of 25 ° C overnight, with the addition of the 0.55 M and 0.75 M. deactivation solution of 3-cyanopyridine, 3 · and 6 hours after the reaction is initiated. '

The yield in nicotinamide is 5.58 M after 18 hours from the initiation of the reaction, consequently, the conversion reached 99.5%, which corresponds to an accumulation of nicotinamide in the amount of 681 g. At this concentration, the reaction product solidifies as a result of nicotinamide storage.

The nicotinamide thus produced was identified by crystalline isolation of the product and analyzed by elemental analysis, IR, NMR and mass spectrometry. No nicotinic acid was detected.

Example 3. The suspension containing the cells (corresponding to 2; 33 mg of dry cells obtained in example. 2) is added Ta 4 ml of reaction solution, which¹ pigs, 10 mM potassium phosphate buffer (pH = 8.0) and cyanopyrazine in different concentrations. Reaction: The temperature is 25 ° C. Four moles of cyanide. pyrazine is converted to pyrazinamide with 100% conversion after 6 liters of reaction and 6 moles of cyanopyrazine after 9 hours of reaction. On the other hand, when a suspension containing bacterial cells in the amount corresponding to 4; 66 mg dry mass, instead of 2.33 mg dry mass mentioned above, is added to the similar reaction solution (4 ml), 7 M of cyanopyrazine is converted to prazinamide with 100% conversion after 6 h of reaction and 8 M of cyanopyrazine after 9 h of reaction. Obtaining pyrazinecarboxylic acid was not recognized.

Pyrazinamide crystallizes from the solution as it is obtained. The crystal deposit is collected directly and recrystallized from methanol. The crystals were identified as pyrazinamide by their analysis by elemental analysis, IR, NMR and mass spectroscopy. &gt;

Analysis of cyanopyrazine, pyrazinamide and pyrazinecarboxylic acid was performed by high performance liquid chromatography.

The same analysis, as in this example, was carried out in the following examples.

'Example 4. The suspension of ri'en bacterial cells (corresponding to 4.66 mg of dry cells), obtained in example 2, is added to 4 ml of a reaction solution containing 10 mM 5 of potassium phosphate buffer (pH = 8.0) and 3 M of methacrylonitrile. The reaction is carried out at 25 ° C with the addition of 3 M methacrylonitrile to the reaction solution, 1 and 3 hours after the initiation of the reaction. After 12 h 0 after the initiation of the reaction, 9 M of methacrylamide were obtained with a 100% conversion.

When additional methacryl IM, lonitril, is added 5 hours after the initiation of the reaction, 10 M methacrylamide is obtained with 100% conversion 24 h after the initiation of the reaction. Concentration. 10 M corresponds to 851 g of methacrylamide obtained and accumulated in a reaction solution filter.^: . 0; The reaction solution is diluted with water and the bacterial cells are removed by centrifugation (12000 rpm for 15 min). The solution released by the cells is concentrated on a rotary evaporator and crystallized.

The crystals are then dissolved and recrystallized from water to obtain the methacrylamide crystals.

Example 5. To a suspension of bacterial cells (corresponding to 4.66 mg of dried cells), obtained as in example 2, add 4 ml of reaction solution containing 10 mM potassium buffer (pH = 8.0) and 1 M of crotonitrile. The reaction is carried out at 25 ° C, with the addition of 5 portions of each crotonitrile IM (5 times in total, 1 hour interval from the initiation of the reaction). After 6h from the initiation of the reaction, they are obtained. 6M crotonamide with 100% conversion. When additional portions of 0 M 1 crotonitrile were added to the reaction solution, after 6 and 10 h, respectively, starting the reaction, 7 M and 8 M crotonamide were obtained with 100% conversion after 10 h and 22 h respectively. The concentration of 8 M 5. corresponds to 681 g of crotonamide produced and accumulated in one liter of reaction solution.

Crotonamide crystallization is performed according to the method described in Example 4.

Example .6. The bacterial cell suspension (corresponding to 4.66 mg of dry cells), obtained according to Example 2, was added in 4 ml of reaction solution containing 10 mM 'potassium phosphate buffer (pH = 8.0) and 3 M acetonitrile . The reaction is carried out at 25 ° C with the addition of 3 M portions of 3 M acetonitrile at 1 h and 3 h respectively, and 5 M acetonitrile at 6 h after starting the reaction.

After 12 hours after starting the reaction, 14 M aeetamide is obtained with 100% conversion. in other order, 827 g of aeetamide. obtain and accumulate to 1 1 solution of 5 reaction.

<img file="RO101578B_D0001.tif" />

/ such that nitrile-hydrase accumulates in bacterial cells.

A culture medium may contain the following ingredients (per liter): 0.1 ml of vitamin mixture, 13.4 g K₂HPO₄, 6.5 g KH₂PO₄, 1.0 g sodium chloride, 0.2 g MgSO₄.7100. distilled water - pH balance = 7.0. ·.

The composition of the vitamin mixture is: 2pg biotin, 0.4 mg calcium pancytenate,

2.6 mg inositol, 0.4 mg nicotinic acid, 0.4 mg thiamine hydrochloride, 0.4 mg pyridoxine hydrochloride, 0.2 mg p-aminobenzoic acid, 0.2 mg riheflavine, 0.01 mg folic acid and up to 1 1 water.

Another culture medium may contain: 10 g glycerin, 5 g pepton, 3 g mallet extract and 3 g yeast extract, pH 7.2 equilibration, distilled water.

Another culture medium may contain: Sg yeast extract, 0.5 g K₂HPO₄, 0.5 g KHL, PO₄, 0.5 g MgSO₄-7₂O, pH balance = 7.0, distilled water.

Enzyme inducers for the induction and production of nitrile-hydrate can be, in the microorganisms of the Rhodocoeciis rhodkfAirous microorganism, any of the corresponding ones and close to the object. '.

Characteristic inducers, for the presence of this isoenzyme are nitriles and starches.

Examples of enzyme inducers whose effect has been confirmed for the above open strain are as follows: crotonamide, areltonitrile, propionitrile, benzamide, propismmide, acetamide, isovaleronitrile, ®-anthironitrile, isobutironitrile, n-caproffiftitrite, pyrrhoffytrite, pyrrhoffytrite, pyrrhoffytrite, isobutyramide and phenylacetamide.

As for the source of cobalt ion, one can say:

Nitrile-hydrase is not obtained, even if the above inducer is present in the culture medium. Because the culture medium is aqueous, cobalt acrylic is usually generated by adding a soluble salt of erihdt to the culture medium by water. The water-soluble cryolate compounds are known from camel. Cobalt compounds are characteristic of those which give the bivalent cobalt ion the trivalent cobalt ion, in parfcnlar those which give the bivalent cobalt ion. Particular examples of such salts may be as follows: cobalt chloride, cofee sulphate! cobalt acetate, cofee bromide! cobalt borate or others. Vitamin Ejjj and metallic cobalt are other examples of cobalt compounds, as long as they produce a cobalt ion in the medium of "Sure by ionization or oxidation; by, through, microorganism during «cryivation.

The culture for the production and accumulation of iHinl-hydrate in the bacterial cell can be carried out by cultivating the microorganism used.

./ The amount of inducing enzyme used is of the order of 2.:.6 g / 1 medium ⁵ culture; The amount of cobalt ions is of the order of 5 to 15 mg / 1 culture medium, calculated as cobalt chloride.

Particular examples of cultural environment composition, as specified, are. data in the following:

(i) AII culture medium:

g acetonitrile (inductor), 10 mg cobalt chloride ₁₅ -. (ii) Culture medium Β 1 Γ:

• 2 g isovaleronitrile, 10 mg CoCl₂ .

(iii) Culture medium C 11: 2g per ton gives me 10 mg CoCl₂. Nitrile-hydrate can be obtained by cutting through the cultivation, stirring, of the working strain, at a temperature from 15 to 50 ° C, better from 20 to 45 ° C, and in particular from 30 ° C at a pill ... 7 ... 9 for about 30 hours or preferably 40 hours or in the upper 25 hours, for example 120 hours.

The enzyme inducer is preferable to be present from the initial stage of cultivation, and it is desirable, for the preparation of the bacterial cells to have high activity, to supplement the inducer. For example, when the agitation is to be performed, it will be conducted at 28 ° C for 78 hours, adding. additionally crotonamide at 26 and 56 h, after the start of the reaction, so that the concentration is 0.2% (w / v) each time.

The process for the biological preparation of the amide in which a nitrile is hydrated to its corresponding amide by the action of nitrile⁴⁰ The hydrasease resulting from a microorganism includes various variants suitable for the pathway for obtaining nitrile-hydrase, acting on the nitrile.

One of these forms is production ⁴⁵ . of amide in culture medium, when a nitrile substrate is present in the culture medium of the microorganism.

Another form for obtaining nitrile₅₀ of the hydrate, which acts on the substrate, is to add a nitrile substrate to the culture medium in which nitrile · ·. hydrase was accumulated for the hydration reaction. The shape change is₅₅ to use the culture medium in which the cells of the microorganism were destroyed as "a culture medium in which nitrile hydrate was accumulated!

Another way to cause nitrile60 to act on the substrate is to isolate the cells in which the nitrilehydrase accumulated from the culture medium, preferably to place the cells on a suitable support or immobilize 65 and then contact the substrate.

The technique in which immobilized cells are used is well known to those working in the specialized field. The method of immobilization, of the microorganism is also related to the use of the microorganism 5 immobilized in the so-called bioreactor.

Another form, of the nitrile-hydrasease acting on the substrate is the method in which a nitrile-hydrasease is prepared and the nitrile is non-biologically prepared enzyme hydrate. It can be said that the hydration reaction in this way must be conducted at such pH and temperature conditions that the activity of the enzyme will be lost (biologically).

The nitrile hydrase, according to the present invention, has a range of 7 to 9 pars, and the optimum pll is 8.0. If the solution of the reaction medium shows a pH below 20 7.0, the activity of the enzyme tends to decrease steeply. Consequently, it is desirable to add a buffer solution to the reaction medium. When using any of the buffer solutions, such as potassium phosphate, Tris / HCl or sodium phosphate, the activity of the nitrile-hydrate enzyme is used.

The concentration of the substrate in the reaction medium or in the solution of the hydration reaction is usually in the range of 4 to 7 30 moles / 1, and the reaction temperature is usually in the range of 10 to 30 ° C. Experimental examples were made, as follows:

The method for measuring the activity of nitro-hydrate and the activity unit of nitrile-hydrate in the experimental examples of the lower hands are defined as follows:

(1.) Method for measuring the activity of 40 nitrile-hydrate

The activity of nitrile-hydrate is measured by reaction with 2 ml of reaction mixture containing 10 mM of benzonitrile, 30 mM of phosphate buffer, potassium (pH = 7.0) and a certain amount of microorganism cells (isolated from culture medium at 10 ° C for 5 min and addition of 2 ml of 1N hydrochloric acid to stop the reaction).

(2) Definition of unity.

One unit (0) of nitrile hydrate activity is defined as the amount of enzyme required to produce benzamide from benzonitrile under the conditions described above. a speed of 1 pmol / min.

... Reference example 1

The working strain, used in the present invention, was cultivated using the culture medium with the composition specified below under cultivation conditions, which are also &lt;specified below, and the expression of nitrile-hydrase activity is examined by adding cobalt chloride and / or ferrous sulphate in the medium. culture during cultivation.

(i) Composition of the culture medium (in 1 1 medium): 3.0 ml of vitamin mixture, 0.5 g K₂HPO₄, 0.5 g KH₂PO₄, 0.5 g MgSO₄· • 7H₂One and 2.0 ml propionitrile and until the pH is balanced with 7.2 distilled water.

(ii) Cultivation conditions: 28 ° CI10 ... 80 h The results obtained are presented below:

Nitrile hydrate does not develop its activity, even if ferrous sulphate is added to the basic environment, but it develops its activity, when cobalt chloride is added, and the addition of ferrous sulfate, to the environment that already contains cobalt chloride, will adversely influence the results. .

<td colspan="11">Metallic ion added:</td>

<td>verdigrised₂ mg</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> ' 0</td><td>: 1Q</td><td> 10</td><td> 10</td><td> 10.</td><td> 10</td>

<td>Ee₂SO₄ mg</td><td> 0</td><td>⁵</td><td> 10</td><td> 20</td><td> 40</td><td> , . 0</td><td> 5</td><td> 10</td><td> 20</td><td> 40</td>

<td>, Quantity of cells (mg / ml) 1 *</td><td> 1,06</td><td> 1,14 </td><td> 1,25</td><td> 1,24</td><td> 1,34</td><td> 2,04</td><td> 1,90</td><td> 2,16</td><td> 2,16</td><td> 2,07</td>

<td colspan="11">Enzyme activity:. '</td>

<td>ϋ / mg cell'2 *</td><td> 0</td><td>ό</td><td> 0</td><td> 0</td><td> 0</td><td> 0,59</td><td> 0,26</td><td> 0,34</td><td> 0,32</td><td> 0,16</td>

<td>U / ml medium</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 1,20</td><td> 0,49</td><td> 0,73</td><td> 0,69.</td><td> 0,33</td>

i * Number of cells per. weight basis in dry state, 2 * U: unit of activity, as defined above, dry state ·

Reference example 2

The effect of the different nitrile or mycelium ₍enzyme inducers on the strain and working it^; dM in table 4, where the results obtained by the preliminary cultivation of the working strain in culture medium B, at the temperature below ₍ and the amount of cells is determined relative to the weight in nitrile or amide feed as an inducer of 0.1% (v / v) or 0.2% (y / v), respectively when the strain has proliferated sufficiently, ^J and, further - inoculating the culture medium strain C - to which was added 0.001% / weight / volume) chloride. cobalt for 5 cultivation time, of 36 .. .48 h.

/

Table 4

<td>nitriles</td><td>Specific activity, U / mg</td><td>Total activity, U / ml</td><td>Total amount of cells mg dry cells per ml</td>

<td> ' 1</td><td> 2</td><td> 3</td><td> 4</td>

<td>Crotonamidă</td><td> 2,22</td><td> 4,48</td><td> 2,02</td>

<td>acetonitrile</td><td> 1,41 </td><td> 3,47</td><td> • 2,46.</td>

<td>propionitrile</td><td> 1,36</td><td> 4,44</td><td> 3,26</td>

<td>benzamide</td><td> 0,84</td><td> 2,75</td><td> 3,26</td>

<td>propionamide</td><td> 0,79</td><td> 2,29</td><td> 2,90</td>

<td>acetamide</td><td> 0,71</td><td> 1,55</td><td> 2,18</td>

<td>day-butyronitrile</td><td> 1,40</td><td> 0,38</td><td> . 3,70 .</td>

<td>isobutyronitrile</td><td> 0,41</td><td> 1,24</td><td> 3,06</td>

<td>Isovaleronitrile.</td><td> 0,34</td><td> 1,05</td><td> 3,07</td>

<td>n-Capronitril</td><td> 0,28</td><td> 1,04</td><td> 3,71</td>

<td>3-pentenenitrile</td><td> 0,32</td><td> . 1,42</td><td> 4,49</td>

<td>Pivelonitril</td><td> 0,35</td><td> 0,24</td><td> 0,69 ‘</td>

<td>N-butyramide</td><td> 0,43</td><td> 1,56</td><td> 3,'62</td>

<td>isobutyramide</td><td> 0,09</td><td> 0,33</td><td> 3,48</td>

<td>Isovaleronitrile '</td><td> 0,44</td><td> 1,08</td><td> 1,81</td>

<td>n-Capronamidă</td><td> 0,30</td><td> 1,06</td><td> 3,52</td>

<td>meth</td><td> 0,20</td><td> 0,62</td><td> 3,12</td>

<td>phenylacetamide</td><td> 0,29</td><td> 0,28</td><td> 0,95</td>

It is found that the nitrile hydration enzyme, for example, nitrile hydrase, resulting from a bacterium of the genus Pseudomorias, contains Fe⁺⁺⁺ in its active centers and, for this reason, the presence of iron ions in the culture medium is essential for the respective culture of the microorganism, consequently it is also assumed, "in the case of the microorganism within the genus Rhodoccocus from the known examples described above, pathways an iron ion in the culture medium for cultivating the micro-organism is essential for the production of the nitrile hydration enzyme.

The present invention has been developed based on the findings that a specific strain of the genus Rhodoccocus, for example, a strain of the Rhodochrous species does not produce ₄₅nitrile hydrate in a culture medium containing iron ion and produces nitrile hydrate in a culture medium containing cobalt ion and that the nitrile hydrate thus produced can also be used in a nitrile _5Qaromatic as a substrate, thus transforming it into amide.

Accordingly, the process for preparing an amide according to the present invention is a process for the biological production of an amide, a process in which a nitrile is hydrated to its corresponding amide by the action of a nitrile-hydrate resulting from a microorganism, a process which is characterized in that a so-called nitrile hydrate is obtained by cultivating a species microorganism at Rhodoccocus rhodoch / ous in the presence of cobalt ion.

In conclusion, according to the present invention.

although there is zero activity of nitrile hydrate in a culture medium containing ion. iron, the activity can be initiated and developed in the culture environment by introducing cobalt ion. It was considered that the unexpected development of the nitrile-hydrase of this specific microorganism would have a critical dependence on the type of metal ion in the culture medium. Furthermore, according to the present invention, the aromatic nitrile hydrase may be advantageously conducted. The effect of the present invention is useful because nicotinamide is of great importance, as it is the hydration product of 3-cyanopyridine, as 15, a crude material for the synthesis of vitamin or pyrazinamide, which is the hydration product of cyanopyrazine, useful as tuberculostatic. , -. ·₂₀ The present invention relates to a process for hydration of nitrile, in order to convert it into the corresponding amide by the action of the resulting nitrile-hydrate _λ from a microorganism, comprising, in ₂₅ mainly, cultivation of the micro-organism, induction of nitrile-hydrase and the production of this nitrile-hydrate thus obtained to act on the nitrile substrate.

These steps as such are known as unit operations and are used in their corresponding form in the present invention. The term "nitrile hydrate is contained by cultivation of the microorganism in the presence of cobalt ion means that induction of nitrile hydrate is a natural premise.

The introductory part of the present invention "a process for hydrating the nitrile to convert it into the corresponding amide by the action of the nitrile hydrate resulting from a microorganism includes any corresponding cases or variations, in order to cause the nitrile hydrate to act on the nitrile. As one of these forms, it is mentioned the method of collecting the enzyme produced by a microorganism and using the enzyme as a preparatory enzyme. This route of use of nitrile hydrate, in which the enzyme is used as enzyme preparers, should be understood as falling within a category of "a biological preparation process according to the present invention.

The process according to the invention has the advantage of obtaining a product with good qualities.