Method of amides manufacture

The subject of the invention is a process for the production of amides, which consists in the hydration of nitrile and its transformation into an appropriate amide as a result of the action of nitrite hydrate produced by small-scale companies. Exactly, it is a method of biological amide sparking, distinguished by the microorganism used and the method of producing nitrile hydrate.

Lower allpharic acid, such as acrylamide, is prepared by hydrating a nitrile, such as acrylorntrile, with a number of methods of hydration proposed by the action of enzymes such as nitrilase or ntryloo hydrate produced by microorganisms / see e.g. Japanese Patent Application Publication No. 21519/87, US Patent No. 4,001 081, not issued, Japanese Patent Application Publication No. 162193/86 1 91189/87, European Patent Applications No. 0 188 316 1 0 204 555 and Japanese Patent Applications No. 17918/81 1 37951/84, as well as US Patent Nos. 4,248,998 and 4,637,982. Such biological production methods for amide have been used on an industrial scale and have been highlighted as preferred methods for producing acrylamide.

To date, a number of microorganisms have been proposed for biological production of amides. However, it has been found that, although these microorganisms are effective in the hydration of lower aliphatic nitriles, they are not always effective in the hydration of aromatic nitriles. For example, in the production of nicotinamide by hydration of 3-cyanopyridine, insufficient performance is obtained from an industrial point of view.

There is a known way of cultivating microorganisms in the presence of iron or mannan ions. This method is also used in the biological process. For example, the culture of Rhodococcus microorganisms in the presence of iron ions is disclosed in Unexamined Published Japanese Patent Applications Nos. 162193/86 and 91189/87.

As a result of the research, it was found that the enzyme nitrile hydration, or filamentous hydratase, found in bacteria of the genus PtJudoeonθ, contains Fe ion in the center of activity, and in this connection the presence of Iron Ions in the culture environment is of decisive importance for breeding of micro-organisms. For the sake of this, in the case of microorganisms of the genus RhodrcrcJut, the known examples described above also assume that Iron ions in the microbial culture environment are of decisive importance for the production of the nitrile hydrate enzyme.

The invention is based on the discovery that a specific strain of the genus, i.e. strains 3-1 of the species R ^ <^ <^ <^ c ^ f ^ rrut does not produce hydrate, mitigates in a culture environment containing iron ion and that this strain produces nitrate hydrate in a culture medium containing Jon kobaHu, and also that the nitrite hydratase produced in this way can be used in reaction with an aromatic nitrite as a substrate, which is then converted to an amide. In this connection, the method for producing the amide according to the invention is a method for producing the amide by biological means in which the nitrile breaks away from the corresponding ammdu as a result of the action of the hydrated enzyme produced by the hydronase, characterized in that the hydrated enzyme obtains in the cultivation of RhodococJut rhodochrous microorganisms in the presence of tobium and kobaHu ions.

In accordance with the invention, it turned out that Although nitrite hydratase has zero activity in a culture medium containing iron ion, this activity is increased by a culture environment containing ion kobaHu. Unexpectedly, it turned out that the development of nitrate hydrate in these particular microorganisms strongly depends on the type of mmeon ion in the culture environment.

Hydration of aromatic nitrile can also be successfully carried out by the method of the invention. The process according to the invention is of practical importance because of the importance of the hyd ^ aacc product! 3-Janrplridine, nicotinic acid - a drug for the synthesis of vitamins, or a product of hyda- aa ^ and cyanopyrazine, a pyrazinamide useful as an anti-tuberculosis drug.

1. General concept of the biological method for amide production.

The invention relates to a method for nitrile hydrates to convert it into the corresponding amide by the action of ytroyro hydrate produced in durbnutrition, the process essentially involving culturing of durrobes, induction of nitrite hydration and trihydration so that the nitrite nitrate hydrate produced in this way acts on the nitrite hydrate.

These stages are known as Unit operations and are used in an appropriate form in the process of the invention. The term nitrate hydratase is obtained in microbial culture in the presence of cobalt ion and obviously also includes induction of nitrile hydrate.

The determination of the nitrile hydacylation method to convert it to the appropriate amine as a result of the action of the nitrile hydrate produced by microorganisms includes any secondary dilutions or post-drug variants that the hydratase acts on nitrile.

□ One of these solutions includes a method of collecting the enzyme produced by durbπoustΓrje and using this enzyme as an enzymatic preparation. It is understood that such a method of using nitrile hydrate, in which the enzyme is used in the form of an enzyme preparation, is a category of the biological production method of the invention.

160 904

2. Hydration reaction details 1 / Microbes

The microorganism used in the method of the invention is the 3-1 gatsnks Rhoducuccst rhodochross strain.

Details about tzczeps 3-1 include the following:

/ 1 / Origin and deposit

Strain 3-1 was taken from soil in Sakyo-ks, Kyoto, Japan and deposited as an international deposit / pursuant to the Budapest Arrangement on the International Use of Microbial Deposits for Patent Purposes / at Fermnt ^ tlon Research Institste, Jepor ^ ia, Agency of Indussrial Sciences and Technology, registered under the number FERM BP-1478.

/ 1 / Property / bacteriological / a / Moufolugia / 1 / Cell shape and size 0.9 - 1.0 sin x 3-10 sn / 2 / The presence of polymorphs from cells is in the shape of long rods at the initial stage of culture, they grow from cracking in a curved shape, after which they are divided into small pałeczki / 3 / Ruchność / 4 / Obecność sp / 5 / Coloring according to Gram / 6 / Resistance to acid / 7 / HertuflUg granulocytes / b / Stany growths in various culture media bslUouowyz / 2 / Agar stox culture z bslUoney / 3 / Hodowla w zagłuliłem / 4 / Hodowla w zagłębianis in gelatin z bulions / 5 / Mleczko ^ Umcs ^ e / 0 / Properties fizUoUoglcznθ / 1 / Reduction of nitrates / 2 / Oanitryfikacja / 3 / Test MR / 4 / Test VP / 5 / Indole production none negative positive detected / 30 ° C / circle with a diameter of 1 mm / 43 hours /, non-uniform and smooth, rather dry, flat, opaque, pale orange pink threads with a smooth surface, rather dry, slightly cross-pale, rather dry, pale pink .

no formation of bacterial tumor membrane, growth accompanied by syrupized clouding, sediment formed with a thin layer on, cone-shaped along part of the cavity, but not in the lower layer, no gelatin flow is observed no change negative positive negative negative positive

160 904

<td> /6/</td><td>Hydrogen sulfide production</td><td colspan="2">positive</td>

<td> /7/</td><td>Starch hydrolysis</td><td>negative</td><td></td>

<td> /0/</td><td>The use of citric acid Kocura culture medium</td><td>negative</td><td></td>

<td></td><td>Christensen culture medium</td><td>positive</td><td></td>

<td> /9/</td><td>Eliminating an unlimited source of nitrogen</td><td></td><td></td>

<td></td><td>nitrate</td><td>positive</td><td></td>

<td></td><td>ammonium salt</td><td>positive</td><td></td>

<td> /10/</td><td>Production of colored mass</td><td>uj emna</td><td></td>

<td> /11</td><td>urease</td><td>positive</td><td></td>

<td> /12/</td><td>oxidase</td><td>negative</td><td></td>

<td> /13</td><td>catalase</td><td>positive</td><td></td>

<td> /14/</td><td>Cellulose hydrolysis</td><td>negative</td><td></td>

<td> /15/</td><td>Range of Growth</td><td>pH 5-10,</td><td>temperature 10 ~⁴¹° C</td>

<td> /15/</td><td>Behavior towards oxygen</td><td>aer ^ spaces non-</td><td></td>

<td> /11/</td><td>Tyrosine degradation</td><td>positive</td><td></td>

<td> /16</td><td>Adenine breakdown</td><td>positive</td><td></td>

<td> /11/</td><td>phosphatase</td><td>positive</td><td></td>

<td> /20/</td><td>Tween 80 hydrolysis</td><td>positive</td><td></td>

<td> /21</td><td>Test 0-F</td><td>negative</td><td></td>

<td> /22/</td><td>Heat resistance / in 10% milk Dark in ¹²° C, 15 minutes /</td><td>lack</td><td></td>

<td> /23/</td><td>Acid and gas production from sugar:</td><td colspan="2">Acid Gas</td>

<td></td><td>L-arabinose</td><td> -</td><td> -</td>

<td></td><td>D-xylose</td><td> -</td><td> -</td>

<td></td><td>O-glucose</td><td> ♦</td><td> -</td>

<td></td><td>0-mannose</td><td> -</td><td> -</td>

<td></td><td>D-fructose</td><td> 4</td><td> -</td>

<td></td><td>mitosis</td><td> 4</td><td> -</td>

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

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

<td></td><td>trabaloze</td><td> -</td><td> -</td>

<td></td><td>D-sorbitol</td><td> ♦</td><td> -</td>

<td></td><td>D-mannitol</td><td> ♦</td><td> -</td>

<td></td><td>glycerine +: positive test, -: negative test</td><td> ♦</td><td></td>

<td> /24/</td><td>Growth in individual carbon sources</td><td></td><td></td>

<td></td><td>rnozyt</td><td> -</td><td></td>

<td></td><td>matoza</td><td> ♦</td><td></td>

<td></td><td>D-mannitol</td><td> ♦</td><td></td>

<td></td><td>rhamnose</td><td> -</td><td></td>

<td></td><td>D-eorbit</td><td> 4</td><td></td>

<td></td><td>m-hydroxybenzoic acid</td><td> 4</td><td></td>

<td></td><td>sodium adipate</td><td>V</td><td></td>

<td></td><td>sodium benzoate</td><td> 4</td><td></td>

<td></td><td>sodium citrate</td><td> 4</td><td></td>

<td></td><td>sodium lactate</td><td> 4</td><td></td>

<td></td><td>testotetron</td><td> 4</td><td></td>

<td></td><td>L-tyrosine</td><td> 4</td><td></td>

<td></td><td>glycerin / 1% / wtagowo / included</td><td> /♦/</td><td></td>

<td></td><td>trehalose p-hydroxybenzoic acid</td><td> /♦/</td><td></td>

<td></td><td>/ 1% / / wtgoworobJ ^ trstowe /</td><td> 4</td><td></td>

160 904 ♦ and positive, negative, / ♦ / «slightly positive / 28 / Fatty receipt 1 cell wall analysis

Contains unsaturated 1 saturated straight chain fatty acids and tuberculostearic acid. TLC mycolic acid gives a single stain.

As a result of the classification of the bacteriological properties described above based on the Berga Manuel of Systamaaic Becteriology * publication, it can be stated that the strain 0-1 is eerobic rods, Gram-doodling, poorly resistant to acid, catalase-positive and not producing endosporins, flageiuum bases. Micaluum has an elongated cochlear shape at the initial stage of growth, with further growth branching occurs and then division into short sticks. For this reason, it is believed that the strain belongs to the Nockeria bakkerH.

Analysis of the constituent acids of the bacteria that the bacterium contains unsaturated and saturated fatty acids, including tubartulo-steßtynic acid. TLC analysis / thin-charlet chromatography / mycolic acid gives a single spot with the same Rf value as for the standard Rhkdockcces rhodochrous / IFO 3338 / bakkeria, which distinguishes it from the genus Mycob ^ tenum. It also differs from the type of Nocardia in the composition / Quality of carbon atoms / mycolic acid. As a result of testing other biochemical properties it was found that it is a Rhodococcus Rhodoccwoue bacterium.

OrolnoketrkJa I am inclined to him in Thai. Therefore, it goes without saying that even a twaltttated mutant of a strain such as strain 0-1 can be used in the process of the present invention, as long as the breeding product produces nitrite hydride in the presence of tobatite ion.

Nitriles, which can be used As substrates for nitroyl hydrate produced by the drkbokutrokone described above, are aromatic and alpha-matric or dihydrate, in particular monohydrate.

The nitriles that are best suited for use in the process of the invention are aromatic nitriles, especially those that contain 4-10 carbon atoms with too much aromatic ring. A number of typical examples of aromatic nitriles include compounds represented by general formulas 1-6, including the ulcer of formula 1, preferably 4-, 3- or 2-yyJ8nzpyridia and the compound for sea 2, wherein R ^ and R ^ are hydrogen, CH groups ^, OH, OCH ^, Cl, I,

ON, NHg or NOg · A typical such compound is ^ η ^ Μΐ ^ Ι, o-, m- and p-y-z-benzenitrile, o-, Z-1 p-fluorolenzitrile, o-1 m-nitrobenzocitrile, p-aminoOlezonlt, o- , m- and ptolunitriles, 4-ct -ankenphenol, anisonitrile, phthalonitrile, isof ^^ πΗΓγΣ, terephthalonntryl, 2,6-dihydroxyl-octitrite, 2,4-douchlorobeaizkitrtl and 2,6-doufluOobeenocltril. Typical examples of compounds of formula (III) are 4 - 1/3 - naphthyl nitriles. These also include compounds of formula 4 in which X is a sulfur or oxygen atom, 2-thiofinotarbonitrtl and 2-furonntryl, the compound of formula 5, 5-yan Yankindzl and compounds of formula 6, examples 6 and ^ and ^ cyanopyrazine.

Another group of trillins useful in the process of the invention are preferably alitatic nitriles, Even more preferably mono- or di-methyl containing 2-6 carbon atoms, most preferably moo-nitriles. Due to the usefulness of the produced amides, a typical example of a nitrile is acrylonmryl, which is transformed into an amide with good hydration. It goes without saying that amides zdpowoadaJąya nlttykmm are compounds obtained as a result of the transformation of the CN group in nitriles into the CONHg group. In the case of two times the corresponding amides are obtained by converting at least one CIJ group into a CONHH group.

3 / Cultivation / nitrous hydrocarbons

The cultivation of microorganisms in the Rhzdoczyyet species can be carried out under any suitable conditions, except that cobalt ions must be present in the culture medium.

It may be practical to introduce into the culture environment an enzyme inducer, described in detail below, so that the hydrothermal nittyheats are heated in bakkerri cells.

/ 1 / basic environment

Examples of kdpowdnelyh culture media are given below. Those skilled in the art can easily change the amounts of the given ingredients, replace one ingredient with another, and ellminoo some ingredients or add other ingredients.

160 904 which nitrite is hydrated with the help of an enzyme preparation, and therefore not by biological means. It is clear that the hydration reaction should be carried out under such pH and temperaure conditions that the enzyme does not lose its activity. It can be said that these are the same conditions under which the reactions described above are carried out^Bby the way biologist *. Gak is described above, a solution according to which micro-organisms do not occur during enzyme activity, should also be considered as the biological production method of the invention.

In the method according to the invention, the preferred pH range for nitrile hydrate is 7-9, at an optimum pH of 8.0. When the pH of the reaction solution is below 7, the enzyme activity tends to drop rapidly. Therefore, it is desirable to add a buffer to the reaction solution. Any of buffers such as press phosphate, Tris / KCl buffer, HEPES / KCH buffer and sodium borate will be used as the enzyme activity, nitrile hydrate enzyme activity will not change.

The substrate concentration in the culture medium or hydration reaction solution is usually 4-7 moles / liter, and the reaction temperature is usually in the range of 10-3Qec.

3. These are the examples

The method of measuring nitrile hydrate activity and the unit of activity in the examples below are defined as follows.

/ 1 / Method for measuring nitrile hydrate activity

Nitrile Hydratase Activity is dreamed by carrying out the reaction in a 2 ml reaction mixture containing 10 millimH benhydritrile, 30 millimH potassium phosphate As buffer (pH 7.0) and a certain amount of fine cells (secreted from the culture medium) at 10 ° C for 5 p. 2 ml of LN KC1 to stop the resection.

/ 2 / Unit definition □ other unit \1j nit 1yJowrj hydrate activity is defined as the amount of enzyme necessary for the production of benzamide from benzoyl under the above-mentioned conditions at a rate of 1 urnol / minute.

Example 1. Strain 3-1 was cultured in the following medium composition, under conditions also defined, with the disclosure of nitrile hydratase activity being tested by adding C0Cl2 and / or FrSO₄ to the medium during breeding.

/ 1 / Composition of the medium

Ingredients mix of vitamins K2HPO4 kh₂after₄

MgS0₄.7H20 distilled pripionitrile

Quantity / in 1 liter of fire ^ in / 3.0 ml 0.5 g 0.5 g 0.5 g 2 ml form / pH 7.2 / / ii / Conditions at 28 ° C / 70 - 80 hours

Obtained in the table in Table 1.

It can be stated that nitrile hydratase activity is not revealed. If FeSO4 is added in two basic media, the active and hydrate activity is revealed by the addition of CoClg together, the addition of FeSO4 by the system, which C0Cl2 added · adversely affects udder.

160 904 / i / Medium A

Ingredients

X1 vitamin blend

K₂HP04 kh₂after₄

NACD

MgS04.7H20 distilled water xl Composition: biotin calcium pantothenate mite nicotinic acid tlamine hydrochloride pyridoxine hydrochloride p-amlbenzoic acid riboflavin folic acid water

Amount / in 1 liter of food / 0.1 ml

13.4 g 6.5 g 1.0 g 0.2 g rest / pH 7.0 / ug 0.4 mg 2 mg 0.4 mg 0.4 mg 0.4 mg 0.2 mg 0.2 mg 0.01 mg to 1 liter / ii / Medium B glycerin peptone malt extract yeast extract distilled water / 111 / Medium C yeast extract kh₂after₄

K₂HP04 g 5 g 3 9 3 g rest / pH 7.2 / g 0.5 g 0.5 g 0.5 g rest / pH 7.2 /

MgS04.7H₂0 distilled water / 2 / Enzyme inducer

The enzyme inducer used to induce and produce nitrile hydratase in Rhodiciccus rhidichrius micro-organisms may be any inducer suitable for the particular micro-Bistro.

Typical inducers suitable for use in the process of the invention are nitriles and amides.

Examples of enzyme dukes. whose action has been confirmed in relation to strain 3-1 are crotonones. acetonitrile. niry prop ^ !. benzamide. «Ϊ́γ ^^^ γΐ. izobutyromtryl. n-kaproontryl. 3-pentillary trill. piwalorntryl. n-butyramide. isobutyramide. n-kaleΓamid_l n-caprinamide, mercrystilide 1 and phenylcetamide.

/ 3 / source of cobalt ions

Nitroyiockey hydrates are not obtained even if the enzyme inducer described above is present in the culture medium anyway. that it is relevant to the method of the invention. that there are kobaat ions in the breeding environment. Because the breeding environment is an aquatic environment. Cobalt ions are usually formed by adding a water-soluble cobalt compound to this environment. Water soluble cobalt compounds are given in chemical textbooks. therefore, it will be easy for a specialist to select and apply an associated compound. in some cases by doing a simple initial blank. About typical cobalt compounds include these. which provide Ion Co ** or Co ^ *. and especially these. which provide Ion Co **. and the particular is not e.g. kobaat chloride. cobalt sulfate. kobaat acetate. cobalt bromide. cobalt borate etc. Other examples of cobalt compounds are vitamin Bj? and metallic cobalt. which produce cobalt ions in situ in

160 904 the culture environment by ionization or oxidative action of the micro-organism during culture.

/ 4 / Kennel

For the production and accumulation of nltryo hydrate in bacterial cells, the strain 3-1 used is cultured in the culture medium described above under appropriate conditions.

The enzyme inducer is used in an amount of 2-6 g / liter of culture medium, and cobalt ion in an amount of 5-15 rng / liter of culture medium, calculated as CoC ^ ·

Specific examples of the composition of the culture medium mixture are as follows and

<td>/ V</td><td>Medium A</td><td>1 liter</td>

<td></td><td>Ace ^ o ^ ry! / Nnduktor /</td><td> 2 9</td>

<td></td><td>CeClg</td><td>10 mg</td>

<td>/ Ϋ /</td><td>Medium B</td><td>1 liter</td>

<td></td><td>Izowa ae ^ om ryl</td><td> 2 9</td>

<td></td><td>C ^ t ^ 3L2</td><td>10 mg</td>

<td>/ N /</td><td>Medium C</td><td>1 liter</td>

<td></td><td>Krwtwnαaid</td><td>2 g</td>

<td></td><td>C0CI2</td><td>10 mg</td>

Nitrite hydrratase can be successfully prepared by shaking the culture of strain 3-1 at 15-50 ° C, preferably 20-45 ° C, and most preferably about 30 ° C at pH 7-9 for about 30 hours or longer, preferably for 40 hours or longer, at the upper limit e.g. 120 hours. It is preferred that the enzyme inducer is present from the initial stage of the dodo, and in addition it is desirable to introduce additional amounts of the inducer if one wants to obtain bacterial cells with high activity. For example, if the shaken culture is carried out at 28 ° C for 76 hours, further amounts of crotonamide are added 26 1 56 hours after the start of the reaction so that its concentration is 0.1% by weight / volume.

4 / Nitrile hydration

The term biological production of the amide used in the specification, according to which nitrile is hydrated to the corresponding amide as a result of the action of nitrile hydratase produced by fine-grained wbejauSr. As stated above, various real solutions and variants ensuring the action of filament hydrate on nitrile.

One of these solutions involves the production of amide in a culture medium when the substrate nitrile is present in the culture of microorganisms.

Another solution that suppresses nitrate hydrate on the substrate is that in order to carry out the hydraacc reaction, a non-fat substrate is added to the culture medium in which nitrile hydrate has accumulated. A variation of this solution is to use the bodGow ^ environment in which com ^! microbes have been destroyed as a breeding environment in which iltryWowd's hydratase^,.

Another solution ensuring the action of nltryWoyrj hydrate on the substrate is to isolate the cells in which the nitroyoy hydratase has accumulated from the culture environment, preferably by introducing cells on the appropriate carrier or immobilizing them, and then bringing them into contact with the substrate. This method, and in particular the preferred solution, according to which immobilized cells are used. It is considered suitable for industrial use just as or more than other solutions described in p ^ ^ i ^. This method of application of immobilized cells is widely used, which is also true for the selection of the type of carrier for the method of immobilizing microorganisms as carriers, such as the use of immobilized microorganisms in a so-called biological reactor.

Yet another solution to fill the substrate with hydrates of nitrate for the substrate relates to the method in which the enzyme preparation of iltrywater and

160 904

Table 1

Metal ion added

<td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>• * T '</td><td></td><td>"Γ</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td></td><td rowspan="2">CoClg</td><td>/ mg / 1</td><td>0 and 0</td><td> 1</td><td> 0 1</td><td> 0</td><td>and</td><td> 0</td><td> ,</td><td> 10</td><td></td><td> 10</td><td></td><td> 10</td><td></td><td> 10</td><td> «</td><td> 10</td><td></td>

<td></td><td>- «» - -I</td><td>- - r -</td><td>"AND</td><td>- "</td><td></td><td> T</td><td></td><td>"</td><td></td><td>• L</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td></td><td>FeS0₄</td><td>/ mg /,</td><td> 0 , 5</td><td> 1</td><td> 10 ,</td><td> 20</td><td> 1</td><td> 40</td><td> 1</td><td> 0</td><td></td><td> 5</td><td></td><td> 10</td><td></td><td> 20</td><td>AND</td><td> 40</td><td></td>

<td></td><td rowspan="2">Majesty</td><td rowspan="2">cell^χΑ '</td><td>- - r -</td><td> •|</td><td>• - Γ</td><td></td><td>"T.</td><td></td><td></td><td></td><td>"AND</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td></td><td></td><td rowspan="2"> 14*</td><td rowspan="2"> 1,25*</td><td></td><td></td><td></td><td></td><td></td><td rowspan="2"></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td></td><td colspan="2">/ mg / ml / '</td><td> 1,06* 1,</td><td> 1.</td><td> 24*</td><td> 1.</td><td> 34</td><td> 2,04</td><td> 1,90</td><td></td><td> 2,16</td><td></td><td> 1,16</td><td></td><td> 2,07</td><td></td>

<td></td><td></td><td></td><td>-___ L-</td><td>__AT</td><td>--- L.</td><td></td><td> —1.</td><td></td><td>-L.</td><td></td><td> .1.</td><td></td><td></td><td></td><td>t</td><td></td><td> 1</td><td></td><td></td>

Enzymatic activity

<td>U / mg</td><td>^kohm^m beds^x^</td><td>T</td><td> 0</td><td>L</td><td> 0</td><td> ’ 0</td><td>at</td><td> 0</td><td></td><td> 0</td><td> . 1</td><td> 0,59 ¹ ,</td><td> 0,26 1</td><td> 0,34</td><td> 0,32</td><td colspan="2"> 0,16 1</td>

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

<td></td><td></td><td> .1.</td><td></td><td>_L.</td><td></td><td>_L___</td><td>_L.</td><td></td><td> ..1.</td><td></td><td>-J.</td><td> ------4.</td><td colspan="2"> - _ 1 _</td><td></td><td> 1</td><td></td>

xl Number of cells based on dry weight x2 U is the unit of activity as defined above, and the number of cells is expressed as dry mms.

Example II · The effect of various nitriles or amides as enzyme inducers on strain 0-1 is given in Table 2 below.

given in Table 2 was obtained by pre-culturing the strain 0-1 in the given medium B at 28 ° C, adding nitrile or emide as an inducer in an amount of 0.1% by volume or .0.0% w / v, respectively, after appropriate strain proliferation, e followed by loculation of the strain in the aforementioned C-culture medium, to which 0 (011% by weight of Weight in CoCl? 1 was added, cultivating for 36-48 hours.

Table 2

<td></td><td></td><td> 1 1</td><td>Activity right / U / mg /</td><td>Activity whole / U / ml /</td><td> 1 “ ,</td><td>Number of cells / mg dry ^ ^ Imórk m /</td><td> 1</td>

<td></td><td></td><td> 1</td><td></td><td></td><td> 4 -</td><td></td><td></td>

<td></td><td>crotonamide</td><td> 1</td><td> 2,22</td><td> 4,48</td><td></td><td> 2,02</td><td></td>

<td></td><td>Αοθ πΙι ^ ^ Ι</td><td> 1</td><td> 1.41</td><td> 3,47</td><td></td><td> 2,46</td><td></td>

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

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

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

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

<td></td><td>n-Butyronite ryl</td><td> ,</td><td> 1,40</td><td> 0,38</td><td></td><td> 3,70</td><td></td>

<td></td><td>Izwbυtyronitryl</td><td> ,</td><td> 0,41</td><td> 1.24</td><td></td><td> 3,06</td><td></td>

<td></td><td>Izow ^ llerom. t ryl</td><td> 1</td><td> 0,34</td><td> 1,05</td><td> ,</td><td> 3,07</td><td></td>

<td></td><td>n-Kappooiiry1</td><td>t</td><td> 0,28</td><td> 1,04</td><td> ,</td><td> 3,71</td><td></td>

<td></td><td>3-P en te mom t ryl</td><td>AND</td><td> 0,32</td><td> 1,42</td><td> ,</td><td> 4,49</td><td></td>

<td></td><td>Beer ^ it ^</td><td> ,</td><td> 0,35</td><td> 0,24</td><td> ,</td><td> 0,69</td><td></td>

<td> 5</td><td>n * - Sut yroarnd</td><td> 1</td><td> 0.43</td><td> 1,55</td><td> 1</td><td> 3,62</td><td></td>

<td></td><td>isobutyramide</td><td> ,</td><td> 0,09</td><td> 0,33</td><td> 1</td><td> 3,48</td><td></td>

<td></td><td>Izwwaierantd</td><td> 1 |</td><td> 0.44</td><td> 1,08</td><td> ,</td><td> 1,81</td><td></td>

<td></td><td>r-Kapronamid</td><td> 1</td><td> 0,30</td><td> 1,06</td><td> ,</td><td> 3,52</td><td></td>

<td></td><td>Rilamide tag</td><td> ,</td><td> 0,20</td><td> 0,62</td><td> ,</td><td> 3,12</td><td></td>

<td></td><td>Fjnylwajetamtd</td><td> 1</td><td> 0,29</td><td> 0,28</td><td></td><td> 0,95</td><td></td>

<td>k</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

Example III. While strain 0-1 is obtained as a result of growing the strain in medium which is above supporting C medium with the addition of C0Cl2, 1 × ^ per ^, suitable in amounts of 0.01 g and 2 g / liter of food. Various nitriles used as suootates were reacted in the medium. The reaction was carried out using a 2 ml reaction solution containing in ml and 10 ml of potassium phosphate buffer as a buffer of pH 8.0 and 200 mm of substrate obtained at 2 ° C for 25 hours for 76 hours. The reaction was stopped by adding 0.2 ml to the mixture

160 904 IN HCl. Nitrile hydrate activity for individual substrates is expressed as the ratio of activity relative to nitrile hydrate activity measured for 3- * cyanopyridine as substrate, i.e. specific activity in%, with the amount of reaction product or remaining substrate analyzed by HPLC / high-performance liquid chrommtoogaphy /. The results obtained are shown in Table 3 below.

Table 3

<td>substratum</td><td>and and</td><td>Specific activity /% /</td><td></td>

<td>• 3-Cyanopyridine</td><td>and</td><td> 100</td><td></td>

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

<td>1 4-Cyanopyridine</td><td>and</td><td> 129</td><td></td>

<td>1 2-Cyanopyridine</td><td>and</td><td> 64</td><td></td>

<td>* 5-Cyanflendol</td><td>and</td><td> 9</td><td></td>

<td>2-TiofBornit style</td><td>and</td><td> 116</td><td></td>

<td>2-Furonettyl</td><td>and</td><td> 71</td><td></td>

<td>1 Benzooitrile</td><td> 1</td><td> 80</td><td></td>

<td>• 4-CyJtioienol</td><td>and</td><td> 24</td><td></td>

<td>¹ p-AmlnobbniiZitryl</td><td>AND</td><td> 16</td><td></td>

<td>m-Ni-benzo-iryl</td><td>and |</td><td> 7</td><td></td>

<td>, o-Nitrobenzenni style</td><td>and</td><td> 16</td><td></td>

<td>1 m-ChloΓobennzzitrile</td><td>and</td><td> 29</td><td></td>

<td>• p-Tolunmtryl</td><td>and</td><td> 5</td><td></td>

<td>o-tolunitrile</td><td>and</td><td> 46</td><td></td>

<td>m-ToZunitryl</td><td>and</td><td> 32</td><td></td>

<td>, Anlzonittyl</td><td>and</td><td> 20</td><td></td>

<td>1 o-Chlorobenzozitrile</td><td>and</td><td> 41</td><td></td>

<td>• p-Chlorobenzonitrile</td><td>and</td><td> 7</td><td></td>

<td>2,4-OoucClorobeeiiOiiryl</td><td>and</td><td> 2</td><td></td>

<td>2,6-DwuchhoΓobeeizoetryl</td><td>and</td><td> 1</td><td></td>

<td>and Cyanopyrazine</td><td> 1</td><td> 80</td><td></td>

Example IV V 400 ml Sakeguchi mine flask was placed with 400 ml of medium being the above medium C with the addition of CoClg 1 crotonamide, in amounts of 0.0¹ g 1 2 ^g/ liter of medium, then fermented ^hodowl^and mixtures on a drier at 28 ° C. Cultivation was continued by adding 0, «% by weight vol. Crotonamide / 800 mg / to the wort.

400 ml / after 30 and 60 hours from the start of breeding. The process was completed after 80 hours from the start of breeding.

Bacterial chambers were separated by centrifugation of the wort at an overload of 12,000 for 15 minutes, in a centrifuge separator (model Hitachi SCR 20 B), then washed with 0.85% Nad, centrifuged again and dispersed in 40 ml of the above-described solution. A small portion of the suspension was taken as a sample, which was used to determine the dry weight of bacterial cells in suspension.

A suspension containing cells (in an amount corresponding to 2.33 mg dry cells) was added to 7 ml of the reaction solution containing 10 m ^ mZ ^. potassium phosphate as buffer / P^{H 8},0/ ^{and 7},⁵⁷ mol ³-cyjanopirydyny. Prooα reaction^dionz at 25 ° C during the night, he adds^j0.55 mole and 0.79 mole of 3-cyampiridine to the reaction solution after 316 hours from the start of the reaction.

18 hours after the start of tetkCIl, 5.58 moles of nicotinic acid amide were obtained. Consequently, the conversion was 99.5%, which corresponds to an accumulation of 681 g nicotinamide. At this concentration, the reaction mixture is deposited as a result of nicotinamide depositing.

160 904

The nicotinic acid amide thus obtained was identified by isolating the product in the form of crystals, which were analyzed by means of elemental analysis, IR / sub-infrared spectroscopy, NMR / nuclear magnetic resonance / and βρβΗηΒ ^ ρϋ massive. No nicotinic acid detected

EXAMPLE V A suspension containing cells (in an amount corresponding to 2.33 mg dry chambers) obtained in Example 4, was added to 4 ml of the reaction solution containing 10 mm potassium phosphate As buffer (pH 0.0) and cyanopyrazine in various concentrations. The reaction was carried out at 25 ° C. 4 moles of cyanopyrazine were converted to pyrazinamide at a 100% conversion rate over 6 hours and 6 my cyanopyrazine within 9 hours. N ^^ spread when the suspension containing the baking agent in an amount of

4.66 mg dry cells instead of 2,: 33 mg dry cells used above were added to 4 ml of similar reaction solution, after 6 hours of reaction 7 my cyanopyrazine was converted to pyrazinamide at 100% conversion, and after 9 hours 9 moH of cyanopyrazine. No pyrazinecarboisilic acid formation

The pyrazinamide crystallized out of the solution in a margin of formation. The crystalline precipitate was collected directly and crystallized from ^^ i ^ and ^ o. Crystals from zJtiatiized as pyrazinamide based on analysis by elJóβJtarnθr, IR, NMR and spectroscopy © ι ^ βοο ^. The analysis of cyanopyrazine, pyrazinamide and pyrazinecarboxylic acid was carried out using high-density immunoassay method. The same analyzes were also made in the following examples.

Example VI. Suspension of bacterial cells / in amount

4.66 mg of dry and which (obtained in Example 4) was added to 4 ml of the reaction solution containing 10 mm of potassium phosphate As buffer (pH 8.0) and 3 mmle mm ^ a / υΉπ ^^ lu, after which the reaction was carried out in ° C by adding 3 moles mytotkytoltiryl to the reaction solution 1 hour and 3 hours after initiation of regression. 12 hours after the onset of reskeces, 9 moH of meracrylamlZ was obtained with a conversion of 100%.

At the above-mentioned dynamics, when an additional 1 ml of methotkytonttryl was introduced 5 hours after initiation of resk, 10 mm me mei ^ t ^ ryl ^ billion was obtained at 100% conversion 24 hours after the initiation of reskec. This means that 851 g of methacrylilld per liter of reaction solution was prepared and accumulated. The reaction solution was diluted with water, whereupon the bacillus was removed by centrifugation at 12,000 g for 15 minutes. The cell-free solution was concentrated in a rotary evaporator, followed by crystalline solution. The crystals were dissolved in crystalline water, yielding crystals ^^ et ^ 1 ^ yl ^ a ^ ^ du.

Example VII. Suspension of bacterial cells / in the amount of equivalent

4.66 mg of dry substance obtained in Example 4, was added to 4 ml of the reaction solution containing 10 mm © potassium phosphate As buffer (pH 8.0) and 1 mole crotonitrile, after which the reaction was carried out at 25 ° C by adding 1 ml of irltonltryl reaction solution making goZdin ^. After 6 hours from the initiation of the reaction, 6 mm © crotonamide was obtained with a conversion of 100%. When additionally 1 ml of crotonitrile was added to the reaction solution after 6 and 10 hours, 7 ml and 8 ml of krltltamiZu were obtained with a conversion of 100%. This corresponds to the production and accumulation of 681 g of blood cells in 1 solution. Crystallization of krktktαylZu was carried out in the same manner as in Example VI.

Example VCXI. Suspension of bacterial cells / in an amount of anthrax response

4.66 mg dry weight / obtained in Example 4, was added to 4 ml of the reaction solution containing 10 ml of my potassium phosphate as buffer (pH 8.0) and 3 moles of acetonmtril, after which the reaction was carried out at 25 ° C. 3 moles acetonitrile to the reaction solution 1 and 3 hours after the start of the reaction and 5 mdi acetonitrile 6 hours after the start of regression. After 12 hours from the start of the reaction, 14 minutes of acetamide were obtained with a conversion of 100%. In other words, 827 g / liter of reaction solution was prepared and accumulated. The reaction solution was diluted with water, followed by centrifugation to kill bacterial cells. The liquid was concentrated to dryness on a rotary evaporator, dissolved in methanol and irritated to give acetamide crystals.

160 904

Example IX. Bacterial cell suspension (in an amount corresponding to 4.66 mg dry kornmrre), obtained in Example 4, was added to 4 ml of the reaction solution containing 10 mmmli potassium phosphate as buffer (pH 8.0) and 3 moles of 3-hydroxypropionitr^slu, followed by reestablishment at 25 ° C ^dgiving away four^krotnie ^p3 moles 3^-HYDO^lxy- * propionitrile at intervals of 1 hour. After 5 hours from the start of the reaction, 15 mdi 3 ^ h ^ dr ^^^ sy ^ pr ^^ amide was obtained with a conversion of 100%. When 3 moles of 3-hydroxypropioitrile were added at this stage, after 11 hours from the start of the reaction, 18 my 3-hydroxyproplonone was obtained with a conversion of 100%. This means that 3-hydro / syprorlOlαold was prepared and accumulated in an amount of 1600 g / liter of reaction solution.

The reaction solution was diluted with water and centrifuged to remove cells. The cell-free solution was concentrated in a rotary evaporator and crystallized at a temperature of -20 ° C. The crystals were dissolved in iso-opam and 3-hydroxyrrorloneoid crystals were obtained after recrystallization from this solvent.

Example X. In a 1-liter Sakaguchi flask, 400 ml of the medium given above, medium C with the addition of C0Cl2 and crotonamide were placed, in amounts of 0.01 g of 1 2 ^ 1-yt nutrient solution, respectively, followed by mixing of the mixture with shaker at 28 ° C. The culture was continued to be added to wort 0.25 iag / vol / crotonamide / 800 mg / 400 ml / 00 36 1 56 hours after inoculation with the culture. The process was completed 76 hours after the beginning of hodl.

While the bacterial cells were separated by centrifugation of the wort at an overload of 10,000 g for 20 minutes in a centrifuge separator (model Hitachi SCR 208), after which they were washed with 0.85 NaCl, the plnlinle was centrifuged and dispersed in 40 ml of the above-described solution. A small portion was taken as sample that was used to determine the cell dry matter. The suspension containing cells (in an amount corresponding to 2.96 mg dry cells) was added to 4 ml of the reaction solution containing 10 potassium phosphate as buffer / pH 8.0 / 1 cyanide at various concentrations. The reaction was carried out in²⁵° C. ⁸ ooh 3-c^Useall^plridine was subjected to thallyloxyl at a conversion of 10C% within 9 hours and 9 m-cyanopyridine within 22 hours. However, when the suspension containing coagulant balls in the amount of ldpowiβdajajaj 5.92 mg dry cells instead of 2.96 mg dry cells were added to 4 ml of similar reaction solution, after 5 hours of reaction 9 moU 3-cyanoriΓydyly was transformed into nickeltin αmίd at 100% conversion, and after 9 hours of reaction, 12 mdi 3-cyano, pyridine, which means that 1.765 g of ni / otin · billion per liter of reaction solution were prepared and accumulated.

Nicotinamide crystallized from the solution into a void of formation. The crystalline precipitate was collected 1 oe / oystallOiwanl from methanol.

Example XI. Bacterial cell suspension / in an amount corresponding to J.

5.92 mg dry cells /, obtained in Example X, were added to 4 ml of the reaction solution containing 10 mmOU of potassium phosphate as buffer (pH 8.0) and 1 mole of benzzouril, followed by^kcJ^ę was conducted in ²⁵° C Pretending to be 1 mole of benzomer ^dabout a real solution ^pat 12,

3, 4, 5 and 7 hours after cutting off the reaction. After 24 hours from the onset of sedimentation, 7 mdi / 848 g / liter / barrel are obtained with a conversion of 100%.

Example XII. Suspension of bacterial cells / in an amount equivalent to;

5.92 mg dry cells / uiys / ene in Example X, was added to 4 ol of the reaction solution containing 10 omoH potassium phosphate as buffer / pH 8.0 / 1 0.5 mole 2,6-difluoro-IcenzooUryl, after which the re-establishment in ²5 ° C ^dodając ⁰»⁵ 2,6- ^ υί J.uorobenzcri.rfydo mole reaction solution 2, 4, 6 and 8 hours after the start of the resketsk. After 22 hours from the decomposed reaction, 2.5 mol / 393 g / liter / 2,6-difluocobenzamide was obtained with a conversion of 100%.

Example XIII. Zewiθsilą bacterial cells / in number corresponding to^,

5.92 mg of dry kod ^ r ^ lc /, obtained in Example X, was added to a 4-ol reaction solution containing 10 potassium phosphate as buffer / pH 8.0 / 1 1 ool of 2-thiophyll, nitrate-nitrile, ^pReaction was carried out at 25 ° C by adding 1 ool of 2-t ^^ no ^ r ^ nitro to the solution.

160 904 reaction after 1 hour from the start of the reaction · After 5 hours from the start of the reaction, 2 moles / 254 g / liter / 2-thiophonocronronide with a conversion of 100% were obtained ·

Example XIV · Suspension of bacterial cells / in an amount corresponding to 5.92 mg dry kimmrθkO, obtained in Example X, was added to 4 ml of the reaction solution containing 10 mmmoi potassium phosphate As buffer / pH 8.0 / and 1 mole 2-0uronitr^slu, then rea^kc^PH was carried out at 25 ° 0 ^dDD^andc 1 mol ²-^furonitr^sinto the real solution 1, 2, 4, 6, 8, 11 and 23 hours after initiation of regression. 30 hours after the start of the reaction, 8/888 g / liter / 2-furanecarbonamide was obtained with a conversion of 100%.

Example XV · Suspension of bacterial cells / in an appropriate amount

5.92 mg dry cells /, obtained in Example X, were added to 4 ml of the reaction solution containing 10 mmmU of potassium phosphate as buffer '(pH 8.0) and 4 moles of 3-indolircetomtr^slu, then rea^kc^PH conducted at 25 ° 0 · After ²4 morning in^h 4 moles / 697 g / liter / 3-indoleacetamide at a conversion of 100% were obtained from initiation of the reaction (o3-cn

Formula 1

Formula 3

<img file="PL160904B1_D0001.tif" />

CN r4o3r²

Formula 2

O-CN

Model 4 (^ CN

Formula 6

Department of Publications of the Republic of Poland. Cakład 90 copies

Price: PLN 10,000