Biological production of amide

[Detailed Description of the Invention]

The Backstage J (technical field) present invention of [invention relates to the method of transforming to the amide which hydrates nitril and corresponds this by the operation of nitril Hydra Tarta of microorganism origin. Specifically, the present invention relates to the biological manufacturing process of the amide which consists the main features in the production method of a microorganism and nitril Hydra Tarta to be used. (Prior art) Although manufactured by the aqueous phase of low-grade fatty series amide, for example, acryl amide, and the nitril which Correspondence , for example, acrylonitrile, The way the operation of Trilase or nitril Hydra Tarta performs this hydration to the production To shake enzyme of a microorganism is proposed. (For example, refer to JP, 62-21519, B, JP, 61-162193, A, JP, 62-91189, A, JP, 56-17918, B, and JP, 59-37951, B). notes l] Also industrially the biological manufacturing process of such amide is enforced, and is carried out as a Competitive manufacturing process of acryl amide. Although some microorganisms are already proposed as what is used for the biological manufacturing process of such amide, when these artificers get to know, even if these microorganisms are effective in hydration of low-grade fatty series nitril, they are not necessarily effective in hydration of aromatic nitrile. For example, the method of hydrating 3-cyano pyridine and manufacturing nicotinic acid amide has low yield , and it cannot enforce it easily industrially. By the way, generally cultivating a microorganism under existence of ferric iron or manganese ion is known, The example which this art is used also for biological manufacture of amide, for example, cultivates the microorganism of Rhodococcus Genus under existence of ferric iron can be seen in JP, 61-162193, A and JP, 62-91189, A each gazette. Although it was indispensable in Fe being included in the nitril hydration enzyme of the Pseudomonas Genus bacterium origin in the active center of trill Hydra Tarta and Its , therefore ferric iron carrying out into a culture medium at cultivation of this microorganism in q In according to the place which these artificers examined, Also in the case of the above-mentioned well-known example about the microorganism of Rhodococcus Genus , it is presumed that the ferric iron in the culture medium in the case of the cultivation is indispensable because of nitril aqueous phase enzyme production. [Outline of an invention] (important point purport) Strain in which the present invention of Rhodococcus Genus is specific against the above-mentioned knowledge, namely, -- in that there is no J-1 share , of the Rod Claus kind about nitril Hydra Tarta at a ferric iron content culture medium in Produce , and a cobalt ion Including H culture medium -- for the first time -- nitril Hydra Tarta -- production To shake -- things, And nitril Hydra Tarta produced by doing in this way is made based on discovery of thing , for which it is changed into amide by using aromatic nitrile as a substrate. Therefore, in the method of changing into the amide which the manufacturing process of the amide by the present invention hydrates nitril by operation of the nitril Hydra Tarta enzyme of microorganism origin, and corresponds this, nitril Hydra Tarta cultivates and obtains the microorganism of a Rhodococcus Genus Rodoku loth kind (Rhodococcusrhodochrous) under cobalt ion existence. (EFFECT OF THE INVENTION) According to the present invention, by a ferric iron content culture medium, nitril Hydra Tarta activity is revealed by a cobalt ion content culture medium for the first time to nitril Hydra Tarta activity being zero. This specific criticality nature of the metal ion demand nature of a microorganism is understood to completely have been unexpected about nitril Hydra Tarta revelation. According to the present invention, aromatic nitrile can be hydrated advantageously. It says from the importance of the nicotinic acid amide (namely, hydrate of 3-cyano pyridine) as vitamin materials etc., or pyrazine amide (namely, hydrate of cyano pyrazine) useful as antituberculosis drugs, and this effect of the present invention is useful. [Detailed description of the invention] 1 and the fundamental contents present invention of biological manufacture of amide are although it is the method of hydrating nitril and changing this into amide by the operation of nitril Hydra Tarta of microorganism origin, This method consists of an operation on cultivation of a microorganism, guidance of nitril Hydra Tarta , and the obtained substrate nitril of nitril Hydra Tarta fundamentally. These can take arbitrary modes [it is publicly known in itself as unit operation, and be-like / the present invention / INL / affinity]. "Cultivating and obtaining a microorganism under cobalt ion existence" sets it as a natural premise to have performed guidance of nitril Hydra Tarta by the present invention. The operation of nitril Hydra Tarta carries out the method of changing into the amide which hydrates nitril and corresponds this by operation of the nitril Hydra Tarta enzyme of [microorganism origin which the present invention makes a premise", and it includes arbitrary modes pertinent about a way. Although the enzyme which the microorganism was made to produce is collected as one of such the modes and there is the method of using this as enzyme Standard product , a case like such an ordinary catalyst reaction shall also be treated as "biological manufacturing process J by the present invention. 2 and details 1 of a hydration reaction The microorganism used by the microorganism present invention is a thing of a Rhodococcus Genus Rodoku loth kind (Rbodococcus rbodocbrous). The number of the typical things of this kind of stock is J-1. J-1 share of details are as follows. (1) The origin and Nearest : these artificers extracted the eJ-1 share from the soil of Sakyo-ku, Kyoto-shi, it was deposited with the Fermentation Research Institute, the Agency of Industrial Science and Technology, on September 18, Showa 62, and has obtained the FERM BP-1478 No. accession number. (2) Mycology character (a) type The form and of a voice (1) cell. It 9~1. O Is and is nothing [ Ladle °] of the polymorphism of a 3~1ou size (2) cell. The shape of long Culm is presented in early stages of cultivation, The shape of a Offer object shows the growth accompanied by snapping without a curve, and it carries out a short bacillus to behind, (3) motility which Tear behind Existence of nothing To 4 spore are. 5 Durham stainability to spread Positive (6) acid-fastness negative (7) different stain -- snug Growth state (30degreeC) (1) meat-juices agar *1'- Losing culture in (b) each culture medium accepted Diameter 11I11 (48 hours) circular and irregularity, It is smooth and they are opacity and light orange pink (2) meat 11 Cold person oblique culture to surface dryness feeling, and Shao and 1. Filar, surface smoothness, and a section get dry in the letter of upheaval a little, and are feeling and light orange pink (3) meat-juices liquid culture. A bacillus film is formed and it grows flourishing. Raw (4) Meat 11 gelatin stab culture (5) Lydmus milk (e) the use (10) pigment of the source of use (9) inorganic-matter nitrogen of the hydrolysis (8) citrate of the generation (7) starch of generation (6) hydrogen sulfide of reduction (2) Denitrification reaction (3) MR test (4) VP test (5) Indore of a physiological property (+) nitrate raw-grows up -- it being alike and following -- middle -- muddiness of a degree, Precipitation is formed. Although it grows on the surface to low 1 and A state along with growth and a puncturing part well, to a lower layer part, it hardly grows. As for gelatin, liquefaction is not accepted. The culture medium of positive negativity negativity negativity positivity positivity negativity Koser not changing: ShadeThe culture medium of sex Christensen: Sun Sex nitrate: SunSex ammonium salt: Sun Decomposition (19) phospha Tarta (20) Twe of decomposition (18) adenine of attitude (17) tyrosine to range (16) oxygen of hydrolysis (15) growth of sex negativity (11) Urease (12) oxidase (13) catalase (14) cellulosee n80 hydrolysis (21) 0-F test (22) heat resistance (72degree [in 10% skim milk] C) 15 minutes (23) From vM to acidAnd generation L-arabinose pH of gas:5~10 temperature: Generation of 10~41 degreeC aerotropism positivity positivity positivity positivity 0 (weak) -less acid As the natural material-xylose D-glucose D-mannose glycerin [D-fructose maltose sucrose milk sugar trehalose D-vine bit D-Mannit] (24) sole carbon source of gas Growth inositol maltose D-Mannit rum north D-sorbitol m-hydroxy benzoic acid ++10+ sodium sodium benzoate sodium-acid-citrate sodium lactate test Tetoron L- Tyrosing resale (1%) ( Vengeance /V) trehalose p-hydroxy +++++++ adipic acid benzoic acid (1%) (nu/V) (25) fatty acid and cell wall analysis (+) -- it is positive although it is weak. Unsaturated, saturation straight chain fatty acid, and label crossing Thearin acid are included. TLC of mycolic acid gives a single spot. When the above mycelia cake character is classified based on a bacteria classification document (Bcrgy's Manual orSystematic I3acteriology) (198G) of a barge, J-1 share is a bacillus which does not produce an inner raw spore of aerotropism, gram-positivity, weak acid-fastness, and a catalase positivity. A flagellum is not grown. The shape of a fungal thread is presented according to the shape of a long bacillus in the initial process of growth, the growth accompanied by a branch (Branching) is shown, and belonging to Nocardia type bacteria from Tear (ing) in the shape of a short bacillus behind is admitted. Analysis of fatty acid composition contains straight chain fatty acid of unsaturated and saturation containing label crossing Thearin acid. Since TLC of mycolic acid gives It-spot which shows the same Rf as standard drawing Rodococcus rhodocl+rous (IFo 3338), MycobacLerluIl Genus is distinguished. It is distinguished from Noeardla Genus from composition (carbon number) of mycolic acid. In addition, from examination of biochemical character, bacteria is accepted to be Rhodococcus rhodoehous. 2) a substrate/nitril -- the nitril used as the substrate of production To shake nitril Hydra Tarta of the above microorganisms -- the mono-nitril of an aromatic series and a fatty series, or dinitrile -- above all -- mono-nitril -- come out. the carbon number in which enjoying the special feature of the present invention best forms aromatic nitrile, especially an aromatic ring -- the thing of 4~10 -- it comes out. If some of examples of aromatic nitrile are illustrated, it will be as follows, and the compound shown by following general formula (1) ~ [VI) is mentioned. For example, they are 4-13-1, 2-cyano pyridine, and That . (It is here and R1 and R- are HlcHOH, OCH3, CI, F, and CN, respectively.) It is 3 In NH or NO2. For example, benzonitrile, 0-5m-, and p-chlorobenzonitrile % O -"-, p-fluorobenzo nitril, O-, and m-nitrobenzo nitril, p-amino benzonitrile, 0-lm-, pl Runitril , 4-cyano phenol, They are Aniso nitril, phtalo nitril, isophthalonitrile, tele phtalo nitril, 2 *6-cyclo Lopenzo nitril, 2.4-dichloro benzonitrile, 2, 6-CIF Luoro benzonitrile, and That . For example, they are alpha-, beta-naphth nitril, and That . They are (here, X isS or 0, for example, 2- Thiophene carb nitril), 2- Fruni trill, and That . They are L~, for example, 5-cyano in Doll , and That . It passes, namely, is cyano pyrazine. Other groups of the target nitril are fatty series nitril in the present invention. mono- Also of carbon number 2~6 -- dinitrile -- mono-nitril , is suitable above all. It says from the usefulness of generation amide, and acrylonitrile is typical, and productivity is also good. as for the amide corresponding to these nitril, CN basis is changed into two CONH (s) (it obtains one and there is also until [no].) In the case of dinitrile, what at least one of CN basis changed into C0N1 shall be considered to be corresponding amide. 3) If cobalt ion removes [cultivation of the microorganism of the production Rhodococcus Genus Rodoku loth kind of cultivation / nitril Hydra Tarta ] existence, then the thing to say to a culture medium, as long as it is pertinent about other conditions, there is no restriction. It just described above making the enzyme induction agent (details 1 postscript) exist in a culture medium, and storing up nitril Hydra Tarta usually into a mycelia cake. (1) a basic culture medium -- it will be as follows if some of suitable culture media are illustrated. Probably, it will be easy for a person skilled in the art to change the quantity of the ingredient of assignment, to replace a certain ingredient with other ingredients, and to omit a certain ingredient, or to add the ingredient of a pond. ( A ) Culture-medium A Formation Quantity (inside of culture medium 1g) vitamin mixture"0.1m1K 2HP 0413, 4 gK H2P O46, 5gNaCI1. OgM G S 0 and 7 H200 or 2G Distilled WaterRemainder (pH 7, 0) *1 Composition (inside of solution III) biotin2microg calcium pantothenateIt is 2HP04MgSO4 to 0.4mg inositol nicotinic acid thiamin hydrochloride pyridoxine hydrochloride p-aminobenzoic acid riboflavin folic acid distilled water ( Ro ) culture-medium B glycerol Peptone malt extract yeast extract distilled water ( C ) culture-medium C yeast extract H2PO4. * 7H 20 distilled-water mg0.4mgQ, The enzyme induction agent which makes the microorganism of 0, 5g0, and a 0 or 5g remainder (pH 7, 2) (2) enzyme-induction agent Rhodococcus Genus Rodoku loth 5g kind 5g of 0g5g of 4mg0.4mg0.2ng0 and 2mg0. Olng remainders 5g5g remainders (pH 7, 2) carry out guidance production of the nitril Hydra Tarta , There may be pertinent arbitrary things. An inducer suitable at the present invention has nitril and typical amide. Some are following if the example of the enzyme induction agent which is checking the effect about J-1 share is given. Croton amide, acetonitrile, a Prop demon trill, benzamide, Prop -on amide, the aceto amide, iso valeronitrile, [1- Butyro nitril, isobutyronitrile, n-capronitrile, 3-pen ten nitril, Vivalo nitril, n-butyl amide, isobutylamide, n-barrel amide, locker prong amide, methacrylamide, phenyl aceto amide. (3) the source of cobalt -- the above enzyme induction agents -- a culture medium (since nitril Hydra Tarta is not obtained even if j In carries out, it is indispensable to make cobalt ion exist in a culture medium in the present invention.) As for cobalt ion, it is more common than the place whose culture medium is water to make it generate by adding a water-soluble cobalt compound to a culture medium. Probably, a person skilled in the art (doing six layers of Easy !11 Such as examination depending on the case) is easy for chemicals dictionaries to be just going to clarify a water-soluble cobalt compound, and to carry out selection use of the suitable thing. the typical cobalt compound can The the thing which gives Co or Co, for example especially the thing which gives Co, and coming out, being and specifically illustrating a cobalt chloride, sulfuric acid cobalt, cobalt acetate, bromination cobalt, boric acid cobalt, and others. In addition, in the present invention, vitamin B1 * and metallic cobalt can also be used. In vitamin B12, cobalt is contained as a complex, and it ionizes in the case of cultivation. Metal Cobal 1~ is ionized by the oxidization power by a machine living thing during cultivation. (4) The useless cultivation which carries out production accumulation of the cultivation nitril Hydra Tarta into a mycelia cake should just carry out a use microorganism, for example, J-1 share, on suitable conditions by the above culture media. The amount of the enzyme induction agent used is about 2~6g among 1 liter of culture media, and the amount of the cobalt ion used is about 5~15 a+g in Co Cl 2 conversion per 1 liter of culture media. It will be as follows if concrete cultivation culture-medium composition is shown. (b) culture-medium A 1-liter acetonitrile ( , ) if5 Guiding agent 2gCoCl2 10mg ( Ro ) -- culture medium 8 1 liter -- iso valeronitrile (inducer) 2gCoC1210101i Ha culture-medium C 1-liter Croton amide (inducer) 2gCoCl2 such [10 mg] a cultivation culture medium -- 15~50 degreeC grade, desirable -- a 20~45 degreeC grade -- if J-1 share is Nourishing (ed) shaking 18 by pH 7~9 for about 30 hours or more, preferably 40 hours or more (a maximum is 120 hours, for example), nitril Hydra Tarta can be made to produce advantageously before and after 30 degreeC preferably especiallyIn order it is preferred to make it exist from the time of cultivation as for an enzyme induction agent and to prepare a highly active mycelia cake especially, for example, 28" -- when carrying out shake culture for C 76 hours, it is desirable to take the method of carrying out additional addition of the Croton amide at a culture medium so that it may become 0.2% (w/v) of concentration in the 26th hour and the 56th hour, respectively. 4) The premised "method of changing into the amide which hydrates nitril and corresponds this by operation of the nitril Hydra Tarta enzyme of microorganism origin", [present invention / of nitril / hydration] It just described above that it was what the operation of nitril Hydra Tarta carries out and includes various modes pertinent about a way. One of such the modes is making the nitril of the substrate exist in the cultivation system of a microorganism, and making amide generate in a culture medium. One of everything [the] but the mode on which nitril Hydra Tarta is made to act is adding substrate nitril to the culture solution in which nitril Hydra Tarta is accumulated, and making a hydration reaction perform to it. The method of using the culture solution which crushed the mycelia cake as an example of change of this mode will be mentioned. One of the modes on which nitril Hydra Tarta is made to act of further others is the method of separating the mycelia cake which accumulated nitril Hydra Tarta from culture solution, "fixing" by making a suitable carrier supporting this preferably, and making a substrate contacting. Especially this desirable mode can be called this method and thing which was located in a line with the second above-mentioned mode, or was suitable for industrial operation more than the second mode. This art also including use of the immobilized microbial cells as what is called a bioreactor is a well-known thing, including Vienna of a carrier, and the support method of a microorganism. One of everything [the] but the mode on which nitril Hydra Tarta is made to act is the method of obtaining nitril Hydra Tarta enzyme Standard product and so to speak hydrating nitril un-biologically with this enzyme. To say nothing of [a hydration reaction] I!IF matter activity being performed by Lost pH and temperature conditions of the range which cannot be broken, generally, these conditions can be said to be the same as it in the above-mentioned biological means. A microorganism (as for j In , it is as having described above also dealing with the mode which is not carried out as a "biological manufacturing process" by the present invention.) at the time of such an enzyme operation, Optimal pH is [7~9 and the optimal pH of nitril Hydra Tarta by the present invention] 8.0. Reaction liquid pH tends to low- Do activity of enzyme rapidly by less than seven. Therefore, it is desirable to add a buffer in reaction liquid. Even if buffers are potassium phosphate A buff 7 tris / MCI buffer, a HEPES/KOH buffer, and a sodium borate buffer, a difference does not arise in the enzyme activity of nitril Hydra Tarta . Although substrate concentration in culture solution thru/or hydration reaction liquid changes also with kinds of substrate, it is usually 0.5~15 mol/liter. Reaction temperature is usually the range of 10~30 degreeC. the example of an experiment below 3 and the example of an experiment -- nitril Hydra Tarta -- active All -- a law -- a method and active 1111fi are defined as follows. (1) Measuring method nitril Hydra Tarta activity of nitril Hydra Tarta activity, Benzonitrile 10mM, phosphoric acid Potassium < Zufa - (pH 7, 0) 30mM, And after making a reaction perform into 2 ml of reaction mixed-solutions containing the mycelia cake (what separated from Culture solution ) of the specified quantity for 5 minutes by One 0 The and 10 degreeC, Ha 1 law is carried out by adding 0.2-ml lNHCl and stopping a reaction. (2) 1 uni-Soto (U) of the definition nitril Hydra Tarta activity of a unit is defined from benzonitrile on the above conditions by making benzamide into the quantity of the enzyme made to generate at 1 g mol/minute speed. J-1 share was cultivated by the culture medium and culture condition of composition of the reference example 1 following, at that time, a CoCI One and (or) F e S O4 were added and revelation of nitril Hydra Tarta activity was investigated. ( A ) Culture-medium Composition Part 2 ml of 3 m of quantity (inside of culture medium 1g) vitamin mixture 1K 2HPO40, 5gKH2PO40, 5gMg50, and 7H200 or 5g Prop demon trill distilled water Result which remained (pH 7, 2) and was obtained at 28 degrees of ( Ro ) culture condition C / 70~80:00 as for IHI, It was as follows. As for it, even if it adds F e S O4 in not revealing nitril Hydra Tarta activity even if it adds F e S O4 to a basic culture medium, that nitril Hydra Tarta activity is revealed by addition of Co Cl 2, and a Co CI 2 addition system, a result turns out that is got worse rather , . fFI mycelia cake ffi: a dried food standard -- 2 U: -- active unit by the above-mentioned definition. The amount of mycelia cakes is as the effect as various kinds of nitril or an enzyme induction agent of amide on dried food machine Sound reference example 2J-1 share being shown in the following table. After the result of the following table cultivates [front] J-1 share by the above-mentioned culture medium B (28degreeC) and the law fully increases, It is a thing when it moves to the above-mentioned culture medium C which added at 0.1% (v/v) about the former, and added nitril or amide at the concentration of O9296 (w/v) about the latter, and also added Co CI 2 0.001% (W/V) and cultivation is performed for 36~48 hours. a ratio -- active Crotonamide acetonitrile Propion nitril benzamide Propionamide acetamide n- Butyronitrile isobutyronitrile -- iso valeronitrile n-capronitrile 3- Pentene nitril Bihiro nitril n-butyl amide isobutylamide rl-barrel amide"]

- Capron amide methacrylamide phenyl Acetamide 2.221. 411.360. 840.7 90.711. 400.410. 340.280. 320.350. 430.090. 440.300. 200.29 full-active 4.483. 474.442. 752.291. Amount of 550.381. 241.051. 041.420. 241.550. 331.081. 060.620. 28 mycelia cakes 2.022. 463.263. 262.902. 183.703. 063.073. 714.49 (1, 693, 623, 481, 813, 120) It is COCl2 to culture medium C of the 95 example 1 above. It was made to react to J-1 share of mycelia cake produced by cultivating by the culture medium which adds a 0. C1g/liter and Croton amide 2g/a liter, using 15 sorts of nitril as a substrate. The reaction was performed by 25 degreeC for 76 hours using the reaction liquid (2 ml) consisting of the mycelia cake obtained from culture solution 21, the potassium phosphate buffer (pH 8, 0) of 10mM, and the substrate of 2 [] OmM. The ratio (Mol Ml) to nitril Hydra Tarta activity when a reaction is made to add and Stop 0.2-ml +NHCl, the nitril Hydra Tarta activity to each substrate measures the amount of generation of a reaction product, or consumption of a substrate by HPLC and 3-cyano pyridine is used as a substrate, namely, a ratio -- it was considered as activity (%) and shown. The result was as follows. Basis Quality Ratio activity (%) 3-cyano pyridine100 acrylonitrile 1064-cyano pyridine1292-cyano pyridine 5-cyano in Doll 2 and Thiophene carb nitril 2- Fruni trill benzonitrile 4-cyano phenol p-amino benzonitrile m-nitrobenzo nitril 0-nitrobenzo nitril m-chlorobenzonitrile p4 Runitril 0- Torni trillm-) Before Ru nitrile aniso nitril 0-chlorobenzonitrile p-chlorobenzonitrile 2.4-dichloro benzonitrile 2.6-dichloro benzonitrile cyano Virazine example 2, ; It is Co Cl 2 to Self-contained 1jjlC. 400 m of culture media which add 0, 01 g / liter, and Croton amide 2g/a liter put 1 in 1-liter hill Lofrasco , and J-1 share was inoculated, and it cultivated by 28 degreeC on the shaker. Cultivation 30 hours, and 60 hours afterward, Croton amide was added 0.2% (w/v) (800 a+g/400m1), cultivation was continued, and cultivation was finished from the cultivation start at the time of 80 hours. The mycelia cake was extracted by centrifuging for 15 minutes in 12tJOOg, was washed by NaCl 0 or 85%, was centrifuged again, and was made again suspended in 40-m1 of the wave with a centrifuge (Hitachi 5CR2alumnus). A small amount of samples of the suspension are taken, and it is dryness of the mycelia cake in it! lI! It used for measuring a melon. The suspension containing an equivalent for 2.33 mg of dry mycelia cakes is added to reaction rl& (4 ml) containing 3-cyano pyridine of 10m M potassium phosphate buffer solution (pH 8, 0) and the concentration of 4.57M, Reaction start 3 hours and, and 6 hours afterward, 3-cyano pyridine of 0.55M and 0.49M was added, respectively, and it reacted by 25 degreeC overnight. The quantity of the generation nicotinic acid amide 18 hours after t8 Start nurturing was 5.58M. Therefore, a conversion ratio is 99.596 and it responds to 681 g per f3i%3tliter of nicotinic acid amide having been carried out as for # product. At this concentration, the reaction mixture was assimilated by precipitation of nicotinic acid amide. Identification of generation nicotinic acid amide separated this as a crystal, and ultimate analysis, IR, NMR, and mass spectrometry performed it. Generation of nicotinic acid was not detected. It is 10mM potassium phosphate R&j liquid (it added to the reaction liquid (4 ml) containing the cyano pyrazine of pf (8, 0) and various concentration.) about the mycelia cake suspension / & (an equivalent for 2 or 33111g of dry mycelia cakes) obtained in example 3 Example 2. The reaction was performed by 25 degreeC and the cyano pyrazine of 6M was converted into pyrazine amide for the cyano pyrazine of 4M at the reaction for 9 hours by the reaction for 6 hours at the conversion ratio of 100% again. When the Suspended EJ liquid which, on the other hand, contains the mycelia cake which is equivalent to dry weight 4.66+ny instead of above 2.33ffg was added to the same reaction liquid (4 ml), the cyano pyrazine of 8M was converted into pyrazine amide for the cyano pyrazine of 7M at the reaction for 9 hours by the reaction for 6 hours at the conversion ratio of 10096. Generation of pyrazine carboxylic acid was not accepted. It was Crystallization (ed) from solution as it generated pyrazine amide. These crystalline sediments were collected directly and +Ir crystal was carried out from methanol. This crystal was identified pyrazine amide by ultimate analysis, IR, NMR, and mass spectrometry. High speed liquid chromatography performed part In of cyano pyrazine, pyrazine amide, and pyrazine carboxylic acid. Also in the following examples, 7 Fr'I was performed to this example, 51 Tan , and 1 t. The mycelia cake suspension (an equivalent for 4.66 mg of dry mycelia cakes) obtained in example 2 of example 4 fruit h& is added to 11011I potassium phosphate buffer solution (pH 8, 0) and the reaction liquid (4 ml) containing the meta- Krylo nitril of 3M, When the meta- Krylo nitril of 3M was added, respectively and it reacted by 25 degreeC reaction start 1 hour and, and 3 hours afterward, the methacrylamide of 9M generated with the conversion ratio of 100% 12 hours after the reaction start. In the above-mentioned reaction, when the meta- Krylo nitril of IM was further added 5 hours after the reaction start, the methacrylamide of 1ON1 generated with the conversion ratio of 100% 24 hours after the reaction start. It means that 851 g per a liter of reaction liquid of methacrylamide is generated, and IOM concentration was accumulated. Diluted this reaction liquid with water, centrifugal separation processing (for [12000Fr] 15 minutes) removed the mycelia cake, it was made to condense and crystallize by an evaporator, and the crystal of methacrylamide was obtained by subsequently to water dissolving and re-crystallizing this crystal. 14 Fungus body suspension (an equivalent for 4.66 mg of dry mycelia cakes) is added to 1011M potassium phosphate buffer solution (pl+8.0) and the reaction liquid (4 ml) containing the Croton nitril of IM in example 5 Example 2, When it reacted by Croton nitril °C of IM for every hour after the reaction start, the Croton amide of 6M generated by the inversion rate of 100% 6 hours after the reaction start. the place which added the Croton nitril of 1~1 reaction start 6 hours and, and 10 hours afterward, respectively -- reaction start 10 hours and, and 22 hours after -- respectively -- the Croton amide of 7M and 8M -- 1 F' -- t-1 It generated with the conversion ratio boiled nine. 8 It means that 681 g per a liter of reaction ifk Croton amide was generated and accumulated M > 0 times. Crystallization of Croton amide was performed like Example 4. The mycelia cake suspension (this [dry mycelia cake 4.66mg Hj]) obtained in example 6 Example 2 is added to ILld potassium phosphate buffer solution (pH 8.0) and the reaction liquid (4 ml) containing the acetonitrile of 3M, 111 or one reaction start and 3+1! When the acetonitrile of 5M1 was added and it reacted by 25 degreeC 3M and 6 hours after the reaction start behind between j, respectively, it generated with the aceto amide of 14M, or the conversion ratio of 100% 12 hours after the reaction start. That is, it means that it is generated [the 827 g per a liter of reaction liquid aceto amide, or], and was accumulated. This reaction liquid was diluted with water, centrifugal separation processing was carried out, the mycelia cake was removed, that supernatant liquid was condensed and hardened by drying by the evaporator, and the crystal of the aceto amide was obtained by dissolving this in methanol and making it crystallize. The mycelia cake suspension (an equivalent for dry mycelia cake 4.66II1g) obtained in example 7 Example 2 is added to the reaction liquid (4 ml) containing 101mM potassium phosphate Loose species il Sho (pH 8, 0) and 3-hydronalium Kimpropi demon trill of 3~1, When 3-hydroxyprop demon trill of 3M was added 4 times for every hour after the reaction start and having been reacted by 25 degreeC, 3-hydroxyprop Oamid of 15M generated with the conversion ratio of 100% 5 hours after the reaction start. here -- further -- when 3-hydronalium Kimpropi demon trill of 3M was added, 3-hydroxyprop Oamid of 18M generated with the conversion ratio of 10096 11 hours after the reaction start. That is, it is 3-hydroxyprop Oamid of 1600 g, or /1 per 1 liter of reaction liquid. - It means that it was Formation (ed) and accumulated. After diluting this reaction liquid with water and removing a mycelia cake by centrifugal separation, condensed by the evaporator, it was made to crystallize by 20 degreeC, and the crystal of 3-hydroxyprop Oamid was obtained by subsequently to an iso proper tool dissolving and re-crystallizing this crystal. 400 m of culture media which add 0.01g/liter example 8 above-mentioned culture-medium C1:CoC1 and Croton amide 2g/a liter put 1 into 1-liter hill Lofrasco II, and J-1 share was inoculated, and it cultivated by 28 degreeC on the shaker. Cultivation 26 hours, and 56 hours afterward, Croton amide was added 0.2% (w/v) (800 mg / 400m1), cultivation was continued, and cultivation was finished from the cultivation start at the time of 76 hours. With a centrifuge (Hitachi 5CR2alumnus), it is a mycelia cake 10 (10 (it extracted by centrifuging for 20 minutes at 1 g, and washed by NaC1 0.85%, and you centrifuged again, and made it again suspended in solid-liquid 40-m1)) A little trials f4 of the Suspended ?4 liquid were taken, and it used for measuring the dry weight of the mycelia cake in it. The suspension containing this [dry mycelia cake 2.96mgt11] was added to 10mM potassium phosphate Warm juice (pH 8, 0) and the reaction liquid (4 ml) containing 3-cyano pyridine of various concentration. The reaction was performed by 25 degreeC and 3-cyano pyridine of 9M was converted into nicotinic acid amide for 3-cyano pyridine of 8M at the reaction for 22 hours by the reaction for 9 hours at the conversion ratio of 100% again. When @ murky solution which, on the other hand, contains the mycelia cake which is equivalent to dried type u5.92omegag instead of above 2.96rAg is added to the same reaction liquid (4 ml), 3-cyano pyridine of 12M was converted into nicotinic acid amide for 3-cyano pyridine of 9M at the reaction for 9 hours by the reaction for 5 hours at the conversion ratio of 100%. It means that generation accumulation of the 1, 465g nicotinic acid amide per a liter of reaction llk was carried out 12~14 degrees. Generation of nicotinic acid was not accepted. It was Crystallization (ed) from solution as it generated nicotinic acid amide. These crystals were collected and it was made to re-crystallize from methanol. The mycelia cake suspension (an equivalent for 5 or 92 mg of dry mycelia cakes) obtained in example 9 Example 8 is added to 101M potassium phosphate buffer solution (pH 8, 0) and the reaction liquid (4 ml) containing the benzonitrile of 1M, When the benzonitrile of IM was added, respectively and it reacted by 25 degreeC reaction start 1.2. 3.4. 5 and 7 hours afterward, the benzamide of 7M (848g/ (liter)) generated with the conversion ratio of 100% 24 hours after the reaction start. The mycelia cake suspension (this [dry mycelia cake 5.92111g Hj]) obtained in example 10 Example 8 is added to 10d potassium phosphate buffer solution (pH 18, 0) and the reaction liquid (4 ml) containing 2 of 0.5M, and 6-CIF Luoro benzonitrile, In reaction start 2.4. 6 and 8 hours, 2 of 0.5M and 6-CIF Ruoropenzo nitril are added, respectively, When reacted by 25 degreeC, 2 of 2.5M (393g/ (liter)) and 6-CIF Ruolopens amide generated with the conversion ratio of 100 % 22 hours after the reaction start. The mycelia cake suspension (an equivalent for dry mycelia cake 5 and 92atg) obtained in example 11 Example 8 is added to 10IIM potassium phosphate buffer solution (pH 8, 0) and reaction liquid (4 ml) including 2- Thiofuen power Leboni ] and Lil of IM, When 2- Thiophene carb nitril of IM was added and it reacted by 25 degreeC 1 hour after the reaction start, 2- Thiophenecarboxamide of 2M (254g/ (liter)) generated with the conversion ratio of 100% 5 hours after the reaction start. The mycelia cake suspension (an equivalent for 5.92 mg of dry mycelia cakes) obtained in example 12 Example 8 is added to 10 a+M potassium phosphate buffer solution (rho118.0) and the reaction liquid (4 ml) containing 2- Fruni trill of IM, When 2- Fruni trill of IM was added, respectively and it reacted by 25 degreeC reaction start 1.2. 4.6. 8.11 and 23 hours afterward, 2-Franka Ruboxamide of 8M (888g/ (liter)) generated with the conversion ratio of 100% 30 hours after the reaction start. This [it was right in example 13 Example 8 / mycelia cake suspension (5.92 mg of dry mycelia cakes) If] is added to 1 On+I4 potassium-phosphate buffer solution (pH 8, 0) and the reaction liquid (4 ml) containing 3-Indore acetonitrile of 4M, When reacted by 25 degreeC, 3-Indore aceto amide of 4M (697g/ (liter)) generated with the conversion ratio of 100% 24 hours after the reaction start. Mycelia cake suspension it&' (an equivalent for 4 or 66111g of dry mycelia cakes) obtained in example 14 Example 2 is added to reaction iffl (4 ml) containing 10mM potassium phosphate buffer solution (pH 8, O) and acrylonitrile of 3M, When acrylonitrile of 2M was added, respectively and it reacted by 25 degreeC reaction starts 0 and 5l and 2 hours afterward, acryl amide of 9M (640g/ (liter)) generated with the conversion ratio of 10096 8 hours after the reaction start.