US20040142447A1 - Microbiological method for producing amides - Google Patents

Microbiological method for producing amides Download PDF

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US20040142447A1
US20040142447A1 US10/465,495 US46549503A US2004142447A1 US 20040142447 A1 US20040142447 A1 US 20040142447A1 US 46549503 A US46549503 A US 46549503A US 2004142447 A1 US2004142447 A1 US 2004142447A1
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nitrile hydratase
rhodococcus
acetonitrile
cyanopyridine
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Karen Robins
Toru Nagasawa
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Lonza AG
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/02—Amides, e.g. chloramphenicol or polyamides; Imides or polyimides; Urethanes, i.e. compounds comprising N-C=O structural element or polyurethanes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y402/00—Carbon-oxygen lyases (4.2)
    • C12Y402/01—Hydro-lyases (4.2.1)
    • C12Y402/01084—Nitrile hydratase (4.2.1.84)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales

Definitions

  • the invention relates to microorganisms which are capable of tolerating acetonitrile concentrations of at least 3 M, to an enzyme having nitrile hydratase activity, to a method for producing amides by using said microorganisms or said enzyme, and to the use of said microorganisms for removing acetonitrile waste.
  • amides such as, for example, nicotinamide, a vitamin of the vitamin B complex, which is essential to animals and humans, are already known.
  • EP-A 0 307 926 describes the conversion of 3-cyanopyridine to nicotinamide by means of Rhodococcus rhodochrous J1.
  • This method has the disadvantage of Rhodococcus rhodochrous J1 being red, as a consequence of which the product is discolored.
  • said microorganism has a high KM value with respect to the substrate 3-cyanopyridine, has low temperature tolerance and low tolerance with respect to 3-cyanopyridine.
  • WO 99/05306 describes, for example, a method for producing nicotinamide, starting from the corresponding nitrile, by means of microorganisms of the genera Rhodococcus, Amycolatopsis and Actinomadura.
  • This method has the disadvantage that the microorganisms used, of the genus Amycolatopsis, are inactivated at elevated temperatures. Furthermore, said microorganisms have low tolerance to 3-cyanopyridine and nicotinamide.
  • the described microorganisms of the genus Rhodococcus have a high K M value with respect to 3-cyanopyridine and low temperature stability. Accordingly, said method is not economical as an industrial process.
  • microorganisms as claimed in Claim 1 by the enzyme as claimed in Claims 5 or 7 , and by the method as claimed in Claim 8 .
  • the microorganisms of the invention can be obtained by appropriate selection, for example from soil samples, sludge or wastewater, with the aid of common micro-biological techniques. Said microorganisms are conveniently selected by cultivation with a pyridinaldoxime of the general formula
  • nitriles as carbon sources in the presence of cobalt ions and, for example, yeast extract and/or ammonium salts. This is followed by selecting from the cultures obtained those microorganisms which are capable of tolerating an acetonitrile concentration of at least 3 M and of converting nitrites such as, for example, 3-cyanopyridine and acetonitrile to the corresponding amide.
  • Pyridinaldoximes which may be used are pyridin-2-, pyridin-3- or pyridin-4-aldoxime.
  • Nitriles suitable for selection are in particular also those which are intended to be used as substrates in the later biotransformation, for example acetonitrile (acetic acid nitrile), propionitrile, butyronitrile, crotonic acid nitrile, adipic acid nitrile and malonic acid nitrile.
  • acetonitrile acetic acid nitrile
  • propionitrile propionitrile
  • butyronitrile crotonic acid nitrile
  • crotonic acid nitrile adipic acid nitrile
  • malonic acid nitrile malonic acid nitrile
  • Preferred sources of the cobalt ions are “cobalt ion-generating cobalt compounds”, for example Co 2+ or Co 3+ salts such as cobalt chlorides, cobalt sulfates and cobalt acetates.
  • the preferred cobalt compound used is a Co 2+ salt such as, for example, CoCl 2 .
  • cultivation may also be carried out in combination with metallic cobalt or with other cobalt compounds.
  • cobalt or cobalt compounds are used in the cultivation medium in an amount of from 1 to 30 mg/l, preferably from 1 to 20 mg/l.
  • ammonium salts which may be used are ammonium phosphates such as (NH 4 ) 2 HPO 4 or (NH 4 )H 2 PO 4 .
  • microorganisms are cultured in appropriate media prior to the actual biotransformation.
  • appropriate culture media are the media described in Tables 3 and 5.
  • the cultivation is usually carried out at a temperature of from 20 to 40° C. and at a pH of between 5 and 8, preferably at a temperature of from 25 to 35° C. and at a pH of between 6 and 7.5.
  • the active enzymes i.e. nitrile hydratases
  • the active enzymes are induced during cultivation by adding an enzyme inducer.
  • Enzyme inducers which may be used are saturated or unsaturated aliphatic nitriles or the corresponding amides.
  • Aliphatic nitriles which may be used are all C 2-7 -alkanenitriles, such as, for example, butyronitrile, isobutyronitrile, valeronitrile or isovaleronitrile, or C 3-7 -alkenenitriles, such as, for example, methacrylonitrile or crotononitrile.
  • Aliphatic amides which may be used are any C 2-7 -alkanamides, such as, for example, butyramide, isobutyramide, valeramide or propionamide, or C 3-7 -alkenamides, such as, for example methacrylamide or crotonamide.
  • Preferred enzyme inducers are methacrylamide, butyramide, isobutyramide, valeramide, methacrylonitrile, crotonamide, butyronitrile and isobutyronitrile. Particular preference is given to using methacrylonitrile as enzyme inductor.
  • microorganisms of the invention tolerate an acetonitrile concentration of at least 3 M, meaning that the enzyme activity is stable after incubation with 3 M acetonitrile in 0.1 M potassium phosphate buffer at pH 7.0 and 20° C. for 1 hour, i.e. that no more than 10% of activity is lost.
  • Preferred microorganisms tolerate an acetonitrile concentration of at least 6 M for 1 hour under the abovementioned conditions, with a loss of activity of no more than 50%.
  • Particularly preferred microorganisms tolerate an acetonitrile concentration of at least 9 M for 1 hour under the conditions mentioned, with a loss of activity of no more than 70%.
  • the enzyme activity is stable even after several minutes of incubation with 15 M and 19 M acetonitrile (corresponding to pure acetonitrile).
  • the loss of enzyme activity after 10 minutes of incubation with 15 M acetonitrile is less than 10%.
  • the microorganisms of the invention have high thermal stability, i.e. higher stability at high temperatures than the microorganisms known to date.
  • the loss of enzyme activity of the microorganisms of the invention is preferably no more than 10% after incubation in 0.1 M potassium phosphate buffer, pH 7.0 at 60° C. for 1 hour, and the loss of enzyme activity after incubation under the conditions mentioned for 2 hours is no more than 40%.
  • Enzyme activity here means nitrile hydratase activity, in particular nitrile hydratase activity with respect to the substrate 3-cyanopyridine.
  • microorganisms of the invention are a high tolerance with respect to the preferably employed substrate 3-cyanopyridine and with respect to the product nicotinamide produced therefrom and a lower K M value with respect to 3-cyanopyridine.
  • Another particularly outstanding property is the fact that they can accumulate acetamide at a higher concentration than that corresponding to the concentration of an acetamide solution saturated at 30° C. (approx. 220-230 g of acetamide in 100 ml of water).
  • Preferred microorganisms belong to the genus Rhodococcus.
  • Particularly preferred microorganisms are the strain Rhodococcus sp. FZ4 and the functionally equivalent variants and mutants thereof.
  • Functionally equivalent variants and mutants mean those variants and mutants which tolerate acetonitrile concentrations of at least 3 M.
  • Very particular preference is given to “pigment-negative” Rhodococcus strains, i.e. strains which lack the red color which may lead to discoloration of the desired product.
  • Such strains can, where appropriate, be readily generated from pigment-producing microorganisms by mutagenesis by means of UV radiation or mutagenic chemicals.
  • the strain Rhodococcus sp. FZ4 was deposited with the Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH (DSMZ), Mascheroder Weg 1b, D-38124 Braunschweig with deposition number DSM 13597 on Jul. 11, 2000, in accordance with the Budapest Treaty. It was not possible to classify this microorganism on the basis of its identification data to any, of the previously known Rhodococcus species, and it was therefore classified as a novel species.
  • DSMZ Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH
  • the functionally equivalent variants and mutants of the strain Rhodococcus sp. FZ4 can be produced either by spontaneous mutation or, for example, by UV irradiation or mutagenic chemicals.
  • Preferred variants and mutants of the Rhodococcus sp. FZ4 strain are “pigment-negative”, i.e. they lack the red color which may lead to discoloration of the desired product.
  • the enzyme extract may be obtained, for example, by disrupting the microorganisms, for example by means of ultrasound, French press or by means of the lysozyme method.
  • the inventive enzymes having nitrile hydratase activity can be obtained from the above-described microorganisms. They are preferably obtainable from the microorganisms of the genus Rhodococcus, in particular from the microorganism Rhodococcus sp. FZ4 (DSM 13597).
  • Said enzymes have, in particular, the following properties:
  • said enzymes have
  • the actual biotransformation may be carried out using the above-described microorganisms, an enzyme extract of said microorganisms or the isolated enzyme. Preference is given to carrying out the biotransformation using the microorganism Rhodococcus sp. FZ4.
  • Substrates which may be used for the biotransformation are nitrites of the general formula
  • the substituent R 1 is a C 1-6 -alkyl group, a C 2-6 -alkenyl group or a group of the general formula IV
  • X is a nitrogen atom or —CH ⁇
  • R 2 and R 3 are, independently of one another, a hydrogen atom, a halogen atom, a C 1-6 -alkyl group or a C 2-6 -alkenyl group.
  • C 1-6 -alkyl groups which may be used are methyl, ethyl, propyl, isopropyl, butyl, tert-butyl, isobutyl, pentyl and its isomers, and hexyl and its isomers.
  • Examples of C 2-6 -alkenyl groups which may be used are vinyl, allyl, 1-propen-1-yl and 1-propen-2-yl.
  • Halogen atoms which may be used are F, Cl, Br or I.
  • Preferred representatives of the nitriles of the general formula II are acetonitrile, butyronitrile, acrylonitrile, propionitrile, crotononitrile, 2-cyanopyridine, 3-cyanopyridine, 4-cyanopyridine, benzonitrile, fluorobenzonitrile, chlorobenzonitrile and bromobenzonitrile.
  • the most preferred substrates are acetonitrile and 3-cyanopyridine.
  • the biotransformation is preferably carried out with a single or continuous addition of substrate.
  • the substrate concentration to be used depends on the solubility of the substrate to be used.
  • Media which may be used for the biotransformation are those known in the art, for example low-molarity phosphate buffers, HEPES buffer, citrate buffer and borate buffer.
  • Low-molarity phosphate buffer means preferably a 0.01 to 0.5 M phosphate buffer, particularly preferably a 0.05 to 0.25 M phosphate buffer.
  • the preferred pH is between 5 and 10, the particularly preferred between 6 and 7.5.
  • R 1 is defined as mentioned above, where appropriate after removing the cells, by using common work-up methods such as, for example, crystallization or spray drying.
  • the present invention furthermore relates to the use of the above-described microorganisms, in particular of the genus Rhodococcus, for removing acetonitrile waste.
  • Acetonitrile is a solvent which is used, for example, in HPLC and which, ultimately, needs to be disposed of as waste.
  • acetonitrile may be present at a concentration of up to a maximum of 19 M, corresponding to pure acetonitrile.
  • a solution or suspension of from 0.25 to 15.0 M, preferably from 1 to 10 M, acetonitrile is used.
  • the microorganisms are advantageously used at a temperature of from 5 to 50° C., preferably at a temperature of from 20 to 40° C.
  • the pH is advantageously between 5 and 10, preferably between 6 and 8.
  • the duration of the conversion of acetonitrile to acetamide for removing waste depends on the acetonitrile concentration and is, for example, approx. 2 hours for producing a 9.5 M acetamide solution/suspension at pH 7.0 and a temperature of approx. 20° C.
  • mycolic acids mycolic acids having a chain length of from C 40 to C 48 are present
  • fatty acid patterns linear, saturated and unsaturated fatty acids and a high proportion of tuberculostearic acid are present.
  • strain FZ4 was identified as a member of the genus Rhodococcus.
  • strain FZ4 The macroscopic appearance and morphology of the cells of strain FZ4 were similar to Rhodococcus rhodochrous.
  • the colonies of strain FZ4 are salmon-red (RAL 3022), and young cultures developed branched hyphae which developed into rods and cocci.
  • strain FZ4 Owing to the chemotaxonomic and conventional markers, strain FZ4 was identified as belonging to the species Rhodococcus rhodochrous, but having a low correlation factor.
  • the sequence of the first 500 bases of the 16S rDNA reveals a similarity of only 97.7% to that of the typical representative strain of the species Rhodococcus rhodochrous, Rhodococcus rhodochrous DSM 43241 and of 99.1% to another Rhodococcus rhodochrous reference strain. Since the similarity of the sequence of the first 500 bases of the 16S rDNA of strain FZ4 to that of strain Rhodococcus rhodochrous DSM 43241 was below 99.5%, it was not possible to identify strain FZ4 as a member of the species Rhodococcus rhodochrous.
  • Strain FZ4 was therefore identified as a novel species within the genus Rhodococcus.
  • FIG. 1 depicts the biotransformation of acetonitrile to acetamide using resting cells of Rhodococcus sp. FZ4.
  • FIG. 2 depicts the temperature optimum of the Rhodococcus sp. FZ4 nitrile hydratase activity in resting cells.
  • FIG. 3 depicts the thermal stability of Rhodococcus sp. FZ4 nitrile hydratase activity in resting cells.
  • FIG. 4 depicts the pH optimum of Rhodococcus sp. FZ4 nitrile hydratase activity in resting cells.
  • FIG. 5 depicts the pH stability of Rhodococcus sp. FZ4 nitrile hydratase activity in resting cells.
  • FIG. 6 depicts 3-cyanopyridine tolerance of Rhodococcus sp. FZ4 nitrile hydratase activity in resting cells.
  • FIG. 7 depicts nicotinamide tolerance of Rhodococcus sp. FZ4 nitrile hydratase activity in resting cells.
  • FIG. 8 depicts the influence of the acetonitrile concentration on Rhodococcus sp. FZ4 nitrile hydratase activity in resting cells in the biotransformation of acetonitrile to acetamide.
  • FIG. 9 depicts the influence of the acetonitrile concentration on Rhodococcus sp. FZ4 nitrile hydratase activity in resting cells.
  • FIG. 10 depicts the Rhodococcus sp. FZ4 nitrile 5 hydratase activity in resting cells as a function of 3-cyanopyridine concentration.
  • FIG. 11 depicts the logarithmic plot of the molecular weights of nitrile hydratase and of reference proteins as a function of the respective HPLC retention time.
  • FIG. 12 depicts the logarithmic plot of the molecular weights of nitrile hydratase subunits and of reference proteins as a function of the respective SDA Page RF value.
  • FIG. 13 depicts the activity of purified Rhodococcus sp. FZ4 nitrile hydratase as a function of the 3-cyanopyridine concentration.
  • FIG. 14 depicts the activity of purified Rhodococcus sp. FZ4 nitrile hydratase as a function of acetonitrile concentration.
  • FIG. 15 depicts the thermal stability of purified Rhodococcus sp. FZ4 nitrile hydratase.
  • FIG. 16 depicts the pH optimum of purified Rhodococcus sp. FZ4 nitrile hydratase.
  • FIG. 17 depicts the pH stability of purified Rhodococcus sp. FZ4 nitrile hydratase.
  • Nitrile hydratase activity was determined by incubating a reaction mixture comprising 3-cyanopyridine (1.0 M; 1.0 ml), potassium phosphate buffer (0.1 M, pH 7.0; 0.5 ml) and cell suspension (0.5 ml) with stirring at 20° C. for 5 min. The reaction was stopped by adding HCl (5 M; 0.1 ml).
  • the enrichment medium of Table 1 was inoculated with various soil samples, followed by incubation at 37° C. for 7 to 10 days. The cultures thus obtained were transferred into the same medium and cultured again at 37° C. for another 7 to 10 days. This procedure was repeated three times. Subsequently, the cultures were diluted and plated out. After incubating the plates at 37° C. for 5 days, individual colonies were obtained. The individual colonies were assayed for the presence of a nitrile hydratase activity according to Example 1. In this way, strain Rhodococcus sp. FZ4 (DSM 13597) was isolated.
  • the preculture medium of Table 2 was inoculated with strain Rhodococcus sp. FZ4 (DSM 13597), followed by incubation with shaking at 28° C. for 1 to 2 days.
  • the preculture was transferred into the basal medium of Table 3, which contains either CoCl 2 or FeSO 4 , and cultured with shaking at 28° C. for 2 to 3 days.
  • Nitrile hydratase activity was determined according to Example 1. The results are summarized in Table 4. A nitrile hydratase activity was present only when culturing Rhodococcus sp. FZ4 in the presence of cobalt.
  • Preculture medium Concentration Components [g/l] Peptone 5.0 Meat extract 5.0 NaCl 2.0 Yeast extract 0.5
  • the preculture medium of Table 2 was inoculated with strain Rhodococcus sp. FZ4 (DSM 13597), followed by incubation with shaking at 28° C. for 1 to 2 days.
  • the preculture was transferred into the culture medium of Table 5, which contained different inducers, and cultured with shaking at 28° C. for 3 days.
  • Nitrile hydratase activity was determined according to Example 1. The results are summarized in Table 6. Rhodococcus sp. FZ4 nitrile hydratase was expressed during cultivation only in the presence of an inducer.
  • the preculture medium of Table 2 was inoculated with strain Rhodococcus sp. FZ4, followed by incubation with shaking at 28° C. for 1 to 2 days.
  • the preculture was transferred into the culture medium of Table 5, containing 6 g/l methacrylamide as inducer, and cultured with shaking at 28° C. for 3 days. After 48 h, additional methacrylamide (0.20 (v/v)) was fed in.
  • Rhodococcus sp. FZ4 nitrile hydratase activity with respect to different substrates was determined according to Example 1, except that the appropriate substrate was used instead of 3-cyanopyridine and that the HPLC conditions were modified according to the substrate used.
  • Table 7 summarizes the substrate specificity of Rhodococcus sp. FZ4 nitrile hydratase activity in comparison with the substrate specificity of Rhodococcus rhodochrous J1 nitrile hydratase activity. TABLE 7 Comparison of substrate specificities of Rhodococcus sp. FZ4 and Rhodococcus rhodochrous J1 nitrile hydratase activities Rhodococcus Rhodococcus sp.
  • Nitrile hydratase activity was determined according to Example 1 at different temperatures in the range from 20 to 70° C. The temperature optimum for nitrile hydratase activity was at 60° C. (FIG. 2).
  • Nitrile hydratase activity was determined according to Example 1 using different buffers (0.1 M) at different pH values in the range from 3 to 12. The pH optimum of nitrile hydratase activity was between 6 and 7 (FIG. 4).
  • nitrile hydratase activity was determined according to Example 1 and, after incubation at pH values in the range from 5 to 10 for 24 hours, corresponded approximately to the original nitrile hydratase activity (FIG. 5).
  • a cell suspension was incubated at different 3-cyano-30 pyridine concentrations in the range from 0 to 10% (w/v) at 20° C. for 60 min.
  • the cells were removed, washed and resuspended in potassium phosphate buffer (0.1 M; pH 7.0).
  • Nitrile hydratase activity was deter-mined according to Example 1 and, after incubation at 3-cyanopyridine concentrations in the range from 0 to 20% (w/v) for 60 minutes, corresponded approximately to the original nitrile hydratase activity (FIG. 6).
  • a cell suspension was incubated at different nicotinamide concentrations in the range from 0 to 20% (w/v) at 20° C. for 24 h.
  • the cells were removed, washed and resuspended in potassium phosphate buffer (0.1 M; pH 7.0).
  • Nitrile hydratase activity was determined according to Example 1 and, after incubation at nicotinamide concentrations in the range from 0 to 20% (w/v) for 24 h, corresponded approximately to the original nitrile hydratase activity (FIG. 7).
  • a reaction mixture comprising 3-cyanopyridine 20 (0.1-1.0 M; 1.0-1.8 ml), aqueous NaCl solution (0.85% (w/v); 0.7 to 0.1 ml), potassium phosphate buffer (0.1 M; pH 7.0; 0.3 ml) and cell suspension (0.01 ml) was incubated with shaking at 30° C. for 10 min.
  • the total volume of the reaction mixture was between 2.0 to 2.2 ml, depending on the 3-cyanopyridine concentration.
  • the reaction was stopped by adding HCl (2 M; 0.1 ml). After centrifugation of the reaction mixture (12 000 rpm; 5 min), the amount of nicotinamide produced was determined by means of HPLC according to Example 1.
  • the K M value determined was 160 mM (FIG. 10).
  • the K M values for the substrate 3-cyanopyridine with respect to the nitrile hydratase activities of Rhodococcus sp. FZ4, Rhodococcus sp. GF270 (DSM 12211; WO 99/05306), Amycolatopsis sp. NA40 (DSM 11617; WO 99/05306) and Rhodococcus rhodochrous J1 were determined according to Example 11 using the respective microorganism.
  • a reaction mixture comprising acetonitrile (0.2-19.0 M; 1.0-1.6 ml), aqueous NaCl solution (0.85% (w/v); 0.6-0.0 ml), potassium phosphate buffer (0.1 M; pH 7.0; 0.3 ml) and cell suspension (0.1 ml) was incubated with shaking at 20° C. for 10 min. The total reaction volume was 2.0 ml. The reaction was stopped by adding MeOH. The reaction mixture was centrifuged (12 000 rpm, 5 min) and the amount of acetamide produced was determined by means of HPLC according to Example 14. The nitrile hydratase activity was nearly constant in the range from 0.1 to 15 M acetonitrile (FIG. 8).
  • nitrile hydratase activity was nearly constant with acetonitrile in the concentration range from 0 to 3 M after 1 hour of incubation. After 1 hour of incubation with 6 M acetonitrile, 60% of the original nitrile hydratase activity was still present. After 1 hour of incubation with 9 M acetonitrile, approx. 35% of the original nitrile hydratase activity was still present (FIG. 9).
  • “Nutrient broth” medium 50 ml was inoculated with strain Rhodococcus sp. FZ4, followed by incubation with shaking at 28° C., until an OD 610 nm of 6.29 was reached. The preculture (10 ml) was then transferred to “nutrient broth” medium (25 ml) and incubated with shaking at 28° C., until an OD 610 nm , of 1.90 was reached. The culture (10 ml) thus obtained was centrifuged (8 000 rpm, 5 min). The supernatant was discarded and the cell precipitate was suspended in phosphate-buffered saline. The cell suspension was centrifuged (8 000 rpm, 5 min).
  • the supernatant was discarded and the cell precipitate was suspended in phosphate-buffered saline (5 ml).
  • the cell suspension was transferred to a glass petri dish (90 mm diameter).
  • the cells were irradiated using a UV lamp (15 W, 254 nm) from a distance of 25 cm for 17 min.
  • the cells were then incubated with shaking in double-concentrated “nutrient broth” medium at 28° C. for 4 days.
  • the culture thus obtained was diluted 100 times, and 100[I aliquots thereof were plated out on “plate count agar” and incubated at 28° C. Almost 150 single colonies grew per plate.
  • the plates were exposed to daylight, in order to induce the formation of red pigments.
  • the colonies of pigment-negative mutants were readily distinguishable from those of the colored mutants and those of the red wild type.
  • Rhodococcus sp. FZ4 was cultured according to Example 3 in a 2-1 fermenter. The culture was centrifuged and the cell precipitate resuspended in aqueous NaCl solution (0.85% (w/v)). The cell suspension was transferred to potassium phosphate buffer (0.1 M; pH 7.0) containing butyric acid (44 mM) and sonicated. Cell debris was removed by centrifugation. The supernatant was used for the purification of nitrile hydratase according to Table 12. Nitrile hydratase activity was determined according to Example 1, using, however, the respective extracts instead of the cell suspension. TABLE 12 Purification of Rhodococcus sp.
  • the molecular weight was determined by means of HPLC (TSK gel G 300 SW (0.75 ⁇ 60 cm); potassium phosphate buffer (0.1 M; pH 7.5) and potassium chloride (0.2 M); 0.7 ml/min; 280 nm).
  • the molecular weight of nitrile hydratase was 465 kDa (FIG. 11).
  • Nitrile hydratase consists of an ⁇ -subunit having a molecular weight of 27.7 kDa and ⁇ -subunit having a molecular weight of 31.2 kDa (FIG. 12).
  • a reaction mixture comprising 3-cyanopyridine (0.5 M; 0.500 ml), nitrile hydratase solution (0.697 ⁇ mol/min; 0.025 ml) and different buffers in the pH range from 4 to 11 (0.1 M; 0.0475 ml) was incubated at 20° C. for 10 min. The reaction was stopped by adding MeOH. The amount of nicotinamide produced was determined by means of HPLC according to Example 1. The pH optimum of nitrile hydratase was in the range from 6.0 to 6.5 (FIG. 16).
  • a reaction mixture comprising an aliquot of the incubated nitrile hydratase solution (0.05 ml), 3-cyanopyridine (0.5 M; 0.5 ml) and the respective buffer (0.1 M; 0.45 ml) was incubated at 20° C. for 10 min.
  • the reaction was stopped by adding MeOH.
  • the amount of nicotinamide produced was determined by means of HPLC according to Example 1. After 60 minutes of incubation at a pH in the range from 6 to 8, nitrile hydratase activity corresponded approximately to the original nitrile hydratase activity (FIG. 17).
  • a reaction mixture comprising nitrile hydratase solution (0.695 ⁇ mol/min; 0.025 ml), different substrates (0.500 ml) and potassium phosphate buffer (0.1 M; pH 7.0; 0.475 ml) was incubated at 20° C. for 5 to 10 min. The concentrations of the substrates used were in the range from 0.015 to 0.250 M. The reaction was stopped by adding MeOH. The amount of amide produced was determined by means of HPLC. The results are summarized in Table 13. With the substrates assayed, nitrile hydratase activity is highest with respect to the substrate acetonitrile.
  • Acetonitrile was added at different concentrations (2.5-80 mM; 0.500 ml) to a solution comprising nitrile hydratase solution (0.0697 ⁇ mol/min; 0.025 ml) and potassium phosphate buffer (0.1 M; pH 7.0; 0.475 ml). The reaction mixture was incubated at 20° C. for 10 min. The reaction was stopped by adding methanol and the amount of acetamide produced was determined by means of HPLC according to Example 14. The K M value for acetonitrile was 2.84 mM (FIG. 14).

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Cited By (4)

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US7288402B2 (en) 2004-03-20 2007-10-30 Degussa Ag Rhodococcus nitrile hydratase
CN102212566A (zh) * 2011-04-11 2011-10-12 江苏大学 一种高纯度异丁酰胺生产方法
US10975401B2 (en) 2016-05-18 2021-04-13 Columbia S.R.L. Biotechnological method for the production of acrylamide and new bacterial strain
CN114686538A (zh) * 2020-12-30 2022-07-01 杭州唯铂莱生物科技有限公司 一种烟酰胺制备中烟酸含量的控制方法

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IT1400395B1 (it) 2010-06-08 2013-05-31 Procos Spa Processo one-pot per la sintesi di dalfampridine.
JP7149337B2 (ja) * 2017-11-14 2022-10-06 コロンビア エス．アール．エル． アミド調製のための微生物プロセス
WO2024195728A1 (ja) * 2023-03-17 2024-09-26 三菱ケミカル株式会社 アミド化合物の製造方法

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AU627648B2 (en) * 1990-02-28 1992-08-27 Teruhiko Beppu Dna fragment encoding a polypeptide having nitrile hydratase activity, a transformant containing the gene and a process for the production of amides using the transformant
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US4432887A (en) * 1980-09-08 1984-02-21 Zajic James E De-emulsification agents of microbiological origin
US4720456A (en) * 1982-12-27 1988-01-19 Wintershall Ag Trehalose lipid tetraesters
US5179014A (en) * 1985-01-08 1993-01-12 Nitto Chemical Industry Co., Ltd. Process for the preparation of amides using microorganisms
US5298414A (en) * 1989-05-12 1994-03-29 British Technology Group Limited Detection of morphine using morphine dehydrogenase
US5827699A (en) * 1993-12-17 1998-10-27 Gosudarstvenny. Nauchno-Issledovatelsky Institut Genetiki I Selektsii Promshlennykh Mikroorganizmov Strain of Rhodococcus rhodochrous as a producer of nitrile hydratase
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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7288402B2 (en) 2004-03-20 2007-10-30 Degussa Ag Rhodococcus nitrile hydratase
US20080057549A1 (en) * 2004-03-20 2008-03-06 Degussa Ag Rhodococcus nitrile hydratase
US7491521B2 (en) 2004-03-20 2009-02-17 Evonik Degussa Gmbh Rhodococcus nitrile hydratase
CN102212566A (zh) * 2011-04-11 2011-10-12 江苏大学 一种高纯度异丁酰胺生产方法
US10975401B2 (en) 2016-05-18 2021-04-13 Columbia S.R.L. Biotechnological method for the production of acrylamide and new bacterial strain
CN114686538A (zh) * 2020-12-30 2022-07-01 杭州唯铂莱生物科技有限公司 一种烟酰胺制备中烟酸含量的控制方法

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