Method for preservation of nitrile hydration activity

Abstract: Nitrile hydration activity of the nitrilase or the im­ mobilized form thereof can be stably preserved by adding as a stabilizer at least one compound selected from nitriles, ami­ des, and organic acids and salts thereof to a solution or sus­ pension of the nitrilase or of the immobilized form thereof.

Description: The present invention relates to a method for prevention of reduction with elapse of time in nitrile hydration activity of nitrilase produced by specific microorganisms and for stable preservation of this activity. In recent years, the technology of immobilized enzymes and microorganisms has developed rapidly, resulting in increasing attempts to utilize microorganisms and enzymes as they are or in the immobilized state as catalysts for various single or complex chemical reactions. Nitrilase has been known as an enzyme capable of hydrating nitriles to produce the corresponding amides. As examples of the utilization of this enzyme, methods for producing (meth)acrylamide from (meth)acrylonitrile with the use of microorganisms of the genus Corynebacterium or Nocardia which produce nitrilase (Reference: Japanese Patent Pub. No. 17918/1981); for producing from C₂₋₄ nitriles the corresponding amides with the use of microorganisms of the genus Rhodococcus (Reference: Japanese Patent Appln. No. 452/1985); and for producing from nitriles the corresponding amides with the use of microorganisms of the genus Bacillus , Bacteridium , Micrococcus or Brevibacterium (Reference: Japanese Patent Laid-Open Pub. No. 86186/1976) have been proposed. As a result of further investigation, however, we have found that the nitrile hydration activity of the above mentioned nitrilase is labile and decreases as the temperature rises or the purity of the enzyme increases. This decrease occurs also with immobilized enzymes. SUMMARY OF THE INVENTION As a result of extensive research effort expended toward solving the above problems and efficiently utilizing the nitrile hydration activity of nitrilase, we have found that specific substances, i.e., amides and organic acids or salts therof, is very effective for this purpose and have arrived at the present invention on the basis of this finding. More specifically, the present invention provides a method for preservation of the nitrile hydration activity of nitrilase produced by a Gram-positive microorganism or of an immobilized form thereof, both having nitrile hydration activity, which method comprises adding as a stabilizer at least one compound selected from the group consisting of amides and organic acids and salts therof to a solution or supension of said nitrilase or of said immobilized form thereof. DETAILED DESCRIPTION OF THE INVENTION Microorganism: The microorganisms used in the present invention are specific ones capable of producing nitrilase and hydrating nitriles, especially acrylonitrile, to produce the corresponding amides, especially acrylamide. Specific examples of such microorganisms are strains N-771 (FERM P-4445) and N-774 (FERM P-4446)of the genus Corynebacterium and strain N-775 (FERM P-4447) of the genus Nocardia disclosed in Japanese Patent Publ. No. 17918/1 81 mentioned above and corresponding to U.S. Patent 4,248,968 (February 3, 1986), strain sp. S-6 (FERM BP-687) of the genus Rhodococcus disclosed in Japanese Patent Appln. No. 452/1985 again mentioned above and corresponding to European Patent Pub. 0 188 316 A1 (July 23, 1986), and strains of the genera Bacillus , Bacterium , Micrococcus and Brevibacterium disclosed in Japanese Patent Laid-Open Pub. No. 86186/1976 and corresponding to U.S. Patent 4,001,081, mentioned previously. Cultivation of these microorganisms is ordinarily carried out under aerobic conditions by inoculating strains of the respective microorganisms into culture media containing: carbon sources such as glucose, sucrose, dextrins and glycerol; nitrogen sources such as ammonium sulfate and urea; organic nutrient sources such as yeast extract, meat extract, malt extract and peptone; and other optional ingredients. The pH of the culture medium is of the order of 5 to 9, preferably of the order of 6 to 8, while the cultivation temperature is of the order of 20 to 37°C, preferably of the order of 20 to 30°C, and the cultivation time is about 1 to 3 days. Cells obtained by the cultivation can be collected for example, by centrifugation. Nitrilase: The nitrilase used in the present invention is an enzyme produced by the aforementioned microorganisms and irradiation with light rays is required in order to induce its activity to the fullest extent (Reference: Japanese Patent Appln. No. 2724/1985). This enzyme can be separated and extracted by the following procedure. First, the cells collected from the culture fluid by centrifugation and the like are suspended in a buffer in a quantity sufficient to reach a given cell concentration and crushed in an ultrasonic cell crusher, Daino mill or French press. Uncrushed cells and cell walls or membranes are removed, for example, by centrifugation to obtain a soluble fraction (cell extract). The cell extract thus obtained is purified by a conventional method such as ammonium sulfate fractionation, DEAE-Sephacel Phenyl-Sepharose® or Sephadex® column chromatography or crystallization whereby a partly purified enzyme solution or a purified enzyme (solution) can be obtained. Both these enzymes can be used in the present invention irrespective of whether they are purified partly or completely. Immobilized Enzyme: The immobilized enzymes of the present invention can be obtained by subjecting the above stated nitrilase to the adsorption method which involves adsorption onto activated carbon and the like, the ionic bond method which involves bonding to ion exchange resins and the like, the covalently bonding method which involves covalently bonding to glass beads, and the entrapping method which involves entrapping with acrylamide, carrageenan, alginate and the like in accordance with a conventional method. Stabilizer: The stabilizers used in the present invention are compounds selected from amides, and organic acids and salts thereof, and these compounds can be used singly or in combination. Specific examples of such compounds are: (1) Aliphatic amides, such as acetamide, propionamide and isobutyramide and the corresponding acids or salts thereof. (2) Aminoacidamides such as glycine amides, α-aminopropionamide and β-aminopropionamide and the corresponding acids or salts thereof. (3) Hydroxy-carboxylic acid amides such as lactamide, hydroxyacetamide and β-hydroxypropionamide and the corresponding acids or salts thereof. (4) Unsaturated amides such as acrylamide and methacrylamide and the corresponding acids or salts thereof. (5) Diamides such as malondiamide, succindiamide and adipic acid diamide and the corresponding dibasic acids or salts thereof. (6) Aromatic amides such as benzamide and phenylacetamide and the corresponding acids or salts thereof. (7) Heterocyclic amides such as nicotinamide and isonicotinamide and the corresponding acids or salts thereof. (8) Halogenated amides such as chloroacetamide and β -chloropropionamide and the corresponding acids or salts thereof. (9) Acids having an aldehyde group such as glyoxylic acid or salts thereof. Preservation of Nitrile Hydration Activity: The preservation of nitrile hydration activity can be attained by adding any of the above enumerated compounds to a solution or suspension of the previously mentioned nitrilase or an immobilized form thereof dissolved or dispersed in various buffers or physiological saline. Ordinarily, the quantity of the stabilizer added is in the range of from 0.01 to 50 g/l but 0.1 to 10 g/l of the stabilizer is preferably added from the point of view of storage stability, cost and the like. The pH of the solution or suspension is 5 to 8, preferably 5.5 to 7, and a variety of buffers such as phosphate buffer and Tris buffer are ordinarily employed. In some cases, it may be effective to control pH by adding an acid such as hydrochloric acid or sulfuric acid or an alkali such as caustic soda or caustic potash. While the solution or suspension thus obtained may be stored at room temperature as long as the storage period is short, storage at a low temperature, especially at a temperature in the vicinity of 0°C is preferred. In accordance with the present invention, nitrile hydration activity can be preserved stably over a long period of time. This effect is observed not only in the storage stability of the enzyme solution at the respective stages of the enzyme purification described previously but also in the preservation of the enzyme activity in each purification step. It is therefore preferable that the stabilizer be added to the enzyme solution or buffer used in each purification step. In order to indicate more fully the nature and utility of this invention, the following specific examples of practice are set forth, it being understood that these examples are presented as illustrative only and not intended to limit the scope of the invention. Experimental Examples In each of the following experimental examples, 0.1 ml of an enzyme solution was charged into a beaker containing 9.9 ml of phosphate buffer and irradiated with a 100-W EYE LAMP (supplied by Toshiba, Japan) positioned at a distance of 50 cm for 30 minutes in a bath maintained at 10°C to induce the enzyme activity to the fullest extent. The resulting solution was then added to 10 ml of M/20 phosphate buffer (pH 7.7) containing 5.0% by weight of acrylonitrile to cause reaction at 10°C for 10 minutes. The nitrile hydration activity of the test enzyme solution was determined by measuring the quantity of acrylamide produced by gas chromatography, the capability of producing 1 µ mole of acrylamide per ml of the enzyme solution per minute being designated as 1 unit (U). Example 1 and Comparison Example 1 A culture medium comprising 10 g/l of glucose, 5 g/l of peptone, 3 g/l of yeast extract, and 3 g/l of malt extract was adjusted to a pH of 7.2, and 100 ml of the resulting culture medium was sterilized in a 500-ml Erlenmeyer flask. After cooling, the sterilized culture medium was inoculated with 1 ml of a culture fluid obtained by precultivating strain N-774(FERM P-4446) of the genus Corynebacterium in the same culture medium as is described above for 2 days, and cultivation was carried out aerobically at 30°C for 2 days. Cells were separated from the culture fluid by centrifugation (3°C, 10,000 rpm, 20 minutes) and washed with a physiological saline. The washed cells were subjected to centrifugation under the same conditions and then suspended in a physiological saline to obtain a suspension of washed cells. Subsequently, 50 ml of this cell suspension was mixed with 50 ml of M/10 phosphate buffer (pH 6.5) containing 10 g/l of ammonium isobutyrate (2.5x10³ U/ml). The resultant cell suspension was pressed in a French press (fabricated by Ohtake Seisaku-sho, Japan) to crush cells under a pressure of 1,000 to 1,500 kg G and then subjected to centrifugation (12,000 rpm, 30 minutes) to remove uncrushed cells and insolubles such as cell walls, whereby a cell extract, i.e. a crude enzyme solution, was obtained. For comparison purposes, a similar cell suspension (2.5x10³ U/ml) which contained no ammonium isobutyrate was prepared and subjected to a French press treatment and centrifugation under similar conditions, whereby a control cell extract was obtained. Both cell extracts were left standing for 2 days at 20°C, and the nitrile hydration activity levels were measured before and after the extracts were left standing. The residual nitrile hydration activity percent (in Table 1, as in all succeeding tables, abbreviated to % residual activity) was calculated on the basis of the data thus obtained. The results are shown in Table 1. Experimental Example Ammonium Isobutyrate Activity Before Being Left Standing (U/ml) Activity After Being Left Standing (U/ml) % Residual Activity Example 1 Added 1.3 x 10³ 1.2 x 10³ 92 Comparison Example 1 None 1.2 x 10³ 0.33 x 10³ 28 Examples 2 through 6 and Comparison Examples 2 through 6 Each aliquot of a suspension of cells of a genus as shown in Table 2 prepared as in Comparison Example 1 was subjected to French press treatment and centrifugation similarly as in Comparison Example 1, whereby a cell extract was obtained. The cell extract thus obtained was divided into two aliquots, 1 ml of one aliquot being mixed with 1 ml of M/10 phosphate buffer (pH 6.5) containing 10 g/l of ammonium isobutyrate, and 1 ml of the other aliquot being mixed with 1 ml of M/10 phosphate buffer (pH 6.5) as a comparison example. Both solutions were left standing for 2 days at 20°C, and the nitrile hydration activity levels were measured before and after each of the solutions was left standing to calculate % residual activity. The results obtained are summarized in Table 2. Experimental Example Genus Strain % Residual Activity Example 2 Bacillus CBS-494 83 3 Bacteridium CBS-496 78 4 Micrococcus CBS-497 91 5 Brevibacterium CBS-717 89 6 Nocardia N-775 93 Comparison Example 2 Bacillus CBS-494 23 3 Bacteridium CBS-496 26 4 Micrococcus CBS-497 30 5 Brevibacterium CBS-717 22 6 Nocardia N-775 24 Examples 7 through 24 and Comparison Example 7 To each 2.5-ml aliquot of a cell extract (0.8x10³ U/ml) prepared as in Comparison Example 1, was added 2.5 ml of M/10 phosphate buffer containing 10 mg of an amide as shown in Table 3. The pH of the solution was adjusted to 6.5, and the resulting solution was then left standing at 20°C for 2 days. The % residual activity was calculated similarly as in the preceding Examples, whereupon the results set forth in Table 3 were obtained. Experimental Example Amide Added % Residual Activity Comparison Example 7 None 21 Example 7 Acetamide 57 8 Propionamide 83 9 n-Butyramide 90 10 Isobutyramide 85 11 n-Valeramide 78 12 n-Capronamide 75 13 Glycine amide 49 14 β-Aminopropionamide 52 15 Lactamide 50 16 β-Hydroxypropionamide 61 17 Acrylamide 63 18 Methacrylamide 70 19 Malondiamide 48 20 Succindiamide 63 21 Benzamide 47 22 Nicotinamide 39 23 Chloroacetamide 60 24 β-Chloropropionamide 68 Examples 25 through 41 and Comparison Example 8 The procedures of Examples 7 through 24 were followed except that the amides added were replaced by organic acids and that the pH of the solution was adjusted to 6.5. The results obtained are set forth in Table 4. Experimental Example Organic Acid Added % Residual Activity Comparison Example 8 None 21 Example 25 Formic acid 48 26 Acetic acid 55 27 Propionic acid 78 28 Isobutyric acid 83 29 Valeric acid 81 30 Caproic acid 78 31 β-Aminopropionic acid 45 32 Malic acid 43 33 Glycolic acid 52 34 DL-Glyceric acid 60 35 Oxalacetic acid 68 36 Succinic acid 59 37 Benzoic acid 48 38 Monochloroacetic acid 39 39 Glyoxylic acid 72 40 Acrylic acid 53 41 Methacrylic acid 59 Examples 42 through 50 and Comparison Examples 9 through 12 . To each 2.5 ml aliquot of a cell extract (1.8x10³ U/ml) prepared as in Comparison Example 1, was added 2.5 ml of M/10 phosphate buffer containing 25 mg of a stabilizer as shown in Table 5. The pH of the solution was adjusted to a predetermined level, and the resulting solution was left standing at 20°C for 2 days. The nitrile hydration activity levels were measured before and after each of the solutions was left standing to calculate % residual activity. The results obtained are summarized in Table 6. Experimental Example Stabilizer Added pH % Residual Activity Comparison Example 9 None 5.0 4 10 " 6.0 13 11 " 7.0 27 12 " 8.0 32 Example 42 Isobutyramide 5.0 4 43 " 6.0 80 44 " 7.0 74 45 " 8.0 48 Example 46 Isobutyric acid 5.0 2 47 " 5.5 82 48 " 6.0 83 49 " 7.0 77 50 " 8.0 42 Examples 51 through 62 and Comparison Example 13 To each 2.5-ml aliquot of a cell extract (0.7x10³ U/ml) prepared as in Comparison Example 1, was added 2.5 ml of M/10 phosphate buffer containing a predetermined quantity of a stabilizer as shown in Table 6. The pH of the solution was adjusted to 6.0 (6.5 for the solution containing no stabilizer), and the resulting solution was then left standing at 20°C for 2 days. The nitrile hydration activity levels were measured before and after each of the solutions was left standing to calculate % residual activity. The results are set forth in Table 6. Experimental Example Stabilizer Added Concentration (g/l) % Residual Activity Comparison Example 13 None - 17 Example 51 Butyramide 0.01 60 52 " 0.1 85 53 " 1.0 85 54 " 5.0 89 55 " 20 78 56 " 50 63 Example 57 Butyric acid 0.01 63 58 " 0.1 87 59 " 1.0 92 60 " 5.0 90 61 " 20 85 62 " 50 67 Concentration: Concentration of the stabilizer in the solution left standing Example 65 and Comparison Example 14 40 ml of a cell extract prepared as in Example 1, 4.5 g of acrylamide, 0.5 g of N,N'-methylenebisacrylamide, and 40 ml of M/20 phosphate buffer (containing 10 g/l of ammonium isobutyrate, pH 6.0) were mixed to form a homogeneous suspension. To this suspension were added 5 ml of a 5% aqueous solution of dimethylaminopropionitrile and 10 ml of a 1.0% aqueous solution of potassium persulfate, and the mixture was maintained at 10°C for 30 minutes to cause polymerization. A mass of cell-containing gel obtained was crushed into small particles, which were thoroughly washed with M/20 phosphate buffer containing 5 g/l of ammonium isobutyrate, pH 7.0, whereby approximately 90 g of an immobilized enzyme are obtained. The washed, immobilized enzyme thus obtained was placed in the same buffer containing 5 g/l of isobutyric acid and left standing at 20°C for 3 days. For comparison purposes, the enzyme was immobilized and washed similarly but with the use of phosphate buffer containing no ammonium isobutyrate, and the resultant immobilized enzyme was left standing at 20°C for 5 days. The nitrile hydration activity levels of the gel samples were measured as follows before and after the respective immobilized enzymes were left standing. 1 g of immobilized gel was mixed with 5 g of acrylonitrile and 97 ml of M/20 phosphate buffer (pH 7.7), and the mixture was subjected to reaction at 0°C for 20 minutes with stirring. The quantities of acrylamide (AA) in the respective reaction solutions were determined by gas chromatography. The results obtained are set forth in Table 7. Experimental Example Ammonium Isobutyrate Quant.of AA produced (%) before enzyme was left standing Quant.of AA produced (%) after enzyme was left standing Example 63 Immobilized with addition of isobutyric acid + left standing in buffer containing isobutyric acid 0.71 0.60 Comparison Example 14 None 0.51 0.26

Claims: A method for preservation of the nitrile hydration activity of nitrilase produced by a Gram-positive microorganism or of an immobilized form thereof, both having nitrile hydration activity, which method comprises adding as a stabilizer at least one compound selected from the group consisting of amides and organic acids and salts thereof to a solution or suspension of said nitrilase or of said immobilized form thereof. A method as claimed in claim 1, wherein the concentration of the amide and organic acid or the salt thereof in the solution or suspension is in the range of from 0.01 to 50 g/l. A method as claimed in claim 1, wherein the pH of the solution or suspension is in the range of from 5 to 8. A method as claimed in claim 1, wherein the microorganism is selected from those of the genera Corynebacterium , Nocardia , Rhodococcus , Bacillus , Bacteridium , Micrococcus , and Brevibacterium .

Inventors: ENOMOTO KANEHIKO, RYUNO KOITCHIRO, SHIMIZU HITOSHI, ENOMOTO, KANEHIKO, RYUNO, KOITCHIRO, SHIMIZU, HITOSHI
Applicants: MITSUBISHI RAYON CO, NITTO CHEMICAL INDUSTRY CO LTD, MITSUBISHI RAYON KABUSHIKI KAISHA, NITTO KAGAKU KOGYO KABUSHIKI KAISHA
Application year: 1987
Main classification: C12N9
IPC codes: C12P13, C12N9, C12N11