Method for preservation of nitrile hydration activity

BACKGROUND OF THE INVENTION

0001The present invention relates to a method for prevention of reduction with elapse of time in nitrile hydration activity of nitrile hydratase produced by microorganisms of the genus <u style="single">Pseudomonas</u> and for stable preservation of this activity.

0002In 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.

0003It is known from EP-A-0 137 076 or EP-A-0 105 083 to culture <u style="single">Pseudomonas</u> bacteria by adding amides or nitriles to the culture medium as "enzyme inducers" during cultivation. The nitriles or amides used as enzyme inducers in this method are decomposed by microorganisms during cultivation.

0004From Monsan et al., World Biotech. Report, The Proceedings of Biotech. '84 Europe, <u style="single">1</u>, pp. 379-390 -(1984) it is known that an enzyme substrate almost always has a positive effect on the stability of the respective enzyme.

0005Nitrile hydratase was discovered by Hideaki Yamada, one of the present inventors, et al., as an enzyme capable of hydrating nitriles to produce the corresponding amides. (Reference: Agric. Biol. Chem. <u style="single">46</u>, 1165-1174 (1982)) As one example of the utilization of this enzyme, a method for producing from C₂-₄ nitriles the corresponding amides with the use of <u style="single">Pseudomonas</u> microorganisms which produce nitrile hydratase has been proposed. (References: U. S. Patent 4,637,982 and Agric. Biol. Chem. <u style="single">46</u>, 1183-1189 (1982))    As a result of further investigation, we have found that the nitrile hydration activity of the abovementioned nitrile hydratase is labile and decreases with elapse of time. This decrease occurs in either case whether the nitrile hydratase stays in microorganism cells or is separated from the cells irrespective of whether or not immobilization is effected, but the separated enzyme shows a greater reduction in nitrile hydration activity and even becomes quite unusable within a short period of time when the reduction is drastic.

SUMMARY OF THE INVENTION

0006As a result of extensive research effort expended toward solving the above problems and efficiently utilizing the nitrile hydration activity of nitrile hydratase, we have found that the use of amides and organic acids or salts thereof is very effective for this purpose and have arrived at the present invention on the basis of this finding.

0007More specifically, the present invention provides a method for preservation of the nitrile hydration activity of nitrile hydratase having been produced by a microorganism of the genus <u style="single">Pseudomonas</u>, of cells of microorganisms of said genus <u style="single">Pseudomonas</u>, or of immobilized forms thereof, all having nitrile hydration activity, which method comprises adding other culturing, at least one compound selected from the group consisting amides and organic acids and salts thereof to a suspension or solution of said nitrile hydratase, od said microorganism cells, or of said immobilized forms thereof.

DETAILED DESCRIPTION OF THE INVENTION

Microorganism:

0008The microorganisms used in the present invention are those of the genus <u style="single">Pseudomonas</u> capable of producing nitrile hydratase and hydrating nitriles, especially acrylonitrile, to produce the corresponding amides, especially acrylamide. Specific examples of such microorganisms are <u style="single">Pseudomonas</u><u style="single">chlororaphis</u> B 23 (FERM BP-187) and <u style="single">Pseudomonas</u> sp. PS 1 (FERM BP-188) disclosed in Japanese Patent Pub. No. 37951/1984 (corresponding to U.S. Patent 4,637,982) and <u style="single">Pseudomonas</u><u style="single">ovalis</u> (IAM 1002), <u style="single">Pseudomonas</u><u style="single">fluorescens</u> (IFO 3081 and IFO 3925) and <u style="single">Pseudomonas</u><u style="single">reptilivora</u> (IFO 3461) listed in JFCC CATALOGUE OF CULTURES, Third Ed. (1979). These microorganisms were deposited with the Fermentation Research Institute, Agency of Industrial Science &amp; Technology (FERM), the Tokyo University Institute of Applied Microbiology (IAM) and the Institute of Fermentation, Incorporated Foundation (IFO) and accorded respectively the above accession numbers.

0009Cultivation 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; enzyme inducers such as propionitrile, isobutyronitrile, propionamide, isobutyramide, and methacrylamide and other optional ingredients.

0010The pH of the culture medium is of the order of 6 to 9, preferably of the order of 7 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.

Nitrile Hydratase

:

0011The nitrile hydratase used in the present invention is an enzyme produced by the aforementioned microorganisms and can be separated and extracted by the following procedure.

0012First, 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.

0013Both these enzymes can be used in the present invention irrespective of whether they are purified partly or completely.

Immobilized Cell-Immobilized enzyme:

0014The immobilized cells and enzymes of the present invention can be obtained by subjecting the above stated microorganism cells and nitrile hydratase 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:

0015The 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.

0016Specific examples of such compounds are: <ul id="ul0001" list-style="none"><li>(1) Aliphatic amides such as acetamide, propionamide and isobutyramide and the corresponding acids or salts thereof.</li><li>(2) Aminoacid amides such as glycinamide, α-aminopropionamide and β -aminopropionamide and the corresponding acids or salts thereof.</li><li>(3) Hydroxyacid amides such as lactzmide, hydroxyacetamide and β -hydroxypropionamide and the corresponding acids or salts thereof.</li><li>(4) Unsaturated amides such as acrylamide and methacrylamide and the corresponding acids or salts thereof.</li><li>(5) Dicarboxylic acid diamides such as malnamide, succinamide and adipamide and the corresponding dibasic acids or salts thereof.</li><li>(6) Monocyanoamides and monocyanic acids such as cyanoacetamide and cyanoacetic acid or salts thereof.</li><li>(7) aromatic acid amides such as benzamide and phenylacetamide and the corresponding acids or salts thereof.</li><li>(8) Heterocyclic amides such as nicotinamide and isonicotinamide and the corresponding acids or salts thereof.</li><li>(9) Halogenated amides such as chloroacetamide and β -chloropropionamide and the corresponding acids or salts thereof.</li><li>(10) Acids having an aldehyde group such as glyoxylic acid or salts thereof.</li></ul>

Preservation of Nitrile Hydration Activity:

0017The preservation of nitrile hydration activity can be attained by adding any of the above enumerated compounds to a suspension or solution of the previously mentioned microorganism cells and nitrile hydratase as they are or in an immobilized state dispersed or dissolved 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.

0018The pH of the suspension or solution 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.

0019While the suspension or solution 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.

0020In accordance with the present invention, nitrile hydration activity can be preserved stably over a long period of time. The addition of the stabilizer of the present invention is particularly effective for enzymes separated from microorganism cells and liable to show a substantial reduction in activity with elapse 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.

0021In 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

0022In each of the following experimental examples, 0.1 ml of an enzyme solution or cell suspension was added to 9.9 ml of M/20 phosphate buffer (pH 7.7) containing 2.5% by weight of acrylonitrile, and the resulting solution was caused to react at 10°C for 10 minutes. The nitrile hydration activity of the test samples 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 or cell suspension per minute being designated as 1 unit (U).

Examples 1 through 18 and Comparison Example 1

0023Culture media each comprising 10 g/l of sucrose, 1 g/l of KH₂PO₄, 1 g/l of K₂HSO₄, 1 g/l of MgSO₄·7H₂O, 10 g/l of chemical soy sauce, 1 g/l of L-cysteine, 10 g/l of FeSO₄·7H₂O, and 8 g/l of methacrylamide were adjusted to a pH of 7.2, and 100 ml of each resulting culture medium was sterilized in a 500-ml Erlenmeyer flask.

0024After cooling, each sterilized culture medium was inoculated with 1 ml of a culture fluid obtained by precultivating <u style="single">Pseudomonas</u><u style="single">chlororaphis</u> B23 (FERM BP-187) in a culture medium (pH 7.8) comprising 5 g/l of sucrose, 5 g/l of peptone, 3 g/l of yeast extract, and 3 g/l of malt extract for one day, and cultivation was carried out aerobically at 25°C for 2 days.

0025Cells 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 (cell concentration: ca. 20 g/l, 1.7x10³ U/ml).

0026To 2.5 ml of this cell suspension was added 2.5 ml of M/10 phosphate buffer containing 10 mg of each of the amides shown in Table 1, the pH of the cell suspension was adjusted to 6.5, and then the cell suspension was left standing for 5 days in ice-cooled state. 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 nitrile hydration activity levels measured before and after the cell suspension was left standing. The results obtained are summarized in Table 1 below. <tables id="tabl0001" num="0001"><table frame="all"><title>Table 1</title><tgroup cols="3" colsep="1" rowsep="0"><colspec colnum="1" colname="col1" colwidth="52.50mm" /><colspec colnum="2" colname="col2" colwidth="52.50mm" /><colspec colnum="3" colname="col3" colwidth="52.50mm" /><thead valign="top"><row><entry namest="col1" nameend="col1" align="center">Experimental Example</entry><entry namest="col2" nameend="col2" align="center">Amide Added</entry><entry namest="col3" nameend="col3" align="center">% Residual Activity</entry></row></thead><tbody valign="top"><row rowsep="1"><entry namest="col1" nameend="col1" align="right">Comparison Example 1</entry><entry namest="col2" nameend="col2" align="left">None</entry><entry namest="col3" nameend="col3" align="right">42</entry></row><row><entry namest="col1" nameend="col1" align="right">Example 1</entry><entry namest="col2" nameend="col2" align="left">Acetamide</entry><entry namest="col3" nameend="col3" align="right">67</entry></row><row><entry namest="col1" nameend="col1" align="right">2</entry><entry namest="col2" nameend="col2" align="left">Propionamide</entry><entry namest="col3" nameend="col3" align="right">93</entry></row><row><entry namest="col1" nameend="col1" align="right">3</entry><entry namest="col2" nameend="col2" align="left">n-Butyramide</entry><entry namest="col3" nameend="col3" align="right">89</entry></row><row><entry namest="col1" nameend="col1" align="right">4</entry><entry namest="col2" nameend="col2" align="left">Isobutyramide</entry><entry namest="col3" nameend="col3" align="right">99</entry></row><row><entry namest="col1" nameend="col1" align="right">5</entry><entry namest="col2" nameend="col2" align="left">n-Valeramide</entry><entry namest="col3" nameend="col3" align="right">94</entry></row><row><entry namest="col1" nameend="col1" align="right">6</entry><entry namest="col2" nameend="col2" align="left">n-Capronamide</entry><entry namest="col3" nameend="col3" align="right">88</entry></row><row><entry namest="col1" nameend="col1" align="right">7</entry><entry namest="col2" nameend="col2" align="left">Glycine amide</entry><entry namest="col3" nameend="col3" align="right">63</entry></row><row><entry namest="col1" nameend="col1" align="right">8</entry><entry namest="col2" nameend="col2" align="left">β-Aminopropionamide</entry><entry namest="col3" nameend="col3" align="right">68</entry></row><row><entry namest="col1" nameend="col1" align="right">9</entry><entry namest="col2" nameend="col2" align="left">Lactamide</entry><entry namest="col3" nameend="col3" align="right">70</entry></row><row><entry namest="col1" nameend="col1" align="right">10</entry><entry namest="col2" nameend="col2" align="left">β-Hydroxypropionamide</entry><entry namest="col3" nameend="col3" align="right">74</entry></row><row><entry namest="col1" nameend="col1" align="right">11</entry><entry namest="col2" nameend="col2" align="left">Acrylamide</entry><entry namest="col3" nameend="col3" align="right">73</entry></row><row><entry namest="col1" nameend="col1" align="right">12</entry><entry namest="col2" nameend="col2" align="left">Methacrylamide</entry><entry namest="col3" nameend="col3" align="right">79</entry></row><row><entry namest="col1" nameend="col1" align="right">13</entry><entry namest="col2" nameend="col2" align="left">Malondiamide</entry><entry namest="col3" nameend="col3" align="right">66</entry></row><row><entry namest="col1" nameend="col1" align="right">14</entry><entry namest="col2" nameend="col2" align="left">Succindiamide</entry><entry namest="col3" nameend="col3" align="right">68</entry></row><row><entry namest="col1" nameend="col1" align="right">15</entry><entry namest="col2" nameend="col2" align="left">Benzamide</entry><entry namest="col3" nameend="col3" align="right">65</entry></row><row><entry namest="col1" nameend="col1" align="right">16</entry><entry namest="col2" nameend="col2" align="left">Nicotinamide</entry><entry namest="col3" nameend="col3" align="right">59</entry></row><row><entry namest="col1" nameend="col1" align="right">17</entry><entry namest="col2" nameend="col2" align="left">Chloroacetamide</entry><entry namest="col3" nameend="col3" align="right">71</entry></row><row rowsep="1"><entry namest="col1" nameend="col1" align="right">18</entry><entry namest="col2" nameend="col2" align="left">β-Chloropropionamide</entry><entry namest="col3" nameend="col3" align="right">71</entry></row></tbody></tgroup></table></tables>

Examples 19 through 38 and Comparison Example 2

0027The procedures of Examples 1 through 10 were followed except that the amides added were replaced by organic acids and that the pH of each cell suspension was adjusted to 6.0. The results obtained are set forth in Table 2. <tables id="tabl0002" num="0002"><table frame="all"><title>Table 2</title><tgroup cols="3" colsep="1" rowsep="0"><colspec colnum="1" colname="col1" colwidth="52.50mm" /><colspec colnum="2" colname="col2" colwidth="52.50mm" /><colspec colnum="3" colname="col3" colwidth="52.50mm" /><thead valign="top"><row><entry namest="col1" nameend="col1" align="center">Experimental Example</entry><entry namest="col2" nameend="col2" align="center">Organic Acid Added</entry><entry namest="col3" nameend="col3" align="center">% Residual Activity</entry></row></thead><tbody valign="top"><row rowsep="1"><entry namest="col1" nameend="col1" align="right">Comparison Example 2</entry><entry namest="col2" nameend="col2" align="left">None</entry><entry namest="col3" nameend="col3" align="right">43</entry></row><row><entry namest="col1" nameend="col1" align="right">Example 19</entry><entry namest="col2" nameend="col2" align="left">Formic acid</entry><entry namest="col3" nameend="col3" align="right">83</entry></row><row><entry namest="col1" nameend="col1" align="right">20</entry><entry namest="col2" nameend="col2" align="left">Acetic acid</entry><entry namest="col3" nameend="col3" align="right">77</entry></row><row><entry namest="col1" nameend="col1" align="right">21</entry><entry namest="col2" nameend="col2" align="left">Propionic acid</entry><entry namest="col3" nameend="col3" align="right">93</entry></row><row><entry namest="col1" nameend="col1" align="right">22</entry><entry namest="col2" nameend="col2" align="left">Isobutyric acid</entry><entry namest="col3" nameend="col3" align="right">97</entry></row><row><entry namest="col1" nameend="col1" align="right">23</entry><entry namest="col2" nameend="col2" align="left">Valeric acid</entry><entry namest="col3" nameend="col3" align="right">84</entry></row><row><entry namest="col1" nameend="col1" align="right">24</entry><entry namest="col2" nameend="col2" align="left">Caproic acid</entry><entry namest="col3" nameend="col3" align="right">83</entry></row><row><entry namest="col1" nameend="col1" align="right">25</entry><entry namest="col2" nameend="col2" align="left">β-Aminopropionic acid</entry><entry namest="col3" nameend="col3" align="right">71</entry></row><row><entry namest="col1" nameend="col1" align="right">26</entry><entry namest="col2" nameend="col2" align="left">Malic acid</entry><entry namest="col3" nameend="col3" align="right">81</entry></row><row><entry namest="col1" nameend="col1" align="right">27</entry><entry namest="col2" nameend="col2" align="left">Citric acid</entry><entry namest="col3" nameend="col3" align="right">77</entry></row><row><entry namest="col1" nameend="col1" align="right">20</entry><entry namest="col2" nameend="col2" align="left">Fumaric acid</entry><entry namest="col3" nameend="col3" align="right">82</entry></row><row><entry namest="col1" nameend="col1" align="right">29</entry><entry namest="col2" nameend="col2" align="left">Glycolic acid</entry><entry namest="col3" nameend="col3" align="right">77</entry></row><row><entry namest="col1" nameend="col1" align="right">30</entry><entry namest="col2" nameend="col2" align="left">DL-Glyceric acid</entry><entry namest="col3" nameend="col3" align="right">87</entry></row><row><entry namest="col1" nameend="col1" align="right">31</entry><entry namest="col2" nameend="col2" align="left">Oxalacetic acid</entry><entry namest="col3" nameend="col3" align="right">83</entry></row><row><entry namest="col1" nameend="col1" align="right">32</entry><entry namest="col2" nameend="col2" align="left">Succinic acid</entry><entry namest="col3" nameend="col3" align="right">80</entry></row><row><entry namest="col1" nameend="col1" align="right">33</entry><entry namest="col2" nameend="col2" align="left">Malonic acid</entry><entry namest="col3" nameend="col3" align="right">63</entry></row><row><entry namest="col1" nameend="col1" align="right">34</entry><entry namest="col2" nameend="col2" align="left">Benzoic acid</entry><entry namest="col3" nameend="col3" align="right">68</entry></row><row><entry namest="col1" nameend="col1" align="right">35</entry><entry namest="col2" nameend="col2" align="left">Monochloroacetic acid</entry><entry namest="col3" nameend="col3" align="right">70</entry></row><row><entry namest="col1" nameend="col1" align="right">36</entry><entry namest="col2" nameend="col2" align="left">Glyoxylic acid</entry><entry namest="col3" nameend="col3" align="right">88</entry></row><row><entry namest="col1" nameend="col1" align="right">37</entry><entry namest="col2" nameend="col2" align="left">Acrylic acid</entry><entry namest="col3" nameend="col3" align="right">70</entry></row><row rowsep="1"><entry namest="col1" nameend="col1" align="right">38</entry><entry namest="col2" nameend="col2" align="left">Methacrylic acid</entry><entry namest="col3" nameend="col3" align="right">81</entry></row></tbody></tgroup></table></tables>

Examples 39 through 50 and Comparison Examples 3 through 8.

0028To each 2.5-ml aliquot of a suspension of washed cells (1.8x10³ U/ml) prepared as in Example 1, was added 2.5 ml of M/10 phosphate buffer containing 25 mg of a stabilizer as shown in Table 3. The pH of the mixture was adjusted to a predetermined level, and the resulting mixture was then left standing for 4 days in ice-cooled state.

0029The nitrile hydration activity levels were measured before and after each of the suspensions was left standing to calculate % residual activity, whereupon the results summarized in Table 3 were obtained. <tables id="tabl0003" num="0003"><table frame="all"><title>Table 3</title><tgroup cols="4" colsep="1" rowsep="0"><colspec colnum="1" colname="col1" colwidth="39.37mm" /><colspec colnum="2" colname="col2" colwidth="39.37mm" /><colspec colnum="3" colname="col3" colwidth="39.37mm" /><colspec colnum="4" colname="col4" colwidth="39.37mm" /><thead valign="top"><row><entry namest="col1" nameend="col1" align="center">Experimental Example</entry><entry namest="col2" nameend="col2" align="center">Stabilizer Added</entry><entry namest="col3" nameend="col3" align="center">pH</entry><entry namest="col4" nameend="col4" align="center">% Residual Activity</entry></row></thead><tbody valign="top"><row><entry namest="col1" nameend="col1" align="right">Comparison Example 3</entry><entry namest="col2" nameend="col2" align="left">None</entry><entry namest="col3" nameend="col3" align="char" char=".">5.0</entry><entry namest="col4" nameend="col4" align="right">2</entry></row><row><entry namest="col1" nameend="col1" align="right">4</entry><entry namest="col2" nameend="col2" align="left">   "</entry><entry namest="col3" nameend="col3" align="char" char=".">5.5</entry><entry namest="col4" nameend="col4" align="right">32</entry></row><row><entry namest="col1" nameend="col1" align="right">5</entry><entry namest="col2" nameend="col2" align="left">   "</entry><entry namest="col3" nameend="col3" align="char" char=".">6.0</entry><entry namest="col4" nameend="col4" align="right">41</entry></row><row><entry namest="col1" nameend="col1" align="right">6</entry><entry namest="col2" nameend="col2" align="left">   "</entry><entry namest="col3" nameend="col3" align="char" char=".">6.5</entry><entry namest="col4" nameend="col4" align="right">52</entry></row><row><entry namest="col1" nameend="col1" align="right">7</entry><entry namest="col2" nameend="col2" align="left">   "</entry><entry namest="col3" nameend="col3" align="char" char=".">7.0</entry><entry namest="col4" nameend="col4" align="right">43</entry></row><row rowsep="1"><entry namest="col1" nameend="col1" align="right">8</entry><entry namest="col2" nameend="col2" align="left">   "</entry><entry namest="col3" nameend="col3" align="char" char=".">8.0</entry><entry namest="col4" nameend="col4" align="right">26</entry></row><row><entry namest="col1" nameend="col1" align="right">Example 39</entry><entry namest="col2" nameend="col2" align="left">Isobutyramide</entry><entry namest="col3" nameend="col3" align="char" char=".">5.0</entry><entry namest="col4" nameend="col4" align="right">3</entry></row><row><entry namest="col1" nameend="col1" align="right">40</entry><entry namest="col2" nameend="col2" align="left">   "</entry><entry namest="col3" nameend="col3" align="char" char=".">5.5</entry><entry namest="col4" nameend="col4" align="right">100</entry></row><row><entry namest="col1" nameend="col1" align="right">41</entry><entry namest="col2" nameend="col2" align="left">   "</entry><entry namest="col3" nameend="col3" align="char" char=".">6.0</entry><entry namest="col4" nameend="col4" align="right">100</entry></row><row><entry namest="col1" nameend="col1" align="right">42</entry><entry namest="col2" nameend="col2" align="left">   "</entry><entry namest="col3" nameend="col3" align="char" char=".">6.5</entry><entry namest="col4" nameend="col4" align="right">87</entry></row><row><entry namest="col1" nameend="col1" align="right">43</entry><entry namest="col2" nameend="col2" align="left">   "</entry><entry namest="col3" nameend="col3" align="char" char=".">7.0</entry><entry namest="col4" nameend="col4" align="right">78</entry></row><row rowsep="1"><entry namest="col1" nameend="col1" align="right">44</entry><entry namest="col2" nameend="col2" align="left">   "</entry><entry namest="col3" nameend="col3" align="char" char=".">8.0</entry><entry namest="col4" nameend="col4" align="right">52</entry></row><row><entry namest="col1" nameend="col1" align="right">Example 45</entry><entry namest="col2" nameend="col2" align="left">Isobutyric acid</entry><entry namest="col3" nameend="col3" align="char" char=".">5.0</entry><entry namest="col4" nameend="col4" align="right">1</entry></row><row><entry namest="col1" nameend="col1" align="right">46</entry><entry namest="col2" nameend="col2" align="left">   "</entry><entry namest="col3" nameend="col3" align="char" char=".">5.5</entry><entry namest="col4" nameend="col4" align="right">100</entry></row><row><entry namest="col1" nameend="col1" align="right">47</entry><entry namest="col2" nameend="col2" align="left">   "</entry><entry namest="col3" nameend="col3" align="char" char=".">6.0</entry><entry namest="col4" nameend="col4" align="right">100</entry></row><row><entry namest="col1" nameend="col1" align="right">48</entry><entry namest="col2" nameend="col2" align="left">   "</entry><entry namest="col3" nameend="col3" align="char" char=".">6.5</entry><entry namest="col4" nameend="col4" align="right">88</entry></row><row><entry namest="col1" nameend="col1" align="right">49</entry><entry namest="col2" nameend="col2" align="left">   "</entry><entry namest="col3" nameend="col3" align="char" char=".">7.0</entry><entry namest="col4" nameend="col4" align="right">78</entry></row><row rowsep="1"><entry namest="col1" nameend="col1" align="right">50</entry><entry namest="col2" nameend="col2" align="left">   "</entry><entry namest="col3" nameend="col3" align="char" char=".">8.0</entry><entry namest="col4" nameend="col4" align="right">49</entry></row></tbody></tgroup></table></tables>

Examples 51 through 62 and Comparison Example 9

0030To each 2.5-ml aliquot of a suspension of washed cells (1.8x10³ U/ml) prepared as in Example 1, was added 2.5 ml of M/10 phosphate buffer containing a predetermined quantity of a stabilizer as shown in Table 5. The pH of the mixture was adjusted to 6.0 (6.5 for the suspension containing no stabilizer), and the resulting mixture was then left standing for 3 days in ice-cooled state.

0031The nitrile hydration activity levels were measured before and after each of the suspensions was left standing to calculate % residual activity. The results obtained are presented in Table 4. <tables id="tabl0004" num="0004"><table frame="all"><title>Table 4</title><tgroup cols="4" colsep="1" rowsep="0"><colspec colnum="1" colname="col1" colwidth="39.37mm" /><colspec colnum="2" colname="col2" colwidth="39.37mm" /><colspec colnum="3" colname="col3" colwidth="39.37mm" /><colspec colnum="4" colname="col4" colwidth="39.37mm" /><thead valign="top"><row><entry namest="col1" nameend="col1" align="center">Experimental Example</entry><entry namest="col2" nameend="col2" align="center">Stabilizer Added</entry><entry namest="col3" nameend="col3" align="center">Concentration (g/l)</entry><entry namest="col4" nameend="col4" align="center">% Residual Activity</entry></row></thead><tbody valign="top"><row rowsep="1"><entry namest="col1" nameend="col1" align="right">Comparison Example 9</entry><entry namest="col2" nameend="col2" align="left">None</entry><entry namest="col3" nameend="col3" align="char" char=".">-</entry><entry namest="col4" nameend="col4" align="right">52</entry></row><row><entry namest="col1" nameend="col1" align="right">Example 51</entry><entry namest="col2" nameend="col2" align="left">Isobutyramide</entry><entry namest="col3" nameend="col3" align="char" char=".">0.01</entry><entry namest="col4" nameend="col4" align="right">82</entry></row><row><entry namest="col1" nameend="col1" align="right">52</entry><entry namest="col2" nameend="col2" align="left">   "</entry><entry namest="col3" nameend="col3" align="char" char=".">0.1</entry><entry namest="col4" nameend="col4" align="right">88</entry></row><row><entry namest="col1" nameend="col1" align="right">53</entry><entry namest="col2" nameend="col2" align="left">   "</entry><entry namest="col3" nameend="col3" align="char" char=".">1.0</entry><entry namest="col4" nameend="col4" align="right">100</entry></row><row><entry namest="col1" nameend="col1" align="right">54</entry><entry namest="col2" nameend="col2" align="left">   "</entry><entry namest="col3" nameend="col3" align="char" char=".">5.0</entry><entry namest="col4" nameend="col4" align="right">100</entry></row><row><entry namest="col1" nameend="col1" align="right">55</entry><entry namest="col2" nameend="col2" align="left">   "</entry><entry namest="col3" nameend="col3" align="char" char=".">20</entry><entry namest="col4" nameend="col4" align="right">100</entry></row><row rowsep="1"><entry namest="col1" nameend="col1" align="right">56</entry><entry namest="col2" nameend="col2" align="left">   "</entry><entry namest="col3" nameend="col3" align="char" char=".">50</entry><entry namest="col4" nameend="col4" align="right">90</entry></row><row><entry namest="col1" nameend="col1" align="right">Example 57</entry><entry namest="col2" nameend="col2" align="left">Isobutyric acid</entry><entry namest="col3" nameend="col3" align="char" char=".">0.01</entry><entry namest="col4" nameend="col4" align="right">80</entry></row><row><entry namest="col1" nameend="col1" align="right">58</entry><entry namest="col2" nameend="col2" align="left">   "</entry><entry namest="col3" nameend="col3" align="char" char=".">0.1</entry><entry namest="col4" nameend="col4" align="right">83</entry></row><row><entry namest="col1" nameend="col1" align="right">59</entry><entry namest="col2" nameend="col2" align="left">   "</entry><entry namest="col3" nameend="col3" align="char" char=".">1.0</entry><entry namest="col4" nameend="col4" align="right">100</entry></row><row><entry namest="col1" nameend="col1" align="right">60</entry><entry namest="col2" nameend="col2" align="left">   "</entry><entry namest="col3" nameend="col3" align="char" char=".">5.0</entry><entry namest="col4" nameend="col4" align="right">97</entry></row><row><entry namest="col1" nameend="col1" align="right">61</entry><entry namest="col2" nameend="col2" align="left">   "</entry><entry namest="col3" nameend="col3" align="char" char=".">20</entry><entry namest="col4" nameend="col4" align="right">95</entry></row><row><entry namest="col1" nameend="col1" align="right">62</entry><entry namest="col2" nameend="col2" align="left">   "</entry><entry namest="col3" nameend="col3" align="char" char=".">50</entry><entry namest="col4" nameend="col4" align="right">87</entry></row><row rowsep="1"><entry namest="col1" nameend="col4" align="justify">Concentration: Concentration of the stabilizer in the solution left standing</entry></row></tbody></tgroup></table></tables>

Example 63 and Comparison Example 10

003250 ml of a suspension of washed cells prepared as in Example 1 was mixed with 50 ml of M/10 phosphate buffer (pH 6.5) containing 10 g/l of ammonium isobutyrate (3.2x10³ U/ml).

0033The resultant cell suspension was pressed in a French press (fabricated by Ohtake Seisakusho, 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.

0034For comparison purposes, a similar cell suspension (3.2x10³ 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.

0035Both cell extracts were left standing for 3 days in ice-cooled state, and the nitrile hydration activity levels were measured before and after the extracts were left standing. The results are shown in Table 5. <tables id="tabl0005" num="0005"><table frame="all"><title>Table 5</title><tgroup cols="5" colsep="1" rowsep="1"><colspec colnum="1" colname="col1" colwidth="31.50mm" /><colspec colnum="2" colname="col2" colwidth="31.50mm" /><colspec colnum="3" colname="col3" colwidth="31.50mm" /><colspec colnum="4" colname="col4" colwidth="31.50mm" /><colspec colnum="5" colname="col5" colwidth="31.50mm" /><thead valign="top"><row><entry namest="col1" nameend="col1" align="center">Experimental Example</entry><entry namest="col2" nameend="col2" align="center">Ammonium Isobutyrate</entry><entry namest="col3" nameend="col3" align="center">Activity Before Being Left Standing (U/ml)</entry><entry namest="col4" nameend="col4" align="center">Activity After Being Left Standing (U/ml)</entry><entry namest="col5" nameend="col5" align="center">% Residual Activity</entry></row></thead><tbody valign="top"><row><entry namest="col1" nameend="col1" align="left">Example 63</entry><entry namest="col2" nameend="col2" align="center">Added</entry><entry namest="col3" nameend="col3" align="char" char=".">2.9x10³</entry><entry namest="col4" nameend="col4" align="char" char=".">2.8x10³</entry><entry namest="col5" nameend="col5" align="right">94</entry></row><row rowsep="1"><entry namest="col1" nameend="col1" align="left">Comparison Example 10</entry><entry namest="col2" nameend="col2" align="center">None</entry><entry namest="col3" nameend="col3" align="char" char=".">2.7x10³</entry><entry namest="col4" nameend="col4" align="char" char=".">1.0x10³</entry><entry namest="col5" nameend="col5" align="right">38</entry></row></tbody></tgroup></table></tables>

Example 64 and Comparison Example 11

003640 ml of washed cells (water content 75%) 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% 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 immobilized cells were obtained.

0037The washed, immobilized cells thus obtained were placed in the same buffer containing 5 g/l of isobutyric acid and left standing at 0°C for 5 days.

0038For comparison purposes, the cells were immobilized and washed similarly but with the use of phosphate buffer containing no ammonium isobutyrate, and the resultant cells were left standing at 0°C for 5 days.

0039The nitrile hydration activity levels of the gel samples were measured as follows before and after the respective immobilized cells were left standing.

00401 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 acrylmide (AA) in the respective reaction solutions were determined by gas chromatography. The results obtained are set forth in Table 6. <tables id="tabl0006" num="0006"><table frame="all"><title>Table 6</title><tgroup cols="4" colsep="1" rowsep="1"><colspec colnum="1" colname="col1" colwidth="39.37mm" /><colspec colnum="2" colname="col2" colwidth="39.37mm" /><colspec colnum="3" colname="col3" colwidth="39.37mm" /><colspec colnum="4" colname="col4" colwidth="39.37mm" /><thead valign="top"><row><entry namest="col1" nameend="col1" align="center">Experimental Example</entry><entry namest="col2" nameend="col2" align="center">Ammonium Isobutyrate</entry><entry namest="col3" nameend="col3" align="center">Quant. of AA produced (%) before cells were left standing</entry><entry namest="col4" nameend="col4" align="center">Quant. of AA produced (%) after cells were left standing</entry></row></thead><tbody valign="top"><row><entry namest="col1" nameend="col1" align="left">Example 64</entry><entry namest="col2" nameend="col2" align="left">Immobilized with addition of isobutyric acid + left standing in buffer containing isobutyric acid</entry><entry namest="col3" nameend="col3" align="char" char=".">1.02</entry><entry namest="col4" nameend="col4" align="char" char=".">0.93</entry></row><row rowsep="1"><entry namest="col1" nameend="col1" align="left">Comparison Example 11</entry><entry namest="col2" nameend="col2" align="left">None</entry><entry namest="col3" nameend="col3" align="char" char=".">0.88</entry><entry namest="col4" nameend="col4" align="char" char=".">0.52</entry></row></tbody></tgroup></table></tables>

Example 65 and Comparison Example 12

004125 ml each of a cell extract containing ammonium isobutyrate and a control cell extract containing no ammonium isobutyrate prepared as in Example 63 and comparison Example 10 were mixed with 15 g each of DEAE-Cellulofine AM (supplied by Seikagaku Kogyo Co., Ltd., Japan) equilibrated with M/20 phosphate buffer (pH 7.7). The mixture was stirred at 0°C for 90 minutes to immobilize the enzymes.

0042Both immobilized enzymes thus obtained were left standing for 3 days in ice-cooled state, and the nitrile hydration activity levels were measured before and after the enzymes were left standing. The results are presented in Table 7 in which the protein levels in the immobilized enzymes were determined in accordance with the procedure of Lowry et al. <tables id="tabl0007" num="0007"><table frame="all"><title>Table 7</title><tgroup cols="5" colsep="1" rowsep="0"><colspec colnum="1" colname="col1" colwidth="31.50mm" /><colspec colnum="2" colname="col2" colwidth="31.50mm" /><colspec colnum="3" colname="col3" colwidth="31.50mm" /><colspec colnum="4" colname="col4" colwidth="31.50mm" /><colspec colnum="5" colname="col5" colwidth="31.50mm" /><thead valign="top"><row><entry namest="col1" nameend="col1" align="center">Experimental Example</entry><entry namest="col2" nameend="col2" align="center">Ammonium Isobutyrate</entry><entry namest="col3" nameend="col3" align="center">Activity Before Being Left Standing</entry><entry namest="col4" nameend="col4" align="center">Activity After Being Left Standing</entry><entry namest="col5" nameend="col5" align="center">% Residual Activity</entry></row></thead><tbody valign="top"><row><entry namest="col1" nameend="col1" align="left">Example 65</entry><entry namest="col2" nameend="col2" align="right">Added</entry><entry namest="col3" nameend="col3" align="char" char=".">19.4</entry><entry namest="col4" nameend="col4" align="char" char=".">18.4</entry><entry namest="col5" nameend="col5" align="right">95</entry></row><row rowsep="1"><entry namest="col1" nameend="col1" align="left">Comparison Example 12</entry><entry namest="col2" nameend="col2" align="right">None</entry><entry namest="col3" nameend="col3" align="char" char=".">4.6</entry><entry namest="col4" nameend="col4" align="char" char=".">1.7</entry><entry namest="col5" nameend="col5" align="right">37</entry></row></tbody></tgroup></table></tables>