Production process of L-alpha-amino acids

0001This invention relates to a process for the production of L-α-amino acids from nitrile compounds, particularly from racemic α-aminonitrile compounds or derivatives thereof by making use of microorganisms.

0002L-α-amino acids are important chemical substances useful in a variety of fields such as foods, feeds, pharmaceuticals and cosmetics.

0003Conventionally, the fermentation, synthetic, enxymatic and extraction processes have been known to produce L-α-amino acids. Among these processes, the synthetic process comprises synthesizing first DL-α-amino acids in accordance with the Strecker process or the modification process thereof and then subjecting the amino acids to optical resolution to produce L-α-amino acids. In the optical resolution, the enzymatic process using aminoacylase or similar processes have been employed (Kagaku Zokan 97, Progress of Asymmetric Synthesis and Optical Resolution, Kagakudojin, 1982, p. 175).

0004However, production of L-α-amino acids according to the above-described synthetic process requires increased costs in the stage of optical resolution. For such economical reasons, the process has recently suffered gradual share reduction in the production of amino acids.

0005An attempt has also been made reportedly to produce α-amino acids from DL-α-aminonitriles, which are intermediates in the synthesis of α-amino acids according to the aforementioned Strecker process and represented by the following general formula: <chemistry id="chem0001" num="0001"><img file="EP0332379B1_D0001.tif" /></chemistry> wherein R is an alkyl group, phenyl group or the like, by making use of microorganisms [Y. Fukuda et al., J. Ferment. Technol. <u>49</u>, 1101 (1971)].

0006According to this report, however, DL-α-aminopropionitrile and DL-α-aminoisovaleronitrile are hydrolyzed in the presence of microorganisms belonging to <u>Corynebacterium</u> sp. to produce respectively DL-alanine and DL-valine, and therefore L-α-amino acids can not be obtained directly.

0007It has also been reported that amino acids are produced by the hydrolysis of DL-α-aminonitriles in the presence of <u>Brevibacterium</u> sp. R312 [J. C. Jallageas et al., Adv. Biochem. Engineer, <u>14</u>, 1 (1980)]. According to the report, only DL-α-amino acids are obtainable from DL-α-aminonitriles. In this connection, although the above report discloses simultaneous production of L-α-amino-acids and D-α-amino acid amides from DL-α-aminonitriles by the use of <u>Brevibacterium</u> sp. A4, a mutant obtained from <u>Brevibacterium</u> sp. B312, no report has been found with regard to the direct and exclusive production of L-α-amino acids from DL-α-aminonitriles. Further, this process necessarily involves such complex operation of separating an L-α-amino acid from a D-α-amino acid amide and hence is unfavoured.

0008It has also been disclosed to produce amino acids by irradiating light to the reaction system before completion of the microbiological hydration of DL-α-aminonitrile compounds (Japanese Patent Laid-Open No. 162191/1986). However, only DL-α-amino acids are obtainable by this process.

0009The use of enantio-preferential enzymes for the conversion of amino nitriles into the corresponding amino acid amides or amino acid, has been disclosed in International Patent Application WO86/07386. The process described in this application involves the conversion of amino nitriles into the corresponding amino acid amides in an enzyme catalysed reaction using a nitrilase which preferentially converts one of the two enantiomeric forms of the amino nitriles into the corresponding amino acid amides. The amino acid amides of said enantiomeric form thus generated is subsequently hydrolysed by chemical means to provide the amino acid of the corresponding enantiomeric form. In the process disclosed in this application, the enzyme-catalyzed reaction is carried out at a pH value of about 7.

0010Thus, no report has been made to produce L-α-amino acids directly from racemic α-aminonitrile compounds.

0011In view of the aforementioned existing state of art, the present inventors have made intensive investigations and found that L-α-amino acids are selectively produced when microorganisms having the ability of nitrile hydrolysis are caused to act on an α-aminonitrile compound at a pH in the range of 8-12 or on an α-(N-alkylideneamino) nitrile comopound. The present invention has been completed on the basis of this finding and makes it the object to provide a process for the direct production of L-α-amino acids from α-aminonitrile compounds or derivatives thereof by making use of microorganisms selected from specific genera and having nitrile-hydrolyzing ability.

0012The process of the present invention comprises causing a microorganism having nitrile-hydrolyzing activity to act on one or more α-aminonitrile compounds represented by the following general formula (I): <chemistry id="chem0002" num="0002"><img file="EP0332379B1_D0002.tif" /></chemistry> and/or the following general formula (II): <chemistry id="chem0003" num="0003"><img file="EP0332379B1_D0003.tif" /></chemistry> at a pH in the range of 8-12 and/or in the presence of an aldehyde, or on one or more α-(N-alkylideneamino)nitrile compounds represented by the following general formula (III): <chemistry id="chem0004" num="0004"><img file="EP0332379B1_D0004.tif" /></chemistry> and/or the following general formula (IV): <chemistry id="chem0005" num="0005"><img file="EP0332379B1_D0005.tif" /></chemistry> to convert the compound or compounds to L-α-amino acids, R and R' in the above formulae being individually an alkyl group, substituted alkyl group, phenyl group, substituted phenyl group, imidazolyl group, substituted imidazolyl group, indolyl group, substituted indolyl group, furyl group, substituted furyl group, pyridyl group, substituted pyridyl group, thiazolyl group or substituted thiazolyl group, and R and R' may be the same or different groups.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE INVENTION

:

0013The microorganisms useful in the practice of the present invention are, as described above, those selected from the group having nitrile-hydrolyzing ability and may be illustrated as in the following Table 1.

0014<tables id="tabl0001" num="0001"><table frame="all"><title>Table 1</title><tgroup cols="2" colsep="1" rowsep="1"><colspec colnum="1" colname="col1" colwidth="78.75mm" /><colspec colnum="2" colname="col2" colwidth="78.75mm" /><thead valign="top"><row><entry namest="col1" nameend="col1" align="center">Strain</entry><entry namest="col2" nameend="col2" align="center">FERM-BP No.</entry></row></thead><tbody valign="top"><row><entry namest="col1" nameend="col1" align="left">Nocardia sp. PC-29</entry><entry namest="col2" nameend="col2" align="center">1561</entry></row><row><entry namest="col1" nameend="col1" align="left">Nocardia sp. PA-34</entry><entry namest="col2" nameend="col2" align="center">1559</entry></row><row><entry namest="col1" nameend="col1" align="left">Nocardia sp. AB-16</entry><entry namest="col2" nameend="col2" align="center">1555</entry></row><row><entry namest="col1" nameend="col1" align="left">Nocardia sp. BA-1</entry><entry namest="col2" nameend="col2" align="center">1557</entry></row><row><entry namest="col1" nameend="col1" align="left">Mycobacterium sp. AB-43</entry><entry namest="col2" nameend="col2" align="center">1556</entry></row><row><entry namest="col1" nameend="col1" align="left">Corynebacterium sp. PA-15</entry><entry namest="col2" nameend="col2" align="center">1558</entry></row><row rowsep="1"><entry namest="col1" nameend="col1" align="left">Corynebacterium sp. PC-3</entry><entry namest="col2" nameend="col2" align="center">1560</entry></row></tbody></tgroup></table></tables>

0015These microorganisms have been deposited to Fermentation Research Institute of the Agency of Industrial Science and Technology under the Budapest Treaty on Nov. 5, 1987 with Accession Numbers given in Table 1.

0016The properties of the above-described microorganisms are illustrated in Table 2. <tables id="tabl0002" num="0002"><img file="EP0332379B1_D0006.tif" /></tables><tables id="tabl0003" num="0003"><img file="EP0332379B1_D0007.tif" /></tables>

0017As seen in Table 2, the microorganisms used in the present invention are all Gram-positive aerobic bacteria and hence do not grow at all in anaerobic conditions. They do not exhibit motility, catalases being positive, and they do not form heat-resistant spores. They show mycelial growth in the early stage of culture. Branching cells and cocci are also observed in older cultures. As to the utilization of sugar, they do not generate gas from sugar, and their acid-forming ability is weak so that when they are incubated in litmus milk, the milk will turn blue indicating alkaline.

0018Among the above-described microorganisms, <u>Nocardia</u> sp. PC-29 forms somewhat dry and wrinkled colonies on nutrient agar and shows markedly branched mycelial growth in the early stage of culture. It grows at 5 - 32°C but does not grow at 37°C.

0019On the other hand, <u>Nocardia</u> sps. PA-34, AB-16 and BA-1 do not grow at 5 - 10°C, but grow at 37 - 42°C. They show mycelial growth in the early stage of culture and in older cultures shorter rods or cocci are formed.

0020<u>Mycobacterium</u> sp. AB-43 is positive in acid-fast stain and does not grow at 45°C, forming viscous yellow-orange colonies. <u>Corynebacterium</u> sps. PA-15 and PC-3 are Gram-positive rods and their shapes are rather 5 irregular. In older cultures, they form granules in the cells and exhibit a negative Gram stain, and shorter rods or cocci are formed. The strains grow at 5 - 10°C, but do not grow at 37°C or higher. The colonies on nutrient agar are yellow-green and creamy, and have strong abilities of hydrolyzing both gelatin and starch but no ability of decomposing cellulose.

0021The above-described microorganisms were identified according to Bergey's Manual of Systematic Bacteriology, 1986, in view of the properties shown in Table 2 and as described above.

0022In the present invention, the α-aminonitrile compounds represented by the foregoing general formulae (I) and (II), the substrates for producing L-α-amino acids by the aforesaid microorganisms, may be synthesized with ease according to the synthetic process of α-aminonitriles, i.e., the first stage reaction in the Strecker process [for example, J. Ferment. Technol., <u>49</u> 1011 (1971) or Chem. Lett., 687 (1987)], or to the process disclosed in Org. Syn. Col. Vols. I, p. 21 and III, p. 84.

0023On the other hand, the α-(N-alkylideneamino)nitrile compounds represented by the general formulae (III) and (IV), which are another substrate, may be obtained in such a way that as soon as the aldehyde used as a raw material is confirmed to disappear by the analysis using gas chromatography in the synthesis of α-aminonitrile compounds or in the first stage reaction in the Strecker process, inorganic salts are separated out by such a procedure as filtration or extraction and the resulting solution is concentrated. The crude products may in some cases contain, as impurities, α-aminonitriles, which are the partial hydrolysis products of α-(N-alkylideneamino)nitrile compounds represented by the foregoing general formulae (III) and (IV), and aldehydes used as a raw material for the synthesis. After these impurifies have been removed by such a procedure as fractional distillation, the resulting products are used as the substrate. However, the crude products containing the impurities may also be used as the substrate.

0024R and R' in the general formulae (I) (II) (III) and (IV) representing the α-aminonitrile compounds and derivatives thereof, i.e., α-(N-alkylideneamino)nitrile compounds, are individually an alkyl group, substituted alkyl group, phenyl group, substituted phenyl group, imidazolyl group, substituted imidazolyl group, indolyl group, substituted indolyl group, furyl group, substituted furyl group, pyridyl group, substituted pyridyl group, thiazolyl group or substituted thiazolyl group. No particular limitations are imposed on the substituents in the substituted alkyl group, substituted phenyl group, substituted imidazolyl group, substituted indolyl group, substituted furyl group, substituted pyridyl group and substituted thiazolyl group, among these Rs. However, as preferred substituents may be mentioned hydroxyl, methoxyl, mercapto, methylthio, amino, halogen, carboxyl, carboxamide, phenyl, hydroxyphenyl and guanyl by way of example.

0025Further, where the compounds of the general formulae (III) and (IV) are used as a substrate, use of the compound having the same groups for both R and R' as a raw material may lead to the formation of an L-α-amino acid, while use of the compound having different groups for R and R' as a raw material can result in the formation of a mixture of two L-α-amino acids.

0026It goes without saying that the aforesaid nitrile compounds and derivatives thereof used as the raw material may be used as a mixture of two or more of them without any inconvenience.

0027Illustrative examples of the above-described α-aminonitrile compound may include 2-aminopropanenitrile, 2-aminobutanenitrile, 2-amino-3-methylbutanenitrile, 2-amino-4-methylpentanenitrile, 2-amino-3-methylpentanenitrile, 2-amino-3-hydroxypropanenitrile, 2-amino-3-hydroxybutanenitrile, 2-amino-5-guanidinopentanenitrile, 2-amino-3-mercaptopropanenitrile, 2,7-diamino-4,5-dithiaoctanenitrile, 2-amino-4-methylthiobutanenitrile, 2-amino-4-methylthiopropanenitrile, 2-amino-3-phenylpropanenitrile, 3-(4-hydroxyphenyl)propanenitrile, 3-amino-3-cyanopropanoic acid, 4-amino-4-cyanobutanoic acid, 3-amino-3-cyanopropanamide, 4-amino-4-cyanobutanamide, 2,6-diaminohexanenitrile, 2,6-diamino-5-hydroxyhexanenitrile, 2-amino-3-(3-indolyl)propanenitrile, 2-amino-3-(4-imidazolyl)propanenitrile, 2-cyanopyrrolidine, 2-cyano-4-hydroxypyrrolidine and 2-amino-2-phenylethanenitrile.

0028On the other hand, as the α-(N-alkylideneamino)nitrile compounds may be mentioned the Schiff base compounds of the α-aminonitrile compounds as illustrated above with the aldehydes which are the raw material for the synthesis of the α-aminonitriles.

0029In the present invention, where the α-aminonitrile compound is used as a substrate, the substrate is brought into contact with a microorganism at a pH in the range of 8 - 12 or else it is brought into contact with a microorganism in the presence of an aldehyde. As the aldehyde used for this purpose, it is possible to use an aldehyde, which is the raw material in the synthesis of the aminonitrile compounds represented by the general formula (I) or (II) illustrated above. In this case, solely an L-α-amino acid is obtained, whereas use of a different aldehyde may result in the formation of a mixture of different L-α-amino acids.

0030In the present invention, in order to produce an L-α-amino acid by causing each of the above-described microorganisms, which have nitrile-hydrolyzing activities, to act on the aforementioned nitrile compound or derivative thereof used as a raw material (hereinafter abbreviated as "the raw nitrile"), it is advisable to use any of the following processes (a) to (c).

0031Specifically, it is possible to employ (a) a process comprising incubating a microorganism in a medium containing a nitrile compound such as propionitrile to obtain a cell mass and bringing the thus-proliferated cell mass into contact with the raw nitrile or the raw nitrile plus an aldehyde to react them with each other, (b) a process comprising cultivating microorganism in advance, bringing the thus-proliferated cell mass into contact with a nitrile compound such as propionitrile, and adding to the resulting cell mass the raw nitrile or the raw nitrile plus an aldehyde to react them with each other, or (c) a process comprising cultivating a microorganism in advance, and bringing the thus-proliferated cell mass into direct contact with the raw nitrile or the raw nitrile plus an aldehyde to react them with each other.

0032In these reaction processes, it is also feasible to use destructed products of proliferated cells, dried cells, enzyme preparations such as cell free extract and purified nitrile-hydrolyzing enzymes, or cells and enzyme preparations immobilized according to conventional procedure.

0033The nitrile compounds useful in the practice of the processes (a) and (b) may embrace acetonitrile, n-butyronitrile, n-capronitrile, methacrylonitrile, isobutyronitrile, glutaronitrile, triacrylonitrile, crotononitrile, lactonitrile, succinonitrile, acrylonitrile, benzonitrile, phenylacetonitrile, etc. as well as propionitrile.

0034In the foregoing process (a), the preculture of each of the above-described microorganisms is inoculated to a medium which has been added with carbon sources such as glucose, sucrose, molasses and starchhydrolyzate, acetic acid and other utilizable carbon sources in addition to the nitrile compound, and further with a nitrogen source such as ammonium chloride, ammonium sulfate, ammonium phosphate, ammonium nitrate, urea, aqueous ammonia, sodium nitrate, and amino acids and other utilizable organic nitrogen compounds, inorganic salts such as calcium phosphate, sodium phosphate, magnesium sulfate, manganese sulfate, iron (II) sulfate, iron (III) chloride, calcium chloride and manganese chloride, salts of boric acid, salts of copper, zinc, etc., what is called micronutrients, and optionally growth-promoting materials such as vitamins, yeast extracts and corn steep liquor. The preculture is incubated therein under aerobic conditions to cause the cells to proliferate. The raw nitrile or the raw nitrile plus an aldehyde is added to the thus-obtained cultured broth, a suspension of cells separated from the cell-culture or enzyme preparations to react them with each other.

0035When an α-aminonitrile compound is reacted in the absence of an aldehyde, the reaction is carried out at a pH in the range of 8 - 12, whereas otherwise it is effected at a pH in the range of 4 - 13, preferably 8 - 12, for 1 - 6 days in both cases. If an α-aminonitrile compound is reacted at a pH outside the range of 8 - 12 in the absence of an aldehyde, an α-amino acid rich in L-configuration is formed, which is however low in optical purity and hence has no practical use. In this reaction, a variety of buffer solutions may be used. Of these, ammoniacal buffer solutions are preferred, including, for example, a mixture of aqueous ammonia and aqueous ammonium chloride solution, a mixture of aqueous ammonia and aqueous ammonium sulfate solution, a mixture of aqueous ammonia and aqueous ammonium phosphate solution, and a mixture of aqueous ammonia and aqueous ammonium acetate solution. Further, as the alkali for use to control pH, aqueous ammonia is preferred.

0036The reaction temperature should preferably fall within the range of 20 - 70°C. Further, the above-described carbon source, nitrogen source and other components used to proliferate the cells may be added properly to the reaction system to maintain and enhance the cell concentration or the cell's ability of nitrile hydrolysis. In the feeding of the raw nitrile, any one of the following methods may be employed: a method of adding at the time of reaction initiation, a method of intermittent addition or a method of continuous addition.

0037In the foregoing process (b), the nitrile compound is not added during incubation and proliferation of the cell mass as in the foregoing process (a), but is added after the cell mass has been proliferated so that the ability of nitrile hydrolysis of the mass of the microorganism is activated. Then, the cell mass is reacted with the raw nitrile or the raw nitrile plus an aldehyde to produce an L-α-amino acid.

0038In the foregoing process (c), as soon as the cell mass has been proliferated as in the process (b), the raw nitrile or the raw nitrile plus an aldehyde is added thereto for effecting reaction to produce an L-α-amino acid.

0039In either of the processes (b) and (c), it is possible to employ the same incubation conditions, reaction conditions and separation and collection procedures of the produced L-α-amino acid as those used in the process (a).

0040As regards the amount of an aldehyde to be used where the reaction is conducted in the presence of the aldehyde, it is preferable to use 0.1 - 10 mols, particularly 0.5 - 3 mols of the aldehyde per mol of an α-aminonitrile compound.

0041The L-α-amino acids formed by the above-described reactions are isolated by the use of well-known procedures such as phase separation, filtration, extraction and column chromatography.

0042The L-α-amino acids obtained according to the present invention as described above can find their applications in a variety of fields including those of foods, feeds, pharmaceuticals and cosmetics.

0043As has been illustrated above, the present invention permits the direct and selective production of L-α-amino acids from racemic α-aminonitrile compounds or derivatives thereof by making use of microorganisms. Thus, the present invention is useful in the production of L-α-amino acids which are utilized in a variety of fields as described above.

0044The present invention will be illustrated more specifically by reference to the following examples. However, it should not be construed that the present invention is limited to or by the examples.

EXAMPLES:

Example 1

:

(1) Preparation of medium:

0045A medium (100 ml) of the following composition was placed in a 500 ml Sakaguchi flask, which was then sterilized at 120°C for 20 minutes in an autoclave. To the medium was added 1 ml of propionitrile having been sterilized by means of a 0.2-µ Millipore Filter. The medium for the preparation of cell mass was thus obtained.

Medium composition:

0046<tables id="tabl0004" num="0004"><table frame="all"><tgroup cols="2" colsep="1" rowsep="0"><colspec colnum="1" colname="col1" colwidth="78.75mm" /><colspec colnum="2" colname="col2" colwidth="78.75mm" /><tbody valign="top"><row><entry namest="col1" nameend="col1" align="left">glucose</entry><entry namest="col2" nameend="col2" align="char" char=".">10 g/l</entry></row><row><entry namest="col1" nameend="col1" align="left">yeast extract</entry><entry namest="col2" nameend="col2" align="char" char=".">0.1 g/l</entry></row><row><entry namest="col1" nameend="col1" align="left">Na₂HPO₄·12H₂O</entry><entry namest="col2" nameend="col2" align="char" char=".">2.5 g/l</entry></row><row><entry namest="col1" nameend="col1" align="left">KH₂PO₄</entry><entry namest="col2" nameend="col2" align="char" char=".">2.0 g/l</entry></row><row><entry namest="col1" nameend="col1" align="left">MgSO₄·7H₂O</entry><entry namest="col2" nameend="col2" align="char" char=".">0.5 g/l</entry></row><row><entry namest="col1" nameend="col1" align="left">FeSO₄·7H₂O</entry><entry namest="col2" nameend="col2" align="char" char=".">0.03 g/l</entry></row><row><entry namest="col1" nameend="col1" align="left">CaCl₂·2H₂O</entry><entry namest="col2" nameend="col2" align="char" char=".">0.06 g/l</entry></row><row rowsep="1"><entry namest="col1" nameend="col1" align="left">pH</entry><entry namest="col2" nameend="col2" align="char" char=".">7.2</entry></row></tbody></tgroup></table></tables>

Strains for use:

0047<tables id="tabl0005" num="0005"><table frame="all"><tgroup cols="2" colsep="1" rowsep="0"><colspec colnum="1" colname="col1" colwidth="78.75mm" /><colspec colnum="2" colname="col2" colwidth="78.75mm" /><thead valign="top"><row><entry namest="col1" nameend="col1" align="center">Name of strain</entry><entry namest="col2" nameend="col2" align="center">FERM-BP No.</entry></row></thead><tbody valign="top"><row><entry namest="col1" nameend="col1" align="left"><u>Nocardia</u> sp. PC-29</entry><entry namest="col2" nameend="col2" align="center">1561</entry></row><row><entry namest="col1" nameend="col1" align="left"><u>Nocardia</u> sp. PA-34</entry><entry namest="col2" nameend="col2" align="center">1559</entry></row><row><entry namest="col1" nameend="col1" align="left"><u>Nocardia</u> sp. AB-16</entry><entry namest="col2" nameend="col2" align="center">1555</entry></row><row><entry namest="col1" nameend="col1" align="left"><u>Nocardia</u> sp. BA-1</entry><entry namest="col2" nameend="col2" align="center">1557</entry></row><row><entry namest="col1" nameend="col1" align="left"><u>Mycobacterium</u> sp. AB-43</entry><entry namest="col2" nameend="col2" align="center">1556</entry></row><row><entry namest="col1" nameend="col1" align="left"><u>Corynbacterim</u> sp. PA-15</entry><entry namest="col2" nameend="col2" align="center">1558</entry></row><row rowsep="1"><entry namest="col1" nameend="col1" align="left"><u>Corynbacterium</u> SP- PC-3</entry><entry namest="col2" nameend="col2" align="center">1560</entry></row></tbody></tgroup></table></tables>

(2) Incubation and proliferation of cell mass:

0048To the medium prepared as described above were inoculated 3 loopfuls of each of the five strains given in Table 3. The resultant medium was incubated under oscillation at 140 rpm at a temperature of 30°C for 48 hours.

0049The cell mass obtained by the incubation was isolated by centrifugation, washed once with a 0.1M NH₄Cl-NH₃ buffer solution (pH 10), and thereafter resuspended in a 0.1M NH₄Cl-NH₃ buffer solution so as to adjust the optical density (OD) at 40.

(3) Reaction:

0050Into a test tube with an inner diameter of 24 mm was admitted 5 ml of each of the cell suspensions obtained as described above. Then, 50 mg of DL-2-amino-2-phenylethanenitrile were added thereto and the test tube was stopcocked. The test tube was shaked at 300 rpm at a temperature of 30°C for 48 hours.

0051After completion of the reaction, the reaction mixture was extracted twice with 5 ml of hexane, and the water phase thus-obtained was analyzed by thigh performance liquid chromatography (HPLC).

0052The quantity of L-α-phenylglycine thus-formed was determined by means of an amino acid analyzer (manufactured by Beckman Co.: Type 7300). Its absolute configuration and optical purity were determined by HPCL using CHIRALPAK WH (product of Daicel Chemical Industries, Ltd.) as a column packing. The results are as shown in Table 3.

0053<tables id="tabl0006" num="0006"><table frame="all"><title>Table 3</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="left">Strain</entry><entry namest="col2" nameend="col3" align="left">L-α-phenylglycine</entry></row><row><entry namest="col1" nameend="col1" /><entry namest="col2" nameend="col2" align="center">Amount (mg/ml)</entry><entry namest="col3" nameend="col3" align="center">Optical purity (% e.e.)</entry></row></thead><tbody valign="top"><row><entry namest="col1" nameend="col1" align="left"><u>Nocardia sp. PC-29</u></entry><entry namest="col2" nameend="col2" align="char" char=".">0.4</entry><entry namest="col3" nameend="col3" align="left">Not less than 99</entry></row><row><entry namest="col1" nameend="col1" align="left"><u>Nocardia sp. PA-34</u></entry><entry namest="col2" nameend="col2" align="char" char=".">0.2</entry><entry namest="col3" nameend="col3" align="left">ditto</entry></row><row><entry namest="col1" nameend="col1" align="left"><u>Nocardia sp. AB-16</u></entry><entry namest="col2" nameend="col2" align="char" char=".">0.1</entry><entry namest="col3" nameend="col3" align="left">ditto</entry></row><row><entry namest="col1" nameend="col1" align="left"><u>Nocardia sp. BA-1</u></entry><entry namest="col2" nameend="col2" align="char" char=".">0.1</entry><entry namest="col3" nameend="col3" align="left">ditto</entry></row><row rowsep="1"><entry namest="col1" nameend="col1" align="left"><u>Mycobacterium sp. AB-43</u></entry><entry namest="col2" nameend="col2" align="char" char=".">0.5</entry><entry namest="col3" nameend="col3" align="left">ditto</entry></row></tbody></tgroup></table></tables>

Example 2

:

0054In the same manner as in Example 1, the cell mass of <u>Nocardia</u> sp. PA-34 was prepared and collected, and then resuspended in a 0.1M NH₄Cl-NH₃ buffer solution so as to adjust the optical density (OD) at 40. Thereafter, 500 ml of the cell suspension thus-obtained was admitted in a 1.2-1 fermenter, to which 4.9 g (50 mmol) of DL-2-amino-3-methylbutanenitrile were added. While maintaining the pH at 10.0 ± 0.1 with 4N aqueous ammonia and 1N HCl, the resulting suspension was submitted to reaction at a temperature of 30°C and at 700 rpm agitation for 48 hours.

0055After completion of the reaction, the reaction mixture was centrifuged to obtain a supernatant, which was then filtered by means of a 0.45-µ Millipore Filter. The resulting water phase was analyzed by HPLC.

0056The accumulated concentration of L-valine was 0.5 mg/ml, while the optical purity thereof was 67% e.e. The quantity of L-valine was determined by HPLC using Asahipak Inertsil ODS (product of Asahi Chemical Industry Company, Ltd.) as a column packing. Its absolute configuration and optical purity were determined similarly by HPLC using CHIRALPAK WH (product of Daicel Chemical Industries, Ltd.)

Comparative Example 1

:

0057Reaction and analysis were conducted by using the same cell mass and in the same manner as described in Example 2 except that the pH was adjusted at 7.0 ± 0.1 with 4N aqueous ammonia and 1N HCl in the procedure described in Example 2. As a consequence, the accumulated concentration of L-valine was found to be 3.5 mg/ml, while its optical purity was 32% e.e.

Comparative Example 2

:

0058The cell mass of <u>Nocardia</u> sp. PA-34 was prepared in the same manner as described in Example 1. After the mass had been collected by centrifugation, it was washed twice with a 0.1M Na₂HPO₄-KH₂PO₄ buffer solution (pH 7.0) and then resuspended in a 0.1M Na₂HPO₄-KH₂PO₄ buffer solution so as to adjust the optical density (OD) at 40. The resulting cell suspension was subjected to reaction and analysis in the same manner as described in Comparative Example 1. The results revealed that L-valine had an accumulated concentration of 4.3 mg/ml and an optical purity of 17% e.e.

Example 3

:

0059The cell mass of Nocardia sp. PA-34 was prepared in the same manner as in Example 1, and was resuspended in a 0.1M NH₄Cl-NH₃ buffer solution so as to adjust the optical density (OD) at 40. Then, 500 ml of the resulting cell suspension was admitted in a 1.2-1 fermenter, to which 4.9 g (50 mmol) of DL-2-amino-3-methylbutanenitrile and 7.2 g (100 mmol) of isobutyraldehyde were added. They were reacted under stirring for 48 hours at a temperature of 30°C, at 700 rpm agitation, and at 0.2 vvm aeration, while maintaining the pH at 10 ± 0.1 with 4N aqueous ammonia and 1N HCl.

0060After completion of the reaction, the reaction mixture was analyzed in the same manner as described in Example 2. As a result, it was found that the accumulated concentration of L-valine was 1.3 mg/ml, while its optical purity was 96% e.e.

Example 4

:

0061Using each of the microorganisms given in Table 4, a cell suspension was prepared in the same manner as described in Example 1. Then, 5 ml of the cell suspension was admitted in a test tube having an inner diameter of 24 mm, to which 49 mg of DL-2-amino-3-methylbutanenitrile and 72 mg of isobutyraldehyde were added. The test tube was stopcocked and the contents were reacted for 48 hours at a temperature of 30°C under 300 rpm oscillation. The reaction mixture thus-obtained was treated and analyzed in the same manner as described in Example 2. The results are given in Table 4.

0062<tables id="tabl0007" num="0007"><table frame="all"><title>Table 4</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="left">Strain</entry><entry namest="col2" nameend="col3" align="left">L-valine</entry></row><row><entry namest="col1" nameend="col1" /><entry namest="col2" nameend="col2" align="center">Amount (mg/ml)</entry><entry namest="col3" nameend="col3" align="center">Optical purity (% e.e.)</entry></row></thead><tbody valign="top"><row><entry namest="col1" nameend="col1" align="left"><u>Nocardia</u> sp. PC-29</entry><entry namest="col2" nameend="col2" align="char" char=".">0.8</entry><entry namest="col3" nameend="col3" align="center">100</entry></row><row><entry namest="col1" nameend="col1" align="left"><u>Nocardia</u> sp. AB-18</entry><entry namest="col2" nameend="col2" align="char" char=".">0.5</entry><entry namest="col3" nameend="col3" align="center">96</entry></row><row><entry namest="col1" nameend="col1" align="left"><u>Nocardia</u> sp. BA-1</entry><entry namest="col2" nameend="col2" align="char" char=".">0.4</entry><entry namest="col3" nameend="col3" align="center">100</entry></row><row rowsep="1"><entry namest="col1" nameend="col1" align="left"><u>Mycobacterium</u> sp. AB-43</entry><entry namest="col2" nameend="col2" align="char" char=".">0.6</entry><entry namest="col3" nameend="col3" align="center">97</entry></row></tbody></tgroup></table></tables>

Example 5

:

0063Using each of the microorganisms given in Table 5, a cell suspension was prepared in the same manner as described in Example 1. Reaction was carried out according to the procedure described in Example 4 except for the addition of 50 µl of DL-2-aminopentanenitrile in place of DL-2-amino-3-methylbutanenitrile and 60 µl of n-butyraldehyde to 5 ml of the cell suspension. The L-norvaline thus-formed was analyzed with HPLC using Asahipak Inertsil ODS and CHIRALPAK WE as column packings. The results are shown in Table 5.

0064<tables id="tabl0008" num="0008"><table frame="all"><title>Table 5</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" /><entry namest="col2" nameend="col3" align="center">L-norvaline</entry></row><row><entry namest="col1" nameend="col1" align="center">Strain</entry><entry namest="col2" nameend="col2" align="center">Amount (mg/ml)</entry><entry namest="col3" nameend="col3" align="center">Optical purity (% e.e.)</entry></row></thead><tbody valign="top"><row><entry namest="col1" nameend="col1" align="left"><u>Mycobacterium</u> sp. AB-43</entry><entry namest="col2" nameend="col2" align="char" char=".">3.4</entry><entry namest="col3" nameend="col3" align="center">92</entry></row><row rowsep="1"><entry namest="col1" nameend="col1" align="left"><u>Nocardia</u> sp. PA-34</entry><entry namest="col2" nameend="col2" align="char" char=".">3.5</entry><entry namest="col3" nameend="col3" align="center">98</entry></row></tbody></tgroup></table></tables>

Example 6

:

0065Using each of the microorganisms given in Table 6, a cell suspension was prepared in the same manner as described in Example 1. Reaction was carried out following the procedure described in Example 4 except for the addition of 50 µl of DL-4-methyl-2-aminopentane nitrile and 60 µl of 3-methylbutyraldehyde to 5 ml of the cell suspension. The L-leucine thus-formed was analyzed as in the same manner as described in Example 5. The results are shown in Table 6.

0066<tables id="tabl0009" num="0009"><table frame="all"><title>Table 6</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" /><entry namest="col2" nameend="col3" align="center">L-leucine</entry></row><row><entry namest="col1" nameend="col1" align="center">Strain</entry><entry namest="col2" nameend="col2" align="center">Amount (mg/ml)</entry><entry namest="col3" nameend="col3" align="center">Optical purity (% e.e.)</entry></row></thead><tbody valign="top"><row><entry namest="col1" nameend="col1" align="left"><u>Mycobacterium</u> sp. AB-43</entry><entry namest="col2" nameend="col2" align="char" char=".">1.4</entry><entry namest="col3" nameend="col3" align="center">97</entry></row><row rowsep="1"><entry namest="col1" nameend="col1" align="left"><u>Nocardia</u> sp. PA-34</entry><entry namest="col2" nameend="col2" align="char" char=".">1.6</entry><entry namest="col3" nameend="col3" align="center">95</entry></row></tbody></tgroup></table></tables>

Example 7

:

0067Using each of the microorganisms given in Table 7, a cell suspension was prepared in the same manner as described in Example 1. Reaction was carried out following the procedure described in Example 4 except for the addition of 50 µl of DL-2-aminohexanenitrile and 60 µl of n-valeraldehyde. The L-norleucine thus-formed was analyzed as described in Example 5. The results are shown in Table 7.

0068<tables id="tabl0010" num="0010"><table frame="all"><title>Table 7</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" /><entry namest="col2" nameend="col3" align="center">L-norleucine</entry></row><row><entry namest="col1" nameend="col1" align="center">Strain</entry><entry namest="col2" nameend="col2" align="center">Amount (mg/ml)</entry><entry namest="col3" nameend="col3" align="center">Optical purity (% e.e.)</entry></row></thead><tbody valign="top"><row><entry namest="col1" nameend="col1" align="left"><u>Mycobacterium</u> sp. AB-43</entry><entry namest="col2" nameend="col2" align="char" char=".">1.1</entry><entry namest="col3" nameend="col3" align="center">100</entry></row><row rowsep="1"><entry namest="col1" nameend="col1" align="left"><u>Nocardia</u> sp. PA-34</entry><entry namest="col2" nameend="col2" align="char" char=".">1.3</entry><entry namest="col3" nameend="col3" align="center">100</entry></row></tbody></tgroup></table></tables>

Example 8

:

0069Three loopfuls of <u>Corynebacterium</u> sp. PA-15 strain were inoculated to 100 ml of an NBG medium in a 500-ml Sakaguchi flask [the NBG medium is a liquid medium prepared by adding deionized water to 10 g of "Lab-Lemco" powder (code L29: product of Oxoid Co., Ltd.), 10 g of Bacteriological peptone (code L37), 10 g of glucose and 5 g of sodium chloride to make the total volume 1,000 ml, adjusting the pH of the resulting liquid at 7.5 with 1N aqueous sodium hydroxide solution, and then sterilizing the liquid with heat at 120°C for 15 minutes in an autoclave]. The cells were cultured under oscillation (150 times/minutes) at 30°C for 48 hours.

0070The cell mass formed by the incubation was collected and washed in the same manner as described in Example 1, and resuspended in a 0.1M NH₄Cl-NH₃ buffer solution so as to adjust the optical density (OD) at 40.

0071The cell suspension was reacted with DL-2-amino-3-methylbutanenitrile in the presence of isobutyraldehyde in the same manner as described in Example 4. The accumulated concentration of L-valine was 0.3 mg/ml and its optical purity was 95% e.e.

Example 9

:

0072Reaction was carried out in the same manner as described in Example 4 except that 50 µl of DL-2-aminopentanenitrile and 60 µl of n-butyraldehyde were added to 5 ml of each of the cell suspensions prepared according to the procedure given in Example 8. The L-norvaline thus-formed was analyzed as described in Example 5. The results are shown in Table 8.

0073<tables id="tabl0011" num="0011"><table frame="all"><title>Table 8</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" /><entry namest="col2" nameend="col3" align="center">L-norvaline</entry></row><row><entry namest="col1" nameend="col1" align="center">Strain</entry><entry namest="col2" nameend="col2" align="center">Amount (mg/ml)</entry><entry namest="col3" nameend="col3" align="center">Optical purity (% e.e.)</entry></row></thead><tbody valign="top"><row><entry namest="col1" nameend="col1" align="left"><u>Nocardia</u> sp. BA-1</entry><entry namest="col2" nameend="col2" align="char" char=".">2.7</entry><entry namest="col3" nameend="col3" align="center">100</entry></row><row><entry namest="col1" nameend="col1" align="left"><u>Corynebacterium</u> sp. PA-15</entry><entry namest="col2" nameend="col2" align="char" char=".">2.7</entry><entry namest="col3" nameend="col3" align="center">84</entry></row><row rowsep="1"><entry namest="col1" nameend="col1" align="left"><u>Corynebacterium</u> sp. PC-3</entry><entry namest="col2" nameend="col2" align="char" char=".">2.8</entry><entry namest="col3" nameend="col3" align="center">82</entry></row></tbody></tgroup></table></tables>

Example 10

:

0074Reaction was carried out in the same manner as described in Example 4 except that 50 µl of DL-4-methyl-2-aminopentanenitrile and 60 µl of 3-methylbutyraldehyde were added to 5 ml of each of the cell suspensions prepared according to the procedure given in Example 8. The L-leucine thus-formed was analyzed as described in Example 5. The results are shown in Table 9.

0075<tables id="tabl0012" num="0012"><table frame="all"><title>Table 9</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" /><entry namest="col2" nameend="col3" align="center">L-leucine</entry></row><row><entry namest="col1" nameend="col1" align="center">Strain</entry><entry namest="col2" nameend="col2" align="center">Amount (mg/ml)</entry><entry namest="col3" nameend="col3" align="center">Optical purity (% e.e.)</entry></row></thead><tbody valign="top"><row><entry namest="col1" nameend="col1" align="left"><u>Nocardia</u> sp. BA-1</entry><entry namest="col2" nameend="col2" align="char" char=".">0.6</entry><entry namest="col3" nameend="col3" align="center">85</entry></row><row><entry namest="col1" nameend="col1" align="left"><u>Corynebacterium</u> sp. PA-15</entry><entry namest="col2" nameend="col2" align="char" char=".">1.2</entry><entry namest="col3" nameend="col3" align="center">94</entry></row><row rowsep="1"><entry namest="col1" nameend="col1" align="left"><u>Corynebacterium</u> sp. PC-3</entry><entry namest="col2" nameend="col2" align="char" char=".">1.1</entry><entry namest="col3" nameend="col3" align="center">91</entry></row></tbody></tgroup></table></tables>

Example 11

:

0076Reaction was carried out in the same manner as described in Example 3 except that 50 µl of DL-2-aminohexanenitrile and 60 µl of n-valeraldehyde were added to 5 ml of each of the cell suspensions prepared according to the procedure given in Example 8. The L-norleucine thus-formed was analyzed as discribed in Example 5. The results are shown in Table 10.

0077<tables id="tabl0013" num="0013"><table frame="all"><title>Table 10</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" /><entry namest="col2" nameend="col3" align="center">L-leucine</entry></row><row><entry namest="col1" nameend="col1" align="center">Strain</entry><entry namest="col2" nameend="col2" align="center">Amount (mg/ml)</entry><entry namest="col3" nameend="col3" align="center">Optical purity (% e.e.)</entry></row></thead><tbody valign="top"><row><entry namest="col1" nameend="col1" align="left"><u>Corynebacterium</u> sp. PA-15</entry><entry namest="col2" nameend="col2" align="char" char=".">0.7</entry><entry namest="col3" nameend="col3" align="center">100</entry></row><row rowsep="1"><entry namest="col1" nameend="col1" align="left"><u>Corynebacterium</u> sp. PC-3</entry><entry namest="col2" nameend="col2" align="char" char=".">0.7</entry><entry namest="col3" nameend="col3" align="center">100</entry></row></tbody></tgroup></table></tables>

Example 12

:

0078Cell suspensions of the strains given in Table 11 were prepared according to the procedure described in Example 1. In a test tube with an inner diameter of 24 mm were admitted 5 ml of each cell suspension, to which 100 µl of DL-3-methyl-2-(N-2-methylpropylideneamino)butanenitrile were added. The test tube was stopcocked, and incubated at 30°C under oscillation at 300 rpm for 48 hours.

0079After completion of the reaction, the resulting reaction mixture was extracted twice with 5 ml of hexane. The remaining water phase was analyzed with HPLC as discribed in Example 2. The results are as shown in Table 11.

0080<tables id="tabl0014" num="0014"><table frame="all"><title>Table 11</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" /><entry namest="col2" nameend="col3" align="center">L-valine</entry></row><row><entry namest="col1" nameend="col1" align="center">Strain</entry><entry namest="col2" nameend="col2" align="center">Amount (mg/ml)</entry><entry namest="col3" nameend="col3" align="center">Optical purity (% e.e.)</entry></row></thead><tbody valign="top"><row><entry namest="col1" nameend="col1" align="left"><u>Nocardia</u> sp. PC-29</entry><entry namest="col2" nameend="col2" align="char" char=".">2.3</entry><entry namest="col3" nameend="col3" align="center">96</entry></row><row><entry namest="col1" nameend="col1" align="left"><u>Nocardia</u> sp. PA-34</entry><entry namest="col2" nameend="col2" align="char" char=".">0.6</entry><entry namest="col3" nameend="col3" align="center">98</entry></row><row><entry namest="col1" nameend="col1" align="left"><u>Nocardia</u>sp. AB-16</entry><entry namest="col2" nameend="col2" align="char" char=".">1.3</entry><entry namest="col3" nameend="col3" align="center">92</entry></row><row><entry namest="col1" nameend="col1" align="left"><u>Nocardia</u> sp. BA-1</entry><entry namest="col2" nameend="col2" align="char" char=".">0.9</entry><entry namest="col3" nameend="col3" align="center">95</entry></row><row><entry namest="col1" nameend="col1" align="left"><u>Mycobacterium</u> sp. AB-43</entry><entry namest="col2" nameend="col2" align="char" char=".">3.0</entry><entry namest="col3" nameend="col3" align="center">97</entry></row><row><entry namest="col1" nameend="col1" align="left"><u>Corynebacterium</u> sp. PA-15</entry><entry namest="col2" nameend="col2" align="char" char=".">2.5</entry><entry namest="col3" nameend="col3" align="center">100</entry></row><row rowsep="1"><entry namest="col1" nameend="col1" align="left"><u>Corynebacterium</u> sp. PC-3</entry><entry namest="col2" nameend="col2" align="char" char=".">1.6</entry><entry namest="col3" nameend="col3" align="center">100</entry></row></tbody></tgroup></table></tables>

Example 13

:

0081The procedure described in Example 12 was followed except for the addition of 50 mg of DL-4-methyl-2-(N-3-methylbutylideneamino)pentanenitrile to 5 ml of the cell suspension of each strain given in Table 12; the cell suspension was prepared according to the procedure described in Example 1. The produced L-leucine was analyzed as described in Example 5. The results are shown in Table 12.

0082<tables id="tabl0015" num="0015"><table frame="all"><title>Table 12</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" /><entry namest="col2" nameend="col3" align="center">L-leucine</entry></row><row><entry namest="col1" nameend="col1" align="center">Strain</entry><entry namest="col2" nameend="col2" align="center">Amount (mg/ml)</entry><entry namest="col3" nameend="col3" align="center">Optical purity (% e.e.)</entry></row></thead><tbody valign="top"><row><entry namest="col1" nameend="col1" align="left"><u>Nocardia</u> sp. PA-34</entry><entry namest="col2" nameend="col2" align="char" char=".">2.5</entry><entry namest="col3" nameend="col3" align="center">93</entry></row><row rowsep="1"><entry namest="col1" nameend="col1" align="left"><u>Mycobacterium</u> sp. AB-43</entry><entry namest="col2" nameend="col2" align="char" char=".">0.6</entry><entry namest="col3" nameend="col3" align="center">83</entry></row></tbody></tgroup></table></tables>

Example 14

:

0083The procedure described in Example 13 was followed except for the addition of 50 µl of DL-2-(N-butylideneamino)pentanenitrile to 5 ml of the cell suspension of each strain given in Table 13, the cell suspension being prepared according to the procedure described in Example 1. The results are shown in Table 13.

0084<tables id="tabl0016" num="0016"><table frame="all"><title>Table 13</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" /><entry namest="col2" nameend="col3" align="center">L-norvaline</entry></row><row><entry namest="col1" nameend="col1" align="center">Strain</entry><entry namest="col2" nameend="col2" align="center">Amount (mg/ml)</entry><entry namest="col3" nameend="col3" align="center">Optical purity (% e.e.)</entry></row></thead><tbody valign="top"><row><entry namest="col1" nameend="col1" align="left"><u>Nocardia</u> sp. AB-16</entry><entry namest="col2" nameend="col2" align="char" char=".">2.6</entry><entry namest="col3" nameend="col3" align="center">90</entry></row><row><entry namest="col1" nameend="col1" align="left"><u>Mycobacterium</u> sp. AB-43</entry><entry namest="col2" nameend="col2" align="char" char=".">2.4</entry><entry namest="col3" nameend="col3" align="center">66</entry></row><row><entry namest="col1" nameend="col1" align="left"><u>Nocardia</u> sp. PA-34</entry><entry namest="col2" nameend="col2" align="char" char=".">2.1</entry><entry namest="col3" nameend="col3" align="center">90</entry></row><row rowsep="1"><entry namest="col1" nameend="col1" align="left"><u>Nocardia</u> sp. PC-29</entry><entry namest="col2" nameend="col2" align="char" char=".">1.5</entry><entry namest="col3" nameend="col3" align="center">60</entry></row></tbody></tgroup></table></tables>

Example 15

:

0085The procedure described in Example 13 was followed except for the addition of 50 µl of DL-2-(N-pentylideneamino)hexanenitrile to 5 ml of the cell suspension of each strain given in Table 14, the cell suspension being prepared according to the procedure described in Example 1. The results are shown in Table 14.

0086<tables id="tabl0017" num="0017"><table frame="all"><title>Table 14</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" /><entry namest="col2" nameend="col3" align="center">L-norleucine</entry></row><row><entry namest="col1" nameend="col1" align="center">Strain</entry><entry namest="col2" nameend="col2" align="center">Amount (mg/ml)</entry><entry namest="col3" nameend="col3" align="center">Optical purity (% e.e.)</entry></row></thead><tbody valign="top"><row><entry namest="col1" nameend="col1" align="left"><u>Nocardia</u> sp. AB-16</entry><entry namest="col2" nameend="col2" align="char" char=".">1.3</entry><entry namest="col3" nameend="col3" align="center">98</entry></row><row><entry namest="col1" nameend="col1" align="left"><u>Mycobacterium</u> sp. AB-43</entry><entry namest="col2" nameend="col2" align="char" char=".">0.8</entry><entry namest="col3" nameend="col3" align="center">86</entry></row><row><entry namest="col1" nameend="col1" align="left"><u>Nocardia</u> sp. PA-34</entry><entry namest="col2" nameend="col2" align="char" char=".">1.1</entry><entry namest="col3" nameend="col3" align="center">96</entry></row><row rowsep="1"><entry namest="col1" nameend="col1" align="left"><u>Nocardia</u> sp. PC-29</entry><entry namest="col2" nameend="col2" align="char" char=".">0.5</entry><entry namest="col3" nameend="col3" align="center">51</entry></row></tbody></tgroup></table></tables>

Example 16

:

0087A cell suspension of <u>Nocardia</u> sp. PA-34 was prepared according to the procedure described in Example 1 except that each of the nitrile compounds given in Table 15 was used in place of propionitrile. To 5 ml of the cell suspension were added 50 µl of DL-3-methyl-2-(N-2-methylpropylideneamino)butanenitrile. Reaction and analysis were conducted following the procedure described in Example 12. The results are given in Table 15.

0088<tables id="tabl0018" num="0018"><table frame="all"><title>Table 15</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" /><entry namest="col2" nameend="col3" align="center">L-valine</entry></row><row><entry namest="col1" nameend="col1" align="center">Nitrile compound</entry><entry namest="col2" nameend="col2" align="center">Amount (mg/ml)</entry><entry namest="col3" nameend="col3" align="center">Optical purity (% e.e.)</entry></row></thead><tbody valign="top"><row><entry namest="col1" nameend="col1" align="left">isobutyronitrile</entry><entry namest="col2" nameend="col2" align="char" char=".">0.8</entry><entry namest="col3" nameend="col3" align="center">94</entry></row><row><entry namest="col1" nameend="col1" align="left">glutaronitrile</entry><entry namest="col2" nameend="col2" align="char" char=".">0.9</entry><entry namest="col3" nameend="col3" align="center">100</entry></row><row><entry namest="col1" nameend="col1" align="left">adiponitrile</entry><entry namest="col2" nameend="col2" align="char" char=".">0.1</entry><entry namest="col3" nameend="col3" align="center">100</entry></row><row><entry namest="col1" nameend="col1" align="left">n-butyronitile</entry><entry namest="col2" nameend="col2" align="char" char=".">0.6</entry><entry namest="col3" nameend="col3" align="center">85</entry></row><row><entry namest="col1" nameend="col1" align="left">methacrylonitrile</entry><entry namest="col2" nameend="col2" align="char" char=".">0.3</entry><entry namest="col3" nameend="col3" align="center">100</entry></row><row rowsep="1"><entry namest="col1" nameend="col1" align="left">crotononitrile</entry><entry namest="col2" nameend="col2" align="char" char=".">0.6</entry><entry namest="col3" nameend="col3" align="center">74</entry></row></tbody></tgroup></table></tables>

Example 17

:

0089Three loopfuls of each of the microorganisms given in Table 16 were inoculated to 100 ml of an NBG medium in a 500-ml flask. The flask was oscillated (150 times/minute) at 30°C for 48 hours to incubate the cells. The cell mass grown by the incubation was collected and washed in the same manner as described in Example 1 and resuspended in a 0.1M NH₄Cl-NH₃ buffer solution so as to adjust the optical density (OD) at 40. The cell suspension was reacted with 50 µl of DL-2-(N-butylideneamino)pentanenitrile in the same manner as described in Example 13. The results are shown in Table 16.

0090<tables id="tabl0019" num="0019"><table frame="all"><title>Table 16</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" /><entry namest="col2" nameend="col3" align="center">L-norvaline</entry></row><row><entry namest="col1" nameend="col1" align="center">Strain</entry><entry namest="col2" nameend="col2" align="center">Amount (mg/ml)</entry><entry namest="col3" nameend="col3" align="center">Optical purity (% e.e.)</entry></row></thead><tbody valign="top"><row><entry namest="col1" nameend="col1" align="left"><u>Nocardia</u> sp. BA-1</entry><entry namest="col2" nameend="col2" align="char" char=".">0.1</entry><entry namest="col3" nameend="col3" align="center">80</entry></row><row><entry namest="col1" nameend="col1" align="left"><u>Corynebacterium</u> sp. PA-15</entry><entry namest="col2" nameend="col2" align="char" char=".">1.8</entry><entry namest="col3" nameend="col3" align="center">94</entry></row><row rowsep="1"><entry namest="col1" nameend="col1" align="left"><u>Corynebacterium</u> sp. PC-3</entry><entry namest="col2" nameend="col2" align="char" char=".">1.5</entry><entry namest="col3" nameend="col3" align="center">91</entry></row></tbody></tgroup></table></tables>

Example 18

:

0091Reaction was conducted in the same manner as described in Example 17 except for the addition of 50 µl of DL-2-(N-pentylideneamino)hexanenitrile to 5 ml of the cell suspension of each strain given in Table 17; the cell suspension was prepared according to the procedure described in Example 17. The results are shown in Table 17.

0092<tables id="tabl0020" num="0020"><table frame="all"><title>Table 17</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" /><entry namest="col2" nameend="col3" align="center">L-norleucine</entry></row><row><entry namest="col1" nameend="col1" align="center">Strain</entry><entry namest="col2" nameend="col2" align="center">Amount (mg/ml)</entry><entry namest="col3" nameend="col3" align="center">Optical purity (% e.e.)</entry></row></thead><tbody valign="top"><row><entry namest="col1" nameend="col1" align="left"><u>Corynebacterium</u> sp. PA-15</entry><entry namest="col2" nameend="col2" align="char" char=".">0.7</entry><entry namest="col3" nameend="col3" align="center">84</entry></row><row rowsep="1"><entry namest="col1" nameend="col1" align="left"><u>Corynebacterium</u> sp. PC-3</entry><entry namest="col2" nameend="col2" align="char" char=".">0.5</entry><entry namest="col3" nameend="col3" align="center">60</entry></row></tbody></tgroup></table></tables>

Example 19

:

0093Reaction was conducted in the same manner as described in Example 17 except for the addition of 50 µl of DL-4-methyl-2-(N-3-methylbutylideneamino)pentenenitrile to 5 ml of the cell suspension of each strain given in Table 18; the cell suspension was prepared according to the procedure described in Example 17. The results are shown in Table 18.

0094<tables id="tabl0021" num="0021"><table frame="all"><title>Table 18</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" /><entry namest="col2" nameend="col3" align="center">L-leucine</entry></row><row><entry namest="col1" nameend="col1" align="center">Strain</entry><entry namest="col2" nameend="col2" align="center">Amount (mg/ml)</entry><entry namest="col3" nameend="col3" align="center">Optical purity (% e.e.)</entry></row></thead><tbody valign="top"><row><entry namest="col1" nameend="col1" align="left"><u>Corynebacterium</u> sp. PA-15</entry><entry namest="col2" nameend="col2" align="char" char=".">0.6</entry><entry namest="col3" nameend="col3" align="center">100</entry></row><row rowsep="1"><entry namest="col1" nameend="col1" align="left"><u>Corynebacterium</u> sp. PC-3</entry><entry namest="col2" nameend="col2" align="char" char=".">0.5</entry><entry namest="col3" nameend="col3" align="center">100</entry></row></tbody></tgroup></table></tables>