Method for producing amide compounds

EXAMPLE 1

Method for Determining Enzymatic Activity

[0092]A standard method for determining the nitrile hydratase activity used in the following Examples was as follows. A standard composition of the reaction solution for the enzyme reaction is shown in Tables 1 and 2. The enzyme reaction is initiated by adding 3-cyanopyridine or 2-hydroxy-4-methylthiobutyronitrile (HMBN) as the substrate compound. In the case of 3-cyanopyridine, incubation was continued at 20° C. for 10 minutes; whereas it was conducted at 20° C. for 15 minutes for HMBN. When 3-cyanopyridine was used in the reaction, 0.1 ml of 2 N hydrochloric acid was added to the reaction and the mixture was shaken vigorously to stop the reaction; when HMBN was used in the reaction, 0.1 ml of the reaction solution was added to 0.9 ml of 0.1% (v / v) phosphoric acid and the mixture was shaken vigorously to stop the reaction. The reaction solution was analyzed by HPLC.

[0093]TABLE 110% (v / v) HMBN in 0.1 M KPB (pH 6.5)0.36 ml0.1 M KPB (

EXAMPLE 2

Culture Conditions

[0099]5 ml pre-culture medium of the following composition was aliquoted into each test tube (25×200 mm); a silicone plug was placed in the tube, followed by sterilization by autoclaving. After the tube was cooled, a bacterial strain was inoculated with a platinum loop, and then cultured with shaking at 28° C. for two days.

[0100]Pre-Culture Medium (pH 7.0):

[0101]Polypeptone5.0 gMeat extract5.0 gNaCl2.0 gYeast extract0.5 g, andDistilled water1.0 L.

[0102]Then, the pre-culture was transferred into 20-ml main-culture medium autoclaved in a 500-ml Sakaguchi flask. In the main culture, 0.75% (v / v) acetonitrile was added after 24-hour culture, and then the incubation was continued with shaking at 33° C. for two days.

[0103]Main-Culture Medium (pH 7.0):

[0104]Acetamide 7.5 gGlucose10.0 gC.S.L.10.0 gYeast extract 1.0 gMgSO47H2O 0.5 gK2HPO4 1.0 gCoCl26H2O20.0 mg, andDistilled water 1.0 L.

EXAMPLE 3

Isolation and Identification of Microorganisms

[0105]XL-1 strain was selected as a strain with particularly high ability of decomposing nitrites from nitrile-decomposing bacteria obtained from soil collected in the campus of Gifu University by enrichment culture with a medium containing various nitrites. Since this strain had the following microbiological characteristics, according to “Bergey's Manual of Determinative Bacteriology (ninth edition, 1994)”, it was identified as Rhodococcus equi, which is a strain belonging to the genus Rhodococcus.

1. Morphological Characteristics

[0106](1) Cellular polymorphism: life cycle comprising bacillar and coccal forms[0107](2) Gram staining: +[0108](3) Spore formation: −[0109](4) Motility: −[0110](5) Colony appearance: round-shaped, smooth-edged, less convex, glossy, pink.

2. Characteristics Involved in Culture[0111](1) Broth liquid culture: suspension[0112](2) Litmus milk: unchanged.

3. Physiological Characteristics[0113](1) Denitrification