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With Cupriavidus sp. KNK-J915 strain (FERM BP-10739); at 30℃; for 35h;pH 7;Aqueous phosphate buffer; Enzymatic reaction;Product distribution / selectivity;
Example 3 Selective Hydrolysis of R-Enantiomer in Racemic <strong>[4138-26-5]Nipecotamide</strong>Cupriavidus sp. KNK-J915 strain (FERM BP-10739) was cultivated by the method of Example 1 to prepare a bacterial cell suspension. The bacterial cell suspension (0.1 ml) was mixed with 100 mM phosphate buffer containing 1.0 to 5.0percent of racemic <strong>[4138-26-5]nipecotamide</strong> (0.1 ml, pH 7.0), and the mixture was shaken at 30° C. for 35 hours. After completion of the reaction, solid substance such as bacterial cells was removed by centrifugal separation, and then a substrate and a product in the reaction mixture were reacted with benzyl chlorocarbonate for derivatization. The resulting derivative was analyzed by high performance liquid chromatography to determine the conversion rate (percent) and optical purity (ee percent). The results are listed in Table 3.High Performance Liquid Chromatographic Analysis ConditionsAnalysis of Conversion RateColumn: YMC-A303 (4.6 mm.x.250 mm, manufactured by YMC Inc., Eluent: 20 mM phosphoric acid aqueous solution (pH 2.5)/acetonitrile=7/3, Flow rate: 1.0 ml/minute, Column temperature: 35° C., Measuring wavelength: 210 nmAnalysis of Optical PurityColumn: CHIRALPAK AD-RH (4.6 mm.x.150 mm, manufactured by Daicel Chemical Industries, Ltd.), Eluent: 20 mM phosphate buffer (pH 2.5)/acetonitrile=7/3, Flow rate: 0.5 ml/minute, Column temperature: room temperature, Measuring wavelength: 210 nm
With sodium hydroxide; at 30℃; for 60h;pH 7;Aqueous phosphate buffer; Enzymatic reaction;
Example 4Selective Hydrolysis of S-enantiomer in Racemic <strong>[4138-26-5]Nipecotamide</strong>Cupriavidus sp. KNK-J915 strain (FERM BP-10739) was cultivated by the method of Example 2 to obtain a 20-fold concentrated bacterial cell suspension. To the bacterial cell suspension (100 ml), racemic <strong>[4138-26-5]nipecotamide</strong> (19.9 g) was added. After the pH of the mixture was adjusted to 7 using NaOH, the mixture was shaken at 30° C. for 60 hours. After completion of the reaction, the reaction mixture was heat-treated at 70° C. for 30 minutes, and solid substance such as bacterial cells was removed by centrifugal separation. The substrate and product in the reaction mixture were analyzed by the method of Example 3; as a result, the conversion rate was 52.7percent, the optical purity of (R)-<strong>[4138-26-5]nipecotamide</strong> was 98.8percent ee and the optical purity of (S)-<strong>[4138-26-5]nipecotamide</strong> was 88.1percent ee.
With amidase from Cupriavidus sp. KNK-J915 (FERM BP-10739); water; In aq. phosphate buffer; at 30℃;pH 7;Enzymatic reaction;
General procedure: During the purification and characterization of amidase from Cupriavidus sp. KNK-J915, an enzyme assay was performed with (R,S)-BNPD as a substrate. The standard reaction mixture (0.2 mL) contained 100 mM potassium phosphate buffer (pH 7.0), 45.8 mM BNPD, and an appropriate amount of the enzyme. After the reaction was performed at 30°C for 0.5?1 h, the amount of BNPA was determined using HPLC. One unit of the enzyme was defined as the amount catalyzing the formation of 1 mol of BNPA per minute under the aforementioned condition. Protein content was determined by the Bradford method [11] with BSA as a standard using a kit from Bio-Rad Laboratories Ltd. (Tokyo, Japan).
With amidase from Cupriavidus necator JMP134; water; In aq. phosphate buffer; at 30℃;pH 7;Enzymatic reaction;
General procedure: During the purification and characterization of amidase from Cupriavidus sp. KNK-J915, an enzyme assay was performed with (R,S)-BNPD as a substrate. The standard reaction mixture (0.2 mL) contained 100 mM potassium phosphate buffer (pH 7.0), 45.8 mM BNPD, and an appropriate amount of the enzyme. After the reaction was performed at 30°C for 0.5?1 h, the amount of BNPA was determined using HPLC. One unit of the enzyme was defined as the amount catalyzing the formation of 1 mol of BNPA per minute under the aforementioned condition. Protein content was determined by the Bradford method [11] with BSA as a standard using a kit from Bio-Rad Laboratories Ltd. (Tokyo, Japan).
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