CC1(C)S[C@@H]2[C@H](NC([C@H:13]([NH2:20])[C:14]3[CH:15]=[CH:16][CH:17]=[CH:18][CH:19]=3)=O)C(=O)N2[C@H]1C(O)=O.CC(S[C@@H]1[O:34][C@H](CO)[C@H](O)[C@H](O)[C@H]1O)C.P([O-])([O-])([O-])=O.[K+].[K+].[K+].Cl>P([O-])([O-])([O-])=O.C(#N)C1C=CC=CC=1>[C:13]([NH2:20])(=[O:34])[C:14]1[CH:15]=[CH:16][CH:17]=[CH:18][CH:19]=1
TG1/pNHJ10H and TG1/pNEJ20L were inoculated into 10 ml of 2×YT medium containing 50 μg/ml of ampicillin and incubated at 30° C. overnight (12 hours). 1 ml of the resultant culture was added to 100 ml of 2×YT medium (50 μg/ml of ampicillin, 0.1 g of CoCl2.6H2O/l). The mixture was incubated at 30° C. for 4 hours. IPTG was added to the mixture to a final concentration of 1 mM. The mixture was incubated at 30° C. for 10 hours. After harvesting the cells, the cells were suspended in 5 ml of 0.1M phosphate buffer (pH 7.5). The suspensions were disrupted by sonification for 5 min and centrifuged at 12,000×g for 30 min. The resulting supernatants were used for the enzyme assay. The enzyme assay was carried out in a reaction mixture (12 ml) containing 50 mM potassium phosphate buffer (pH 7.5), 6 mM benzonitrile and an appropriate amount of the enzyme. The reaction was carried out at 20° C. for 30 min and stopped by the addition of 0.2 ml 1M HCl. The amount of benzamide formed in the reaction mixture was determined by HPLC. As a control, the mixture obtained by the same procedure as described above but from E. coli TG1 was used. The levels of nitrile hydratase activity in cell-free extracts of E. coli containing pNHJ10H and pNHJ20L were 1.75×10-3 and 6.99×10-3 units/mg, respectively, when cultured in 2×YT medium in the presence of CoCl2 and IPTG. Benzamide was found in the reaction mixture of TG1/pNHJ10H and pNHJ20L, whereas no benzamide was found in the reaction mixture of TG1. A number of references are cited herein, the disclosures of which are incorporated in their entirities, by reference herein.
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