Reaktion #2781

Reaktionsgleichung

potassium phosphate

ampicillin

IPTG

HCl

ampicillin

→

benzamide

Lösungsmittel

benzonitrile

phosphate

Reaktionsbedingungen

Aufarbeitung

1. 1
   Sonstige(12 hours)
2. 2
   workup.WAITThe mixture was incubated at 30° C. for 4 hours
3. 3
   workup.WAITThe mixture was incubated at 30° C. for 10 hours
4. 4
   workup.WAITThe suspensions were disrupted by sonification for 5 min
5. 5
   workup.WAITcentrifuged at 12,000×g for 30 min
6. 6
   workup.WAITThe reaction was carried out at 20° C. for 30 min

Vorschrift

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.

Quelle