Permeabilization of Corynebacterium glutamicum for NAD(P)H-dependent intracellular enzyme activity measurement

摘要

Permeabilization of Corynebacterium glutamicum cells permits direct determination of enzyme activity measuring NAD(P)H after appropriate calibration and correction for cell density. The optimized conditions found were the treatment of 10 mg cells/ml (dry weight) by Triton X-100 at a final concentration of 0.05% for 5 min at room temperature. Glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase activities were very close to those determined in the cell-free extract. In cell-free extracts malic enzyme activity was about double and that of isocitrate dehydrogenase about triple the values in permeabilized cells. K_m values were similar in cell-free extract and permeabilized cells, but larger than literature values obtained with purified enzymes.