2.4 GDH activity assay
To measure GDH activity, cells from 1 mL of fermentation broth were
collected by centrifuging at 1,500 ×g for 10 minutes at 4℃. The culture
supernatant was discarded, and the cells were resuspended in 1 mL of 100
mM Tris-HCl (pH 7.5). The cell suspensions was lysed by sonication on
ice: 200 W, working 3 s, interval 5 s for 40 times, to yield the crude
enzyme solution. The reaction mixture contained 100 mM glucose, 100 mM
Tris-HCl (pH 7.5), 2 mM NAD+, and an 8000-fold
dilution of crude enzyme solution. The enzyme activity was assayed at 25
℃, by recording the increase of the absorbance at OD340.
The enzyme activity was calculated using the formula U/mL = [ΔA/min]
× [1/ε] × 8000 (ε = A/cL = 6.402 mL / (mol * cm)) (Bucher et al.,
1974).