[OH:1][P:2]([O-:5])([OH:4])=[O:3].[K+].C(O)(=O)CC(CC(O)=O)(C(O)=O)O.[O:20]=[CH:21][C@@H:22]([C@H:24]([C@@H:26]([C@@H:28]([CH2:30]O)[OH:29])[OH:27])[OH:25])O.[NH4+].[OH-].C(O)C([NH2:40])(CO)CO.[Na+].[Cl-]>[O-]S([O-])(=O)=O.[Mn+2].O>[P:2]([O:5][CH2:30][C@H:28]1[O:29][CH:21]([OH:20])[C@H:22]([NH2:40])[C@@H:24]([OH:25])[C@@H:26]1[OH:27])([OH:4])([OH:1])=[O:3]coli cells from frozen vials were inoculated into a flask culture containing 12 L fermentation seed medium. The medium contained the following chemicals (g/L): corn steep liquor, 10; KH2PO4, 2.5; MgSO4.7H2O, 2.0; (NH4)2SO4, 0.5; citric acid, 0.192; FeSO4.7H2O, 0.03; MnSO4.H2O, 0.021; antifoam 6000K, 0.5; dextrose, 84. The seed fermentor was operated at 39° C. at an agitation speed of 700 rpm and an airflow of 3.5 LPM. The pH of the fermentation was maintained at 6.9 with 21% NH4OH. After 24 hours of growth, the broth was centrifuged and the pellets were resuspended in extract buffer (100 mM Tris, pH 7.5; 100 mM NaCl). The cells were centrifuged and the pellets were resuspended in the extract buffer at a concentration of OD660 about 200. Sonication was applied to the cells four times for 30 seconds with intermissions of 1 minute. Centrifugation was used to separate the cell debris and the supernatant was maintained as enzyme extract. Ammonium sulfate was added to the extract to 226 g/L (40% saturation) and the extract was centrifuged. 120 g/L ammonium sulfate (60% saturation) was added to the supernatant. After centrifugation, the pellets were collected and resuspended in the extract buffer. Most of the glucosamine-6-phosphate activity was in this fraction.
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