反応 #10221

反応方程式

KH2PO4

NaCl

citric acid

dextrose

Tris

NH4OH

→

Glucosamine-6-Phosphate

反応条件

後処理

1. 1
   温度coli cells from frozen vials
2. 2
   その他were inoculated into a flask
3. 3
   workup.ADDITIONculture containing 12 L fermentation seed medium
4. 4
   その他was operated at 39° C. at an agitation speed of 700 rpm
5. 5
   その他Sonication
6. 6
   workup.WAITwas applied to the cells four times for 30 seconds with intermissions of 1 minute
7. 7
   その他to separate the cell debris
8. 8
   温度the supernatant was maintained as enzyme
9. 9
   抽出extract
10. 10
    workup.ADDITIONAmmonium sulfate was added to the
11. 11
    抽出extract to 226 g/L (40% saturation)
12. 12
    workup.ADDITION120 g/L ammonium sulfate (60% saturation) was added to the supernatant
13. 13
    その他After centrifugation, the pellets were collected

実験手順

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.

出典