Citric Acid → D-glucosamine 6-phosphate

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.

1. temperaturecoli cells from frozen vials
2. customwere inoculated into a flask
3. additionculture containing 12 L fermentation seed medium
4. customwas operated at 39° C. at an agitation speed of 700 rpm
5. customSonication
6. waitwas applied to the cells four times for 30 seconds with intermissions of 1 minute
7. customto separate the cell debris
8. temperaturethe supernatant was maintained as enzyme
9. extractionextract
10. additionAmmonium sulfate was added to the
11. extractionextract to 226 g/L (40% saturation)
12. addition120 g/L ammonium sulfate (60% saturation) was added to the supernatant
13. customAfter centrifugation, the pellets were collected

ORDレコード

ord-5934e900187f46f2962c30933ab22f3e

出典データセット

uspto-grants-2006_08

DOI

10.6084/m9.figshare.5104873.v1

特許番号

US07094582B2