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Ammonia → O-Acetyl-L-serine

HRID 1369367
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Reaktionsgleichung

Reaktionsgleichung von HRID 1369367
C[C@@]1(O)C2C=CC=C(O)C=2C(O)=C2[C@@H]1[C@H](O)[C@@H]1[C@](O)(C2=O)C(O)=[C:9]([C:26](N)=[O:27])C(=O)[C@H]1N(C)C.[O:34]=C[C@@H]([C@H]([C@@H]([C@@H](CO)O)O)O)O.OP([O-])(O)=O.[K+].[Na+].[Cl-].[O-]S([O-])(=O)=O.[Mg+2].[Cl-].[Cl-].[Ca+2].CC1[N+](CC2C=NC(C)=NC=2N)=CSC=1CCO.Cl.[Cl-].C[C:84]1(C)S[C@@H]2[C@H](NC([C@H](N)C3C=CC=CC=3)=O)C(=O)[N:86]2[C@H:85]1[C:103]([OH:105])=[O:104].N.O=O>O>[C:26]([O:27][CH2:84][C@@H:85]([C:103]([OH:105])=[O:104])[NH2:86])(=[O:34])[CH3:9]
KomponentenVersuchsvorschriftAufarbeitungQuellen und NachweiseNotizen zur Reaktion

Komponenten

12

Edukt 10

HCID 222Ammonia2×HCID 977Oxygen2×HCID 5234Sodium ChlorideHCID 6202Thiamine HydrochlorideHCID 6249AmpicillinHCID 24083Sulfuric acid magnesium salt (1:1)
HCID 107526
(2R,3S,4R,5R)-2,3,4,5,6-pentahydroxyhexanal
2×
HCID 516951Monobasic potassium phosphate
HCID 5284359Calcium Chloride
HCID 54675779Terramycin

Produkt 1

HCID 99478O-Acetyl-L-serine

Reagenzien, Katalysatoren, Lösungsmittel 1

HCID 962WaterLösungsmittel4×

Versuchsvorschrift

The fermenter employed was a Biostat M appliance which was supplied by Braun Biotech (Melsungen, Germany) and which had a maximum culture volume of 2 l. The fermenter containing 900 ml of fermentation medium (15 g of glucose/l; 10 g of tryptone/l; 5 g of yeast extract/l; 5 g of (NH4)2SO4/l; 1.5 g of KH2PO4/l; 0.5 g of NaCl/l; 0.3 g of MgSO4×7 H2O/l; 0.015 g of CaCl2×2 H2O/l; 0.075 g of FeSO4×7 H2O/l; 1 g of Na3citrate×2 H2O/l and 1 ml of trace element solution/l, see above, 5 mg of vitamin B1/l and 100 mg of ampicillin/l, adjusted to pH 7.0 with 25% ammonia) was inoculated with the above-described preliminary culture (optical density of approx. 2 at 600 nm). During the fermentation, the temperature was set at 32° C. and the pH was kept constant at a value of 7.0 by metering in 25% v/v ammonia. The culture was gassed with sterilized compressed air at 1.5 vol/vol/min and stirred with a rotational stirrer speed of 200 rpm. After the oxygen saturation had fallen to a value of 50%, the rotational speed was increased to a value of 1 200 rpm, by way of a monitoring device, in order to maintain 50% oxygen saturation (determined using a pO2 probe which was calibrated to 100% saturation at 900 rpm). A 56% w/v solution of glucose was metered in as soon as the glucose concentration in the fermenter had fallen from the initial value of 15 g/l down to approx. 5-10 g/l. The feeding took place at a flow rate of 3-6 ml/h, with the glucose concentration in the fermenter being kept at between 0.5 and 10 g/l. The glucose was determined using a glucose analyzer supplied by YSI (Yellow Springs, Ohio, USA). When an optical density of 30 had been reached, enzyme production was induced by adding 3 mg of tetracycline/l. The duration of the induction was 7 hours. After this time, samples were removed and the cells were separated off from the culture medium by centrifugation and washed. The resulting cell suspension was analyzed as described in example 3. The O-acetylserine sulfhydrylase activity measured with thiosulfate, AS-Cys, was 12 units/ml OD.

Aufarbeitung

  1. customwas set at 32° C.
  2. temperaturethe rotational speed was increased to a value of 1 200 rpm, by way of a monitoring device, in order
  3. concentrationsoon as the glucose concentration in the fermenter
  4. concentrationwith the glucose concentration in the fermenter
  5. additionby adding 3 mg of tetracycline/l
  6. customAfter this time, samples were removed
  7. customthe cells were separated off from the culture medium by centrifugation
  8. washwashed

Quellen und Nachweise

ORD-Eintrag
ord-b55cf1f1440d43dd96a82f9af5335dda
Quelldatensatz
uspto-grants-2003_06
DOI
10.6084/m9.figshare.5104873.v1
Patentnummer
US06579705B2

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