Ammonia → O-Acetyl-L-serine

Participants

Reaction Steps

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.

Provenance & Evidence