2.5. Determination of an optimal medium for prochymosin expression

Cultivation of the Pichia pastoris GS115/pGAPZαA/ProchymCB strain was evaluated in eight media (Table 1). The cultivation was carried out according to the EasySelect™ Pichia pastoris Kit (Invitrogen, USA) recommendations in 250 mL flasks with 50 mL of a medium at 28°С and 250 rpm for 72 h. Then, 1 mL the culture was collected every 24 h and centrifuged at 13200 × g, for 20 min, at 4 °C (Eppendorf Centrifuge 5415R, Hamburg, Germany). The supernatants were stored at 4 °C and were subjected to the milk-clotting assay.

Composition of the media tested in this work.

YP – yeast extract and peptone; D – dextrose; CAS – citrate-phosphate buffer with ascorbic acid and sorbitol; CrMz – corn molasses; BMz – beet molasses.