Abstract A recombinant Pichia pastoris containing enterokinase light chain gene from bovine was cultured in flasks firstly at different pH, methanol addition, and cell mass concentration for enterokinase production. Activity of enterokinase increased as cell mass concentration increased at more than φ = 6 % methanol added. No significant change of activity of enterokinase was observed when methanol added was less than φ = 4 %. No activity of enterokinase lost after 120 h conservation at different pH. Secondly, high-level enterokinase production was achieved in 3.7 L bioreactor at pH 4.0, φ = 0.6 % methanol, p = 52.5 kPa. Activity of enterokinase was not cell mass concentration dependent at φ = 0.6 % methanol in bioreactor. Yield of enterokinase was 479.99 mg L–1 after 97.5 h induction. Maintenance coefficient and methanol consumed were calculated to analyse the enterokinase production.
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