The optimization of enterokinase fermentation was performed with a recombinant Saccharomyces cerevisiae. The maximum enterokinase yield and specific yield were obtained when the ratio of yeast extract to bacto peptone was 5:5. However, the fraction of plasmid-containing cells was approximately 65% for most nitrogen source ratios. From batch cultures, the minimum DOT level was found to be a key factor. Through DO-stat cultivations, the value was further optimized. As the minimum DOT level increased, the product yield and plasmid-containing cell fraction increased, whereas the ethanol concentration decreased. The optimum agitation speed and aeration rate in a 51 jar were determined as 700 rpm and 2 vvm, respectively. Under these conditions, a high enterokinase yield of 3.8 mg/l was obtained with the final ethanol concentration of 0.4 g/l. The minimum DOT level was maintained to be 60% or more. Scale-up of enterokinase fermentation from a 51 jar to a 3001 jar was successfully accomplished based on strategies to maintain a constant impeller tip speed and a minimum DOT level at 60% or more. (C) 2004 Elsevier Ltd. All rights reserved.