This study is the first utilizing Vitreoscilla hemoglobin in a heterologous bacterium, Enterobacter aerogenes, to determine the effect of such a highly efficient oxygen-uptake system on the production of L-asparaginase, an enzyme that has attracted considerable attention due to its anti-tumor activity. Here, we show that the Vitreoscilla hemoglobin expressing strain has from 10-fold to more than two orders of magnitude lower L-asparaginase activity than the wild type or the control without the Vitreoscilla hemoglobin gene under different aeration conditions. Aeration and agitation were also determining factors for enzyme production. The enzyme activity was reduced considerably under both full aerobic and anaerobic conditions, while the highest enzyme activity was determined in cultures under low aeration and low agitation. Also, the effect of different concentrations of glucose on enzyme production showed catabolic repression. Glucose at 1% caused almost total inhibition of enzyme activity, while at 0.1% it showed a slightly stimulatory effect on enzyme production, compared with glucose-free medium.