l-asparaginase is an enzyme that catalyzes the deamination free l-asparagine to yield aspartic acid and ammonia, and it is used in the food industry to reduce the levels of acrylamide that are formed when starchy foods are fried, baked and cooked. In this work, a sequential optimization strategy was used to determine the effect of carbon sources, nitrogen sources, l-asparagine inducer, inoculum concentration, initial medium pH, temperature and agitation rate in the production of l-asparaginase from Aspergillus oryzae CCT 3940 in submerged fermentation. The micro-organism was fermented at 30 °C in modified Czapeck medium composed of proline (2%), glucose (0.5%), l-asparagine inducer (0.2%), yeast extract (0.5%), KH2PO4 (0.152%), KCl (0.052%), MgSO4·7H2O (0.052%), CuNO3·3H2O (0.0001%), ZnSO4·7H2O (0.0001%) and FeSO4.7H2O (0.0001%) adjusted to pH 8.0 using a concentration of 3×107 spores and 72 h of fermentation at 150 rpm at 30 °C. A final activity of 67.49 U mL−1 was achieved, which represents an increase of 225% in relation to the initial non-optimized conditions Ver menos