The lipase produced by the Aspergillus niger strain AC-54 has been widely studied due to its enantioselectivity for racemic mixtures. This study aimed to optimize the production of this enzyme using statistical methodology. Initially a Plackett-Burman (PB) design was used to evaluate the effects of the culture medium components and the culture conditions. Twelve factors were screened: Water content, glucose, yeast extract, peptone, olive oil, temperature, NaH 2P0 4, KH 2P0 4, MgS0 4-7H 20, CaCl 2, NaCI, and MnS0 4. The screening showed that the significant factors were water content, glucose, yeast extract, peptone, NaH 2P0 4, and KH 2P0 4, which were optimized using response surface methodology (RSM) and a mathematical model obtained to explain the behavioral process. The best lipase activity was attained using the following conditions: Water content (20%), glucose (4.8%), yeast extract (4.0%), and NaH2P04 (4.0%). 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