The production optimization of alpha-amylase (E.C.3.2.1.1.) from Aspergillus niger ATCC 16404 fungus is obtained with statistical experimental designs. The methodology was based on the Plackett-Burman design (N = 12) [12 experiments and 11 factors in which seven are real ones (agitation, pH, starch, yeast extract, "corn steep liquor", CaCl2, and salts: MgCl2, 6H(2)O, MnSO4 and FeSO4, 7H(2)O) and four errors]. This design has been chosen in order to select the factors favoring enzyme production. The production medium is prepared using orange waste powder (2% w/v) at 30 degrees C under agitation. The statistical analysis has revealed that variation in agitation from 150 rpm to 200 rpm has no effect on the production of alpha-amylase and that of protein; whereas such as a variation increases the biomass. As for the variation in the pH from 5 to 6 has a positive effect on the production of both alpha-amylase and proteins, while its effect on the biomass is negative. The addition of starch at 10 g/l to the fermentation medium (an inductive substrate and a carbon source) stimulated the production of alpha-amylase, while it is without any effect on the production of other protein and that of the biomass. The presence of yeast extract (5 g/l) as a growth factor has a positive effect on the production of protein; whereas it has a negative effect on the production of alpha-amylase and no effect on the growth of mould. The "corn steep liquor", at 20 ml/l does not affect the production of alpha-amylase; whereas, it has a biomass stimulation on the protein synthesis. Calcium chlorides at 1 g/l (a structural and stabilizing element for the alpha-amylase) solely affect the enzyme production. The use of other salts (manganese and iron sulfates as well as magnesium chloride) seems to increase the production of alpha-amylase, but does not affect either the production of proteins or the biomass. The reaction of the explained factor chi xis adjusted by a multiple linear regression and the investigation has allowed the selection of the following factors: pH, starch, yeast extract, CaCl2 and salts (FeSO4, 7H(2)O, MnSO4 and MgCl2, 6H(2)O) (c) 2005 Elsevier Ltd. All rights reserved.