AbstractA study was conducted to purify and characterize a novel protease produced from Aspergillus niger using different lignocellulosic agro-based by-products including corncobs, wheat bran, and rice bran as substrates under SSF. Maximum protease activity was recorded on wheat bran fermented culture media after the 3rd day of incubation. The optimal conditions found for protease production using wheat bran were cultivation period (3 days), substrate concentration (10 g), pH (7), incubation temperature (45oC), inoculum size (4 mL), and 3% surfactant Tween-80 (2 mL). A purification fold of 2.41 with 29 U/mg specific activity and 70.73 % recovery was achieved after purification. Purified protease from A. niger had a molecular weight of 47 kDa on SDS-PAGE. The enzyme activity profile showed that purified protease was optimally active at pH 7 and 45oC as optimum values. A. niger protease was reasonably stable in the pH range 5-8 and 35-60oC for up to 1 h incubation. Protease was activated by various metal ions/inhibitors tested, Mn2+, Cd2+, Mg2+, Cu2+, PMSF, Pepstatin and Iodoacetic acid at 1 mM, proving the enzyme as metalloprotease, whereas an inhibitory effect was shown by certain agents including EDTA and SDS. The purified protease was compatible with five local detergents with up to 25 days of shelf life at room temperature. The maximum production of protease in the presence of a cheaper substrate at low concentration and its potential as a detergent additive for improved washing makes the strain and its enzymes potentially useful for industrial purposes, especially for the detergent and laundry industry.