A novel protease secreted by Brevibacillus sp. KH3 isolated from excess sludge at 50 degrees C and used as a sludge-lysing strain was investigated in this study. Sludge reduction was minimized by protease inhibitors and a 40-kDa protease, which significantly contributed to this sludge-reducing activity, was purified as the target protein. The final purified protease demonstrated 92-fold higher specific activity than the initial crude extracts. The sludge-reducing efficiency deteriorated relative to decreased protease activity triggered by EDTA: thus, the purified protease was a causative agent in reducing excess sludge. The ;40-kDa protease was a serine metalloprotease and showed the highest activity at 50 degrees C and pH 8.0, and the activity was enhanced in the presence of calcium ions, indicating that the purified protease contained calcium ion. Furthermore, this 40-kDa protease inhibited biofilm formation in excess sludge. These results imply that sludge reduction is because of reduction of biofilm formation in excess sludge. (C) 2011 Elsevier Ltd. All rights reserved.