In this study, animal fleshing (ANFL) was utilized as a substrate for the production of extracellular protease by Clostridium limosum through central composite rotatable design (CCRD) and response surface methodology (RSM). Optimum protease production of 433 U/ml was achieved and the purified enzyme was identified as acidic metalloprotease, a monomeric protein. The molecular weight of the enzyme was 71 kDa, whose activity was enhanced by bivalent metals such as Zn2+ and Mg2+. Scanning electron microscopy (SEM) examination also revealed the hydrolysis/microbial degradation of ANFL through protease activity in the anaerobic fermentation process. Simultaneous hydrolysis of ANFL and production of an enzyme with the potential for different industrial applications provide an attractive methodology for the disposal of tannery solid waste. (C) 2016 Elsevier Ltd. All rights reserved.