Commercially valuable biomolecules could be produced using enormous quantities of organic solid waste (OSW) generated from agricultural and food processing industries due to their nutrient-rich organic composition. In this study, a multi-hydrolytic biocatalyst (MHB) was produced from OSW by optimizing the composition, ultrasonic pre-treatment, and parameters for anaerobic digestion. RSM was used to design the experiments for optimization of anaerobic digestion parameters. Optimum experimental conditions for MHB production which yielded maximum protease, amylase and lipase activities. The experimental results were validated using RSM and ANN modeling for accuracy. The kinetic parameters and the influence of pH and temperature on the activity and stability of the partially purified enzyme were also evaluated. To investigate the multi-activity and practical applicability of MHB, it was used for pre-treatment of municipal waste activated sludge (MWAS) because the sludge contains different complex organic molecules and this pre-treatment would help to improve the usability of the huge quantity of sludge for industrial purpose. The enzymatic pre-treatment of the sludge at optimized conditions yielded SS reduction (29.2%), an increase of sCOD (32%), sProtein (53.57%) and sCarbohydrate (76.71%). Thus, a potential low-cost multi-purpose biocatalyst could be efficiently produced from OSW and used for the enhanced treatment of MWAS. (C) 2018 Institution of Chemical Engineers. Published by Elsevier B.V. All rights reserved.