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Enzymes derived from microbial sources have gained increasing popularity in industrial applications over the past decades. Despite the high production cost, alkaline proteases have wide applications in industries such as tanneries, food production, and detergents. In recent years, there has been a shift towards utilizing natural carbon sources for cultivating microorganisms and extracting proteases in order to reduce production costs. This study aimed to investigate the biochemical and kinetic properties of protease enzymes obtained from Aspergillus niger cultivated in a paper waste medium and compare with the enzyme produced in a basal medium. Glucose is a more favorable carbon source compared to cellulose, so paper waste was pretreated with cellulose-degrading bacteria to convert cellulose into smaller carbohydrates. After the growth of A. niger in basal and combinational media, the enzymatic properties were compared between the extracted enzymes by using casein as substrate. The results demonstrated that A. niger could produce protease enzymes in the paper waste medium similar to the basal medium with more than 5-fold cost saving. The specific activity of the enzymes isolated from the basal and paper waste media was calculated to be 184.95 ± 10.56 U ml−1 and 169.88 ± 11.05 U ml−1, respectively. Carbon sources did not affect the optimum pH and temperature of the protease enzyme, which were found to be 8 and 37 °C, respectively. This study provides valuable insights into the production of alkaline protease from A. niger using a combinational medium (paper waste pretreated by cellulose-degrading bacteria), offering a cost-effective approach for industrial applications. [ABSTRACT FROM AUTHOR]