High-Level Production of Aspartic Proteinase in Aspergillus niger via Combinatorial Engineering Strategies for the Bioconversion of Agro-Food Protein Byproducts

Aspergillus niger is a significant microbial cell factory for food enzyme production. Here, a novel aspartic proteinase ( Mo A1) was identified and expressed at a high level in A. niger FBL-B via combinatorial strategies. In a 5 L fermenter, the engineered strain produced an extracellular proteinase activity of up to 31,104.0 U/mL with a protein content of 27.5 g/L. Mo A1 exhibited maximum activity at pH 3.0 and 55 °C. It remained stable within pH 2.5–6.0 and up to 45 °C. Mo A1 showed broad substrate specificity and the highest specific activity (1,934.8 U/mg) toward hemoglobin. Furthermore, 10 protein byproducts were efficiently hydrolyzed by Mo A1 to produce bioactive hydrolysates. Walnut meal hydrolysates demonstrated the highest XOD inhibitory activity (IC 50, 4.70 mg/mL). Cottonseed meal hydrolysates exhibited the highest ACE inhibitory activity (IC 50, 0.18 mg/mL). This study offers effective strategies for high-level production of aspartic proteinases in A. niger and valorization of protein byproducts.