Abstract: A novel aspartic protease (AnproA1) was cloned from Aspergillus niger in this study. Multiple amino acid sequence alignments revealed that the protease belonged to the pepsin-like aspartic protease A1 family and shared the highest amino acid sequence identity of 42.6% with the aspartic protease from Penicillium rubens. The high-level secretory expression of AnproA1 in Komagataella phaffii was successfully achieved using multiple strategies. After high-cell density fermentation in a 5 L fermenter, the fermentation supernatant showed a protease activity of 15 250.0 U/mL and a protein concentration of 14.0 mg/mL. The optimum pH and temperature for the purified AnproA1 were 2.5 and 55 ℃, respectively. It was stable within the pH range of 2.5–5.5 and up to 50 ℃. AnproA1 displayed broad substrate specificity and the highest hydrolysis ability towards κ-casein followed by hemoglobin. Furthermore, AnproA1 could hydrolyze duck hemoglobin and plasma proteins into angiotensin-converting enzyme (ACE) inhibitory peptides with half maximal inhibitory concentration (IC50) values of 0.084 and 0.042 mg/mL, respectively. This study offers valuable theoretical insights for the high-level expression of aspartic proteases in K. phaffii and the high-value bioconversion of duck blood proteins.

Key words: Aspergillus niger; aspartic protease; Komagataella phaffii; secretory expression; duck blood proteins