Objectives: The efficient release of bioactive peptides (BPs) from bovine milk proteins is of great significance. This study aimed to explore the potential of using the soluble proteinase of lactic acid bacteria (LAB) for production of BPs. Methods: A LAB strain exhibiting high proteolytic activity was screened and identified. The gene encoding the proteinase in the selected LAB was amplified via PCR and then modified by removing the sequence encoding the cell-wall-associated region， enabling it to be a soluble protein. Subsequently， the modified gene prtB3 was cloned and expressed in the food-grade expression strain Lactococcus lactis NZ9000. After purification， a comprehensive evaluation was conducted on the enzymatic properties of the recombinant proteinase PrtB3 and the biological activities of the hydrolysates resulting from casein degradation. Results: The screened LAB strain was identified as Lactobacillus delbrueckii subsp. lactis. A soluble， secreted， C-terminally Myc and His-tagged derivative PrtB3 was constructed and expressed in Lactococcus lactis via the NICE■ Expression System. Compared with the wild-type proteinase， PrtB3 demonstrated a significant expansion in substrate specificity. It was proteolytically active in the hydrolysis of α-， β-， and κ-casein. Regarding biological activity， the casein hydrolysate produced by PrtB3 showed remarkable angiotensin-converting enzyme (ACE) inhibitory activity. The maximum ACE inhibition rate reached 78%， which was twice that of the hydrolysate produced by the wild-type proteinase. The half-maximal inhibitory concentration (IC50) was 1.69 mg/mL， and the ACE inhibitory activity remained stable over an 8-week period. Furthermore， the enzymatically produced hydrolysate showed antimicrobial activity against Staphylococcus aureus and Escherichia coli. Conclusions: In this study， the soluble proteinase PrtB3 was successfully cloned and expressed. It effectively enhanced the degradation of casein and significantly improved the biological activity and stability of the casein hydrolysate. PrtB3 demonstrated great potential in producing BPs by degrading casein， providing a novel approach for the preparation of bioactive peptides.

bioactive peptides  /  casein  /  lactic acid bacteria  /  soluble proteinase  /  ACE inhibitory peptides  /  antibacterial peptides