Valorizing the Limit Proteins of Soy Sauce Residue Using Halotolerant Alkaline Proteases from Endogenous Bacillus velezensis J2

• A novel endogenous strategy for SSR valorization is established. • Protease J2 from B. velezensis J2 targets recalcitrant limit proteins. • The protease retains high activity in broad pH (6-11) and high salt (4 M NaCl). • Outperforms commercial proteases in hydrolyzing SSR. Soy sauce residue (SSR), a high-salt industrial waste, contains a recalcitrant protein system characterized by low solubility (<15%) and a complex composition (7% salt, lipids, fiber), resulting in inefficient resource valorization (<60% protein recovery). This study established a circular bioresource approach using halotolerant alkaline proteases from an endogenous strain, Bacillus velezensis J2, newly isolated from SSR. Partial purification of the extracellular proteases via ammonium sulfate precipitation (60-90%) and centrifugal ultrafiltration resulted in a protease system where a 25 kDa metallo-serine protease was identified as the principal component and designated as Protease J2. Protease J2 enabled direct SSR valorization, exhibiting optimal activity at pH 8.5 and 50°C. Remarkably, it maintained high stability under alkaline conditions (>80% activity at pH 6-11) and high salinity (>50% activity at 4 M NaCl). Protease J2 significantly outperformed commercial alternatives in hydrolyzing SSR’s "limit proteins". Scanning electron microscopy (SEM) confirmed microstructure modification of SSR from dense aggregates to loosened fibrous matrices. Collectively, the B. velezensis J2 proteases provide a sustainable, chemical-free route for SSR valorization, enabling high-value protein recovery without secondary pollution.