Characterization of Glutamyl Aminopeptidase with Novel Degradation Activity of Soybean Trypsin Inhibitor and Salt-Tolerance from <i>Bacillus amyloliquefaciens</i> phb03 in Soy Sauce Residue

Soybean is acknowledged as an excellent plant-based protein source; however, its nutritional efficacy is compromised by the presence of soybean trypsin inhibitor (STI), which diminishes bioavailability and may induce metabolic disorders. To mitigate the inhibitory effects of STI, this study focused on the purification and characterization of STI-degrading protease (SDP) from <i>Bacillus amyloliquefaciens</i> phb03. The results revealed that SDP is an alkaline salt-tolerant protease with optimal activity at 40 °C, pH 9.0, and 2 M NaCl, retaining over 67% activity in 3 M NaCl after 4 d. SDP was identified as a glutamyl aminopeptidase phb03 (GAPP) through LC-MS Q-E analysis, with a molecular weight of approximately 39 kDa and demonstrating an efficient degradation of 95% STI. Subsequently, heterologous expression of GAPP in <i>Bacillus subtilis</i> WB800 yielded a peak enzyme activity of 411.94 ± 0.37 U·L^-1, exhibiting significant STI degradation activity, particularly under high-salinity conditions. It is speculated that the structural compactness and enhanced flexibility in the catalytic center of GAPP contribute to its stability and catalytic activity under high-salt conditions. This degradation protease offers a promising solution to enhancing protein utilization in soybeans affected by STI, with the potential for industrial applications.