Functional modification of thermostable alkaline protease from Bacillus halodurans SE5 for efficient production of antioxidative and ACE-inhibitory peptides from sericin

In this work, the hydrolysis efficiency of recombinant protease_SE5 (r_SE5) on sericin was improved by protein engineering. The S166D variant, obtained from the first round of site-directed mutagenesis, exhibited a 1.4-fold increase in protease activity (602 U/mg) and sericin hydrolysis efficiency (123 mu g peptide/mL). Further substitution of Ser166, located on a low-affinity calcium-binding loop, by Glu consequently improved sericin hydrolysis capability. The S166E variant showed remarkable protease activity (671 U/mg) and sericin hydrolysis efficiency (140 mu g peptide/mL) with no changes in thermostability compared to r_SE5. Interestingly, the purified S166E efficiently hydrolyzed sericin, with a maximal peptide yield of 540 mu g/mL within 60 min. The S166E-derived sericin peptide also exhibited higher ABTS radical scavenging activity (6470 mu mol TEAC/L) and angiotensin-I-converting enzyme (ACE) inhibition activity (40%). Additionally, the identification of peptides by LC-MS/MS revealed that two smaller peptides (HHSGVNR and GWWWNSD) with a higher proportion of key amino acid residues were produced by S166E. These peptides were the potential bioactive peptides predicted by the BIOPEP data-base. Therefore, this study demonstrates the structure-function relevance between the low-affinity calcium-binding loop and hydrolysis of sericin by protease_SE5. S166E is a more powerful protease for producing value-added sericin hydrolysate, which can be used as an active ingredient for food and pharmaceutical applications.