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

Abstract

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 μg 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 μg 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 μg/mL within 60 min. The S166E-derived sericin peptide also exhibited higher ABTS radical scavenging activity (6470 μmol 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 database. 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.