Design, expression and functional assessment of novel engineered serratiopeptidase analogs with enhanced protease activity and thermal stability

Novel serratiopeptidase exhibits different affinities to the substrates and inhibitors

“…Interestingly, in the practical phase of the study, resistance of the T344[8‐339ss] to EDTA as a chemical inhibitor and higher activity toward Bradykinin as a specific substrate, in contrast to the native enzyme has been confirmed. It is worth to mention that disulfide bond formation in engineered serratiopeptidases resulted to higher thermal stability (Rouhani et al, 2021), which is reflected higher tolerance to the environmental conditions; however, the effect of truncation on enhancing cellular penetration should be assessed further in future studies.…”

“…Based on the results of our previous study, the native enzyme and the most reactive and stable truncated form were expressed and purified according to the previously explained procedure (Rouhani et al, 2021). Briefly, the overnight cultures of bacterial suspension possessing recombinant pQE30‐Serra 473 and pET28a‐ T344[8‐339ss] were inoculated into LB broth containing appropriate antibiotics (based on the vector) with shaking (180 rpm) at 37°C until OD 600 was reached to 0.6–0.8.…”

“…Three engineered forms showed higher enzyme activity in contrast to the native enzyme (Rouhani et al, 2021). Interestingly, one of the truncated forms, T344[8‐339ss], with 344 amino acid length and a disulfide bond between residues 8 and 339 showed the highest proteolytic activity and thermal stability in comparison to the native enzyme and the other engineered forms (Rouhani et al, 2021). However, in the mentioned investigation, functional assays were performed using casein, as a nonspecific substrate and there is not any information about the impact of such alternations on the receptor‐binding affinity of these truncated forms for different substrates and inhibitors.…”

“…Moreover, Gibbs free energy (Δ G ) of docked structures was calculated to enlighten the interactions of novel designed variants compared with the native enzyme. Based on the results obtained from docking analysis and our previous non‐specific enzyme activity (Rouhani et al, 2021), the native enzyme and the best variant in terms of interaction pattern and binding score have been expressed and purified as a recombinant protein and the affinities toward Bradykinin (substrate) and EDTA (inhibitor) was examined using HPLC and radial casein hydrolysis test, respectively.…”

“…Based on the results obtained from this study, four engineered forms were expressed as recombinant proteins in Escherichia coli cells, then purified and were subjected to functional assessment. Three engineered forms showed higher enzyme activity in contrast to the native enzyme (Rouhani et al, 2021). Interestingly, one of the truncated forms, T344 [8-339ss], with 344 amino acid length and a disulfide bond between residues 8 and 339 showed the highest proteolytic activity and thermal stability in comparison to the native enzyme and the other engineered forms (Rouhani et al, 2021).…”