Novel serratiopeptidase exhibits different affinities to the substrates and inhibitors

The clinical application of serratiopeptidase as an anti-biofilm and anti-inflammatory agent is restricted due to the enzyme sensitivity to the environmental conditions. In our previous study, six enzyme variants were designed by introducing different mutations and truncations that exhibited higher thermal stability. In the present study, the interaction pattern and affinity of variants to substrates and inhibitors were studied using molecular docking and in vitro studies. CABS-dock and Swiss-dock servers were used for substrate (Bradykinin and Substance-P) and inhibitor (Lisinopril and EDTA) docking, respectively. The interactions were analyzed using LigPlot, UCSF Chimera, and visual molecular dynamics packages. Free energy calculations were performed using PRODIGY. Finally, the native enzyme and the best variant in terms of interaction pattern and binding score were selected for in-vitro affinity analysis toward Bradykinin and EDTA using HPLC and casein hydrolysis test, respectively. Molecular docking revealed that T344 [8-339ss] variant showed a different pattern for both substrates and inhibitors in the way that none of the native active site residues were involved in the receptor binding. As revealed by in vitro studies, T344 [8-339ss] displayed the highest number of hydrogen bond formation in docking with Bradykinin and remarkable decrement in the binding affinity for EDTA. This was the first report on the design of novel serratiopeptidase with higher activity to Bradykinin and improved resistance to EDTA as an inhibitor.