Improved anti-biofilm activity and long-lasting effects of novel serratiopeptidase immobilized on cellulose nanofibers.

Today, enzymatic treatment is a progressive field in combating biofilm producing pathogens. In this regard, serratiopeptidase, a medicinally important metalloprotease, has been recently highlighted as an enzyme with proved anti-biofilm activity. In the present study, in order to increase the long-lasting effects of the enzyme, serratiopeptidase and the novel engineered forms with enhanced anti-biofilm activity were immobilized on the surface of cellulose nanofibers (CNFs) as a natural polymer with eminent properties. For this, recombinant serratiopeptidases including the native and previously designed enzymes were produced, purified and conjugated to the CNF by chemical and physical methods. Immobilization was confirmed using different scanning and microscopic methods. The enzyme activity was assessed using casein hydrolysis test. Enzyme release analysis was performed using dialysis tube method. Anti-biofilm activity of free and immobilized enzymes has been examined on Staphylococcus aureus and Pseudomonas aeruginosa strains. Finally, cytotoxicity of enzyme-conjugated CNFs was performed by MTT assay. The casein hydrolysis results confirmed fixation of all recombinant enzymes on CNFs by chemical method; however, inadequate fixation of these enzymes was found using cold atmospheric plasma (CAP). The AFM, FTIR, and SEM analysis confirmed appropriate conjugation of enzymes on the surface of CNFs. Immobilization of enzymes on CNFs improved the anti-biofilm activity of serratiopeptidase enzymes. Interestingly, the novel engineered serratiopeptidase (T344 [8-339ss]) exhibited the highest anti-biofilm activity in both conjugated and non-conjugated forms. In conclusion, incorporation of serratiopeptidases into CNFs improves their anti-biofilm activities without baring any cytotoxicity. KEY POINTS: • Enzymes were successfully immobilized on cellulose nanofibers using chemical method. • Immobilization of enzymes on CNFs improved their anti-biofilm activity. • T344 [8-339ss] exhibited the highest anti-biofilm activity in both conjugated and non-conjugated forms.