-Chymotrypsin Immobilized on a Low-Density Polyethylene Surface Successfully Weakens Escherichia coli Biofilm Formation

The protease -chymotrypsin (-CT) was covalently immobilized on a low-density polyethylene (LDPE) surface, providing a new non-leaching material (LDPE--CT) able to preserve surfaces from biofilm growth over a long working timescale. The immobilized enzyme showed a transesterification activity of 1.24 nmol/h, confirming that the immobilization protocol did not negatively affect -CT activity. Plate count viability assays, as well as confocal laser scanner microscopy (CLSM) analysis, showed that LDPE--CT significantly impacts Escherichia coli biofilm formation by (i) reducing the number of adhered cells (-70.7 +/- 5.0%); (ii) significantly affecting biofilm thickness (-81.8 +/- 16.7%), roughness (-13.8 +/- 2.8%), substratum coverage (-63.1 +/- 1.8%), and surface to bio-volume ratio (+7.1 +/- 0.2-fold); and (iii) decreasing the matrix polysaccharide bio-volume (80.2 +/- 23.2%). Additionally, CLSM images showed a destabilized biofilm with many cells dispersing from it. Notably, biofilm stained for live and dead cells confirmed that the reduction in the biomass was achieved by a mechanism that did not affect bacterial viability, reducing the chances for the evolution of resistant strains.