Immobilized hydrolyticenzymes for antibiofilm applications

The bacterial planktonic growth and the removal of bacterial cells grown on polypropylene surface coated with covalently immobilized proteases (subtilisin Carlsberg or ?-chymotrypsin) was investigated for Enterococcus hirae, Staphyloccocus epidermidis and Eschericha coli. Immobilization of both proteases on plasma-treated polypropylene was carried out using as cross-linking agent i) glutaraldehyde or ii) N'-diisopropylcarbodiimide and N-hydroxysuccinimide. In the presence of immobilized proteases a higher bacterial planktonic growth (up to 40 %) was observed. Instead, a different effect was observed on cell removal, and it varied according to the bacteria strain, the immobilized protease and the immobilization procedure. In particular, the presence of subtilisin in the polypropylene coating increased the cell removal of E. hirae by simple washing of the polypropylene surface and both subtilisin and ?-chymotrypsin immobilized by N'-diisopropylcarbodiimide and N-hydroxysuccinimide favored the removal of S. epidermidis after sonication. No significant differences compared to the control where observed in all the other cases. In conclusion this study indicates that proteases can be an enhancer of microbial biomass (a phenomena that could be exploited for industrial fermentation) and can affect the strength of cell adhesion for some bacteria. bacterial adhesion. In addition, the confinement of enzymes on a solid surface through immobilization can be a procedure to maximize the enzyme activity just where the biofilm grows. Herein we present how two commercial and readily available proteases (aCT and SubC), immobilized on polypropylene surface [6], affect bacterial planktonic growth and the removal of cells grown on the plastic surface itself. To this end, Enterococcus hirae (gram positive and it causes sepsis in humans), Staphyloccocus epidermidis (gram positive and opportunistic human pathogen) and Eschericha coli (a gram negative component of human microflora and an opportunistic human pathogen) were chosen as model bacteria.