Covalent Immobilization of Subtilisin A onto Thin Films of Maleic Anhydride Copolymers

Abstract

Enzymes cleaving the biopolymer adhesives of fouling organisms are attracting attention for the prevention of biofouling. We report a versatile and robust method to confine the serine protease Subtilisin A (or Subtilisin Carlsberg) to surfaces to be protected against biofouling. The approach consists of the covalent immobilization of the protease onto maleic anhydride copolymer thin film coatings. High-swelling poly(ethylene-alt-maleic anhydride) (PEMA) copolymer layers permitted significantly higher enzyme loadings and activities than compact poly(octadecene-alt-maleic anhydride) (POMA) films. Substantial fractions of the immobilized, active enzyme layers were found to be conserved upon storage in deionized water for several hours. Ongoing studies explore the potentialities of the developed bioactive coatings to reduce the adhesion of various fouling organisms.

Immobilized enzymes affect biofilm formation

“…Recently, we have developed a well-defined model system to investigate the influence of immobilized subtilisin A on the adhesion of major marine foulers (Tasso et al 2009a;Tasso et al 2009b). Our studies showed that the adhesion strength of Ulva linza spores and Navicula perminuta cells decreased in the presence of the active enzyme, and that the effect of the enzyme was significantly enhanced when the enzyme was surfaceconfined (Tasso et al 2009b), revealing the important role of enzyme location at the surface-fouler interface on antifouling efficacy.…”

“…Reactive immobilization of enzymes onto MA copolymer films was achieved by exposing the copolymer layers to enzyme solutions of variable concentration, as described in Tasso et al 2009a). …”

“…Subtilisin A solutions were prepared by dissolving the enzyme in PBS modified by the addition of CaCl 2 and NaCl at 2 mM and 0.1 M, respectively; the pH of the solution was adjusted to 8.6 with NaOH (Tasso et al 2009a). The immobilization was performed using in-house constructed immobilization chambers.…”

“…The immobilization was performed using in-house constructed immobilization chambers. After overnight exposure of the copolymer films to the enzyme solution the samples were rinsed 10 times with deionized water to remove unbound protein (Tasso et al 2009a). …”

“…The immobilization and characterization of subtilisin A onto MA copolymer films are described in detail elsewhere (Tasso et al 2009a;2009b).…”