Potential biological role of laccase from the sponge Suberites domuncula as an antibacterial defense component

Background: Laccases are copper-containing enzymes that catalyze the oxidation of a wide variety of phenolic substrates. Methods: We describe the first poriferan laccase from the marine demosponge Suberites domuncula. Results: This enzyme comprises three characteristic multicopper oxidase homologous domains. Immunohistological studies revealed that the highest expression of the laccase is in the surface zone of the animals. The expression level of the laccase gene is strongly upregulated after exposure of the animals to the bacterial endotoxin lipopolysaccharide. To allow the binding of the recombinant enzyme to ferromagnetic nanoparticles, a recombinant laccase was prepared which contained in addition to the His-tag, a Glu-tag at the N-terminus of the enzyme. The recombinant laccase was enzymatically active. The apparent Michaelis constant of the enzyme is 114 ÊM, using syringaldazine as substrate. Exposure of E. coli to the nanoparticles, coated with Glu-tagged laccase, and to the mediator 2,2Œ-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) in the presence of lignin, as the oxidizable substrate, resulted in an almost complete inhibition of colony formation. Quantitative studies of the effect of the laccase-coated iron oxide nanoparticles were performed using E. coli grown in suspension in reaction tubes within a magnetic nanoparticle separator. Conclusions: This newly designedmagnetic nanoparticle separator allowed a removal of the nanoparticles after terminating the reaction. Using this system, a strong dose-dependent inhibition of the growth of E. coli by the laccase iron oxide nanoparticles was determined. General significance: Fromour data we conclude that the sponge laccase is involved in the anti-bacterial defense of the sponge organism.