Purification and characterization of a periplasmic laccase produced by Sinorhizobium meliloti

Sinorhizobium meliloti CE52G strain produces a periplasmic laccase that has been purified by a two-step procedure involving heat treatment and immobilized metal affinity chromatography (IMAC). The fraction with laccase activity retained its original activity after 24 h of incubation at pH between 4.0 and 8.0 and after 3 h of incubation at 70 °C, pH 7.2 and supplemented with 1.3 M (NH4)2SO4. It proved to be a homodimeric protein with an apparent molecular mass of 45 kDa each subunit and an isoelectric point of 6.2. CE52G laccase was inhibited by halides (NaF and NaCl), ions (Fe3+, Mn2+, and Cu2+), sulfhydryl organic compounds (β-mercaptoethanol and reduced glutathione), and electron flow inhibitors (NaCN and NaF). Laccase activity was strongly enhanced by (NH4)2SO4, Na2SO4, and K2SO4. The effects of all these agents, as well as the probability of a partially unfolding polypeptide chain to enhance the interaction between the substrate and the active site, are discussed. CE52G laccase is a pH- and thermo-stable protein with promising biotechnological applications.