Purification and characterization of the first bacterial laccase in the rhizospheric bacterium Azospirillum lipoferum

Azospirillum lipoferum is a soil bacterium known for its ability to colonize roots and to promote plant growth. Recently, a laccase-like polyphenol oxidase activity has been reported in a strain isolated from the rhizosphere of rice. Purification to apparent homogeneity of A. lipoferum laccase was achieved after cell disruption by sonication, protein solubilization by Triton X-100, acetone and protamine precipitation, hydroxyapatite column chromatography in the presence of urea (5 M) and NaCl (2 M), and finally by conventional hydroxyapatite column chromatography. After this final step, laccase was in an inactive form but could be reactivated by adding sodium dodecyl sulfate (SDS). Based on gel electrophoresis results, we suggest that native A. lipoferum laccase is composed of a catalytic polypeptide chain with low molecular mass (16.3 kDa) and one or two regulatory/structural chains with high molecular mass (81.5 kDa). The purified enzyme was found to be thermostable up to 70°C for 10 min, had an optimal pH of 6.0, and was inhibited by tropolone, a known inhibitor of metal-containing enzymes. Although the natural substrate of this enzyme is unknown, its biochemical characterization may facilitate further investigations on the ecological role of this laccase in the process of root colonization by A. lipoferum.