Molecular Characterization of a Recombinant Manganese Superoxide Dismutase from Lactococcus lactis M4

Abstract EN

A superoxide dismutase (SOD) gene of Lactococcus lactis M4 was cloned and expressed in a prokaryotic system.

Sequence analysis revealed an open reading frame of 621 bp which codes for 206 amino acid residues.

Expression of sodA under T7 promoter exhibited a specific activity of 4967 U/mg when induced with 1 mM of isopropyl-β-D-thiogalactopyranoside.

The recombinant SOD was purified to homogeneity by immobilised metal affinity chromatography and Superose 12 gel filtration chromatography.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blot analyses of the recombinant SOD detected a molecular mass of approximately 27 kDa.

However, the SOD was in dimer form as revealed by gel filtration chromatography.

The purified recombinant enzyme had a pI of 4.5 and exhibited maximal activity at 25°C and pH 7.2.

It was stable up to 45°C.

The insensitivity of this lactococcal SOD to cyanide and hydrogen peroxide established that it was a MnSOD.

Although it has 98% homology to SOD of L.

lactis IL1403, this is the first elucidated structure of lactococcal SOD revealing active sites containing the catalytic manganese coordinated by four ligands (H-27, H-82, D-168, and H-172).