Molecular Characterization of a Recombinant Manganese Superoxide Dismutase fromLactococcus lactisM4

Author:

Tan Boon Hooi1ORCID,Chor Leow Thean12,Foo Hooi Ling23,Abdul Rahim Raha12

Affiliation:

1. Department of Cell and Molecular Biology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia (UPM), 43400 Serdang, Selangor, Malaysia

2. Institute of Biosciences, Universiti Putra Malaysia (UPM), 43400 Serdang, Selangor, Malaysia

3. Department of Bioprocess Technology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia (UPM), 43400 Serdang, Selangor, Malaysia

Abstract

A superoxide dismutase (SOD) gene ofLactococcus lactisM4 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 ofsodAunder 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 ofL. lactisIL1403, 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).

Funder

Ministry of Science, Technology and Innovation

Publisher

Hindawi Limited

Subject

General Immunology and Microbiology,General Biochemistry, Genetics and Molecular Biology,General Medicine

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