Limits...
Molecular characterization of a recombinant manganese superoxide dismutase from Lactococcus lactis M4.

Tan BH, Chor Leow T, Foo HL, Abdul Rahim R - Biomed Res Int (2014)

Bottom Line: 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).

View Article: PubMed Central - PubMed

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

ABSTRACT
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).

Show MeSH
Effect of temperature on SOD activity (a) and stability (b). The purified SOD was assayed at different temperatures (5 to 65°C). Thermostability of SOD was measured by incubating SOD at various temperatures (25–60°C) for 60 min prior to SOD assay at its optimum temperature.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3921932&req=5

fig6: Effect of temperature on SOD activity (a) and stability (b). The purified SOD was assayed at different temperatures (5 to 65°C). Thermostability of SOD was measured by incubating SOD at various temperatures (25–60°C) for 60 min prior to SOD assay at its optimum temperature.

Mentions: The SOD was highly active between 20°C and 30°C with an optimum temperature at 25°C. The enzyme was thermostable up to 45°C by retaining more than 85% of SOD activity. Further treatment above 45°C caused the activity to decrease drastically and completely deactivated at 60°C (Figure 6), indicating it was susceptible to thermal inactivation. These observations suggest that SOD may not be involved in the heat shock or cold shock regulation in L. lactis. Lactococcal cells grow at low temperatures by merely slowing down biological processes whereas growth at high temperature is deleterious to the cell [26]. In support of the results obtained in this work, most of the MnSODs are stable in the range from 25 to 45°C, except for MnSODs derived from the thermophiles which exhibited higher stability [27] due to thermal adaptation at functional environment.


Molecular characterization of a recombinant manganese superoxide dismutase from Lactococcus lactis M4.

Tan BH, Chor Leow T, Foo HL, Abdul Rahim R - Biomed Res Int (2014)

Effect of temperature on SOD activity (a) and stability (b). The purified SOD was assayed at different temperatures (5 to 65°C). Thermostability of SOD was measured by incubating SOD at various temperatures (25–60°C) for 60 min prior to SOD assay at its optimum temperature.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3921932&req=5

fig6: Effect of temperature on SOD activity (a) and stability (b). The purified SOD was assayed at different temperatures (5 to 65°C). Thermostability of SOD was measured by incubating SOD at various temperatures (25–60°C) for 60 min prior to SOD assay at its optimum temperature.
Mentions: The SOD was highly active between 20°C and 30°C with an optimum temperature at 25°C. The enzyme was thermostable up to 45°C by retaining more than 85% of SOD activity. Further treatment above 45°C caused the activity to decrease drastically and completely deactivated at 60°C (Figure 6), indicating it was susceptible to thermal inactivation. These observations suggest that SOD may not be involved in the heat shock or cold shock regulation in L. lactis. Lactococcal cells grow at low temperatures by merely slowing down biological processes whereas growth at high temperature is deleterious to the cell [26]. In support of the results obtained in this work, most of the MnSODs are stable in the range from 25 to 45°C, except for MnSODs derived from the thermophiles which exhibited higher stability [27] due to thermal adaptation at functional environment.

Bottom Line: 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).

View Article: PubMed Central - PubMed

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

ABSTRACT
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).

Show MeSH