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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).

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IEF PAGE analysis and SOD activity staining of purified SOD. Each lane contained 1 μg of purified SOD. Lane 1: broad range pI marker; lane 2: purified SOD with pI value of 4.5; lane 3: purified SOD without any treatment as control; lane 4: SOD treated with 2 mM KCN; lane 5: SOD treated with 3 mM H2O2; and lane 6: SOD treated with 2 mM KCN and 3 mM H2O2. The achromatic zones of the control and the treated enzyme showed no significant differences.
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fig5: IEF PAGE analysis and SOD activity staining of purified SOD. Each lane contained 1 μg of purified SOD. Lane 1: broad range pI marker; lane 2: purified SOD with pI value of 4.5; lane 3: purified SOD without any treatment as control; lane 4: SOD treated with 2 mM KCN; lane 5: SOD treated with 3 mM H2O2; and lane 6: SOD treated with 2 mM KCN and 3 mM H2O2. The achromatic zones of the control and the treated enzyme showed no significant differences.

Mentions: Isoelectric focusing analysis on the purified SOD has shown a pI of 4.5, which is closely related to the calculated pI of L. lactis sp. cremoris MG1363 [9]. When tested with the inhibitors, the results showed that there was no significant difference between the achromatic zones in activity stained gel of the enzyme before and after treatment with cyanide and H2O2, indicating the activity of this enzyme was not inhibited by cyanide or H2O2 (Figure 5). The isoforms of SOD can be distinguished by their different sensitivities to cyanide and H2O2. FeSOD is irreversibly inactivated by H2O2 [24], while CuZnSOD is inhibited by cyanide [25]. MnSOD is resistant to both cyanide and H2O2 [15, 24, 25]. Therefore, the insensitivity of this lactococcal SOD to these two inhibitors confirmed that it was a manganese SOD (MnSOD).


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)

IEF PAGE analysis and SOD activity staining of purified SOD. Each lane contained 1 μg of purified SOD. Lane 1: broad range pI marker; lane 2: purified SOD with pI value of 4.5; lane 3: purified SOD without any treatment as control; lane 4: SOD treated with 2 mM KCN; lane 5: SOD treated with 3 mM H2O2; and lane 6: SOD treated with 2 mM KCN and 3 mM H2O2. The achromatic zones of the control and the treated enzyme showed no significant differences.
© Copyright Policy
Related In: Results  -  Collection

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

fig5: IEF PAGE analysis and SOD activity staining of purified SOD. Each lane contained 1 μg of purified SOD. Lane 1: broad range pI marker; lane 2: purified SOD with pI value of 4.5; lane 3: purified SOD without any treatment as control; lane 4: SOD treated with 2 mM KCN; lane 5: SOD treated with 3 mM H2O2; and lane 6: SOD treated with 2 mM KCN and 3 mM H2O2. The achromatic zones of the control and the treated enzyme showed no significant differences.
Mentions: Isoelectric focusing analysis on the purified SOD has shown a pI of 4.5, which is closely related to the calculated pI of L. lactis sp. cremoris MG1363 [9]. When tested with the inhibitors, the results showed that there was no significant difference between the achromatic zones in activity stained gel of the enzyme before and after treatment with cyanide and H2O2, indicating the activity of this enzyme was not inhibited by cyanide or H2O2 (Figure 5). The isoforms of SOD can be distinguished by their different sensitivities to cyanide and H2O2. FeSOD is irreversibly inactivated by H2O2 [24], while CuZnSOD is inhibited by cyanide [25]. MnSOD is resistant to both cyanide and H2O2 [15, 24, 25]. Therefore, the insensitivity of this lactococcal SOD to these two inhibitors confirmed that it was a manganese SOD (MnSOD).

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