Molecular characterization of a recombinant managanese superoxide dismutase from Lactococcus lactis M4
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 induce...
| Main Authors: | , , , |
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| Format: | Article |
| Language: | English |
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Hindawi Publishing Corporation
2014
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| Online Access: | http://psasir.upm.edu.my/id/eprint/34572/1/Molecular%20Characterization%20of%20a%20Recombinant%20Manganese.pdf |
| _version_ | 1825948476082487296 |
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| author | Tan, Boon Hooi Leow, Adam Thean Chor Foo, Hooi Ling Abdul Rahim, Raha |
| author_facet | Tan, Boon Hooi Leow, Adam Thean Chor Foo, Hooi Ling Abdul Rahim, Raha |
| author_sort | Tan, Boon Hooi |
| collection | UPM |
| description | 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). |
| first_indexed | 2024-03-06T08:29:24Z |
| format | Article |
| id | upm.eprints-34572 |
| institution | Universiti Putra Malaysia |
| language | English |
| last_indexed | 2024-03-06T08:29:24Z |
| publishDate | 2014 |
| publisher | Hindawi Publishing Corporation |
| record_format | dspace |
| spelling | upm.eprints-345722015-12-16T01:41:14Z http://psasir.upm.edu.my/id/eprint/34572/ Molecular characterization of a recombinant managanese superoxide dismutase from Lactococcus lactis M4 Tan, Boon Hooi Leow, Adam Thean Chor Foo, Hooi Ling Abdul Rahim, Raha 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). Hindawi Publishing Corporation 2014 Article PeerReviewed application/pdf en http://psasir.upm.edu.my/id/eprint/34572/1/Molecular%20Characterization%20of%20a%20Recombinant%20Manganese.pdf Tan, Boon Hooi and Leow, Adam Thean Chor and Foo, Hooi Ling and Abdul Rahim, Raha (2014) Molecular characterization of a recombinant managanese superoxide dismutase from Lactococcus lactis M4. BioMed Research International, 2014. art. no. 469298. pp. 1-9. ISSN 2314-6133; ESSN: 2314-6141 http://www.hindawi.com/journals/bmri/2014/469298/abs/ 10.1155/2014/469298 |
| spellingShingle | Tan, Boon Hooi Leow, Adam Thean Chor Foo, Hooi Ling Abdul Rahim, Raha Molecular characterization of a recombinant managanese superoxide dismutase from Lactococcus lactis M4 |
| title | Molecular characterization of a recombinant managanese superoxide dismutase from Lactococcus lactis M4 |
| title_full | Molecular characterization of a recombinant managanese superoxide dismutase from Lactococcus lactis M4 |
| title_fullStr | Molecular characterization of a recombinant managanese superoxide dismutase from Lactococcus lactis M4 |
| title_full_unstemmed | Molecular characterization of a recombinant managanese superoxide dismutase from Lactococcus lactis M4 |
| title_short | Molecular characterization of a recombinant managanese superoxide dismutase from Lactococcus lactis M4 |
| title_sort | molecular characterization of a recombinant managanese superoxide dismutase from lactococcus lactis m4 |
| url | http://psasir.upm.edu.my/id/eprint/34572/1/Molecular%20Characterization%20of%20a%20Recombinant%20Manganese.pdf |
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