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Cloning, Expression, and Characterization of Thermotolerant Manganese Superoxide Dismutase from Bacillus sp. MHS47

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Author(s): Supatra Areekit | Pornpimon Kanjanavas | Paisarn Khawsak | Arda Pakpitchareon | Kajeenart Potivejkul | Gaysorn Chansiri | Kosum Chansiri

Journal: International Journal of Molecular Sciences
ISSN 1422-0067

Volume: 12;
Issue: 1;
Start page: 844;
Date: 2011;
Original page

Keywords: thermotolerant | MnSOD | cloning | expression | Bacillus sp.

ABSTRACT
A superoxide dismutase gene from thermotolerant Bacillus sp. MHS47 (MnSOD47) was cloned, sequenced, and expressed. The gene has an open reading frame of 612 bp, corresponding to 203 deduced amino acids, with high homology to the amino acid sequences of B. thuringiensis (accession no. EEN01322), B. anthracis (accession no. NP_846724), B. cereus (accession no. ZP_04187911), B. weihenstephanensis (accession no. YP_001646918), and B. pseudomycoides. The conserved manganese-binding sites (H28, H83, D165, and H169) show that MnSOD47 has the specific characteristics of the manganese superoxide dismutase (MnSOD) enzymes. MnSOD47 expressed an enzyme with a molecular weight of approximately 22.65 kDa and a specific activity of 3537.75 U/mg. The enzyme is active in the pH range 7–8.5, with an optimum pH of 7.5, and at temperatures in the range 30–45 °C, with an optimum temperature of 37 °C. Tests of inhibitors and metal ions indicated that the enzyme activity is inhibited by sodium azide, but not by hydrogen peroxide or potassium cyanide. These data should benefit future studies of MnSODs in other microorganisms and the biotechnological production of MnSOD47, and could also be used to develop a biosensor for the detection of antioxidants and free radical activity. In the future, this basic knowledge could be applicable to the detection of cancer risks in humans and therapeutic treatments.
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