ORIGINAL ARTICLE The characterization of a thermostable and cambialistic superoxide dismutase from Thermus filiformis F. Mandelli 1,2 , J.P.L. Franco Cairo 1 , A.P.S. Citadini 1 , F. B€ uchli 1 , T.M. Alvarez 1 , R.J. Oliveira 3 , V.B.P. Leite 3 , A.F. Paes Leme 4 , A.Z. Mercadante 2 and F.M. Squina 1 1 Laborat orio Nacional de Ci^ encia e Tecnologia do Bioetanol (CTBE), do Centro Nacional de Pesquisa em Energia e Materiais (CNPEM), Campinas, Brazil 2 Departamento de Ci^ encia de Alimentos da Faculdade de Engenharia de Alimentos, UNICAMP, Campinas, Brazil 3 Departamento de F  ısica, Instituto de Bioci^ encias, Letras e Ci^ encias Exatas, Universidade Estadual Paulista, S~ ao Jos e do Rio Preto, Brazil 4 Laborat orio Nacional de Bioci^ encias (LNBio), do Centro Nacional de Pesquisa em Energia e Materiais (CNPEM), Campinas, Brazil Significance and Impact of the Study: This manuscript describes the expression and characterization of a superoxide dismutase (SOD) from Thermus filiformis with thermophilic and cambialistic characteristics. The SODs are among the most potent antioxidants known in nature, and their stability and pharmacoki- netics can vary widely in accordance to their biological source. Although the currently clinical research work has been focused on human and bovine SODs, alternative sources may become more biotechno- logical attractive in the near future. Our study brings new insights for the research field of antioxidant enzymes with potential application on pharmaceutical, cosmetics and food formulations. Keywords 3-D model, cambialistic enzyme, circular dichroism, nitroblue tetrazolium, pyrogallol autoxidation. Correspondence F.M. Squina, Laborat orio Nacional de Ci^ encia e Tecnologia do Bioetanol (CTBE), do Centro Nacional de Pesquisa em Energia e Materiais (CNPEM), Rua Giuseppe M aximo Scolfaro, nº 10000, Campinas, SP-13083-970, Brazil. E-mail: fabio.squina@bioetanol.org.br 2012/2317: received 27 December 2012, revised 15 March 2013 and accepted 15 March 2013 doi:10.1111/lam.12071 Abstract The superoxide dismutase (TfSOD) gene from the extremely thermophilic bacterium Thermus filiformis was cloned and expressed at high levels in mesophilic host. The purified enzyme displayed approximately 25 kDa band in the SDS-PAGE, which was further confirmed as TfSOD by mass spectrometry. The TfSOD was characterized as a cambialistic enzyme once it had enzymatic activity with either manganese or iron as cofactor. TfSOD showed thermostability at 65, 70 and 80°C. The amount of enzyme required to inhibit 50% of pyrogallol autoxidation was 0Á41, 0Á56 and 13Á73 mg at 65, 70 and 80°C, respectively. According to the circular dichroism (CD) spectra data, the secondary structure was progressively lost after increasing the temperature above 70°C. The 3-dimensional model of TfSOD with the predicted cofactor binding corroborated with functional and CD analysis. Introduction Antioxidant enzymes such as superoxide dismutase (SOD) and catalase protect living cells from reactive oxygen and nitrogen species that are responsible for oxidative damage of essential components of cell structure (Pantazaki et al. 2002). Superoxide dismutases are a class of antioxidant defence metalloenzymes that disproportionate superoxide radical ion into molecular oxygen and hydrogen peroxide (Whittaker and Whittaker 1999). Due to its antioxidative effects, SOD has been widely applied in medical treatments, as well as cosmetic, food, agricultural and chemical indus- tries (Liu et al. 2011). SODs have recently found applica- tions as supplementation to prevent or reverse the adverse effect of cardiovascular diseases, ageing, infertility, neuro- logical disorders, ischaemia–reperfusion injury, transplant rejection, autoimmune diseases, rheumatoid arthritis, diabetes, asthma, septic shock-induced tissue injury and cancer, as well as in the pharmaceutical and cosmetic industries (Bafana et al. 2011). Once thermal denaturation is a common cause of enzyme inactivation in the industry, one of the major 40 Letters in Applied Microbiology 57, 40--46 © 2013 The Society for Applied Microbiology Letters in Applied Microbiology ISSN 0266-8254