Purification and partial characterization of superoxide dismutase from the thermophilic bacteria Thermothrix sp

Abstract

Superoxide dismutase (SOD; EC 1.15.1.1), a high molecular weight component of the antioxidant defense system, provided promising results in the treatment of oxidative damage. Thermothrix sp., isolated from thermal spa water in Serbia, showed high superoxide dismutase activity. The SOD, from cell free extract, was purified to homogenity by ammonium sulfate precipitation, Sephadex G 75 gel filtration chromatography and QAE Sephadex ion exchange chromatography. The specific activity of the purified enzyme was 9191 U/mg. The purified enzyme was analyzed and partially characterized. SOD was localized in polyacrylamide gel by activity staining, based on the reduction of nitroblue tetrazolium (NBT) by superoxide. The enzyme molecular weight determined by gel chromatography is 37 kD. According to SDS PAGE it is composed of two subunits of equal size, joined by noncovalent interactions. The isoelectric point, assessed by isoelectric focusing is 5.3. The optimum pH for enzyme activity was in the range of 8 to 10. The optimum temperature for SOD activity was 60 ?C. After one hour of incubation at 40, 50 and 60 ?C the SOD activity increases, but at 80 ?C, the SOD is denaturated. After 24 hours of incubation at 25 ?C SO Dactivity only slightly decreases.

Cloning and characterization of a thermostable superoxide dismutase from the thermophilic bacterium Rhodothermus sp. XMH10

“…When the temperature went up to 80°C, the RhSOD could retain its activity for more than 4 h. However, the SODs from Thermothrix sp. [20], and Thermomyces lanuginosus [7], were totally inactivated only after 1-h incubation at 80°C. Although the mechanism responsible for protein thermostability is not clear yet, the increased number of charged residues and hydrophobic residues, increased number of ion-pairs, and the increased buried surface area are deWnitely involved [8,26].…”

“…Superoxide dismutase assay SOD activity was assayed based on its ability to inhibit the reduction of nitroblue tetrazolium (NBT) [22]. One unit of SOD activity was deWned as the amount of enzyme which causes 50% of maximum inhibition of NBT reduction [20,22]. The SOD will show a grey band under the blue background in the nondenaturing PAGE gel after staining in the PMS-MTT solution in daylight for half an hour [9].…”

“…SODs exist in most aerobic bacteria in the form of dimer or tetramer [1,20,21] and found to be mainly responsible for the oxidative stress resistance, controlling the oxygen concentration at an appropriate level [5,13,27]. SODs have also been proved to be involved in acid tolerance in Staphylococcus aureus [4].…”

“…[20], have been investigated due to their potential application in industry. The thermostability is one of the most important properties that have been discussed since thermal denaturation is a common cause of enzyme inactivation in industry [7].…”

Hide Dehairing and Laundry Detergent Compatibility Testing of Thermostable and Solvents Tolerant Alkaline Protease from Hot Spring Isolate <i>Bacillus cohnii</i>U3

“…Furthermore, they are more resistant to chemical reagents and extreme pH in comparison to their mesophilic homologues (Hough and Denson, 1999). Their thermostability is associated with performing the enzymatic reaction at high temperature allows higher substrate concentrations, lower viscosity, the fewer risk of contamination and often higher reaction rates (Seatovic et al, 2004). Therefore, thermophiles were the promising choice for massive enzyme production for industrial applications.…”