Purification and biochemical characterization of manganesecontaining superoxide dismutase from deep-sea thermophile Geobacillus sp. EPT3

摘要

Thermostable SOD is a promising enzyme in biotechnological applications. In the present study, thermophile Geobacillus sp. EPT3 was isolated from a deep-sea hydrothermal field in the East Pacific. A thermostable superoxide dismutase(SOD) from this strain was purified to homogeneity by steps of fractional ammonium sulfate precipitation, DEAE-Sepharose chromatography, and Phenyl-Sepharose chromatography. SOD was purified 13.4 fold to homogeneity with a specific activity of 3 354 U/mg and 11.1% recovery. SOD from Geobacillus sp. EPT3 was of the Mn-SOD type, judged by the insensitivity of the enzyme to both KCN and H2O2. SOD was determined to be a homodimer with monomeric molecular mass of 26.0 k Da. It had high thermostability at 50°C and 60°C. At tested conditions, SOD was relatively stable in the presence of some inhibitors and denaturants, such as β-mercaptoethanol(β-ME), dithiothreitol(DTT), phenylmethylsulfonyl fluoride(PMSF), urea, and guanidine hydrochloride. Geobacillus sp. EPT3 SOD showed striking stability across a wide p H range from 5.0 to 11.0. It could withstand denaturants of extremely acidic and alkaline conditions, which makes it useful in the industrial applications.