排硫硫杆菌D6中硫化氢脱氢酶的纯化及性质

从烟气生物脱硫系统的好氧产硫磁性稳态流化床反应器中,经反复纯化分离出脱硫优势菌排硫硫杆菌菌株D6,采用四步工艺纯化出膜结合型硫化氢脱氢酶。SDS-PAGE测定显示其由α1β1亚基组成,光谱分析表明含有1 mol FAD/mol酶,血红素染色揭示小亚基上结合有1 mol血红素c/mol酶,该酶属于氧还蛋白家族。该酶的最适pH为8.6,对马心细胞色素c和硫化物的表观Km分别为2.5μmol/L和6.1μmol/L,反应计量实验表明其氧化产物为元素硫。硫化氢脱氢酶受到硫和亚硫酸盐的抑制,100μmol/L的氰化钾对该酶抑制率达72%。

Purification and characterization of hydrogen sulfide dehydrogenase from Thiobacillus thioparus D6

A novel membrane-bound hydrogen sulfide dehydrogenase was purified to homogeneity by a four-step procedure from Thiobacillus thioparus D6,an neutrophilic,obligately chemolithoautotrophic bacteria obtained from aerobic magnetic stabilized fluidized bed reactor of flue gas biodesulfurization system.The natural hydrogen sulfide dehydrogenase had a molecular mass of 95 kD and comprised two subunits named α and β with molecular masses of 42.6 kD and 51.3 kD determined by exclusion chroma-tography and SDS-PAGE.Spectral and pyridine hemochrome analysis revealed that the enzyme contained 1 mol flavin and 1 mol haem c per mol αβ hydrogen sulfide dehydrogenase,assumed a characteristics of the member of redox proteins.Biochemical determination and nonlinear regression analysis showed that the sulfide dehydrogenase catalyzed sulfide-dependent horse heart cytochrome c reduction at the optimum pH of 8.6 with a kcat of 32.4 s?1,a Km of 6.1 μmol/L for sulfide,and a Km of 2.5 μmol/L for cytochrome c.The yield of 1.9 mol of cytochrome c reduction per mole of sulfide suggested that the product was sul-fur or polysulfide.The activity of the sulfide dehydrogenase was inhibited by sulfur and sulfite like that cyanide(100 μmol/L) inhibited sulfide dehydrogenase activity at pH 6.0 by 72%.