排硫硫杆菌D6中硫化氢脱氢酶的纯化及性质

Purification and characterization of hydrogen sulfide dehydrogenase from Thiobacillus thioparus D6

摘要:

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2.5 μmol/L和6.1 μmol/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, 100 μmol/L的氰化钾对该酶抑制率达72%。

Abstract:

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 chromatography 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 sulfur 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%.