硫化氢脱氢酶的分离纯化及性质研究
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摘要
从来源于烟气生物脱硫系统的好氧产硫磁性稳态流化床反应器(aMSFB)中取磁性多孔珠固定化生物膜,采用排硫硫杆菌培养基在好氧条件下,经富集培养、反复分离纯化获得纯分离物。对分离物进行形态、生理生化分析,结果如下:呈短杆状,大小为0.3～0.5×1.1～1.5μm,最适生长温度为28℃,最适pH为7.0;革兰氏染色阴性,端生鞭毛;在硫代硫酸盐琼脂上的菌落为圆形,表面隆起,边缘完整,直径1mm-2mm左右;菌落开始透明,随后因硫沉淀而呈白色,进一步菌落中心变为褐色;液体长期培养pH值会下降,且有单质硫产生;液体静置培养时形成粘膜,粘膜具有下垂的纤丝;严格自养,通过氧化硫代硫酸盐、硫化物、连四硫酸盐获取能量,积累硫粒,缓慢氧化元素硫,以硝酸盐、铵盐作为氮源。对应伯杰氏细菌鉴定手册,初步鉴定该分离物为硫杆菌属(thiobacillus)的排硫硫杆菌(thiobacillus thioparus)。
将反复纯化分离得到的排硫硫杆菌细胞经超声破碎后,采用四步工艺分离纯化出一种新型的膜结合型的硫化氢脱氢酶。光谱分析表明该酶含有1分子的血红素c和一分子的FAD; SDS-PAGE表明该酶由分子量42.6kDa和51.3 kDa的两个亚基构成;该酶属于氧还蛋白家族,其最适pH为8.5,最大反应速度为4.9±0.1μM cytc (mg蛋白) ~(-1)min~(-1),对马心细胞色素c和硫化物的表观Km分别为2.1±0.3μM和6.1±0.8μM;电子转移化学计量关系表明该酶的氧化产物为元素硫。
Isolation of the bacteria was performed from aerobic magnetic stabilized fluidized bed reactor of flue gas biodesulfurization system. The immobilized biofilm samples were cultured on Thiobacillus enrichment media under aerobic conditions. The isolated cultures were obtained through repeated separation and purification. The isolated bacteria was identified to find out that: the morphology of the bacteria was rod-shaped; the average size of the bacteria was 0.3～0.5×1.1～1.5μm; the optimum growth temperature was 28oC; the optimum PH was 7.0. The results of gram staining for the bacteria indicated that the bacteria were negative with flagella on its side. The colonies on Thiosulphate agar media were round-shaped with bump surface, integrated fringe and the diameter of them is about 1mm-2mm. The colony was transparent first, then it became white for the sulphur sediment and at last the centre of the colony turned brown. After the long-term culture, the media PH dropped and the elemental sulphur was yielded. The film with sag fibers was formed when the samples were cultured stably in the liquid. The bacteria were strictly autotrophic and they derived energy for growth from the oxidation of thiosulphate, sulphide, Na_2S_4O_6 with nitrate and ammonium as nitrogen source to accumulate elemental sulphur particles. Then elemental sulphur particles may be oxidated continually. According to Bergey's Manual of Determinative Bacteriology, the culture was elemently identified as Thiobacillus thioparus of Thiobacillus genus.
A novel membrane-bound sulfide dehydrogenase enzyme was purified from the neutrophilic, obligately chemolithoautotrophic Thiobacillus thioparus by means of a four-step procedure. Spectral analysis revealed that the enzyme contains haem c and flavin. SDS-PAGE showed the presence of two types of subunit with molecular masses of 42.6 and 51.3 kDa. A combination of spectral analysis and the pyridine haemochrome test indicated that the sulfide dehydrogenase heterodimer contains one molecular of haem c and one molecular of flavin. It appeared that the sulfide dehydrogenase is a member of a small class of redox proteins. The pH optimum of the enzyme is 8.5. The Vmax was 4.9±0.1μmol cytochrome c(mg protein×min)~(-1), and the Km values for cytochromes and sulfide were 2.1±0.3μM and 6.1±0.8μM, respectively. On the basis of electron transfer stoichiometry, it seems likely that sulfur is the oxidation product.
将反复纯化分离得到的排硫硫杆菌细胞经超声破碎后,采用四步工艺分离纯化出一种新型的膜结合型的硫化氢脱氢酶。光谱分析表明该酶含有1分子的血红素c和一分子的FAD; SDS-PAGE表明该酶由分子量42.6kDa和51.3 kDa的两个亚基构成;该酶属于氧还蛋白家族,其最适pH为8.5,最大反应速度为4.9±0.1μM cytc (mg蛋白) ~(-1)min~(-1),对马心细胞色素c和硫化物的表观Km分别为2.1±0.3μM和6.1±0.8μM;电子转移化学计量关系表明该酶的氧化产物为元素硫。
Isolation of the bacteria was performed from aerobic magnetic stabilized fluidized bed reactor of flue gas biodesulfurization system. The immobilized biofilm samples were cultured on Thiobacillus enrichment media under aerobic conditions. The isolated cultures were obtained through repeated separation and purification. The isolated bacteria was identified to find out that: the morphology of the bacteria was rod-shaped; the average size of the bacteria was 0.3～0.5×1.1～1.5μm; the optimum growth temperature was 28oC; the optimum PH was 7.0. The results of gram staining for the bacteria indicated that the bacteria were negative with flagella on its side. The colonies on Thiosulphate agar media were round-shaped with bump surface, integrated fringe and the diameter of them is about 1mm-2mm. The colony was transparent first, then it became white for the sulphur sediment and at last the centre of the colony turned brown. After the long-term culture, the media PH dropped and the elemental sulphur was yielded. The film with sag fibers was formed when the samples were cultured stably in the liquid. The bacteria were strictly autotrophic and they derived energy for growth from the oxidation of thiosulphate, sulphide, Na_2S_4O_6 with nitrate and ammonium as nitrogen source to accumulate elemental sulphur particles. Then elemental sulphur particles may be oxidated continually. According to Bergey's Manual of Determinative Bacteriology, the culture was elemently identified as Thiobacillus thioparus of Thiobacillus genus.
A novel membrane-bound sulfide dehydrogenase enzyme was purified from the neutrophilic, obligately chemolithoautotrophic Thiobacillus thioparus by means of a four-step procedure. Spectral analysis revealed that the enzyme contains haem c and flavin. SDS-PAGE showed the presence of two types of subunit with molecular masses of 42.6 and 51.3 kDa. A combination of spectral analysis and the pyridine haemochrome test indicated that the sulfide dehydrogenase heterodimer contains one molecular of haem c and one molecular of flavin. It appeared that the sulfide dehydrogenase is a member of a small class of redox proteins. The pH optimum of the enzyme is 8.5. The Vmax was 4.9±0.1μmol cytochrome c(mg protein×min)~(-1), and the Km values for cytochromes and sulfide were 2.1±0.3μM and 6.1±0.8μM, respectively. On the basis of electron transfer stoichiometry, it seems likely that sulfur is the oxidation product.
引文
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[6] Beffa, T., Berczy, M., and Aragno, M. Inhibition of respiratory oxidation of elemental sulfur (S0) and thiosulfate in Thiobacillus versutus and another sulfur-oxidizing bacterium. FEMS Microbiol. Lett, 1992a, 90: 123–128.
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[8] Chan, C.W., and Suzuki, I. Thiosulfate oxidation by sulfur grown Thiobacillus thiooxidans cells, cell-free extracts, and thiosulfate-oxidizing enzyme. Can. J. Microbiol, 1994, 40: 816–822.
[9] Beffa, T., Fischer, C., and Aragno, M. Growth and respiratory oxidation of reduced sulfur compounds by intact cells of Thiobacillus novellus (Type strain) grown on thiosulfate. Curr. Microbiol, 1993a, 26: 323–326.
[10] Suzuki, I., Lee, D., MacKay, B., Harahuc, L., and Oh, J.K. Effect of vairous ions, pH, and osmotic pressure on oxidaiton of elemental sulfur by Thiobacillus thiooxidans. Appl. Environ. Microbiol, 1999, 65: 5163–5168.
[11] 廖嘉陵,苏士军,丁桑岚. 微生物烟气脱硫技术研究进展[J]. 四川环境,2006,25(1):79–83.
[12] Uisman C J N. Biotechnological sulfide removal from effiuent [J]. Wat Sci &Tech, 1991, 24(3-4):347–356.
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