【作者】 夏乐先；

【导师】 柳建设；

【作者基本信息】 中南大学 ， 微生物学， 2008， 博士

【摘要】 本论文比较了不同能量代谢的嗜酸硫杆菌A.ferrooxidans菌、A.thiobacillus菌对闪锌矿单独和混合浸出行为:单独的氧化亚铁硫杆菌和氧化硫硫杆菌浸出18天,锌浸出率分别为61%和20%,浸出速度分别为0.17g/L.day0.06g/L.day。混合浸出时,锌浸出率和浸出速度分别达到了97%和0.3g/L.day。浸出残渣分析表明:在无菌或仅有氧化亚铁硫杆菌的浸出体系的残渣表面存在着元素硫膜。然而,在氧化硫硫杆菌的浸出体系中矿物残渣表面没有硫的覆盖。结合各自的浸出率表明,这些硫膜阻碍了细菌与矿物表面的直接接触,降低了锌的浸出。氧化亚铁硫杆菌能够不断地补充浸出过程中被消耗的高价铁,使溶液保持一个高的氧化还原电位,从而使生物浸出能得以继续。氧化硫硫杆菌的角色是使元素硫膜氧化成为硫酸,消除了元素硫膜这一“钝化层”,加速酸浸效果,降低浸出体系pH,抑制铁矾的形成,有利于闪锌矿的化学性浸出。通过A.caldus在Cu²⁺胁迫环境下生长行为表明,Cu²⁺毒性能够表现在抑制细菌的呼吸作用及参与能量代谢的酶活性。在铜离子加入培养基后,对呼吸作用的抑制有约30分钟的延迟效应。当A.caldus生长在以硫单质为能源时,Cu²⁺影响细菌硫代谢中的亚硫酸氧化酶和APS还原酶的活性,对其他几种被调查的酶没有明显的抑制作用,进一步的试验表明Cu²⁺对酶的抑制作用是一种间接行为,表现为一种负相关性。分离和测定A.caldus各亚细胞结构部分的酶活及随pH的酶活性变化表明,所研究的五种硫代谢酶均位于A.caldus的细胞质或细胞内膜上。蛋白酶和缬氨霉素处理细菌表明,A.caldus能够通过膜上的跨膜转运蛋白的作用将胞内Cu²⁺转运出胞外,从而维持着胞内较低的Cu²⁺浓度,并且这种转运过程是与能量的消耗相关联。其转运蛋白类似于ATP泵的作用。从A.ferrooxidans ATCC 23270基因组中克隆并证实了一个尚未注明功能的基因,其表达产物能将硫化物中电子传递给cytochromec,因而证明该基因为硫化物脱氢酶基因。该酶在中性pH活性最高,暗示可能位于细胞质或细胞内膜。该基因在起始密码子的上游7个碱基处有典型的GGAG序列（核糖体结合位点）但无典型的-10区和-35区结构。编码的氨基酸序列与其它细菌来源的同源蛋白均含有结合FAD辅基的保守结合区和含保守的两个半胱氨酸,后者通过形成和解除二硫键,从而结合硫化氢使之成为活性位点。与SQR在氨基酸序列上有很高的同源性（39%）,在N末端包含βαβ结构域、保守的FAD结合区及与硫化物氧化有关的保守的半胱氨酸活性位点。半定量和实时定量PCR结果显示:相同能源下,FCSD的mRNA表达丰度明显高于SQR。在不同能源下,他们在亚铁培养下的mRNA表达丰度均高于单质硫和硫代硫酸钠培养下的mRNA表达丰度。然而,他们的蛋白酶的活性,以单质硫培养的酶活性最高,其次是亚铁培养的,活性最小的为硫代硫酸钠培养。这表明两个基因的表达即受到mRNA转录水平的调控,更重要的受到蛋白质翻译水平的调控。最终的蛋白酶活性测定显示FCSD和SQR在以单质硫培养的菌体内含有较其它能源培养高的酶活性,表明两者均参与了单质硫的氧化代谢。结合同工酶SQR的定位推测,两种酶在催化硫化氢的氧化时可能扮演了不同的角色,前者负责胞内硫化氢氧化,后者负责胞外硫化氢氧化。基于16s序列的系统发育分析,两个基因均出现在从古菌到真细菌的广大分布领域。他们均是一个出现较古老的参与硫代谢酶基因。FCSD的分布大于SQR的分布范围。从分布广度和mRNA表达丰度分析,FCSD在进化地位和对细菌硫代谢的贡献可能大于SQR。基于Chromatium vinosum的硫化物脱氢酶的同源模建表明,在A.ferrooxidans菌中硫化物脱氢酶中的氨基酸Cys158和Cys331参与二硫键的形成,该二硫键的成键与解键对酶与硫化物的结合与酶的催化扮演重要角色。另外,在FAD周围,推测Gly298,Ser299,Phe330,Phe332参与了蛋白质与辅基FAD之间的电子传递,Glu164与接收电子有关。对定点突变酶E164A,S299A活性测定表明:突变酶活性与非突变酶相比有很大的降低。证实上述推理的合理性,这几个氨基酸在硫化物的氧化过程中发挥了关键作用。构建了硫化物脱氢酶/纳米金/半胱氨酸/金修饰电极。该修饰电极在0.02mol/L PBS buffer（pH7.4）溶液中循环伏安扫描可见一对稳定、准可逆的氧化还原峰,其阳极峰电位（Epa）和阴极峰电位（Epc）分别为0.13V和-0.034V,式电位E0′=（Epa+Epc）/2=63mV。加入硫化钠到测试底液后,修饰电极氧化峰电流增强,还原峰电流减弱,表明固定在电极表面的硫化物脱氢酶对Na₂S有电化学催化作用。对硫化钠浓度的计时响应表明,修饰电极对底物硫化物电化学响应迅速,在平均4s内达到稳态电流95%。在2×10^-4-2.5×10^-3 mol/L范围内,响应电流与硫化钠浓度呈线性关系,其线性回归方程:i_p=0.4819+2.64c,相关系数为0.992,最低检测下限1.1×10^-5mol/L。固定在电极表面的酶的km^app为4.5mmol/L。Fe³⁺(1.5×10^-3mol/L)、K⁺(1.5×10^-3mol/L)、NO₃^-(1.5×10^-3mol/L)、SO₄^2-(1.5×10^-3mol/L)、柠檬酸(1.5×10^-3mol/L)、葡萄糖(1.5×10^-3mol/L)等六种物质对硫化钠的干扰很小,电极选择性高。考察了不同时间和批次的修饰电极对同一浓度的硫化钠(6.0×10^-4 mol/L)的响应,相对标准偏差RSD＜5%,表明修饰电极有良好的重现性和较好的稳定性。更多还原

【Abstract】 This thesis compares the different energy metabolism of A.ferrooxidans Thiobacillus acidophilus bacteria and A.thiobacillus bacteria on a separate sphalerite and mixed leaching.A higher bioleaching ratio（61%） was reached when using Acidithiobacillus ferriooxidans mixed bacteria compared to that using Acidithiobacillus thiooxidans（20%） after bioleaching for 18 days.What’s more,the bioleaching ratio and rate were reached 97%and 0.3g/L.day when using mixed bacteria,respectively.The bioleaching rates were only reached 0.17 g/L.day for Acidithiobacillus ferriooxidans and 0.06g/L.day for Acidithiobacillus thiooxidans.Elemental sulfur was overlayed on the surface of solid residues from system with sterile or Acidithiobacillus ferriooxidans.However,there was not detected about elemental sulfur on residues surface from system with Acidithiobacillus thiooxidans due to its oxidizing-sulfur property.The elemental sulfur may inhibit the bioleaching when considering the different bioleaching rates.A high redox potential can be maintained by Acidithiobacillus ferriooxidans which can oxidize ferrious iron to ferric iron.Thus,ferric iron can be continuously generated,as a result,a continuous leaching be maintained.The role of A.thiooxidans is to oxidize and dissolve the sulfur layer（passivation） formed on mineral surface which is benifical to enhance the chemical leaching.Also,acid leaching can be improve and jarosite be inhibited due to the oxidization of elemental sulfur to sulfuric acid.The toxicity of Cu²⁺ was reflected in the inhibition of bacterial respiration.However,the inhibition becomes obvious only after Cu²⁺ was added into medium for 30 minters.When bacterias are grown in sulfur medium containing copper ions,copper ions inhibit the activities of sulfite oxidase and APS reductase.Little inhibition is happen to other involved enzymes in sulfur metabolism.Further experiments show that copper ions have an indirect influence on enzymatic activities.The cellular location of enzymes was determined by measuring enzymatic activities under the condition of different pH and cellular components.As was testified by the treatment of protease and valinomycin on bacteria,Acidithiobacillus caldus can maintain the low concentration of copper ions in cytoplasm by the transportation of membrane protein from inside cell to outside cell.The membrane protein is similar to ATP-pump associated with the consumpation of ATP.The analysis on enzymatic activities showed that a gene can encode the sulfide dehydrogenase which can oxidize sulfide to elemental sulfur and transport the electron from sulfide to cytochrome c.Its enzymatic activity is highest at about pH 7 when measued in different pH and different cellular components,this suggests that the protein should be located in cytoplasm.A ribosome-binding site（GGAG） is detected at 7 bp upstream of start code from the above gene.The -10 and -35 domains,however,are not found in FCSD.Similar to other homogenous proteins,there are some high conserved amino acid sequences binding FAD in sulfide dehydrogenase.In addition,two cysteamine can form disulfide bond which is in relation to sulfide binding to protein.There are high homogenous（39%） in amino acid sequences between SQR and FCSD. There are also conserved FAD-binding domains,βαβdomain and disulfide bond site in the amino acid sequences of both SQR and FCSD.When bacteria are grown in medium with ferrious irons as growth energy,the mRNA expression levels of FCSD and SQR are higher than that of bacteria grown in medium with sulfur and thiosulfate.However, their enzymatic activites are highest in sulfur medium,and lowest in thiosulfate.This indicates that the expression levels of FCSD and SQR are regulated on transcribe and translation,especially,the latter are strongly affect the expression of FCSD and SQR.Based on the analysis on expression levels,FCSD and SQR perform the function of sulfur metabolism.It is analysed and compared about FCSD,SQR and corresponding bacterial 16srDNA uploaded in NCBI from various species.The results show that SQR and FCSD are evolutionary in ancient epoch and extentive distribution from archaea to bacteria.FCSD are more important than SQR to sulfur metabolism and sulfur cycles on ancient earth due to the higher expression level and more extentive distribution of FCSD.A preliminary model for the FCSD protein was constructed using the approach of comparative protein modeling on the basis of the sequences of the FCSD protein from Chromatium vinosum.Cyc 158 and cyc 331 can form the disulfide bond.The formation and disconnection of disulfide bond have a very important influence on binding sulfide to protein and catalyzing the sulfide oxidization.In addition,Gly298, Ser299,Phe330 and Phe332 perform the electron transfer between protein and FAD.Further,Glul64 is in relation to accepting electron from FAD.We constructed the mutant expression plasmids of $299A and G164A residues using site directed mutagenesis.Mutant proteins were expressed in E.coli and purified.Enzymatic activities of mutation S299A and G164A are very lower than wild protein.This reveals that ratiocination based on model are true and these residues play key roles to enzymatic activity.FCSD was successfully assembled on a gold electrode by means of nano-gold and cyctemise.The Cyclic voltammmetric response of the modified gold electrode was investigated under the condition of 0.02mol/L PBS buffer（pH 7.4）.A pair of stable and quasi-reversible redox peak was observed from cyclic voltammograms.Epc and Epa are 0.13 and-0.034v,respectively.The form potential is 63my.Epa increase and Epc decrease when Na₂S is added into electrolyte.This indicates that the immobilized FCSD can electrochemically catalyze the oxidization of sulfide.The i-t experiment shows that the modified enzymatic gold electrode is very sensitive to response to sulfide.The response time is about 4s when attaining a stable current of 95%.The response current is linely proportionable to the concentration of sulfide at the range of sulfide from 2×10^-4 to 2.5×10^-3 mol/L.Tthe equation is ip=0.4819+2.64c with a correlation coefficient of 0.992.The response sensitiablity to sulfide is 1.1×10^-5 mol/L and km^app is 4.5mmol/L.To estimate the stability and repetition of modified electrode, electrochemical behavior was investigated after some reagents bein g added into electrolyte such as Fe³⁺(1.5×10^-3mol/L),K⁺(1.5×10^-3mo 1/L),NO₃^-(1.5×10^-3mol/L),SO₄^2-(1.5×10^-3mol/L),Citric acid(1.5×10^-3mol/L),Glucose(1.5×10^-3mol/L).The results show that these reag ents have not influence on response to sulfide.In addition,the mo dified electrodes show also high stability and repetition（RSD＜5%） about the electrodes from various constructed time.更多还原