【作者】 柳广飞；

【导师】 周集体；

【作者基本信息】 大连理工大学 ， 环境工程， 2009， 博士

【摘要】 近年来偶氮染料的生物降解受到广泛关注,国内外学者陆续分离并考察了众多可脱色偶氮染料的微生物,纯化并研究了多种具有偶氮还原酶活性的蛋白质,但对于偶氮还原酶的结构与功能缺乏深入认识。本论文对细菌脱色偶氮染料的能力,细菌FMN依赖型偶氮还原酶的结构模型与功能等展开研究。考察沼泽红假单胞菌Rhodopseudomonas palustris AS1.2352细胞及其细胞提取物脱色偶氮染料的能力。对于生长过程中的R.palustris AS1.2352,在静置缺氧条件下,可在17 h内脱色80%以上活性艳红X-3B(50 mg L^-1)。细菌在30-35℃和pH 6-9具有较高脱色能力。脱色速率与染料初始浓度间可用米门方程描述,V_max和K_m分别为65 mg gcell^-1h^-1和978 mg L^-1。其细胞提取物具有偶氮还原酶活性,脱色速率远高于未破碎的细菌细胞。对比考察两株光合细菌Rhodobacter sphaeroides AS1.1737,R.palustris AS1.2352和基因工程菌Escherichia coli YB静止期早期细胞脱色含磺酸取代基的偶氮染料的能力。光合细菌表现出较基因工程菌更强的脱色能力。R.sphaeroides AS1.1737,R.palustrisAS1.2352和E.coli YB的V_max/K_m值分别为0.2,0.15和0.06 Lg cell^-1h^-1。两株光合细菌具有较好的脱色混合染料的能力,E.coli YB脱色混合染料的效果不佳。利用同源建模方法建立了R.sphaeroides AS1.1737的偶氮还原酶AZR的结构模型。AZR的单体具有类似黄素氧化还原蛋白的α/β型结构,五个相互平行的β-折叠位于分子中央形成一个平面,五个α-螺旋分别位于平面两侧,一侧两个,另一侧三个;分子中结合的FMN辅基可能作为氧化还原反应中电子传递的中介。尽管依序列可将FMN依赖型偶氮还原酶归为两个家族,但它们的单体都具有类似黄素氧化还原蛋白的三级结构。利用NAD（P）H提供电子,AZR可还原多种硝基化合物。以硝基呋喃为底物,AZR硝基还原酶的最适pH=7,最适温度为50℃。NADPH为更适合的辅酶,反应遵循双底物乒乓动力学机理。比较AZR对2,4,6-三硝基甲苯（TNT）、2,4-二硝基甲苯（2,4-DNT）、2,6-二硝基甲苯（2,6-DNT）等几种多硝基取代甲苯的还原发现,TNT为最适底物;2,4-DNT比2,6-DNT更适合降解。经HPLC/MS检测,TNT的还原产物可能为羟胺二硝基甲苯。AZR能遵循双底物乒乓动力学机理还原外加FMN。NADPH和FMN的米氏常数分别为0.5和14.2 mM,V_max为172.4μmol mg protein^-1min^-1;外加FMN抑制AZR的偶氮还原酶和硝基还原酶活性,是NADPH、甲基红和硝基呋喃的竞争性抑制剂,抑制常数K₁分别为1.7、2.3和6.4μM。AZR具有广泛的底物范围,不但可以还原偶氮化合物、硝基化合物和FMN等有机物,还可以还原铁氰化钾和重铬酸钾等无机底物,是一种新的硝基/FMN还原酶家族的成员。在序列对齐分析和结构模型分析的基础上,选择AZR分子中FMN辅基附近两个环状结构中的Tyr⁷⁴、His⁷⁵和Lys¹⁰⁹等氨基酸位点,利用多次PCR的方法进行了K109A,K109H,Y74W和H75N等定点突变研究。利用E.coli异源表达并纯化几种突变型AZR。对比考察野生型与突变型AZR的活性。以酸性红B为底物,野生型AZR的最适pH为7-8,K109H和H75N的最适pH=6,而K109A和Y74W的最适pH=9。突变的引入可能改变了AZR分子表面的电荷情况,从而改变了其最适pH。与野生型AZR相比,几种突变型AZR的酶活都出现下降,表明Y⁷⁴、H⁷⁵和K¹⁰⁹对酶活有重要影响。分析底物结合常数表明,Y74W和H75N突变对NADPH的结合无影响,而只影响AZR对甲基红和硝基呋喃的结合;H75N突变后AZR完全丧失硝基还原酶活性。第109位氨基酸荷正电对甲基红的结合有重要影响,而对硝基呋喃的结合无影响;K109H对NADPH的结合并非保守突变,其结合不仅需要该处荷正电,可能还与空间结构有关。K¹⁰⁹可能只参与对NADPH的2’-磷酸基团的结合,而对NADH的结合无影响。自然界中天然存在的偶氮类和硝基类芳香化合物并不常见,因此还原酶AZR可能具有其他功能。醌类化合物广泛存在于自然界和各种生物体的代谢过程中。AZR与来自不同域的醌还原酶的氨基酸序列中都较为保守地存在一段40-50个氨基酸构成的参与结合底物与辅酶的序列。AZR与醌还原酶NQO1的单体具有类似的α/β型结构（rmsd=9.5 A,467 atoms）和黄素辅基结合方式。AZR具有醌还原酶活性,遵循乒乓机理,可还原甲萘醌、2-羟基-1,4-萘醌（LQ）、葸醌-2-磺酸和葸醌-2,6-二磺酸等萘醌和葸醌化合物,甲萘醌为最适底物,而对1,4-苯醌无活性。与偶氮和硝基化合物相比,醌类化合物为AZR更适合的底物。双香豆素抑制AZR的醌还原酶活性,为NADPH的竞争性抑制剂,抑制常数K₁=87.6μM。胞内过量表达的醌还原酶AZR提高了E.coli YB对氧化应激的耐受。在H₂O₂、甲萘醌、百草枯等应激源存在条件下和经热休克处理后,E.coil YB的存活率都高于对照株E.coli JM109。氧化还原介体LQ的加入可提升E.coli JM109和E.coliYB培养物脱色偶氮染料的能力。E.coil YB的介导脱色能力较E.coli JM109更为突出:在0.2 mM LQ存在下,E.coli YB 2 h内可脱色75%的苋菜红（1 mM）,为目前已报道的最佳的细菌脱色表现,体现了基因工程菌降解能力的优势,为胞内异源表达的还原酶的应用提供了新的思路。本研究深化了对FMN依赖型偶氮还原酶的结构与功能的认识,为AZR等还原酶的应用奠定基础。更多还原

【Abstract】 Biodegradation of azo dyes has attracted a lot of attention in recent years.Many azo-dye-degrading microorganisms were isolated and characterized.In addition,several efficient azoreductases were obtained and studied.However,in-depth understanding of the structure and function of azoreductase is limited.Bacterial decolorization of azo dyes and the structure model and function of FMN-dependent azoreductase are studied in this dissertation.Decolorization of azo dyes by Rhodopseudomonas palustris AS1.2352 and its cell extracts were investigated.Under anoxic conditions,the growing strain could decolorize over 80%of 50 mg L^-1 reactive brilliant red X-3B（RBR X-3B） in 17 h.The optimal temperature and pH were around 30-35℃and pH 6-9,respectively.The correlation between specific decolorization rate and dye concentration could be described by Michaelis-Menten kinetics. The kinetic constants estimated were 65 mg g cell^-1 h^-1 for V_max and 978 mg L^-1 for K_m. Azoreductase activity was found in its cell extracts,which demonstrated more effective decolorization performance than whole cells.Decolorization of sulfonated azo dyes was investigated with early-stationary-phase cells of two photosynthetic bacteria（PSB, Rhodobacter sphaeroides AS1.1737 and R.palustris AS1.2352） and a gene-engineered strain （Escherichia coli YB）.The two PSB strains demonstrated higher decolorization abilities than the recombinant one.Values of V_max/K_m were 0.2,0.15 and 0.06 L g cell^-1 h^-1 for R. sphaeroides AS1.1737,R.palustris AS1.2352 and E.coli YB,respectively.The two PSB strains showed higher decolorization abilities for mixed azo dyes than E.coli YB.Structure model of azoreductase AZR of R.sphaeroides AS 1.1737 was constructed using homology modeling method.AZR monomer demonstrates a flavodoxin-like globularα/βstructure with the central five-stranded parallelβ-sheet flanked by fiveαhelices.An FMN prosthetic group which lies on top of the molecule may act as redox and reactive centers. Although FMN-dependent azoreductases can be classified into two goups according to amino acid sequence,their monomers demonstrated similar flavodoxin-like 3D structures.Many nitroaromatics could be reduced by AZR using NADPH as electron donor.The optimal temperature and pH for its nitroreductase activity was 50℃and pH 7,respectively. Compared with NADH,NADPH was a better electron donor.The reaction followed Ping-Pong Bi Bi kinetic mechanism.It was demonstrated that,among 2,4,6-trinitrotoluene （TNT）,2,4-dinitrotoluene（2,4-DNT） and 2,6-dinitrotoluene（2,6-DNT）,TNT was the best substrate and 2,4-DNT was a better substrate than 2,6-DNT.Hydroxylamino-dinitrotoluene was detected with HPLC/MS as reduction products of TNT.AZR could also reduce FMN following Ping-Pong Bi Bi kinetic mechanism.K_m values for NADPH and FMN were 0.5 and 14.2 mM,respectively.V_max was calculated to be 172.4μmol mg protein^-1 min^-1.Externally added FMN was a competitive inhibitor of NADPH,methyl red and nitrofurazone.The inhibition constants for NADPH,methyl red and nitrofurazone were 1.7,2.3 and 6.4μM, respectively.AZR has a wide range of substrates,including azo and nitro compounds,FMN and metal ions.It is a member of a new nitro-/FMN reductase family.On the basis of model analysis and multiple sequence alignment,several amino acid residues(Tyr⁷⁴、His⁷⁵ and Lys¹⁰⁹) in the two loops around FMN were chosen as mutation targets.Site-directed mutagenesis including K109A,K109H,Y74W and H75N were performed with multiple steps of PCR.Wild-type and mutant AZR proteins were heterogeously overexpressed in E.coli and purified before characterization.Using acid red B as substrate,the optimal pH value of wild type AZR was pH 7-8.While those of K109H and H75N were pH 6,and those of K109A and Y74W were pH 9.The mutations might alter the surficial charge of AZR and resulted in the variation of optimal pH values.Compared to the wild-type AZR,decreased activities were found with all the mutant ones,which indicated that K¹⁰⁹,Y⁷⁴ and H⁷⁵ are essential residues for the activity of AZR.According to the analysis of kinetic parameters,the following conclusions were obtained.Positively charged residues at the 109^th site is necessary for the binding of methyl red,but not for nitrofurazone.Mutations of Y74W and H75N do not affect the binding of NADPH,however they decrease the binding of methyl red and nitrofurazone.The H75N mutant loses its nitroreductase activity completely. K109H is not a conserved mutation for the binding of NADPH,which may also be affected by local spatial structures.K¹⁰⁹ may only be involved in the binding of the 2’-phosphate group of NADPH and have no effect on the binding of NADH.There are very few natural azo and nitro aromatics,thus AZR may have some other functions rather than azo- or nitro- reductase.Quinone compounds are widly found in natural environments and metabolism processes of many different lives.A short partial sequence （40-50 amino acid residues） involved in the binding of substrate and coenzyme was found conserved among AZR and several other quinone reductases from different domains.Similarα/βmonomer structures and ways of flavin cofactors binding were found between AZR and quinone reductase NQO1.It was demonstrated that AZR is a two-electron quinone reductase following the Ping-Pong Bi Bi kinetics.It could reduce 2-methyl-1,4-naphthoquinone （menadione）,2-hydroxy-1,4-naphthoquinone（lawsone,LQ）,anthraquinone-2-sulfonate and anthraquinone-2,6-disulfonate.Menadione was the best substrate among quinones investigated.No activity was detected with 1,4-benzoquinone.Compared to azo and nitro compounds,quinones are better substrate of AZR.The quinone reductase activity of AZR was inhibited by dicoumarol,which was a competitive inhibitor of NADPH with a K_i of 87.6μM. The overexpressed cellular quinone reductase AZR significantly increased the resistance of E. coli YB to oxidative stress.When treated with stressors such as H₂O₂,menadione,paraquat and heat shock,the survival rates of E.coli YB were higher than those of the control strain E. coli JM109.The addition of redox mediator LQ significantly enhanced the decolorization performances of E.coli JM109 and E.coli YB.The recombinant strain possessed better mediated decolorization ability than the control one.In the presence of 0.2 mM LQ,E.coli YB removed 75%amaranth（1 mM） in 2 h,which is the best bacterial decolorization performance ever reported.This demonstrated the advantage of gene-engineered strain and provided a new strategy for the application of heterogeneously expressed intracellular reductase.The study provides a deeper understanding of the structure and function of FMN-dependent azoreductase.It lays the foundation for the application of AZR and other reductases.更多还原