【作者】 蓝靖；

【导师】 黄锡荣；

【作者基本信息】 山东大学 ， 分析化学， 2008， 博士

【摘要】 木素是自然界中仅次于纤维素的第二大有机碳源。木素的降解已构成自然界碳素循环的限速步骤。白腐菌是自然界中唯一能够彻底降解木素的微生物,黄孢原毛平革菌是其典型菌种。白腐菌降解木素主要依靠细胞分泌的木素降解酶系,其中木素过氧化物酶（LiP）是最重要的一种。藜芦醇（VA）是白腐菌的次级代谢产物,也是LiP的生理底物。有关藜芦醇在木素降解中的作用多年来一直是学术界争论的焦点。白腐菌降解木素主要是过氧化物酶参与的自由基氧化过程,这种独特的自由基氧化机制引起了环境科学工作者的关注。1985年,Bumpus等首次报道纯培养的黄孢原毛平革菌能降解多环芳烃化合物,此后,利用白腐菌降解环境污染物的研究成了环境科学领域里的研究热点。本论文以黄孢原毛平革菌分泌的具有高氧化性能的LiP酶为氧化剂,酚型或非酚型芳香化合物为模型化合物,通过H₂O₂酶法缓释、反胶束中LiP活力的介质调控以及添加电子介体等手段和方法提高了LiP对疏水性芳香化合物的降解效率,阐明了不同介质中VA介导LiP催化氧化芳香化合物的介导机制。上述一系列较为系统深入的工作对于提高疏水性污染物的LiP酶解效率、进一步弄清VA在白腐菌降解木素过程中的作用与机制等具有十分重要的意义。本论文的具体研究内容与结果如下:一.通过双酶耦联及介质调控实现疏水性芳香化合物的高效降解1.葡萄糖氧化酶与LiP耦联,使H₂O₂缓释,实现芳香染料的高效降解H₂O₂的提供方式是影响LiP对污染物降解效率的关键因素之一。在本文中,我们用葡萄糖氧化酶催化氧化葡萄糖来产生和控制H₂O₂,并将之与LiP催化氧化芳香化合物的反应相耦合。通过调节反应体系的pH及葡萄糖氧化酶的加入量可以方便的控制体系H₂O₂的生成速率。由于H₂O₂缓释,避免了一次性加入H₂O₂时H₂O₂对LiP的失活现象,从而使LiP在较长时间内保持较高的催化活力。芳香染料降解实验表明酶法H₂O₂缓释的方法可以明显提高LiP对芳香染料的降解效率。2.介质调控LiP高效降解疏水性芳香化合物研究疏水性芳香化合物在水中溶解度很小,而LiP是水溶性酶,因此水介质中LiP降解疏水性芳香化合物的效率低。反胶束可以提高疏水性芳香化合物的溶解度,而且LiP在反胶束中也可以保持一定的催化活力,因此用反胶束介质代替水介质应该可以提高LiP对疏水性芳香化合物的降解效率。反胶束介质中疏水性芳香化合物的降解效率取决于LiP在反胶束中的活力及H₂O₂的含量。在先前工作基础上,通过一定量的AOT与Brij30复配,形成了一种新的AOT/Brij30反胶束体系。该体系克服了传统的单一表面活性剂体系低LiP活力的缺点,通过反胶束介质电性质及反胶束大小等条件优化,获得了AOT/Brij30反胶束中高的LiP活力。高浓度的H₂O₂对LiP的催化活力起抑制作用,而且反胶束介质中这种抑制作用更加突出。鉴于这种情况,我们将前文的酶法H₂O₂缓释方法移植到AOT/Brij30反胶束体系中,进一步改善了LiP在反胶束中的催化活力。三苯甲烷类染料降解实验表明在AOT/Brij30反胶束体系中,用GOD/LiP双酶耦联的方式可以显著提高染料的降解效率,为反胶束介质中疏水性芳香化合物的LiP酶转化奠定了基础。二.不同介质中,VA介导LiP催化氧化芳香化合物反应的双底物机制1.水介质中H₂O₂调控VA介导LiP催化氧化邻苯三酚红（PR）反应研究LiP催化H₂O₂氧化邻苯三酚红（PR）反应的氧化产物受H₂O₂与PR的摩尔比控制,H₂O₂与PR的摩尔比不同,所得降解产物不一样。分析表明,H₂O₂在LiP催化氧化PR过程中的双重作用（即低浓度的H₂O₂是LiP的激活剂,高浓度的H₂O₂是LiP的抑制剂）是导致上述现象的根本原因。藜芦醇（VA）对LiP催化氧化PR的反应有促进作用,尤其是当H₂O₂与PR的摩尔比较高时这种促进作用更为明显;然而PR对LiP催化氧化VA的反应却有抑制作用。后者可以用来解释为什么在用白腐菌降解染料时在培养液中常常检测不到LiP的藜芦醇活力。分析表明,VA的存在不但促进了LiP酶中间体LiP（Ⅱ）和/或LiP（Ⅲ）向LiP的转化,使LiP的催化循环加速,VA生成的VA^+·也间接氧化了染料PR,从而使PR的氧化速率提高。2.反胶束介质中VA介导LiP催化氧化PR反应的机制白腐菌降解木素及环境污染物是在与本体水差别盛大的胶体介质中进行的,反胶束更接近LiP的天然环境,因此反胶束介质中VA介导LiP催化氧化反应机制的研究将更有助于阐明VA在木素及环境污染物降解中的作用。AOT/Brij30反胶束中VA介导LiP催化氧化PR反应机制的研究表明:反胶束介质中VA对LiP催化氧化PR的反应有促进作用,即使是在低H₂O₂浓度下。此外,VA的促进作用随VA浓度的增大而增大,直至最大值。对一系列设计实验所得结果的综合分析表明,与水介质中不同,反胶束介质中VA对LiP催化氧化PR反应的促进作用主要是VA^+·对PR的间接氧化作用,VA对LiP的保护作用并不显著。3.介质对VA^+·寿命的影响VA^+·自由基的寿命是阐明VA在木素降解初始过程中的作用的关键,也是阐明VA介导LiP催化氧化芳香化合物反应机制的关键。以往关于VA^+·自由基寿命的研究都是在水介质中进行的,而LiP的天然环境与水介质差别盛大。不同介质中VA^+·的寿命应该是不一样了。因此,在这一部分工作中,我们研究了介质对几种自由基寿命的影响。结果表明,与水介质中相比,反胶束介质中自由基的寿命均明显变长,且变化的程度与自由基的结构有关。以苯酚阳离子自由基为例,水介质与反胶束介质中,苯酚自由基的衰减动力学常数分别为k_{aqueousmedium}=0.023s^-1,k_{reversed micelles}=5.8×10^-3s^-1。反胶束介质介电常数的减小有可能是导致阳离子自由基衰减变慢的主要原因。LiP活性中间体而非LiP可以稳定阳离子自由基,延长自由基寿命。三.色氨酸对LiP催化氧化芳香化合物的促进作用色氨酸（TRP）对LiP催化氧化芳香化合物的促进作用与芳香化合物的氧化还原电位有关。对于氧化还原电位高于TRP的物质,TRP对其酶促反应有抑制作用;而对氧化还原电位低于TRP的物质,TRP则可以促进其氧化。以PR为模型化合物,我们研究了TRP促进LiP催化氧化PR反应的反应机制。研究表明:TRP可以促进LiP催化氧化PR的反应。与VA一样,TRP对LiP催化氧化PR反应的促进也是通过两种方式进行的:1)TRP与LiP（Ⅱ）和/或LiP（Ⅲ）反应,帮助LiP完成其催化循环。与VA相比,TRP还原LiP（Ⅲ）似乎更有效。（2）TRP^+·对PR有间接氧化作用。TRP^+·是具有氧化还原活性的中间体,它可以间接氧化像PR这样的芳香类化合物。更多还原

【Abstract】 Lignin is the second most abundant renewable aromatic polymer on earth.Its biodegradation is the rate-determining step in the carbon cycle.White rot fungi are the only known organisms that can completely break down the lignin to carbon dioxide and water.The ligninolytic system of Phanerochaete chrysosporium,a white rot fungus,has been widely studied as a model strain.White rot fungi was studied for the degradation of lignin at first.The degradation of lignin by white rot fungi mainly depended on the lignin degradative system,of which lignin peroxidase（LIP）was the most important enzyme.Veratryl alcohol（VA）was the physiological substrate of LiP. It is the secondary metabolite of Phanerochaete chrysosporium.The role of veratryl alcohol in the degradation of lignin has been the subject of numerous studies and considerabe debates.The degradation mechanism of white rot fungi was nonspecific and the nonspecific mechanism aroused the interest of the environment scientist.In 1985,Bumous et al.found that Phanerochaete chrysosporium could degrade polycyclic aromatic hydrocarbon and from then on,the degradation of aromatic pollutants using white rot fungi and their enzymes has been a hot topic in the fields of environmental science and technology.In this paper,by using LiP from Phanerochaete chrysosporium as the oxidant and phenolic or nonphenolic compound as the model compounds,we studied the LiP catalyzed oxidation of aromatic compounds and the related mechanism.Attempts have been made to improve the degradation efficiency of aromatic compounds with LiP,such as coupling glucose oxidase with LiP,medium regulation and addition of small molecular mediator.The mechanism of VA mediated LiP catalyzed oxidation of aromatic compound in different medium was also studied in detail to elucidate the role of VA in the degradation of lignin and aromatic compound.All these will not only help to clarify the role of VA in lignin degradation,but also contribute to find a new way to improve the degradation efficiency of aromatic pollutants with LiP.The innovative research results were acquired as following: Ⅰ.Improvement of the degradation efficiency of hydrophobic aromatic compounds by coupling glucose oxidase with LiP and medium regulation.1.High efficient degradation of aromatic dyes by coupling of glucose oxidase with LiP.The H₂O₂ supply strategy was one of crucial factors for high efficient degradation of pollutants with lignin peroxidase（LiP）.In this section,an attempt was made to couple a H₂O₂ producing enzymatic reaction to the LiP catalyzed oxidation of dyes. H₂O₂ needed was generated by glucose oxidase（GOD）and its substrate glucose.The generation rate of H₂O₂ could be easily controlled by adjusting the pH of the degradation system and the amount of GOD added.Due to the controlled release of H₂O₂,a sustainable constant activity of LiP was observed.The inhibition of LiP by high level H₂O₂ supplied externally by a single addition at the beginning of the experiments could be avoided.Degradation of three dyes（xylene cyanol,fuchsine and rhodamine B）with LiP coupled with GOD indicated that the present H₂O₂ supply strategy was very effective for improvement of the efficiency of the decolourization of dyes.2.Improvement of the degradation efficiency of aromatic compounds with LiP by medium regulation.Anionic surfactant sodium bis（2-ethylhexyl）sulfosuccinate（AOT）had an inhibiting effect on lignin peroxidase（LiP）.To improve the catalytic activity of LiP in an AOT reversed micelle in isoctane,a nonionic surfactant polyoxyethylene lauryl ether（Brij30）was incorporated into the interfacial membrance.H₂O₂ played dual roles in the LiP catalyzed oxidation of substrates.To obtain a sustainable high activity of LiP,a coupled enzymatic reaction,i.e.,the glucose oxidase（GOD）catalyzed oxidation of glucose was used an H₂O₂ source.Due to modification of the charge density of the interfacial membrane,the activity of LiP in an optimized AOT/Brij30 reversed micellar medium(xB（the molar percentage of Brij30）=0.53,ω₀（[H₂O]/ （[AOT]+[Brij30]）=23,pH=4.8）was ca.40 times that in a single AOT reversed micelle.Due to the controlled release of H₂O₂,the concentration of H₂O₂ in the mixed reversed micellar medium could be always kept at a moderate high level,which made the LiP catalyzed oxidation of substrates go at higher conversion than the counterpart in which H₂O₂ was supplied externally in one batch at the beginning of the reaction. Decolourization of two water-less-soluble aromatic dyes（pyrogallol red and bromopyrogallol red）using LiP coupled with GOD in the medium also demonstrated that a higher decolourization percentage was obtained if H₂O₂ was supplied enzymatically,our proposed measures（both physicochemicai and biochemical）were very effective for significant improvement of the catalytic performance of LiP in a single AOT reversed micelle in isooctane,which helped to degrade or transform hydrophobic aromatic compounds with LiP in reversed micelles more efficiently.Ⅱ.Mechanism studies on the VA mediated LiP catalyzed oxidation of aromatic compound in aqueous medium and reversed micelles1.Studies on the hydrogen peroxide regulated VA mediated oxidation of pyrogallol red（PR）catalyzed by LiPThe oxidation reaction of PR by H₂O₂ in the presence of LiP was studied at different concentrations of H₂O₂.Experiments showed that the oxidation products depended on the molar ratio of H₂O₂ to PR,suggesting that the LiP catalyzed oxidation products of PR should be controllable.This phenomenon was caused by the dual roles of H₂O₂;i.e.,at lower concentrations it was an activator of LiP,while at higher concentrations it was an inhibitor.VA could stimulate the oxidation of PR catalyzed by LiP,especially at higher molar ratios of H₂O₂ to PR,however,PR inhibited the LiP catalyzed oxidation of VA.The inhibition should be used to explain a phenomenon that no veratryl alcohol activity was detected in the culture of white rot fungi where dye was effectively decolorized.Kinetics analysis suggested that VA should accelerate the conversion of LiP（Ⅱ）and/or LiP（Ⅲ）to LiP,and therefore the catalytic cycle of LiP.Indirect oxidation of PR by the veratryl alcohol cationic radical was also contributed to the increase in the oxidation rate of PR.2.Mechanistic studies on the effect of veratryl alcohol on the lignin peroxidase catalyzed oxidation of pyrogaliol red in reversed micelles.The LiP catalyzed oxidation of PR in the absence and presence of VA was carried out in AOT/Brij30 reversed micelles to elucidate the role of VA.Results indicated that VA could accelerate the LiP catalyzed oxidation of PR,especially at low H₂O₂ concentrations.Unlike in bulk aqueous medium,the protection of LiP by VA in the present medium was relatively not prominent,even at high H₂O₂ concentrations. Analysis of data from a series of well designed experiments showed that the enhancement of the PR oxidation caused by VA was mainly due to the indirect oxidation of PR by VA^+·from the LiP catalyzed oxidation of VA.It was also found that at low concentrations,VA（the psychological substrate of LiP）was less effective than PR（a phenolic compound）in protecting LiP from the H₂O₂ derived inactivation. This novel phenomenon deserves further study.3.Effect of medium on the life time of VA^+·The life time of VA^+·was a key factor in the elucidation of the role of VA in the degradation of lignin and environmental pollutant.Previous studies on the life time of VA^+·were all carried out in aqueous medium.However,natural degradation of lignin by the fungus was carried out in a colloidal medium which is very similar to reversed micelles but quite different from bulk aqueous medium.Therefore,the difference of medium may cause the inaccuracy of the determining of the life time of VA^+·.In this section we studied the effect of medium on the life time of several radicals.Results indicated that the decay of all the radical studied in reversed micelles were slower that that in the aqueous medium.For example,the first-order rate constant of the phenol radical in aqueous and reversed micelles were k_{aqueous medium}=0.023s^-1and k_{reversed micelles}=5.8×10^-3s^-1,respectively.The difference of the dielectric constant between the two medium may be the reason that caused the above results.Ⅲ.Tryptophan enhanced oxidation of aromatic compounds with lignin peroxidaseTryptophan（TRP）could accelerate the LiP catalyzed oxidation of some aromatic compounds with redox potential lower than that of TRP.To explore the role of TRP and the related mechanism,PR was chosen as a model compound and the LiP catalyzed oxidations of PR in the absence and presence of TRP were investigated in detail.Our results indicated that the acceleration of the LiP catalyzed oxidation of PR was owed to the protection of TRP against the H₂O₂-derived inactivation of LiP and the indirect oxidation of PR by TRP^+·of high redox potential.TRP was a good substrate of LiP.In the presence of both PR and TRP,TRP was preferentially oxidized by LiP to form a TRP cation radical(TRP^+·).TRP^+·could oxidize PR and make the LiP catalyzed oxidation of PR proceed at a larger rate,but the LiP catalyzed oxidation of TRP be inhibited.The present study also showed that TRP had a better reactivity towards LiP（Ⅲ）than VA,the physiological substrate of LiP.By reacting with LiP（Ⅲ）, TRP could convert LiP（Ⅲ）to its native state and therefore protect LiP from the H₂O₂-derived inactivation.The present study helps to find a new effective way to improve the degradation efficiency of aromatic pollutants with LiP.更多还原