Lignin Degradation by Phanerochaete Chrysosporium: Biochemical Characterization of Extracellular Peroxidases

;​R o o t E n t y F -, 4 : C o m p o b j b W o r d d o c u m e n t O b j e c t P O O l 我* * 4:4:A, b, c, d， I, jF Microsoft Word 6.0文档MSWordDoc。6;​本项目的目的是进一步了解降解木质素的担子菌Phanerochaete chrysosponum的锰过氧化物酶（MnP）和木质素过氧化物酶（LiP）。潜在的目标是进一步表征这些酶的结构、机制和功能。MnP、Mn和草酸盐之间的结合相互作用将通过动力学光谱和平衡透析方法进行研究。草酸盐被MnP氧化以及草酸盐在H2O2使MnP失活中的作用将被研究。利用同源表达系统，P.I.打算构建、表达和表征MnP的Mn结合位点的位点定向突变体。为了了解LiP如何氧化聚合木质素，将研究几种模型聚合物底物的LiP氧化，包括细胞色素c和核糖核酸酶。特别地，私家侦探将研究戊曲醇在促进这些反应中的作用。亲核取代和二聚化反应将用于监测在二甲氧基苯的LiP氧化过程中稳定阳离子自由基的形成。通过对一系列二甲氧基苯和二甲氧基苯醇底物的考察，确定了该反应中产生和稳定阳离子自由基中间体的要求%%%木质素是生物圈中含量最多的可再生芳香族聚合物。它构成了木本植物细胞壁的15.30%，形成了包围纤维素的基质。这种覆层基质显著延缓了纤维素的微生物解聚，因此木质素在地球的碳循环中起着关键作用。利用微生物将木质纤维素转化为有用的材料、化学品和燃料是一个重要的研究领域。大多数关于木质素降解的研究都集中在白腐担子菌上，因为这些真菌比其他生物更快地降解这种聚合物。此外，它们显然是唯一能将木质素完全降解为CO2和H2O的生物。研究得最好的木质素降解真菌是黄孢平革菌。自1983年以来，对黄孢霉木质素降解系统的研究取得了重大突破。两种参与这一过程的胞外过氧化物酶的发现和纯化激发了大量的进一步研究。重要的是最近的报道，LiP和MnP都能够在体外解聚合成木质素，并氧化几种芳香族污染物。对LiP和MnP的结构和机理的研究也具有重要意义。本课题的主要目标是：(1)阐明木质素降解过氧化物酶的结构和作用机制；(2)确定这些酶在木质素和芳香污染物降解中的作用；(3)研究编码这些酶的基因的调控和表达。*** @ ....（）（） （）（） （）（） （）（） （）（） （）（） （）（） （）（） （）（） （）（） （）（） （）（） （）（） （）（） （）（） （）（） （）（） （）（） (Oh +' 0 $ H l D H R:\WWUSER\TEMPLATE\NORMAL。DOT marcia steinberg marcia steinberg @ a 1: @ M 4：@ Microsoft Word 6.0e = e w " " " " " " " L L L L L L d n L C x | | | | | | | #

; R o o t E n t r y F -,4: C o m p O b j b W o r d D o c u m e n t O b j e c t P o o l i *4: i *4: A B C D E F G H I J F Microsoft Word 6.0 Document MSWordDoc Word.Document.6 ; 9506338 Gold The aim of this project is to further our understanding of manganese peroxidase (MnP) and lignin peroxidase (LiP) from the lignin degrading basidiomycete Phanerochaete chrysosponum. The underlying objectives are to further characterize the structure, mechanism and function of these enzymes. The binding interactions between MnP, Mn and oxalate will be studied by kinetic spectroscopic and equilibrium dialysis methods. The oxidation of oxalate by MnP and the role of oxalate in the inactivation of MnP by H2O2 will be examined. Using a homologous expression system, the P.I. intends to construct, express and characterize site directed mutants of the Mn binding site of MnP. In order to understand how LiP oxidizes polymeric lignin, the LiP oxidation of several model polymeric substrates, including cytochrome c and ribonuclease, will be examined. In particular, the P.I. will examine the role of veratryl alcohol in facilitating these reactions. Nucleophilic substitution and dimerization reactions will be used to monitor the formation of stable cation radicals during the LiP oxidation of dimetho xybenzenes. By examining a series of dimethoxybenzene and dimethoxybenzyl alcohol substrates, the requirements for producing and stabilizing the cation radical intermediate in this reaction will be determined %%% Lignin is the most abundant renewable aromatic polymer in the biosphere. It constitutes 15 30% of woody plant cell walls, forming a matrix that surrounds the cellulose. This encrusting matrix significantly retards the microbial depolymerization of cellulose and thus lignin plays a key role in the earth's carbon cycle. The potential utilization of microorganisms for transforming lignocellulose into useful materials, chemicals and fuels is a significant field of research. Most studies on lignin degradation have focused on white rot basidiomycetes because these fungi degrade this polymer much more rapidly than other organisms. In addition, apparently they are the only organisms that completely degrade lignin to CO2 and H2O. The best studied lignin degrading fungus is Phanerochaete chrysosporium. Since 1983 major breakthroughs have occurred in the study of the lignin degradative system of P. chrysosporium. The discovery and purification of two extracellular peroxidases involved in this process have stimulated a great deal of further research. Of major significance are the recent reports that both LiP and MnP are able to depolymerize synthetic lignin in vitro) and to oxidize several aromatic pollutants. Also of major significance is the work on the structure and mechanism of LiP and MnP. Three major goals of this project are: (1) to elucidate the structure and mechanism of the lignin degrading peroxidases; (2) to establish the role of these enzymes in lignin and aromatic pollutant degradation; and (3) to study the regulation and expression of the genes encoding these enzymes. *** @ ....()()))()() S u m m a r y I n f o r m a t i o n ( @ Oh +' 0 $ H l D h R:\WWUSER\TEMPLATE\NORMAL.DOT marcia steinberg marcia steinberg @ a 1: @ @ M 4: @ Microsoft Word 6.0 4 e = e w " " " " " " " L L L L L d n L C x | | | | | | | #