Wood Rot Fungi Secrete Low Molecular Weight Substances In Lignin Biodegradation Mechanisms Of Action

Lignin is the most abundant renewable aromatic polymer on earth.The biodegradation of lignin is an oxidative and nonspecific process mainly performed by a group of basidiomycetes known as white-rot fungi.Coriolus versicolor is one of the most efficient wood-degrading white-rot basidiomycetes and causes the simultaneous decay of lignin,cellulose,and hemicellulose.This fungus simultaneously produces large amounts of extracellular laccase and a manganese-dependent peroxidase(MnP). Neither MnP nor laccase are able to degrade non-phenolic structures,which are the primary components(80-90%)of lignin macromolecules.Lignin peroxidase(LiP)can oxidize non-phenolic linkages of lignin molecules,but LiP activity was not detected in many ligninolytic fungi.Laccase can oxidize nonphenolic lignin in the presence of a suitable mediator,but few mediators have been found in nature.This suggested that white-rot fungi also possess another way to oxidize lignin.Although many wood decaying basidiomycetes secrete oxidative and hydrolytic enzymes that participate in lignocellulose biodegradation,it is generally recognized now that these enzymes cannot penetrate sound wood.Studies with electron microscopy have confirmed that the size of the enzymes is too large to penetrate into the wood cell wall micropores during the early stages of decay to initiate the lignin degradation,so it is thought that the fungi must employ smaller agents to initiate decay.We previously found that a low molecular weight peptide(termed short fiber generating factor,SFGF)produced by cellulolytic fungi can degrade cellulose into short fibers through an oxidative reaction and after this it became susceptible to hydrolysis by cellulases.The purified peptide from Gloeophyllum trabeum can chelate Fe³⁺,reduce it to Fe²⁺,form hydroxyl radicals via the Fenton reaction,and the hydroxyl radicals can oxidize cellulose and lignin in a nonspecific manner.Another low-molecular-weight peptide from Phanerochaete chrysosporium(named PC factor) has phenol oxidase activity,and may function as an electron carrier in the oxidation-reduction system.But our understanding of the fungal oxidation-reduction mechanism is by no means complete,and more work is needed to assess these possibilities.The research in this dissertation was the further study based on the previous results.Using 25 wood-decay fungi,we researched their production of those extracellular low molecular weight substances that have special activity during their growth and those substances' catholicity in those fungi also were discussed.In this work,new low-molecular-weight organic compounds,which serve as the iron-binding chelator,were isolated and partially purified from the cultures of the white-rot basidiomycete Coriolus versicolor.The oxidative properties of the compounds chelated with iron on cellulase-treated lignin(CEL)were studied.The primary attempts had been done to organize a durative oxidative system to degrade lignin in vitro.All these enriched the understanding on the composing and function of low-molecular-weight systems from white-rot fungi.Research strategies and analytic methods in the dissertation were positive to further study in this field.Our goal was to expand our knowledge of the non-enzymatic mechanisms involved in the white rot fungal degradation of lignin.1.Production of low molecular weight substances by wood-rot fungi is a universal phenomenonPhenol oxidase activity,Fe³⁺chelating ability,the ability of reducing Fe³⁺and the generation of hydroxyl radicals were detected from the low-molecular-weight substances(Mr＜5000)which were secreted from 25 wood-decay fungi.Our research showed that most of these substances have the phenolic oxidase activity,the ability of producing the hydroxyl radical,and also has high affinity for Fe³⁺and could reduce Fe³⁺to Fe²⁺.Some of these low molecular weight substances have the characteristic of siderophore,some of them can produce OH·and some of them can reduce Fe³⁺to Fe²⁺.And we also found that different fungi produced different kinds of low molecular weight substances.Some of them produced two kinds of low molecular weight substances but some of them could produce more kinds of low molecular weight substances.The study also revealed that the substances could put an oxidative attack on lignin and make the lignin susceptible to attack by correlative enzymes.So these extracellular substances seem to play a role in the initial degradation of the lignin in wood by the fungus.2.Function of the iron-binding chelator produced by Coriolus Versicolor in lignin biodegradationAn ultrafiltered low-molecular-weight preparation of chelating compounds was isolated from a wood-containing culture of the white-rot basidiomycete Coriolus versicolor.This preparation could chelate Fe³⁺and reduce Fe³⁺to Fe²⁺,demonstrating that the substance may serve as a ferric chelator,oxygen-reducing agent,and redox-cycling molecule,which would include functioning as the electron transport carrier in Fenton's reactions.Lignin was treated with the iron-binding chelator and the changes in structure were investigated by ¹H-NMR,¹³C-NMR,difference spectrum caused by ionization under alkaline conditions and nitrobenzene oxidation.The most important finding of this study was the effects of the iron-binding chelator on lignin,involving destruction theβ-O-4 bonds in etherified lignin units. Furthermore,the iron-binding chelator can insert phenolic hydroxyl groups in the lignin polymer without enzymes.This reaction was supposed to be important because the lignin units with phenolic hydroxyl groups could be oxidized by MnP or Laccase. It may be one of the lignin oxidative mechanisms of the lignolytic fungi,which cannot secrete LiP.Lignin degradation is a long-distance reaction,which involves many factors. From our results,the iron-binding chelator was demonstrated to serve as one of the factors in the lignin degradation system.This work improves our knowledge of the non-enzymatic mechanisms involved in the white rot fungal degradation of lignin.3.Function of the iron-binding chelator produced by Coriolus Versicolor in native lignocellulose biodegradationThe effects on native wood of the low-molecular-weight compounds and ligninolytic enzymes were estimated on the SEM,AFM and GC-MS methods.Images produced by SEM and AFM revealed that after treatment with the iron-binding chelator and lignocellulolytic enzymes the surface of an aspen slice was more coarse than when treated with only the enzymes.The images showed that the iron-binding chelator and the lignocellulolytic enzymes can destroy the compact structure of wood.It was believed that the synergic effects of the iron-binding chelator and enzymes could split the fibers of the aspen slice.The smaller fragments were more accessible for degradation by the enzymes.Furthermore,the holes in the aspen slice after treatment could allow the large enzyme molecules to penetrate into the cell wall.Thus,we proposed the hypothesis that the iron-binding chelator may initiate the breakdown of natural lignocellulose.Wood biodegradation products were extracted and analyzed by GC-MS.The results indicated that lower acidic fraction products but more neutral fraction and phenolic fraction products were created by the system between lignin enzymes and the low molecular weight substances.4.Characteristic determinations of the low molecular weight substances with phenol oxidase activity by Lentinus lepideusWe determined the qualities of low molecular weight substances from Lentinus lepideus.Its absorption spectra obtained for partially purified the low molecular weight substances dissolved in buffer of acetic acid(pH 4.6)showed one absorption band only at around 254 nm.The characteristic determination of siderophore of low molecular weight substances was carried out using the universal CAS assay.We found that it react with CAS positively within a few hours.We also found its optimum pH is a wide and acidic acid range(4.0-4.8)for 2,6-DMP oxidation.The optimum temperature for 2,6-DMP oxidation by low molecular weight substances was reached at 70℃. Our results shown that the low molecular weight substances from Lentinus lepideus could oxidate Guaiacol,2,6-DMP and Syringaldazine,the oxidation for these components needn't the H₂O₂.It is uncertain if low molecular weight substances can oxdiate 1,2,4-Trimethoxybenzene.It can't oxidate vanilly aldehyde, 1,2,3-Trimethoxybenzene and VA.