No description available.

No description available.

For the first time, the bonding patterns of specific carbon atoms in woody plant lignin have been identified in situ. This was accomplished by administering and incorporating into the lignin fraction of Leucaena leucocephala, a tropical hardwood, ferulic acid enriched with ¹³C at either the 1-, 2-, or 3-C atom of the side chain. The plants were grown hydroponically over extended periods of time (28 days) under aseptic conditions in media containing the ferulic acid precursor, and then the tissues were examined by solid-state ¹³C NMR spectroscopy. Consequently, resonances due to the bonding patterns of the specific carbon atoms were determined. These resonances differ substantially from similarly labelled synthetic dehydrogenatively polymerized (DHP) lignin in both spectral profile and relative peak intensities. Subsequent studies using phenylalanine as precursor showed that it was better translocated into the aerial portions of the plant, and that its uptake did not result in distortion of lignification in those tissues, both in amount and monomeric composition. Consequently, the difference spectra obtained by ¹³C NMR analyses of phenylalanine-treated plants confirmed and extended the results obtained with ferulic acid. Evidence for the conversion of both precursors to the monolignols was shown by the difference spectra of [1-¹³C]-precursor-fed tissues, where the dominant resonance at 61-63 ppm is consistent with substructures containing the hydroxymethyl functionality. The spectrum obtained with roots administered [1-¹³C] ferulic acid showed the presence of a minor resonance (170-174 ppm) attributable to carboxylic acids/esters. By allowing the plant to undergo further metabolism by growing in hydroponic media without the precursor, these signals disappeared from the resulting spectrum. The first direct evidence for the dominance of the β-O-4’ linkage of lignin in situ was shown by the appearance of the resonance at 83 ppm corresponding to this substructure in both [2-¹³C] ferulic acid-treated roots and [2-¹³C] phenylalanine-treated roots and stems. Evidence for the occurrence of α-O-carbohydrate or α-O-aryl linkage in intact plant tissues was obtained in the spectra of tissues administered [3-¹³C] ferulic acid and [3-¹³C] phenylalanine. The effect of horseradish peroxidase/H₂O₂ in organic medium (dioxane/aqueous acetate buffer, pH 5, 95:5) on dehydrogenatively polymerized (DHP) lignin was reinvestigated. We found no evidence for vigorous depolymerization of DHP lignin under these conditions, contrary to claims made by Dordick, Marletta and Klibanov (1986, Proc. Natl. Acad. Sci. USA 83:6255-6257). Furthermore, we did not detect ferulic acid as a degradation product following treatment of DHP lignin with HRP/H₂O₂. Both coniferyl alcohol and DHP lignin were used in incubation experiments to determine effects of lignin peroxidase from the white-rot fungus Phanerochaete chrysosporium and H₂O₂ on these substrates. Gel filtration chromatography showed that polymeric materials of high molecular weights were the result of these treatments. Incubation of [1-¹³C], [2-¹³C] and [3-¹³C] coniferyl alcohol with lignin peroxidase/H₂O₂ resulted in products similar to-DHP lignins prepared by horseradish peroxidase/H₂O₂ with respect to occurrence of identical resonances in corresponding solution-state ¹³C NMR spectra. Consequently, the role of polymerization of low molecular weight phenolics as a mechanism for detoxification was ascribed to these fungal peroxidases. / Ph. D.

Beech (<u>Fagus grand1folia</u> Ehrh.> bark contains appreciable quantities of Z- (cis) con1feryl and Z-s1napyl alcohols and not the corresponding E- (trans) alcohols. Previous rad1otracer experiments suggested that the Z-coniferyl alcohol in beech bark is formed by isomerization of E-coniferyl alcohol which proceeds either directly or through the corresponding aldehydes. In the work conducted in this thesis, is has been found that crude cinnamy1 alcohol dehydrogenase isolated from beech bark shows a strong substrate preference for E-coniferyl alcohol (as opposed to Z-coniferyl alcohol) thereby suggesting that the E to Z isomerization described occurs directly at the alcohol level. Administration of (2-¹⁴C) ferul1c acid to feland wheat (<u>Triticum aest1yum</u> L.) over extended durations (21 days) and subsequent isolation of the lignin from the root tissue as its acetal derivative demonstrated the incorporation of the labelled feru1ic acid into the lignin component of the tissue. Through sim1lar administrations of (1-¹³C, 2-¹³C and 3-¹³C) ferul1c acid and subsequent analysis of the root tissues by solid state ¹³C nuclear magnetic resonance (NMR) spectroscopy, it was possible to determine the bonding patterns of lignin in situ. The lignin component of each ¹³C feru1ic acid enriched root tissue was then isolated as its acetal derivative and analyzed by solution state ¹³C NMR. Through comparison it was shown that the enhanced resonances observed in the solution state ¹³C NMR spectra of the ¹³C ferulic acid enriched acetal lignins corresponded to the enhanced resonances in the respective spectra of the intact root tissues. This indicated that minimal changes to the lignin bonding patterns occurred during the isolation procedure. The dominant presence of ¹³C NMR resonances corresponding to hydroxycinnamic acid functionalities in the solid and solution state NMR spectra demonstrates the important role of hydroxyc1nnamic acids in wheat root lignin. However, no evidence of the formation of dimers such as 4,4'-dihydroxytruxillic acid was noted. Thus, such structures do not represent an important bonding pattern in wheat root lignin. / Master of Science

Lignin degradation, is quite common in nature and is an important link in the natural carbon cycle. A large variety of microorganisms are know to degrade lignin in nature as well as in contrived fermentation systems. White-rot and soft-rot fungi, as well as Actinomycetes, are apparently the most active lignin degraders in nature. The large, cross-linked, polymeric structure of the lignin macromolecule makes its direct uptake, during the initial stages of its degradation, by microbial cells improbable. Moreover, the fact that the lignin macromolecule is composed of different monomeric units linked by a large variety of non-hydrolyzable intermonomeric bonds precludes hydrolytic cleavage of the biopolymer. Despite the fact that many extracellular and membrane-bound enzymes have been suspected in the initial breakdown of lignin, such activities have not yet been found. A close review of the literature indicates that the initial breakdown of the lignin macromolecule may be nonenzymatic. In addition, the degradation of the lignin polymer appears to follow an exo-degradation mechanism. That is, many lignin degrading microorganisms are apparently incapable of splitting the lignin molecule into intermediate molecular weight polyphenolic moieties which are further degraded; instead, they attack the periphery of the macromolecule. The possible involvement of reduced oxygen species produced by white-rot fungi in the initial breakdown of the lignin macromolecule, during its biodegradation, was investigated. Using Coriolus versicolor as a representative of white-rot fungi, I demonstrated that C. versicolor exports superoxide radical and hydrogen peroxide during lignin degradation, into the lignolytic medium. Results presented in this study indicate that a correlation between the concentration of extracellular superoxide radical in the medium and the extent of lignin degradation may exist. Moreover, I have shown that superoxide radical is produced in the cell membrane, and not the organism's mitochondria. This precludes the possibility that such reduced oxygen species are produced as a result of normal respiration by the organism. An investigation of the effects of aeration and agitation indicated that agitation has a detrimental effect on the extent of lignin degradation. On the other hand, increased oxygen tension in lignolytic cultures appeared to enhance the extent of lignin degradation. Another interesting finding was the fact that conditions leading to the formation of reproductive fruits in the lignolytic microorganism favored the degradation of the lignin fraction in lignocellulosic materials. A comparative study of two different fennentation schemes, designed to degrade lignin in 1ignocellu1osic materials on a large scale, indicated that solid state fermentation of such materials led to greater lignin degradation. Fluidized bed fermentations, on the other hand, appeared to favor the degradation of the carbohydrates rather than the lignin fraction of lignocellulosic materials. Studies of the biodegradation of monomeric lignin model compounds do not shed light on the initial step(s) involved in the breakdown of the lignin polymer. Such studies assume that microbial breakdown of lignin model compounds is similar to microbial breakdown of lignin an assumption that may not be correct. It is true that degradation of monomeric lignin model compounds can conceivably elucidate the mode of degradation of low molecular weight moieties resulting from initial breakdown of the lignin macromolecule. However, the chemical identities of these low molecular weight intermediates are not yet known. The efficacy of studies using aromatic, monomeric lignin model compounds in attempts to identify intracellular pathways for metabolism of lignin depends on the assumption that lignin breakdown products are indeed mononuclear phenolic materials. Careful analysis of soluble and insoluble residual lignin resulting from lignin fermentations is a critical step in assessing the lignolytic ability of microorganisms. Furthermore, such analyses are essential in understanding the steps involved in lignin metabolism by microorganisms. To date the methods for residual lignin analyses are complex, time consuming and error prone. There is an urgent need to develop a quick and simple method for residual lignin analysis that will yield accurate and reproducible results capable of elucidating structural changes in residual, biodegraded lignin. The development of such an analysis technique will undoubtedly lead to a better understanding of the complex problem of lignin biodegration. / Ph. D.

Call number: LD2668 .T4 1985 S38 / Master of Science

Thesis (MSc)- Stellenbosch University, 2001. / ENGLISH ABSTRACT: Wood is composed of three main components: cellulose, hemicellulose and lignin. Cellulose is the main structural polymer, whereas the function of lignin in plants is to impart rigidity to the cells, to waterproof the vascular system, and to protect the plant against pathogens. A group of microorganisms, called white-rot fungi, are able to selectively degrade the lignin and hemicellulose from wood leaving the cellulose virtually untouched. The most widely studied fungus of this group is the basidiomycete Phanerochaete chrysosporium, which has become a model organism in studies of lignin degradation. Lignin is a large, heterogenous and water insoluble polymer and therefore the enzymes needed to degrade it have to be extracellular and non-specific. There are a number of enzymes that are involved in the degradation of lignin, including lignin peroxidases, manganese dependent peroxidases and laccases. Laecases are blue copper oxidases that require molecular oxygen to function, whereas lignin peroxidases and manganese peroxidases are heme proteins that require hydrogen peroxide. Phanerochaete chrysosporium has all three of these enzymes, as well as a system for producing the hydrogen peroxide that is necessary for peroxidases to function. For both scientific and industrial purposes, it is important to obtain linkage maps of the positions of genes in the genome of an organism. Most fungi, including P. chrysosporium, lack easily identifiable phenotypical markers that can be used to map the position of genes relative to each other on the genome. Previous methods of mapping genes in P. chrysosporium involved auxotrophic mutants, radioactivity, or the use of hazardous chemicals. Here we describe an automated DNA-sequencing based mapping technique that eliminates many of the problems associated with previous techniques. Portions of the genes to be mapped were amplified from homokaryotic single basidiospore cultures using gene specific primers using the polymerase chain reaction (PCR) technique. The PCR products were sequenced to determine the segregation of alleles. Two previously mapped lignin peroxidases, lipA and lipC, were used to develop this method, and the results obtained corresponded to the known genetic linkage. A newly characterised 13-glucosidase encoding gene from P. chrysosporium was also mapped. Linkage was found between the 13-glucosidase gene and a histone (Hl) encoding gene. In P. chrysosporium the lignin peroxidase isozymes are encoded by a family of at least ten genes. Previous studies with P. chrysosporium BKM-F-1767 in defined media, wood and soil have shown differential expression of the lignin peroxidase isozymes. In this investigation the levels of expression of lignin peroxidases in P. chrysosporium ME446 cultures grown in nitrogen or carbon limited defined liquid media, as well as on aspen wood chips were determined by competitive reverse transcriptase polymerase chain reaction (RT-peR). These results were compared to those previously obtained from P. chrysosporium BKM-F-1767 to evaluate strain specific variation in the expression of lignin peroxidases. The results indicate that, although there were many similarities in the patterns of lignin peroxidase expression, there were also enough differences to conclude that there were strain specific variations in the temporal expression of the lignin peroxidases. To conclude, a fast and cost effective method for mapping genes in P. chrysosporium was developed. Also, we showed that strain specific variation in temporal expression of lignin peroxidases occurs. / AFRIKAANSE OPSOMMING: Hout bestaan uit drie hoof komponente nl. sellulose, hemisellulose en lignien. Sellulose is die hoof strukturele polimeer, terwyl die funksie van lignin in plante is om die selle te versterk, die vaskulêre sisteem waterdig te hou, en die plant teen patogene te beskerm. 'n Groep mikroërganisms, bekend as witvrotswamme, kan lignien en hemisellulose selektief uit die hout verwyder, terwyl die sellulosevesels oorbly. Vanuit hierdie groep swamme is die meeste navorsing op die basidiomiseet Phanerochaete chrysosporium gedoen Lignien is 'n groot, heterogene polimeer en is onoplosbaar in water. Die ensieme wat benodig word om lignien afte breek is daarom nie-spesifiek en kom ekstrasellulêr voor. 'n Aantal ensieme is by die afbraak van lignien betrokke, insluitend lignienperoksidase, mangaanperoksidase en lakkase. Lakkase is 'n blou koperoksidase wat suurstof benodig vir aktiwiteit. Lignienperoksidase en mangaanperoxidase is heemproteïene en benodig waterstofperoksied. Phanerochaete chrysosporium het al drie van hiedie ensieme, sowel as 'n sisteem wat waterstofperoksied produseer. Vir beide wetenskaplike en nywerheidsdoeleindes is koppelingskaarte wat die posisie van gene in die genoom van 'n organisme aandui noodsaaklik. Die meeste swamme, P. chrysosporium ingesluit, het geen fenotipiese merkers wat maklik van mekaar onderskei kan word nie, en dit is dus moeilik om 'n kaart van die ligging van gene op die genoom te bepaal. Vorige metodes om gene in P. chrysosporium te karteer het auksotrofiese mutante, radioaktiwiteit of gevaarlike chemikalieë gebruik. Ons beskryf 'n metode wat van automatiese DNA-volgordebepaling gebruik maak en wat baie van die tekortkominge van die ou metodes oorkom. Dele van die gene is met geen-spesifieke PKR-amplifikasie uit kulture van homokariotiese enkel basidiospore verkry en die DNA-volgorde is bepaal om die segregasie van die allele te ondersoek. Twee gene waarvoor 'n koppelingskaart alreeds uitgewerk is, fipA en lipt), was gebruik om hierdie metode te ontwikkel. Die resultate stem ooreen met die bekende genetiese koppeling tussen hierdie gene. 'n Geen wat onlangs in P. chrysosporium ontdek is, nl. I3-glucosidase, is ook met hierdie metode gekarteer. Koppeling is met 'n histoon (Hl) geen gevind. Die lignienperoksidase isoensieme in P. chrysosporium word deur 'n familie van ten minste tien gene gekodeer. Vorige navorsing met P. chrysosporium BKM-F-1767 in gedefineerde media, hout en grond het getoon dat 'n variasie in die uitdrukking van lignienperoxidase isoensieme voorkom. In hierdie ondersoek is 'n kultuur van P. chrysosporium ME446 in stikstof- of koolstof-beperkende vloeibare media opgegroei, as ook op aspen houtblokkies. Die vlak van uitdrukking van die lignienperoksidases is deur middel van die omgekeerde transkripsie polimerasekettingreaksie (RT-PKR) bepaal. Die resultate vir P. chrysosporium ME446 is vergelyk met vorige resultate van P. chrysosporium BKM-F-1767 om te bepaal of stamspesifieke variasies in die uitdrukking van lignienperoksidases voorkom. Daar is 'n aanduiding dat, alhoewel soortgelyke patrone in die vlakke van lignienperoksidase uitdrukking voorkom, daar ook noemenswaardige verskille is. Hieruit kan afgelui word dat stamverwante variasie van lignienperokisdase uitdrukking voorkom. Ten slotte, ons het 'n vinnige, goedkoop metode om die gene in P. chrysosporium te karteer ontwikkel. Ons het ook bewys dat stam-spesifieke variasie in die uitdrukking van die lignienperoxidase gene voorkom.

Thesis (MSc)--University of Stellenbosch, 2004. / ENGLISH ABSTRACT: The microbial degradation of the lignin carbohydrate complex of plant material is only partially understood. Lignin carbohydrate complex was extracted from wheat straw and subsequently analysed. An adjustment to the standard protocol was required to increase the amount of lignin carbohydrate complex extracted from wheat straw. Characterization of the lignin carbohydrate complex after trifluoacetic acid hydrolysis was done by capillary electrophoresis. HPLC proved ineffective, as baseline separation could not be achieved. Characterization of the lignin carbohydrate complex revealed that a large portion (68 %) consisted of carbohydrate and lignin (20 %). Capillary electrophoresis of the trifluoroacetic acid hydrolysates of the lignin carbohydrate complex revealed that the carbohydrates consisted of mannose, xylose, arabinose, galactose, glucose and rhamnose. The major monosaccharide present in the lignin carbohydrate complex was mannose which made up 34 % of the total carbohydrate composition. Ferulic and p-coumaric acid were present in the lignin carbohydrate complex, but in concentrations less than 1 % of the lignin carbohydrate complex. The lignin carbohydrate complex of wheat straw probably had a heterogenous structure consisting of a variety of molecules, as molecular weight determination could not be accurately determined. An estimated molecular weight of 5.9 kOa was determined. Ten fungal strains (Aspergillus niger, Aureobasidium pul/u/ans, Bjerkandera adusta, Corio/us versicolor, Lenzitus betu/ina, Phanerochaete chrysosporium, Pycnoporus coccineus, Pycnoporus sanguineus 294, Pycnoporus sanguineus K5-2-3 and Trichoderma reeseï; were evaluated for growth on the lignin carbohydrate complex. All strains except B. adusta showed growth after 5 days with A. niger, A. pul/u/ans, C. versicolor, P. chrysosoporium and T. reesei showing the best growth on the lignin carbohydrate complex. The culture fluid revealed a number of proteins secreted by these organisms. The protein determination was confirmed by SOS-PAGE which revealed an array of proteins ranging from 8 kOa to 180 kOA. Prominent bands between 26 kOa and 80 kOa could be observed in the culture fluid of A. niger, A. pul/ulans and T. reesei, but not in C. versicolor. Activity studies on the culture fluid of these four strains revealed activity for xylanase, xylosidase, arabinofuranosidase, ferulic acid esterase and laccase, with vast differences between the activities of the various fungi. The enzymes of these fungal strains were also evaluated for their ability to degrade xylan and sugar cane bagasse using capillary electrophoresis. It appeared that all the organisms produced enzymes to degrade birchwood xylan. However, the electropherograms revealed that the degradation patterns of birchwood xylan differed among these organisms over the same time interval, as xylotetraose, xylotriose, xylobiose and xylose were released in various concentrations. The electropherograms obtained from the enzyme hydrolysates of sugar cane bagasse, pointed to the substrate being inaccessible. Electropherograms of the culture fluid of A. niger, A. pul/ulans, C. versicolor and T. reesei, when incubated on the lignin carbohydrate complex indicated similar peaks to those obtained and identified in the trifluoroacetic acid hydrolysates. However, the electropherograms of the culture fluid of these organisms revealed additional smaller peaks which could not be identified. The electropherograms of the culture fluid of the various organisms also indicated a complete release of some sugars, using the trifluoacetic acid hydrolysate of the lignin carbohydrate complex as a control for the amount of sugars present. HPLC analyses revealed that after 72 h, no apparent degradation of the lignin carbohydrate complex took place as peak height and areas appeared to be similar. These peaks could however not be identified due to a lack of standards as well as baseline separation which could not be achieved. / AFRIKAANSE OPSOMMING: Tans word die mikrobiese afbraak van die lignienkoolhidraatkompleks van plant materiaal slegs gedeeltelik verstaan. Lignienkoolhidraatkompleks was vanaf koringstrooi geïsoleer en gevolglik geanaliseer. Daar moes van die standaard prosedure vir die ekstraksie van lignienkoolhidraatkompleks afgewyk word ten einde beter lignienkoolhidraatkompleks opbrengs te lewer. Karakterisering van die lignienkoolhidraatkompleks na trifluoroasynsuurvertering was deur kapillêre elektroforese bepaal. Dit wou voorkom asof kapillêre elektroforese "n beter opsie vir die analise van die verteerde monster van lignienkoolhidraatkompleks is, vergeleke met hoëdruk vloeistof chromatografie. Daar was gevind dat die lignienkoolhidraatkompleks uit 68 % koolhidraat en 20 % lignien bestaan. Kapillêre elektroforese het die teenwoordigheid van die volgende suikers bevestig naamlik, mannose, xilose, arabinose, glukose, galaktose en ramnose. Mannose was die dominerende suiker in die lignienkoolhidraatkompleks wat 34 % van die totale koolhidraat opbrengs uitgemaak het. Ferulien- en p-kumaarsuur kon ook identifiseer word, maar die twee sure het minder as 1 % van die totale inhoud van die lignienkoolhidraatkompleks uitgemaak. Vanuit resultate bekom wil dit voorkom dat die lignienkoolhidraatkompleks "n heterogene molekuul is omdat die molekulêre gewig daarvan nie akkuraat bepaal kon word nie. 'n Geskatte molekulêre grootte van ongeveer 5.9 kDa was bepaal met verwysing na die hoogste piek wat in die chromatogram waargeneem was. Tien fungus kulture was in die studie gebruik om hul vermoë te toets om op die lignienkoolhidraatkompleks te groei, naamlik Aspergillus niger, Aureobasidium pullulans, Bjerkandera adusfa, Goriolus versicolor, Lenziius betuline. Phanerochaefe chrysosporium, Pycnoporus coccineus, Pycnoporus sanguineus 294, Pycnoporus sanguineus K5-2-3 en Trichoderma reesei. B. eauste het nie groei na 5 dae getoon nie, en dit wou voorkom asof A. niger, A. pul/ulans, G. versicolor, P. chrysosoporium en T. reesei die beste kon groei op die substraat na 5 dae. Die kultuurvloeistof van die vier kulture het getoon dat proteïene deur hierdie organisms uitgeskei was. Hierdie proteinbepaling was ook bevestig deur SOS-PAGE, wat bande tussen 8 kDa en 180 kDa gelewer het. Prominente bande tussen 26 kDa en 80 kDa kon waargeneem word in die kultuurvloeistof van A. niger, A. pul/ulans, en T. reesei, maar nie in die kultuurvloeistof van C. versicolor nie. Aktiwiteitstudies op die kultuur vloeistowwe het getoon dat daar aktiwiteit was vir die volgende ensieme, naamlik xilanase, xilosidase, arabinofuranosidase en feruliensuur esterase. Hierdie aktiwiteit het aansienlik verskil tussen die verskillende organismes. Die ensieme van die vier organismes was ook gebruik om hul vermoë te toets om xilaan en suikerriet af te breek. Daar was gevind dat xilaanafbraak verskillend was vir die organisms oor dieselfde tydperk. Xilotetraose, xilotriose, xilobiose en xilose was in verskillende konsentrasies gevind vir die verskillende organismes. Die elektroferogramme van die kultuurvloeistof op suikerriet van die verskillende organismes het getoon dat die substraat nie toeganklik vir die ensieme was nie. Die elektroferogramme van die kultuurvloeistof op lignienkoolhidraatkompleks van die verskillende organismes het dieselfde pieke getoon soos geïdentifiseer in die elektroferogramme van die trifluoroasynsuur vertering. Die elektroferogramme met die ensiem vertering het egter addisionele pieke getoon wat nie sigbaar op die elektroferogramme van die trifluoroasynsuur vertering was nie. Hierdie pieke het min of meer dieselfde tyd ge-elueer as die monosakkariede. Kapillêre elektroforese het ook getoon dat die ensiematiese afbraak van die lignienkoolhidraatkompleks gelei het tot algehele vrystelling van sommige suikers, wanneer die trifluoroasynsuur vertering as maatstaaf dien vir die hoeveelheid suikers teenwoordig in die lignienkoolhidraatkompleks. Hoëdruk vloeistof chromatografie het getoon dat geen sigbare afbraak na 72 h van inkubasie met die ensieme op die lignienkoolhidraatkompleks plaasgevind het nie aangesien die piek hoogtes konstant gebly het. Hierdie pieke kon egter nie geïdentifiseer word nie as gevolg van lae resolusie van die pieke asook standaarde wat nie beskikbaar was nie.

by Wang Pui. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (leaves 115-128). / Abstracts in English and Chinese. / Acknowledgments --- p.i / Abstract --- p.ii / List of Figures --- p.vi / List of Tables --- p.viii / Abbreviations --- p.ix / Chapter 1. --- Introduction Pg no / Chapter 1.1 --- Ligninolytic enzyme systems --- p.1 / Chapter 1.2 --- Three main ligninolytic enzymes --- p.3 / Chapter 1.2.1 --- Lignin peroxidases (LiP) --- p.3 / Chapter 1.2.2 --- Gene structure and Amino acid sequence structure --- p.7 / Chapter 1.2.3 --- Regulation of expression --- p.8 / Chapter 1.3. --- MnP --- p.8 / Chapter 1.3.1 --- General properties --- p.8 / Chapter 1.3.2 --- Gene structure and Amino acid sequence --- p.9 / Chapter 1.3.3 --- Regulation of Expression --- p.12 / Chapter 1.4 --- Laccase --- p.12 / Chapter 1.4.1 --- General Properties --- p.12 / Chapter 1.4.2 --- Gene structure and Amino acid sequence --- p.14 / Chapter 1.5 --- Pentachlorophenol (PCP) --- p.16 / Chapter 1.5.1 --- Production --- p.16 / Chapter 1.5.2 --- Toxicity --- p.15 / Chapter 1.5.3 --- Persistence --- p.19 / Chapter 1.6 --- Oyster mushroom --- p.22 / Chapter 1.7 --- Application of ligninolytic enzymes in bioremediation --- p.23 / Chapter 1.7.1 --- Genetic modification --- p.23 / Chapter 1.7.2 --- Characterization of enzymes properties --- p.25 / Chapter 1.7.3 --- Ligninolytic enzymes Purification and extraction --- p.26 / Chapter 1.7.4 --- Immobilization of ligninolytic enzymes --- p.26 / Chapter 1.8 --- Fermentation --- p.29 / Chapter 1.8.1 --- Different types of fermentation --- p.29 / Chapter 1.8.1.1 --- Submerged fermentation (SF) --- p.29 / Chapter 1.8.1.2 --- Solid State Fermentation (SSF) --- p.30 / Chapter 1.9 --- Proposal and experimental plan of the project --- p.33 / Chapter 1.9.1 --- Objectives --- p.34 / Chapter 2. --- Methods --- p.36 / Chapter 2.1 --- Materials / Chapter 2.1.1 --- Culture maintenance --- p.36 / Chapter 2.1.2 --- Preparation of Pentachlorophenol (PCP) stock solution --- p.36 / Chapter 2.2 --- Optimization of production of ligninolytic enzymes by effective PCP concentration --- p.37 / Chapter 2.2.1 --- Preparation of mycelial homogenate --- p.37 / Chapter 2.2.2 --- Incubation --- p.37 / Chapter 2.2.3 --- Specific enzyme assays --- p.38 / Chapter 2.2.3.1 --- Laccase --- p.38 / Chapter 2.2.3.2 --- Manganese peroxidase (MnP) --- p.39 / Chapter 2.2.3.3 --- Lignin peroxidase (LiP) --- p.39 / Chapter 2.2.3.4 --- Protein --- p.39 / Chapter 2.3 --- Cloning of specific PCP-degradative laccase cDNA --- p.40 / Chapter 2.3.1 --- Isolation of total RNA --- p.41 / Chapter 2.3.2 --- Spectrophotometric quantification and qualification of DNA and RNA --- p.41 / Chapter 2.3.3 --- First strand cDNA synthesis --- p.42 / Chapter 2.3.4 --- Amplification of laccase cDNA --- p.43 / Chapter 2.3.4.1 --- Design of primers for PCR reaction --- p.43 / Chapter 2.3.4.2 --- Polymerase chain reaction --- p.44 / Chapter 2.3.5 --- Agarose gel electrophoresis of DNA --- p.44 / Chapter 2.3.6 --- Purification of PCR products --- p.45 / Chapter 2.3.7 --- TA cloning of PCR products --- p.46 / Chapter 2.3.8 --- Preparation of Escherichia coli competent cells --- p.46 / Chapter 2.3.9 --- Bacterial transformation by heat shock --- p.47 / Chapter 2.3.10 --- Colony screening --- p.48 / Chapter 2.3.11 --- Mini-preparation of plasmid DNA --- p.48 / Chapter 2.3.12 --- Sequencing --- p.49 / Chapter 2.3.13 --- Identification of sequence --- p.51 / Chapter 2.4 --- Study of regulation temporal expression of laccase genes by PCP --- p.51 / Chapter 2.4.1 --- Semi-quantitative PCR --- p.51 / Chapter 2.4.1.1 --- Design of gene-specific primers --- p.51 / Chapter 2.4.1.2 --- Determination of suitable PCR cycles --- p.54 / Chapter 2.4.1.3 --- Normalization of the amount of RNA of each sample --- p.54 / Chapter 2.5 --- Quantification of residual PCP concentration --- p.55 / Chapter 2.5.1 --- Extraction of PCP --- p.55 / Chapter 2.5.2 --- High performance liquid chromatography --- p.55 / Chapter 2.5.3 --- Assessment criteria --- p.56 / Chapter 2.6 --- Effect of other componds on laccase activity and laccase expression --- p.56 / Chapter 2.6.1 --- Study of different isoform of laccase --- p.57 / Chapter 2.6.2 --- SDS-PAGE analysis of proteins --- p.58 / Chapter 2.7 --- Study of laccase expression and laccase activity in fruiting process of oyster mushroom --- p.59 / Chapter 2.8 --- Statistical analysis --- p.60 / Chapter 3. --- Results --- p.61 / Chapter 3.1 --- Production of Ligninolytic Enzymes by oyster mushroom / Chapter 3.1.1 --- Optimization of laccase production --- p.62 / Chapter 3.1.2 --- Optimization of MnP production --- p.64 / Chapter 3.1.3 --- Change of Protein content at different PCP concentration and time --- p.64 / Chapter 3.1.4 --- Change of specific activity at different PCP concentration and time --- p.64 / Chapter 3.1.5 --- Toxicity of PCP towards mycelial growth --- p.67 / Chapter 3.1.6 --- Enzyme productivities of laccase and MnP --- p.67 / Chapter 3.1.7 --- Change of % of residual PCP concentrations during 14 days --- p.70 / Chapter 3.2. --- Cloning of PCP-degradative laccase genes --- p.70 / Chapter 3.3 --- Regulation of expression of the laccase genes by PCP --- p.74 / Chapter 3.3.1 --- Determination of suitable PCR cycles --- p.74 / Chapter 3.3.2 --- Normalization of total RNA amount of different samples --- p.74 / Chapter 3.3.3 --- Regulation of temporal expression of the laccase genes by PCP --- p.74 / Chapter 3.4 --- Effect of other compounds and physiological status on laccase activity and expression --- p.81 / Chapter 3.5 --- Study of different forms of laccase --- p.86 / Chapter 4. --- Discussion --- p.93 / Chapter 4.1 --- Production of Ligninolytic enzymes by Pleurotus pulmonarius / Chapter 4.1.1 --- Optimization of laccase and MnP production by PCP --- p.95 / Chapter 4.2 --- Cloning of laccase genes --- p.97 / Chapter 4.2.1 --- Cloning strategy --- p.97 / Chapter 4.2.2 --- Analysis of Nucleotide sequence of Lac1 - Lac3 --- p.99 / Chapter 4.2.3 --- Characterization and comparison of deduced amino acid sequences of Lacl-Lac3 --- p.99 / Chapter 4.3 --- Regulation of expression of the laccase genes by PCP --- p.100 / Chapter 4.3.1 --- Regulation of temporal expression by PCP --- p.100 / Chapter 4.4 --- Effect of the potential inducers on laccase activity and expression --- p.103 / Chapter 4.5 --- Effect of the physiological status on laccase activity and expression --- p.105 / Chapter 4.5.1 --- Production of PCP-degradative laccase by Solid-state fermentation --- p.107 / Chapter 4.5.2 --- Uses of molecular probe in bioremediation --- p.107 / Chapter 4.6 --- Different isoforms of laccase --- p.109 / Chapter 4.7 --- Conclusion --- p.112 / Chapter 4.8 --- Further studies / Chapter 4.8.1 --- Confirmation of PCP-degradation by gene product of Lac1 and Lac2 --- p.114 / Chapter 4.8.2 --- Optimization of PCP-degradative laccases production by solid-state fermentation --- p.114 / Chapter 5. --- References --- p.115

Yau Sze-nga. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 155-177). / Abstracts in English and Chinese. / Acknowledgement --- p.i / Abstract --- p.ii / 摘要 --- p.v / Table of Contents --- p.vii / List of Figures --- p.xi / List of Tables --- p.xiv / Chapter 1 --- INTRODUCTION --- p.1 / Chapter 1.1 --- Organopollutants and environment --- p.1 / Chapter 1.2 --- Pentachlorophenol --- p.3 / Chapter 1.2.1 --- Application of pentachlorophenol --- p.3 / Chapter 1.2.2 --- Characteristics of PCP --- p.4 / Chapter 1.2.3 --- Toxicity of PCP --- p.5 / Chapter 1.2.4 --- Environmental exposure of PCP --- p.6 / Chapter 1.3 --- Wastewater treatments of organopollutants --- p.9 / Chapter 1.3.1 --- Physical treatment --- p.10 / Chapter 1.3.2 --- Chemical treatment --- p.10 / Chapter 1.3.3 --- Bioremediation --- p.11 / Chapter 1.4 --- Biodegradation of PCP --- p.13 / Chapter 1.4.1 --- Biodegradation of PCP by bacteria --- p.13 / Chapter 1.4.2 --- Biodegradation of PCP by fungi --- p.14 / Chapter 1.5 --- Ligninolytic enzyme --- p.16 / Chapter 1.5.1 --- Lignin peroxidase --- p.16 / Chapter 1.5.2 --- Manganese peroxidase --- p.19 / Chapter 1.5.3 --- Laccase --- p.21 / Chapter 1.5.4 --- Biodegradation of PCP and other organopollutants by ligninolytic enzymes --- p.25 / Chapter 1.6 --- Structure and gene regulation --- p.27 / Chapter 1.6.1 --- MnP gene and structure --- p.27 / Chapter 1.6.1.1 --- Structure of MnP --- p.27 / Chapter 1.6.1.2 --- MnP gene regulation --- p.30 / Chapter 1.6.2 --- Laccase gene and structure --- p.31 / Chapter 1.6.2.1 --- Structure of laccase --- p.31 / Chapter 1.6.2.2 --- Laccase gene regulation --- p.32 / Chapter 1.7 --- Pleurotus pulmonarius --- p.36 / Chapter 1.8 --- Aims of study --- p.37 / Chapter 2 --- MATERIALS & METHOD --- p.39 / Chapter 2.1 --- Optimization of PCP induction in broth system --- p.39 / Chapter 2.1.1 --- Specific enzyme assays --- p.41 / Chapter 2.1.1.1 --- Assay for laccase activity --- p.41 / Chapter 2.1.1.2 --- Assay for manganese peroxidase (MnP) activity --- p.41 / Chapter 2.1.1.3 --- Assay for protein assay --- p.41 / Chapter 2.1.2 --- PCP effect on biomass gain --- p.42 / Chapter 2.1.3 --- Extraction of PCP --- p.42 / Chapter 2.1.3.1 --- Preparation of PCP stock solution --- p.43 / Chapter 2.1.3.2 --- Extraction efficiency of PCP --- p.43 / Chapter 2.1.3.3 --- Quantification of PCP by HPLC --- p.43 / Chapter 2.1.3.4 --- Study of PCP degradation pathway using GC-MS --- p.44 / Chapter 2.2 --- Isolation of laccase and manganese peroxidase coding genes --- p.46 / Chapter 2.2.1 --- Preparation of ribonuclease free reagents and apparatus --- p.46 / Chapter 2.2.2 --- Isolation of RNA --- p.46 / Chapter 2.2.3 --- Quantification of total RNA --- p.47 / Chapter 2.2.4 --- First strand cDNA synthesis --- p.47 / Chapter 2.2.5 --- Polymerase Chain Reaction (PCR) --- p.48 / Chapter 2.2.6 --- Gel electrophoresis --- p.50 / Chapter 2.2.7 --- Purification of PCR products --- p.50 / Chapter 2.2.8 --- Preparation of Escherichia coli competent cells --- p.51 / Chapter 2.2.9 --- Ligation and E. coli transformation --- p.51 / Chapter 2.2.10 --- PCR screening of E. coli transformation --- p.52 / Chapter 2.2.11 --- Isolation of recombinant plasmid --- p.52 / Chapter 2.2.12 --- Sequence analysis --- p.53 / Chapter 2.2.13 --- Construction of dendrogram for Pleurotus sp. laccase and manganese peroxidase dendrogram --- p.54 / Chapter 2.2.13.1 --- Dendrogram of laccase genes --- p.55 / Chapter 2.2.13.2 --- Dendrogram of manganese genes --- p.55 / Chapter 2.3 --- Differential regulation profiles of laccase and manganese peroxidase genes --- p.57 / Chapter 2.3.1 --- Time course of the effects of PCP on levels of laccase and manganese peroxidase mRNAs --- p.57 / Chapter 2.3.1.1 --- Isolation of RNA --- p.57 / Chapter 2.3.1.2 --- RT-PCR --- p.57 / Chapter 2.3.2 --- The effect of different stresses --- p.65 / Chapter 2.3.2.1 --- Pollutant removal analysis --- p.66 / Chapter 2.3.2.2 --- Differential gene expression under different stresses --- p.69 / Chapter 2.4 --- Construction of full-length cDNA --- p.69 / Chapter 2.4.1 --- Primer design --- p.69 / Chapter 2.4.2 --- First-strand cDNA synthesis --- p.71 / Chapter 2.4.3 --- RACE PCR reactions --- p.71 / Chapter 2.5 --- Statistical analysis --- p.73 / Chapter 3 --- RESULT --- p.74 / Chapter 3.1 --- Optimization of PCP induction in broth system --- p.74 / Chapter 3.1.1 --- Enzyme Assay --- p.74 / Chapter 3.1.1.1 --- Protein content --- p.74 / Chapter 3.1.1.2 --- Specific laccase activity --- p.74 / Chapter 3.1.1.3 --- Specific MnP activity --- p.76 / Chapter 3.1.1.4 --- Laccase productivity --- p.78 / Chapter 3.1.1.5 --- MnP productivity --- p.78 / Chapter 3.1.2 --- PCP effect on biomass development --- p.80 / Chapter 3.1.3 --- PCP removal --- p.80 / Chapter 3.2 --- isolation of laccase and manganese peroxidase coding genes --- p.83 / Chapter 3.2.1 --- Dendrogram construction for heterologous MnP and laccase coding genes --- p.83 / Chapter 3.2.2 --- Phylogeny of ligninolytic enzyme coding genes of P. pulmonarius --- p.85 / Chapter 3.2.2.1 --- Phylogeny of MnP coding genes --- p.88 / Chapter 3.2.2.2 --- Phylogeny of laccase coding genes --- p.88 / Chapter 3.3 --- differential regulation profiles of laccase and MnP genes --- p.91 / Chapter 3.3.1 --- Time course of the effects of PCP on levels of MnP and laccase mRNAs --- p.91 / Chapter 3.3.1.1 --- Time course of the effects of PCP on levels of MnP mRNAs --- p.91 / Chapter 3.3.1.2 --- Time course of the effects of PCP on levels of laccase mRNAs --- p.97 / Chapter 3.3.2 --- The effects of different stresses and two lignocellulosic substrates --- p.99 / Chapter 3.3.2.1 --- The effect on laccase and MnP enzyme activities --- p.99 / Chapter 3.3.2.1.1 --- Protein content --- p.99 / Chapter 3.3.2.1.2 --- Specific laccase activity --- p.100 / Chapter 3.3.2.1.3 --- Specific MnP activity --- p.102 / Chapter 3.3.2.1.4 --- Dry weight of P. pulmonarius --- p.102 / Chapter 3.3.2.1.5 --- Laccase productivity --- p.105 / Chapter 3.3.2.1.6 --- MnP productivity --- p.105 / Chapter 3.3.2.2 --- Organopollutant removal --- p.107 / Chapter 3.3.2.3 --- Differential gene expression under different stresses --- p.107 / Chapter 3.3.2.3.1 --- The effect on MnP mRNAs --- p.107 / Chapter 3.3.2.3.2 --- The effect on laccase mRNAs --- p.115 / Chapter 3.4 --- Construction of full-length cDNA --- p.116 / Chapter 3.4.1 --- PPMnP5 --- p.117 / Chapter 3.4.2 --- PPlac2 --- p.120 / Chapter 3.4.3 --- PPlac6 --- p.120 / Chapter 4 --- DISCUSSION --- p.123 / Chapter 4.1 --- Optimization of PCP induction in broth system --- p.123 / Chapter 4.2 --- Isolation of MnP and laccase coding genes --- p.126 / Chapter 4.3 --- Differential regulation profiles of MnP and laccase genes --- p.128 / Chapter 4.3.1 --- The effects incubation time and PCP on levels of MnP and laccase mRNAs --- p.128 / Chapter 4.3.1.1 --- MnP --- p.129 / Chapter 4.3.1.2 --- Laccase --- p.129 / Chapter 4.3.2 --- Regulation of MnP and laccase by different substrates --- p.130 / Chapter 4.3.2.1 --- Regulation of MnP and laccase activities --- p.131 / Chapter 4.3.2.2 --- Organopollutant removal --- p.132 / Chapter 4.3.2.3 --- Regulation of MnP coding genes --- p.136 / Chapter 4.3.2.4 --- Regulation of laccase coding genes --- p.137 / Chapter 4.4 --- "Characterization of full length cDNAs of PPMnP5, PPlac2 and PPLAC6" --- p.140 / Chapter 4.4.1 --- PPMnP5 --- p.140 / Chapter 4.4.2 --- PPlac2 and PPlac6 --- p.144 / Chapter 4.4.3 --- Real-time PCR --- p.146 / Chapter 4.4.3.1 --- Methodology for SYBR-Green real-time PCR --- p.146 / Chapter 4.4.3.2 --- Comparison of conventional PCR and real-time PCR --- p.148 / Chapter 4.5 --- APPLICATION AND FURTHER INVESTIGATION --- p.150 / Chapter 5 --- CONCLUSION --- p.152 / Chapter 6 --- REFERENCES --- p.155