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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.

A post-genomic study of Lentinula edodes laccases: from basic sciences to potential applications and molecular engineering. / CUHK electronic theses & dissertations collection

January 2012 (has links)
擔子菌的漆酶以及其漆酶介體系統(LMSs)均是具廣泛『色』應用的多功能生物催化劑,能應用於生物修和生物製等方面。可惜缺乏酶的源及簡單的表達平臺、各界一致的酶學研究方法、以及清晰的分子進化方向均是進一步應用及研究漆酶的當前挑戰。本研究以香菇的基因組序為依歸,逐一克服以上的漆酶問題。首先本研究從香菇的基因組中別出漆酶家族,並從其菌絲克出同工酶(共十個同工酶及個等位基因變體)。然後以畢赤酵母建一個穩健的源表達平臺,用以產出五個重組漆酶(Lcc1A、Lcc1B、Lcc4A、Lcc5和Lcc7)。Lcc1A 和Lcc1B 的試點研究首先提供標準化的酶測試方法,以供比較各重組酶在酶學及應用層面上的效能。結果發現Lcc1A 及其LMSs 能最有效地生物解合成染和多環芳烴,亦能把經蒸汽預處的軟木的酶化效能提高37%至46%。2,2'-氮雙(3-乙基苯並噻唑啉-6-磺酸)二銨鹽(ABTS)是最常用於篩選漆酶的基準底物。然而Lcc7 對此基準底物的活性才是眾重組酶中最高(比Lcc1A 高七倍)。鑒於這些重組漆酶對ABTS 和應用性底物活性的一致,我們有需要找出另一種具指標性的基準底物驅動真菌漆酶的蛋白質工程。本研究用相關係分析發現愈創木酚比ABTS 和2,6-二甲氧基苯酚這種常用的基準底物,能有效反映漆酶解合成染的能。氨基酸序分析進一步顯示出可能影響重組漆酶特性的保守基序和底物結合位置的氨基酸殘基。本研究僅為香菇漆酶及新酶提供一個可靠的酵母表達平臺,還總括漆酶蛋白序與其功能的重要關係以及提供具指標性的基準底物。這些都為日後演發出具應用性的漆酶奠定重要的基礎。 / Laccases from basidiomycetes, and their laccase-mediator systems (LMSs), are versatile biocatalysts for "green" applications including bioremediation and biorefinery. Scarcity of sources of enzymes, lack of simple expression platforms, inconsistent enzymology methods, and blurred directions of molecular evolution are major challenges to exploitations of laccases. The present study addressed these challenges using our genome sequence of Lentinula edodes. A laccase family of ten isozymes plus two allelic forms was cloned from L. edodes mycelia. The yeast Pichia pastoris expression platform was then established to achieve robust heterologous expression of five laccases (Lcc1A, Lcc1B, Lcc4A, Lcc5 and Lcc7). Pilot study on Lcc1A and Lcc1B provided standardised methodology for enzymological and application comparisons among the recombinant laccases. Lcc1A and its LMSs were the most efficient in biodegradation of synthetic dyes and polyaromatic hydrocarbons, and could improve the enzymatic saccharification f steam-pretreated softwoods by 37% to 46%. On the other hand, Lcc7 had the highest activity (~7-fold higher than that of Lcc1A) on 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) which is the most commonly employed benchmark substrate for laccase screening. The inconsistent performance of recombinant laccases on ABTS and applications showed the need for a more indicative benchmark substrate for application-oriented engineering of fungal laccases. A correlation analysis revealed that the laccase activity on guaiacol associated much better with the enzyme performance on decolourisation of structurally different dyes than commonly employed ABTS and 2,6-dimethoxyphenol. Sequence analyses also suggested potential amino acid residues in conserved motifs and substrate binding loops that could be responsible for variations of enzymatic properties among the recombinant laccases. This study reported not only a robust yeast expression platform of L. edodes laccases and novel enzymes, but also important sequence-function relationship and an indicative substrate for engineering of laccases for efficient industrial applications. / Detailed summary in vernacular field only. / Wong, Kin Sing. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 158-177). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / ABSTRACT (ENGLISH) --- p.i / ABSTRACT (CHINESE) --- p.iii / ACKNOWLEDGEMENTS --- p.v / DECLARATION --- p.vii / ABBREVIATIONS --- p.viii / TABLE OF CONTENTS --- p.xi / LIST OF TABLES --- p.xv / LIST OF FIGURES --- p.xvi / Chapter CHAPTER 1 --- INTRODUCTION / Chapter 1.1. --- Genomics and next-generation sequencings --- p.1 / Chapter 1.2. --- Laccases and their industrial relevance --- p.4 / Chapter 1.3. --- Lentinula edodes, a white-rot basidiomycete, is a potential sink of laccases --- p.14 / Chapter 1.4. --- Heterologous expression of L. edodes laccases --- p.17 / Chapter 1.5. --- Yeasts as expression hosts for recombinant laccases --- p.21 / Chapter 1.6. --- Objectives of the study --- p.25 / Chapter CHAPTER 2 --- ESTABLISHMENT OF A YEAST EXPRESSION PLATFORM FOR L. edodes LACCASES / Chapter 2.1. --- Introduction --- p.27 / Chapter 2.2. --- Materials and methods / Chapter 2.2.1. --- Prediction of laccase genes from the genome of L. edodes --- p.29 / Chapter 2.2.2. --- Molecular cloning and sequence analysis of laccase genes from L. edodes --- p.30 / Chapter 2.2.3. --- Sub-cloning of laccase genes into expression vectors --- p.35 / Chapter 2.2.4. --- Transformation of P. pastoris and activity screening --- p.36 / Chapter 2.2.5. --- Optimisation of induction regime --- p.37 / Chapter 2.2.6. --- Protein quantification and standard enzymatic assay --- p.40 / Chapter 2.2.7. --- Shake flask fermentation --- p.40 / Chapter 2.2.8. --- Fed-batch fermentation --- p.41 / Chapter 2.2.9. --- Purification of recombinant Lcc1A and Lcc1B --- p.42 / Chapter 2.2.10. --- SDS-PAGE and activity staining --- p.43 / Chapter 2.2.11. --- Determination of kinetic parameters on benchmark substrates --- p.43 / Chapter 2.2.12. --- Evaluation of stability against temperature, pH and organic solvents --- p.44 / Chapter 2.3. --- Results / Chapter 2.3.1. --- Cloning and heterologous expression of pioneering Lcc1 --- p.45 / Chapter 2.3.2. --- Optimisation of the expression titer of Lcc1A --- p.47 / Chapter 2.3.3. --- Characterisations of purified Lcc1A and Lccc1B --- p.54 / Chapter 2.4. --- Discussion --- p.57 / Chapter CHAPTER 3 --- "GREEN" APPLICATIONS OF RECOMBINANT LACCASES / Chapter 3.1. --- Introduction --- p.71 / Chapter 3.2. --- Materials and methods / Chapter 3.2.1. --- Dye decolourisation assay --- p.74 / Chapter 3.2.2. --- PAH degradation assay --- p.75 / Chapter 3.2.3. --- Laccase treatments on steam-pretreated lignocellulosic feedstocks / Chapter --- Steam pretreatment of lignocellulosic feedstocks --- p.76 / Chapter --- Enzymatic hydroloysis of steam-pretreated substrates --- p.77 / Chapter --- Influence of laccase/LMS on substrate hydrolysability --- p.77 / Chapter --- Optimisation of sequential reaction strategy --- p.78 / Chapter --- Evaluation of catalytic contribution of Lcc1A --- p.79 / Chapter 3.3. --- Results / Chapter 3.3.1. --- Decolourisation of synthetic dyes --- p.80 / Chapter 3.3.2. --- Biodegradation of PAHs --- p.80 / Chapter 3.3.3. --- Improved hydrolysis of steam-pretreated softwoods --- p.83 / Chapter 3.4. --- Discussion --- p.91 / Chapter CHAPTER 4 --- A NOVEL LACCASE, COMPARATIVE ENZYMOLOGY, AND INSIGHTS INTO MOLECULAR ENGINEERING / Chapter 4.1. --- Introduction --- p.108 / Chapter 4.2. --- Materials and methods / Chapter 4.2.1. --- Gene prediction and molecular cloning --- p.110 / Chapter 4.2.2. --- Heterologous expression and purification of recombinant laccases --- p.114 / Chapter 4.2.3. --- Characterisations of recombinant laccases --- p.115 / Chapter 4.2.4. --- Bioinformatic analyses of recombinant laccases --- p.116 / Chapter 4.2.5. --- Phylogenetic analysis of recombinant laccases --- p.117 / Chapter 4.2.6. --- Correlation analyses between oxidation of benchmark substrates, dye decolourisation and PAH degradation --- p.118 / Chapter 4.3. --- Results / Chapter 4.3.1. --- Novel laccases and sequence characteristics --- p.118 / Chapter 4.3.2. --- Preparation of purified Lcc4A, Lcc5 and Lcc7 --- p.128 / Chapter 4.3.3. --- Kinetics and stability of Lcc4A, Lcc5 and Lcc7 --- p.128 / Chapter 4.3.4. --- "Green" applications by Lcc4A, Lcc5, Lcc7 and their LMSs --- p.135 / Chapter 4.3.5. --- Correlations between catalyses on benchmark substrates, dyes and PAHs --- p.142 / Chapter 4.4. --- Discussion --- p.145 / Chapter CHAPTER 5 --- CONCLUSIONS AND PERSEPECTIVES --- p.156 / BIBLIOGRAPHY --- p.158

Zur Effizienz der Anreicherung und Isolierung von Oxidoreduktasen mit Hilfe der Zerschäumungsanalyse

Gerken, Birte Maja. January 2005 (has links) (PDF)
München, Techn. Universiẗat, Diss., 2005.

Enzymatic treatment of pharmaceuticals and personal care products (PPCPs) in municipal wastewater

Sharkey, Margaret E 29 October 2013 (has links)
Conventional wastewater treatment plants do not effectively remove pharmaceuticals and personal care products (PPCPs). As a result, PPCPs enter the environment via treated wastewater discharge. Enzymatic treatment, using the laccasemediator system, is a novel biochemical process that has been shown to effectively treat some PPCPs. This study investigates the efficacy of the laccase-mediator system to treat PPCPs using a process that can be easily implemented at an existing wastewater treatment plant. Enzymatic treatment will be most beneficial after primary sedimentation and before conventional biological treatment, where unoxidized PPCPs and byproducts could have the opportunity for further degradation in biological treatment. In this work, two enzymatic treatment configurations were studied. A step-wise optimization process was used that alternately varied treatment conditions: pH, enzyme activity, mediator concentration, and reactor detention time. In the optimization process of each configuration, successful oxybenzone removal (~90%) was achieved in municipal primary effluent. In a direct comparison of treatment configurations, both resulted in vi similar percent removals of oxybenzone. Therefore, the configuration with the simpler operation and reactor design was chosen for further study. During the optimization process, several noteworthy conclusions were made that might have full-scale enzymatic treatment implications. Specifically, successful oxybenzone removal occurred at unadjusted pH and without aeration, but increased biological oxygen demand of the wastewater increased the required mediator concentration. While the first finding would decrease enzymatic treatment costs, the latter would increase the costs associated with the mediator. Thus, an alternative mediator source, specifically one high in phenolic compounds, is desired. The use of wine, as a surrogate of winery wastewater, was in investigated and proved ineffective. Further investigation of alternative mediator sources is required. Treatment of another PPCP, sulfamethoxazole, was less efficient (65% removal) than that of oxybenzone, but nevertheless, the substantial removal might indicate that other PPCPs can be treated with the laccase-mediator system. The most promising result of this work was the simultaneous treatment of multiple PPCPs, oxybenzone and sulfamethoxazole. Simultaneous treatment proved to be as effective as when each PPCP was treated individually. / text

Kinetics of the laccase-catalyzed oxidation of aqueous phenol

Soegiaman, Selvia Kurniawati. January 2006 (has links)
Laccase (E.C catalyzes the oxidation of aromatic substrates with the simultaneous reduction of molecular oxygen to water. It has significant potential for use in many applications due to its high reaction rates, broad substrate-specificity, and use of oxygen as an inexpensive co-factor. The objective of this research was to investigate the ability of laccase from Trametes versicolor to catalyze oxidation reactions under a variety of reaction conditions and to model the kinetics of these transformations. Phenol was selected as a model substrate. / Laccase was very stable when incubated at temperatures less than 30°C and pHs between 6 and 7. The optimum pH for phenol transformation was 6, but when present in sufficient quantities, laccase was able to significantly transform phenol at pHs from 4 to 7 and temperatures from 10 to 60°C. Laccase stability was negatively impacted by the presence of four common redox mediators. Of these, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) and 2,2',6,6'-tetramethylpiperidine-N-oxyl (TEMPO) significantly enhanced phenol transformation but large quantities were required, which may limit the feasibility of the use of these mediators in many applications. / A series of kinetic models was developed in order to achieve a better understanding of the mechanisms and kinetics of laccase-catalyzed reactions and to eventually assist in the choice and design of suitable reactor systems. These models were designed to predict the transient oxygen and phenol concentrations during laccasecatalyzed reactions at pH 6 and 25°C. Over the course of developing and validating these models, it was observed that: (1) the rate-limiting step in the catalytic reactions is the reaction between the oxidized form of laccase and phenol; (2) the stoichiometric ratio, which is defined as the molar ratio of phenol transformed to oxygen consumed in the catalytic reaction, was found to increase with phenol concentration in the reaction mixture from a theoretical lower limit of 1 and to approach a theoretical upper limit of 4; and (3) laccase inactivation occurs over the course of the reaction and was found to be dependent on the rate of substrate transformation. / Ultimately, these findings were incorporated into a comprehensive kinetic model to predict transient species concentrations in an open-system environment where the degree of substrate transformation was not limited by oxygen availability. The model accounts for enzyme kinetics, oxygen mass-transfer, variable reaction stoichiometry, and inactivation related to reaction products. Excellent agreement was observed between measured and modeled phenol and oxygen concentrations for a wide range of initial phenol concentrations and enzyme activities. Simplified models were also developed by incorporating an assumption, referred to as the pseudo-steady-state assumption, that at any instant during the reaction, the enzyme achieves an approximate steady-state distribution of its various forms around the catalytic cycle. The pseudo-steady-state assumption had the advantage of reducing the complexity of model equations without sacrificing their predictive abilities and allowing enzyme quantities to be expressed in activity units instead of molar concentrations.

Insolubilisation de laccase et de tyrosinase pour la transformation enzymatique de produits pharmaceutiques phénoliques dans les eaux usées

Ba, Sidy January 2014 (has links)
Au cours des dernières décennies, un nombre croissant de micropolluants organiques a été détecté et quantifié dans l'environnement à de faibles concentrations échappant aux procédés conventionnels de traitement des stations d'épuration dont les effluents constituent des sources majeures de déversement. Parmi ces micropolluants bons nombres sont des molécules pharmaceutiques à structure aromatique ou phénolique. Ces molécules pharmaceutiques restent généralement biologiquement actives même dans les matrices environnementales d'où les nombreuses préoccupations au sujet de leurs risques et impacts avérés ou inconnus sur les écosystèmes et la santé. Plusieurs méthodes de traitement incluant des méthodes biologiques, adsorption, filtrations membranaires et oxydations avancées ont été expérimentées pour l'élimination de ces contaminants pharmaceutiques dans les eaux usées. Bien que dans de nombreux cas ces méthodes fournissent une efficacité élevée d'élimination des composés récalcitrants ciblés, elles génèrent souvent des sous-produits de toxicité plus élevée que le composé parent ou sont associées à des coûts élevés d'investissement ou d'exploitation. En outre, plusieurs enzymes oxydatives dont la laccase et la tyrosinase ont démontré leurs capacités à transformer des micropolluants aromatiques et phénoliques en solution en des macromolécules qui précipitent et qui peuvent être ensuite séparées de la solution. Ces deux phénoloxidases catalysent la transformation des composés phénoliques en n'utilisant que l'oxygène moléculaire comme unique cofacteur avec production d'eau. Une revue littéraire approfondie a été faite sur les caractéristiques des laccases en relation avec l'élimination des composés phénoliques en solution. Ce qui a permis de faire ressortir un consensus général exprimé chez différents auteurs sur la nécessité d'immobiliser la laccase sur des supports, de la claustrer dans des capsules ou de l'insolubiliser sous forme d'agrégats d'enzymes réticulées (cross-linked enzymes aggreagtes ou CLEAs). Cette dernière technique offr avantageusement un meilleur ratio de masse par volume de biocatalyseur tout en améliorant ses caractéristiques catalytiques. Elle permet aussi de remédier à la dilution de l'activité de l'enzyme sur le support ou dans la capsule observée avec les deux premières techniques. Finalement, une nouvelle technique d'insolubilisation en combinant deux ou plusieurs enzymes de caractéristiques différentes (combination of cross-linked enzymes aggreagtes ou combi-CLEAs) est proposée comme nouvelle approche permettant l'élimination d'une gamme de substrats plus élargie. Ainsi, la laccase fongique de la souche de Trametes versicolor (TvL) et une laccase bactérienne metzyme (MZL) ayant leur pHs optima à 4 et 8, respectivement, ont été insolubilisées sous forme de combi-CLEA. Le combi-CLEA demeure actif aussi bien en pH acide qu'alcalin. Les études de températures ont permis de déterminer que la température optimale du combi-CLEA est à 50 °C et que sa stabilité thermique est supérieure à chacune des deux laccases libres. L'application du combi-CLEA à des échantillons d'eau usée d'usine de pâte et papier a permis de réduite de 70% sa DCO totale. Finalement, le recyclage du combi-CLEA a aussi été mis en évidence par son activité résiduelle après deux cycles d'utilisation dans le traitement des échantillons de l'eau usée. Subséquemment, les caractéristiques catalytiques de la tyrosinase sont examinées et des exemples de son insolubilisation en CLEA trouvés dans la littérature sont fournis. Il apparaît aussi que plusieurs composés phénoliques et aromatiques y compris les chlorophénols, fluorophénols, les alkylphénols, les colorants azoïques ont fait l'objet d'élimination dans les eaux usées par la tyrosinase. Ceci démontre donc le fort potentiel qu'a cette enzyme aussi à éliminer les micropolluants pharmaceutiques phénoliques. Ensuite, la tyrosinase de champignon avec un pH optimum à 7 a été substituée à la MZL pour former un combi-CLEA avec la TvL. Le combi-CLEA a exhibé une grande activité enzymatique aux pH 5-8 et températures 5-30 °C, une résistance significative à la dénaturation. Aucune altération des performances catalytiques du combi-CLEA comparées à celles des enzymes libres qui la constituent n'a en outre été observée en se basant sur l'étude des paramètres cinétiques de Michaelis-Menten. L' application en mode cuvée du combi-CLEA a permis de transformer plus de 80% à près de 100% l'acetaminophène dans l'eau usée municipale et à plus de 90% dans l'eau usée hospitalière. Une étude d'identification des produits de transformation de l'acetaminophène a montré la formation de ses oligomères sous formes de dimères, trimères et tétramères due à la laccase et de 3-hydroxyacetaminophen due à la tyrosinase. Finalement, les travaux de cette thèse suggèrent un réel potentiel d'application de la laccase et de la tyrosinase insolubilisées pour l'élimination de micropolluants phénoliques dans les eaux usées.

Production of laccase by the phytopathogenic fungus Rhizoctonia solani /

Bora, Pranjal. January 2003 (has links)
Thesis (Ph.D.)--Murdoch University, 2003. / Thesis submitted to the Division of Science and Engineering. Bibliography: leaves 138-173.

Enzyme-catalyzed oxidation of 17[beta]-estradiol using immobilized laccase from T. versicolor

Cardinal-Watkins, Chantale. January 1900 (has links)
Thesis (M.Eng.). / Written for the Dept. of Civil Engineering and Applied Mechanics. Title from title page of PDF (viewed 2008/01/14). Includes bibliographical references.

Growth studies of marine and terrestrial lignicolous fungi with special reference to laccase and other lignin-modifying enzyme activities of xylariaceous fungi /

Luo, Wen. January 2005 (has links) (PDF)
Thesis (Ph.D.)--City University of Hong Kong, 2005. / "Submitted to Department of Biology and Chemistry in partial fulfillment of the requirements for the degree of Doctor of Philosophy" Includes bibliographical references (leaves 216-256)

Isolierung und Charakterisierung von Kupferhomeostase-Faktoren aus Trametes versicolor und deren Einfluss auf die Laccase-Expression

Uldschmid, Andreas. Unknown Date (has links) (PDF)
Universiẗat, Diss., 2002--München.

Characterization and heterologous production of a novel laccase from Melanocarpus albomyces /

Kiiskinen, Laura-Leena. January 1900 (has links) (PDF)
Thesis (doctoral)--Helsinki University of Technology, 2005. / Includes bibliographical references. Also available on the World Wide Web.

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