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 3.2.3.1. --- Steam pretreatment of lignocellulosic feedstocks --- p.76 / Chapter 3.2.3.2. --- Enzymatic hydroloysis of steam-pretreated substrates --- p.77 / Chapter 3.2.3.3. --- Influence of laccase/LMS on substrate hydrolysability --- p.77 / Chapter 3.2.3.4. --- Optimisation of sequential reaction strategy --- p.78 / Chapter 3.2.3.5. --- 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
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_328292 |
| Date | January 2012 |
| Contributors | Wong, Kin Sing., Chinese University of Hong Kong Graduate School. Division of Life Sciences. |
| Source Sets | The Chinese University of Hong Kong |
| Language | English, Chinese |
| Detected Language | English |
| Type | Text, bibliography |
| Format | electronic resource, electronic resource, remote, 1 online resource (xvii, 177 leaves) : ill. (some col.) |
| Rights | Use of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/) |
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