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Canada Archives Canada Published Heritage Direction Du Branch Patrimoine De I'edition DISCOVERY AND CHARACTERIZATION OF HYDROLYTIC DEHALOGENASES FROM GENOMIC DATA by Max Wong A thesis submitted in conformity with the requirements for the degree of Master of Applied Science Graduate Department of Chemical Engineering and Applied Chemistry University of Toronto Copyright Q 2008 by Max Wong Library and Bibliotheque et 1*1 Archives Canada Archives Canada Published Heritage Direction du Branch Patrimoine de I'edition 395 Wellington Street 395, rue Wellington Ottawa ON K1A0N4 Ottawa ON K1A0N4 Canada Canada Your file Votre reference ISBN: 978-0-494-38866-2 Our file Notre reference ISBN: 978-0-494-38866-2 NOTICE: AVIS: The author has granted a non­ L'auteur a accorde une licence non exclusive exclusive license allowing Library permettant a la Bibliotheque et Archives and Archives Canada to reproduce, Canada de reproduire, publier, archiver, publish, archive, preserve, conserve, sauvegarder, conserver, transmettre au public communicate to the public by par telecommunication ou par I'lnternet, prefer, telecommunication or on the Internet, distribuer et vendre des theses partout dans loan, distribute and sell theses le monde, a des fins commerciales ou autres, worldwide, for commercial or non­ sur support microforme, papier, electronique commercial purposes, in microform, et/ou autres formats. paper, electronic and/or any other formats. The author retains copyright L'auteur conserve la propriete du droit d'auteur ownership and moral rights in et des droits moraux qui protege cette these. this thesis. Neither the thesis Ni la these ni des extraits substantiels de nor substantial extracts from it celle-ci ne doivent etre imprimes ou autrement may be printed or otherwise reproduits sans son autorisation. reproduced without the author's permission. In compliance with the Canadian Conformement a la loi canadienne Privacy Act some supporting sur la protection de la vie privee, forms may have been removed quelques formulaires secondaires from this thesis. ont ete enleves de cette these. While these forms may be included Bien que ces formulaires in the document page count, aient inclus dans la pagination, their removal does not represent il n'y aura aucun contenu manquant. any loss of content from the thesis. •*• Canada Abstract Discovery and characterization of hydrolytic dehalogenases from genomic data Max Wong Master of Applied Science Graduate Department of Chemical Engineering and Applied Chemistry University of Toronto 2008 Halogenated organic compounds are prevalent environmental contaminants as a result of their widespread use in industry, and they are cited for deleterious health effects. In situ bioremediation offers an alternative to existing processes for removing these substances from contaminated sites. Hydrolytic dehalogenases catalyze the reaction of organohalogens with water, replacing halide with a hydroxyl group. This project's principle objective was to find new dehaloge­ nases from genomic data, using previously characterized dehalogenases as search templates. Putative haloalkane, haloacid and fluoroacetate dehalogenases were identified by BLAST search of a selection of genomes from enviromental bacteria and scrutinized for the presence of known critical residues. They were recombinantly expressed and screened for activity. Out of 27 targets examined, eleven were true dehalogenases. Three haloacid dehaloge­ nases were discovered with previously-uncharacterized activity against fluoroacetate, and one haloalkane dehalogenase exhibited moderate activity against 1,2-dichloroethane. The success rate was family-dependent, and sequence similarity to characterized dehalogenases was gen­ erally a good indicator of a target's dehalogenation ability. 11 Acknowledgements First, my supervisor, Dr. Elizabeth Edwards: thank you for taking me onto the team and allowing me to work on this unique project. It's been quite a ride! Second, the talented people I worked with these two years. There's quite a list... • In the Best lab: Drs. Alexander Iakounine and Alexei Savchenko, Greg Brown and Michael Proudfoot for taking me into their lab and offering their expertise with protein expression and purification - and Thursday night bouts of sanity. • In EdLab: in alphabetical order, Winnie Chan, Angelika Duffy, Melanie Duhamel, Ariel Grostern, Laura Hug, Ahsan Islam, Eve Moore, Allie Simmonds, Alison Waller, Jennifer Wang and Cheryl Washer for acting as sounding boards for ideas and for giving me a home in Wallberg. • In Emil Pai's lab: Peter Chan for assistance in target selection and numerous training sessions, and Terence To for guidance with kinetic assays. Then there are the people who kept me (mostly) happy and hale during my stay... • Nadine Lam, for making sure I knew when to work, when not to work, for making sure I was always fed, for tending to the things I didn't, for listening to senseless ranting, and for letting a lot of my mistakes slide. • Jen Wang, for treating me like family and for giving me a wisp of Calgary. • Raymond Choi, for injecting sardonic realism into my life. • Stanley Wong, for listening to my frustrations on this side of the country. Most importantly, I would like to thank my parents, Paul and Eliza, for standing by me during the past six years. It wouldn't have happened this way without their support. 111 Contents 1 Motivation 1 1.1 Introduction 1 1.2 Existing remediation methods 2 1.2.1 Excavation 3 1.2.2 Pump-and-treat 3 1.2.3 Bioremediation 4 1.3 Opportunities from genomic data 4 1.4 Research objectives 7 1.5 Outline of document 7 2 Background 8 2.1 Haloalkane dehalogenases 8 2.2 L-2-haloacid dehalogenases 13 2.3 Fluoroacetate dehalogenases 15 3 Materials and Methods 17 iv 3.1 Selection of gene targets 17 3.2 Cloning and expression of gene targets 18 3.3 Expression vector preparation 19 3.4 Protein expression 21 3.5 Protein purification by affinity chromatography 21 3.6 Rapid colourimetric dehalogenation assay 22 3.7 Further purification of confirmed dehalogenases 23 3.8 Optimization of enzyme reaction conditions 24 3.9 Determination of enzyme kinetic parameters 25 3.9.1 Quantitative determination of halide production 25 4 Results and Discussion 28 4.1 Selection and purification of targets 28 4.2 Biochemical screening 33 4.2.1 Identification of HADs with defluorination activity 36 4.3 Kinetic characterization of haloacid dehalogenases 37 4.4 Rate estimate of defluorination 39 4.5 Rate estimate of 1,2-DCA dechlorination by Jann2620 41 4.6 Discussion 43 4.6.1 Confirmation of HAD annotations 45 4.6.2 Confirmation of HAn dehalogenase annotations 48 v 4.6.3 Confirmation of fluoroacetate dehalogenase annotations 53 5 Conclusions 58 5.1 Contributions 60 5.2 Future Work 60 Appendices 62 A Standard curves 63 A. 1 Standard curves for spectrophotometric assay 63 A.2 Standard curves for ion chromatographic assay 64 B Kinetic data for HADs 68 B.l Kinetics against chloroacetate 68 B.2 Kinetics against fluoroacetate 68 VI List of Tables 4.1 List of source organisms 29 4.2 List of putative dehalogenase targets 30 4.3 Existing annotations of putative dehalogenases 32 4.4 Protein yields 34 4.5 Results of general screens 35 4.6 Kinetic characteristics of HADs with ClAc 37 4.7 Comparison of turnover rates of HAD-FAcs and FAc dehalogenases 40 4.8 Pairwise alignment statistics of HAD targets 46 4.9 Pairwise alignment statistics of HAn targets 50 4.10 Pairwise alignment statistics of FAc dehalogenase targets 57 Vll List of Figures 2.1 Structure of DhaA of Rhodococcus rhodocrous NCIMB 13064 10 2.2 Reaction scheme of 1,2-DCA with DhlA 11 2.3 Structure of L-DEX YL from Pseudomonas sp. YL 14 3.1 pl5TV-L cloning plasmid 19 3.2 IC elution profile for separating fluoride and fluoroacetate 26 4.1 Visualization of proteins by SDS-PAGE 31 4.2 Example of colourimetric screening results 33 4.3 Defluorination by HAD dehalogenase Adeh3811 36 4.4 Example of kinetic characterization, here of Adeh3811 of Anaeromyxobacter dehalogenans 2CV-C. O.l^g/mL enzyme in 25mM CAPS-Na pH 10.5 38 4.5 Example of defluorination as observed via IC 39 4.6 IC confirmation of Jann2620 dechlorination of 1,2-DCA 42 4.7 Alignment of all HAD dehalogenase targets 44 4.8 Unrooted phylogenetic tree of HAD targets 47 viii 4.9 Alignment of positive HAn dehalogenases and closely related negatives .... 49 4.10 Unrooted phylogenetic tree of HAn targets 51 4.11 Domain structure annotation of Bpro2447 52 4.12 Unrooted phylogenetic tree of FAc dehalogenase targets 54 4.13 Alignment of FAc dehalogenase targets 56 B.l Michaelis-Menten curve for BC2051 69 B.2 Michaelis-Menten curve for Bpro0530 70 B.3 Michaelis-Menten curve for Bpro4516 71 B.4 Michaelis-Menten curve for GMI1362 72 B.5 Michaelis-Menten curve for Jannl658 73 B.6 Defluorination of FAc by Adeh3811 74 B.7 Defluorination of FAc by Bpro0530 74 B.8 Defluorination of FAc by Bpro4516 75 IX Chapter 1 Motivation 1.1 Introduction Industrial societies produce and consume many halogenated organic compounds. Chlori­ nated organic compounds are common feedstocks for industrial chemical synthesis: for ex­ ample, over one million tonnes of 1,2-dichloroethane (1,2-DCA) is produced every year, much of which is used in the synthesis of vinyl chloride (VC), the monomer of the com­ mon plastic PVC. Other chlorinated compounds are used as solvents, fumigants and pesti­ cides, among other applications. Brominated organic compounds are used as flame-retardant materials. Fluorinated organics have been used extensively in refrigeration (chlorofluoro- carbons; CFCs), stain-resistant coatings, non-stick coatings (such as polytetrafluoroethylene, commonly known as Teflon^) and lubricants. Humans were not the first to incorporate halogens into organic compounds. Over 4000 naturally-ocurring chlorinated compounds have been identified of both physicochemical and biological origin (1).
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