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Glucuronic Acid Derivatives in Enzymatic Biomass Degradation: Synthesis and Evaluation of Enzymatic Activity

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Glucuronic Acid Derivatives in Enzymatic Biomass Degradation: Synthesis and Evaluation of Enzymatic Activity Downloaded from orbit.dtu.dk on: Oct 08, 2021 Glucuronic Acid Derivatives in Enzymatic Biomass Degradation: Synthesis and Evaluation of Enzymatic Activity d'Errico, Clotilde Publication date: 2016 Document Version Publisher's PDF, also known as Version of record Link back to DTU Orbit Citation (APA): d'Errico, C. (2016). Glucuronic Acid Derivatives in Enzymatic Biomass Degradation: Synthesis and Evaluation of Enzymatic Activity. DTU Chemistry. General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. Users may download and print one copy of any publication from the public portal for the purpose of private study or research. You may not further distribute the material or use it for any profit-making activity or commercial gain You may freely distribute the URL identifying the publication in the public portal If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. GLUCURONIC ACID DERIVATIVES IN ENZYMATIC BIOMASS DEGRADATION: SYNTHESIS AND EVALUATION OF ENZYMATIC ACTIVITY PHD THESIS – MAY 2016 CLOTILDE D’ERRICO DEPARTMENT OF CHEMISTRY TECHNICAL UNIVERSITY OF DENMARK ACKNOWLEDGEMENTS This dissertation describes the work produced during my PhD studies. The work was primarily conducted at the DTU Chemistry in the period between November 2012 and January 2016 with an external stay at Novozymes A/S between September 2013 and March 2014. First and foremost I would like to thank my supervisor Robert Madsen for giving me the opportunity to be part of this project, for the guidance during these years, for the valuable advices and the inspiring discussions. I am very grateful to Rune Nygaard Monrad for being a great advisor during my experience at Novozymes A/S. Thanks for the patience, for the motivation and for sharing priceless knowledge about both carbohydrate chemistry and biotechnology. Thanks to Kristian B. R. M. Krogh for the stimulating meetings and for providing the glucuronoyl esterases that I characterized during my project. The Robert Madsen group, both present and former members, has been special to me, making the daily routine enjoyable and never boring! Especially, I need to thank my officemates Carola, Dominika and Gyrithe for the interesting talks, the sweets in the office, the reciprocal encouragement and for contributing to make the last three years (and a half) unforgettable. A special thank goes to my colleague Maximilian Böhm for sharing with me his precious knowledge on the glycosylation reactions, and for taking over the glucuronoxylans project making some very remarkable progress. i Thanks to the fellow PhD students at the organic chemistry department for creating a pleasant and lively working environment and for never missing an occasion to share a cake! My sincere gratitude goes to the technical staff of the chemistry department, without whom working in the lab would be simply impossible. Thanks to the sweet Anne Hector, to Brian Dideriksen, Brian Ekman-Gregersen, Charlie Johansen and Tina Gustafsson for their inestimable help, especially during the chaos of the moving to the new building. This thesis would be still a formless draft without the invaluable contribution of Martin Jæger Pedersen, Jens Engel-Andreasen, Christine Kinnaert, Lee Fisher and Beatrice Bonora. Thanks for your time and for the thoughtful comments and suggestions. My greatest thank goes to Beatrice. This experience in Denmark gave me the most amazing gift: your friendship. Thanks for listening, for supporting me unconditionally, for making me feel like home, always. You will always be my favorite vegetarian. Grazie! Thank you Martin, for your love, for being always on my side and for your endless patience. Your presence in my life just made me a better person. Last but not least, my deepest gratitude goes to my family for their infinite support and love, for their constant presence in my life and for always believing in me. Vi amo! Clotilde d’Errico May 2016 ii ABSTRACT An essential tool for biotechnology companies in enzyme development for biomass delignification is the access to well-defined model substrates. A deeper understanding of the enzymes substrate specificity can be used to address and optimize enzyme mixtures towards natural, complex substrates. Hence, the chemically synthesized substrates often outcompete those isolated from natural sources in terms of reproducibility, homogeneity and purity. The first part of this work was the synthesis of two glucuronoxylan fragments designed as model substrates for xylanases. The synthesis involved the use of thioxyloside building blocks in an iterative, linear glycosylation strategy. Two sidechain glucuronate building blocks were synthesized via a divergent strategy from the same thioethylglucose derivative. iii In the second part of the project three alkylaromatic and aromatic esters have been prepared as mimics of lignin-carbohydrate complexes found in lignocellulosic biomass, as model substrates for glucuronoyl esterases (GEs). These esters have been used to characterize a novel GE from Cerrena unicolor (CuGE), produced by Novozymes, to obtain insights into the substrate specificity of the enzymes. HPLC analysis of the enzymatic reactions led to the determination of kinetic parameters that gave information about both bonding affinity and catalytic efficiency. iv RESUMÉ Et vigtigt redskab for bioteknologiske virksomheder, i enzymudvikling til biomasse delignificering er adgang til veldefinerede model substrater. En dybere forståelse af enzymers substratspecificitet kan anvendes til at evaluere og optimere enzymblandinger til naturlige, komplekse substrater. Derfor udkonkurrerer kemisk syntetiserede substrater ofte tilsvarende isoleret fra naturlige kilder i form af reproducerbarhed, homogenitet og renhed. Den første del af dette arbejde var syntesen af to glucuronoxylanfragmenter udformet som modelsubstrater for xylanaser. Syntesen involverede anvendelsen af thioxylosider som byggeklods i en iterativ lineær glycosylerings strategi. To sidekæder af glucuronat byggeklodser blev syntetiseret via en divergerende strategi fra samme thioethylglucose derivat. v I anden del af projektet blev tre alkylaromatiske og aromatiske estere forberedt som efterligninger af de lignin-kulhydrat komplekser, der findes i lignocellulose biomasse, som modelsubstrater til glucuronoyl esteraser (GE). Disse estere er blevet anvendt til at karakterisere en hidtil ukendt GE fra Cerrena unicolor (CuGE), produceret af Novozymes, for at opnå indsigt i substratspecificiteten af enzymet. HPLC-analyse af enzymatiske reaktioner førte til bestemmelsen af kinetiskeparametre, der gav oplysninger om både limning affinitet og katalytisk effektivitet. vi LIST OF ABBREVIATIONS 1-BBTZ 1-(Benzoyloxy)benzotriazole Ac Acetyl acac Acetylacetonate Ar Aromatic Ara Arabinose All Allyl Bn Benzyl br Broad BSP 1-benzenesulfinyl piperidine Bu Butyl Bz Benzoyl CE Carbohydrate esterase CSA Camphorsulfonic acid Cu Cerrena unicolor d Dublet DAST (Diethylamino)sulfur trifluoride DCC N,N'-dicyclohexylcarbodiimide DDQ 2,3-dichloro-5,6-dicyano-1,4-benzoquinone DMAP N,N-dimethyl-4-aminopyridine DMF N,N-dimethylformamide DMTST Dimethyl(methylthio)sulfonium triflate DTBMP 2,6-di-tert-butyl-4-methylpyridine EDC N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide ESI Electrospray ionization Et Ethyl vii Fmoc Fluorenylmethyloxycarbonyl FT-ICR Fourier transform ion cyclotron resonance Gal Galactose gCOSY Gradient-selected correlation spectroscopy GE Glucuronoyl esterase GH Glycoside hydrolase Glc Glucose GlcA Glucuronic acid Glu Glutamic acid HexA Hexenuronic acid His Histidine HMBC Heteronuclear multiple bond correlation HPLC High-performance liquid chromatography HRMS High-resolution mass spectrometry HSQC Heteronuclear single quantum correlation i iso IDCP Iodonium dicollidine perchlorate LCC Lignin-carbohydrate complex Lev Levulinyl m Multiplet; meta MALDI Matrix-assisted laser desorption/ionization Man Mannose mCPBA m-chloroperbenzoic acid Me Methyl MS Mass spectrometry Nap 2-Naphthylmethyl NBS N-bromosuccinimide NIS N-iodosuccinimide viii NMR Nuclear magnetic resonance p para PDC Pyridinium dichromate Ph Phenyl PMB p-methoxy benzyl Pr Propyl Py Pyridine Rf Retention factor RRV Relative reactivity value s Singlet Sc Schizophyllum commune Ser Serine SET Single electron transfer t Triplet TBAF Tetrabutylammonium fluoride TBAI Tetrabutylammonium iodide TBS tert-butyldimethylsilyl TEMPO 2,2,6,6-tetramethyl-1-piperidinyloxy Tf Triflyl; trifluoromethanesulfonyl TFA Trifluoroacetic acid TFAA Trifluoroacetic anhydride THF Tetrahydrofuran TLC Thin-Layer Chromatography TMS Trimethylsilyl Tol Tolyl; p-methylphenyl Tr Trityl; triphenylmethyl UV Ultraviolet Xyl Xylose ix PUBLICATIONS “Enzymatic Degradation of Lignin-Carbohydrate Complexes (LCCs): Model studies Using a Fungal Glucuronoyl Esterase from Cerrena unicolor” “Improved Biomass Degradation using Fungal Glucuronoyl Esterases – Hydrolysis of Natural Corn Fiber Substrate” x CONTENTS ACKNOWLEDGEMENTS .............................................................................................. i ABSTRACT ................................................................................................................ iii RESUMÉ ...................................................................................................................
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