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55856192.Pdf Members of the jury: Prof. dr. ir. Frank Devlieghere (chairman) Prof. dr. ir. Wim Soetaert (promotor) Prof. dr. ir. Erick Vandamme (promotor) Prof. dr. Els Vandamme Prof. dr. Savvas Savvides Dr. Tom Desmet Dr. Henk-Jan Joosten Promotors: Prof. dr. ir. Wim SOETAERT (promotor) Prof. dr. ir. Erick VANDAMME (promotor) Centre of expertise – Industrial Biotechnology and Biocatalysis Department of Biochemical and Microbial Technology Ghent University, Belgium Dean: Prof. dr. ir. Guido Van Huylenbroeck Rector: Prof. dr. Paul Van Cauwenberge The research was conducted at the Centre of expertise - Industrial Biotechnology and Biocatalysis, Department of Biochemical and Microbial Technology, Faculty of Bioscience Engineering, Ghent University (Ghent, Belgium) ir. An CERDOBBEL ENGINEERING THE THERMOSTABILITY OF SUCROSE PHOSPHORYLASE FOR INDUSTRIAL APPLICATIONS Thesis submitted in fulfilment of the requirements for the degree of Doctor (PhD) in Applied Biological Sciences Dutch translation of the title: Engineering van de thermostabiliteit van sucrose phosphorylase voor industriële toepassingen Cover illustration: “Three-dimensional structure of sucrose phosphorylase colored by B-factor.” Printed by University Press, Zelzate To refer to this thesis: Cerdobbel, A. (2011). Engineering the thermostability of sucrose phosphorylase for industrial applications. PhD thesis, Faculty of Bioscience Engineering, Ghent University, Ghent, 200 p. ISBN-number: 978-90-5989-414-3 The author and the promoter give the authorization to consult and to copy parts of this work for personal use only. Every other use is subject to the copyright laws. Permission to reproduce any material contained in this work should be obtained from the author. WOORD VOORAF Er wordt wel eens gezegd dat het woord vooraf het meest gelezen stukje is van een proefschrift. Dit is dus een uitgelezen kans om hier de personen te bedanken die rechtstreeks of onrechtstreeks meegeholpen hebben aan dit doctoraat. In de eerste plaats zou ik mijn promotoren Prof. dr. ir. Wim Soetaert en Em. Prof. dr. ir. Erick Vandamme willen bedanken voor het vertrouwen dat ze de voorbije jaren in mij stelden. Ze hebben me toegelaten mijn onderzoek uit te voeren in een zeer goed uitgerust labo en mij voldoende vrijheid gegeven om naast mijn taken als assistent ook mijn doctoraat te kunnen afwerken. Professor, je bent al enige tijd weg van het labo maar ik apprecieer het enorm dat je me tot het einde bent blijven volgen. Voor jou wordt het je laatste doctoraatsverdediging als promotor, het is een eer voor mij om die rol te mogen vervullen! Wim, over jou kan ik boeken schrijven, waar moet ik beginnen? Jij hebt ervoor gezorgd dat ik in teamverband aan fosforylasen kon werken binnen het SBO project “Glycodirect”. Hierdoor is mijn doctoraat in een stroomversnelling geraakt, waarvoor dank! Een speciaal woord van dank gaat ook naar mijn projectleider dr. Tom Desmet voor de wetenschappelijke ondersteuning tijdens de voorbije jaren. Bedankt voor het geduld die je opbracht om mij nieuwe inzichten bij te brengen en mijn oneindige reeks van vragen te beantwoorden. Je bleef geloven in mijn doctoraat, ook toen ik geen uitweg meer zag. Je hebt me tot het laatste moment met raad en daad bijgestaan en dat waardeer ik enorm! De ganse “Glycodirect” groep wil ik bedanken voor de leuke sfeer en de vele wetenschappelijke discussies. Dirk, bedankt voor de nauwe samenwerking rond sucrose fosforylase, ik zal ons overleg missen. Jef, jij bent de volgende in de rij, ik weet zeker dat het je lukt. Koen, ik heb nog steeds een demonstratie van je danspasjes te goed. Chao, I’ll miss the wonderful smell of Chinese food in the hallway. Karel en Tom, als groentjes wens ik jullie een vlotte start toe, jullie zijn nu de toekomst van Glycodirect. Barbara, ik vind het super dat jij me opvolgt als assistent, ik weet zeker dat je dat schitterend gaat doen. Giang, we’ll see each other definitely again in one of the many Moroccan restaurants in Ghent and hopefully also at the hair dresser! Een oprecht woord van dank gaat ook naar mijn thesisstudenten die me in de loop van mijn doctoraat geholpen hebben. Ben en Evelien, jullie bleven volharden ook na vele maanden tegenslag tot de grote doorbraak er eidenlijk kwam.. Dirk, Eveline en Nick, jullie onvermoeibare inzet tijdens de vele uren op het labo is voor mij onvergetelijk. Karel, jij hebt me door mijn laatste hectisch jaar geholpen, we waren een onklopbaar team! Verder wil ik natuurlijk ook het ganse labo bedanken voor de leuke jaren samen. Marjan en Sofie, jullie hebben me opgevangen in de eerste jaren van mijn doctoraat maar zelfs tot vandaag kan ik altijd bij jullie terecht. Hopelijk zetten we binnenkort onze etentjes verder. Margriet, bedankt voor de vele fijne babbels, de lekker thee en voor het pilates-gezelschap. Katja, alhoewel nu niet meer bij Inbio, bedankt voor de vele zotte momenten samen. Sofie R., sorry dat ik je nu al in de steek moet laten, you can do it, girl! Lien, wanneer mag ik eens komen relaxen in je sauna? Hendrik, Jef en Nicolas, bedankt voor de leuke fietstochten in de Vlaamse Ardennen, ik heb mij toen reuze geamuseerd. Isabelle, ik zal jou gewoon missen! Inge, bedankt om mijn thesis-trauma te delen. Kwok, bedankt voor de korte maar fijna periode als collega’s, je bracht leven in de brouwerij! Maarten, bedankt om even samen met rode nagels door het leven te zijn gegaan. Jo, bedankt voor je statistische hulp. Gilles, streng maar rechtvaardig... maar toch een beetje zot. Karen, gelukkig waren we met twee. Joeri, bedankt om me door de laatste stappen van mijn doctoraat te loodsen. Veerle, je moet me dat spel van jou toch nog eens uitleggen. Pieter, aan jou moet ik mijn excuses nogmaals aanbieden. Dries, stille waters maar... Wim, hou vol met je studies! Simon, ik duim met je mee. Jinxin, good luck in Lille! Catherine, bedankt voor je hulp met de HPLC’s. Sofie en Tom, ook zonder praktisch werk zorgden jullie mee voor de toffe sfeer in het labo. Julia, keep on running! Eric, welkom bij Inbio. Ook het coördinatiecentrum wil ik bedanken voor alle hulp! En als trouwe voetbalsupporter wil ik het Inbio-voetbal-team bedanken voor de leuke voetbalmatchen. Bart en Katrien, ex-collega’s maar ondertussen vrienden voor het leven! Als laatste, wil ik mijn familie en vrienden bedanken voor alles wat ze voor mij gedaan hebben. Mama en papa, bedankt voor jullie liefde en onvoorwaardelijke steun. Broertjes, sorry dat ik weinig tijd voor jullie heb gehad het afgelopen jaar, vanaf nu zal dit wel terug beteren! Vrienden en vriendinnekes, bedankt voor alles, jullie steun, jullie eeuwig optimisme, bedankt voor het luisteren naar mijn doctoraatsgeklaag maar eindelijk is het einde in zicht en is het tijd voor een feestje! An, 2 januari 2011 TABLE OF CONTENTS INTRODUCTION ............................................................................................................................... 1 CHAPTER 1. LITERATURE REVIEW ................................................................................................ 6 1. CAZymes ................................................................................................................................. 7 1.1 Introduction ...................................................................................................................... 7 1.2 Classification .................................................................................................................... 8 1.3 Reaction mechanisms ..................................................................................................... 10 1.4 Glycoside hydrolases...................................................................................................... 12 1.5 Transglycosidases........................................................................................................... 15 1.6 Glycosyl transferases...................................................................................................... 17 1.7 Glycoside phosphorylases .............................................................................................. 18 2. Sucrose phosphorylase .......................................................................................................... 21 2.1 Mechanism and classification ........................................................................................ 21 2.2 Properties and applications............................................................................................. 22 2.3 Crystal structure ............................................................................................................. 24 3. Thermostability ...................................................................................................................... 27 3.1 Introduction .................................................................................................................... 27 3.2 Kinetic versus thermodynamic stability ......................................................................... 28 3.3 Improve the protein stability .......................................................................................... 29 4. Immobilization....................................................................................................................... 36 4.1 Immobilization techniques ............................................................................................. 37 4.2 Immobilization of sucrose phosphorylase ...................................................................... 44 CHAPTER 2. ENZYME
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