University of Groningen Exploring (Per)Oxidases As Biocatalysts for the Synthesis of Valuable Aromatic Compounds Habib, Mohamed

University of Groningen

Exploring (per)oxidases as biocatalysts for the synthesis of valuable aromatic compounds Habib, Mohamed H M

DOI: 10.33612/diss.109693881

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Exploring (per)oxidases as biocatalysts for the synthesis of valuable aromatic compounds

Exploring (per)oxidases as

biocatalysts for the

synthesis of valuable

aromatic compounds

PhD thesis

The research described in this thesis was carried out in the department of Molecular to obtain the degree of PhD at the Enzymology of the Groningen Biomolecular Sciences and Biotechnology Institute University of Groningen according to the requirements of the Graduate School of Science, Faculty of on the authority of the Mathematics and Natural Sciences, University of Groningen, The Netherlands. Rector Magnificus Prof. C. Wijmenga

Mohamed Habib received funding from the Cultural Affairs and Missions Sector, and in accordance with Ministry of Higher Education in Egypt. the decision by the College of Deans.

Cover design: Mohamed Habib and Nina Lindhout This thesis will be defended in public on Printed by: Ipskamp Drukkers B.V. Monday, January 20, 2020 at 12:45 pm hours ISBN:ISBN (printed version): 978-94-034-2334-0 ISBN:ISBN (electronic version): 978-94-034-2335-7 by Copyright © Mohamed Habib 2020, Groningen, The Netherlands. All rights reserved. No part of this thesis may be reproduced, stored in a retrieval system, or transmitted in any form or by any means without prior permission of the Mohamed Habib author.

born on 8 April 1987 in Riyadh, Saudi Arabia

Exploring (per)oxidases as

biocatalysts for the

synthesis of valuable

aromatic compounds

PhD thesis

Mohamed Habib received funding from the Cultural Affairs and Missions Sector, and in accordance with Ministry of Higher Education in Egypt. the decision by the College of Deans.

Cover design: Mohamed Habib and Nina Lindhout This thesis will be defended in public on Printed by: Monday, January 20, 2020 at 12:45 pm hours ISBN: ISBN: by Copyright © Mohamed Habib 2020, Groningen, The Netherlands. All rights reserved. No part of this thesis may be reproduced, stored in a retrieval system, or transmitted in any form or by any means without prior permission of the Mohamed Habib author.

born on 8 April 1987 in Riyadh, Saudi Arabia

Supervisors Table of Contents Chapter I General introduction: Oxidases and peroxidases and their roles in biocatalysis 7 Prof. M.W. Fraaije Prof. D.B. Janssen Chapter II Bacterial enzymes involved in lignin degradation 26

Chapter III A biocatalytic one pot approach for the preparation of lignin oligomers using an oxidase/peroxidase cascade enzyme system 51 ‐ Assessment Committee Chapter IV The biocatalytic synthesis of syringaresinol from 2,6-dimethoxy-4-allylphenol in one-pot using a tailored oxidase/peroxidase system 70 Prof. W.J.H. van Berkel Chapter V Characterization of a new DyP-peroxidase from the alkaliphilic cellulomonad, Prof. G. Maglia Cellulomonas bogoriensis 82 Prof. F. Hollmann Chapter VI Expression and characterization of a resurrected DyP-type peroxidase 100

Summary, Conclusion and Future Outlook 120

Nederlandse Samenvatting 126

Curriculum vitae 131

Acknowledgements 132

Supervisors Table of Contents Chapter I General introduction: Oxidases and peroxidases and their roles in biocatalysis 76 Prof. M.W. Fraaije Prof. D.B. Janssen Chapter II Bacterial enzymes involved in lignin degradation 2426

Chapter III A biocatalytic one pot approach for the preparation of lignin oligomers using an oxidase/peroxidase cascade enzyme system 4851 ‐ Assessment Committee Chapter IV The biocatalytic synthesis of syringaresinol from 2,6-dimethoxy-4-allylphenol in one-pot using a tailored oxidase/peroxidase system 6670 Prof. W.J.H. van Berkel Chapter V Characterization of a new DyP-peroxidase from the alkaliphilic cellulomonad, Prof. G. Maglia Cellulomonas bogoriensis 7882 Prof. F. Hollmann Chapter VI Expression and characterization of a resurrected DyP-type peroxidase 10096

Summary, Conclusion and Future Outlook 116120 Nederlandse Samenvatting 122126

Curriculum vitae 128131

Acknowledgements 129132

General introduction: Oxidases and peroxidases and their roles in biocatalysis

Mohamed Habib and Marco W. Fraaije

1 Oxidases and peroxidases and their roles in biocatalysis Chapter I

1.1 Introduction redox enzymes is shown in Table 1. Redox enzymes have found numerous 1.1.1 Background applications which include: textile and food processing, synthesis and modification 1.1.1.1 Redox enzymes of polymers, oxidative degradation of pollutants, (asymmetric) synthesis of fine In redox reactions, electron transfer takes place from one reactant to another. In chemicals and pharmaceuticals, and as part of biosensors for a variety of analytical [3,4] synthetic chemistry, these reactions often require harsh conditions (e.g. elevated and clinical applications. temperatures and high pressure) and proceed with low chemo-, regio- and enantioselectivity. A large number of enzymes on the other hand are capable of performing redox reactions with an astonishing high degree of selectivity. These biological catalysts typically require cofactors for catalysis because the building blocks of proteins, amino acids, are not well suited for redox chemistry. Only in some rare cases, redox enzymes perform catalysis without any cofactor [1,2]. Not only redox enzymes, but also many other enzymes rely on cofactors: it is estimated that about half of all enzymes depend on one or more cofactors. Cofactors come in many flavors, ranging from metal ions to highly complex molecules such as vitamin B12 and prenylated FMN (Figure 1). They enable enzymes to catalyze a plethora of reactions that would simply be impossible without a cofactor. The importance of cofactors in nature can also be deduced from the dedicated, complex and energy-consuming biosynthetic pathways of many cofactors. Cofactors can be classified into two cofactor types: prosthetic groups and coenzymes. Prosthetic groups are cofactors that are tightly, sometimes even covalently, bound. A well-known example is the heme cofactor bound in hemoglobin. Coenzymes are cofactors that are loosely bound to the target enzymes and which dissociate upon being converted, to be used by another coenzyme- dependent enzyme. Well-known examples are the NAD(P)+-dependent alcohol dehydrogenases. When considering enzymes as biocatalysts, a dependency on coenzymes presents a challenge as it will require an approach that will recycle the coenzyme. This is not the case when dealing with enzymes that contain a prosthetic group which can be regarded as an integral part of the biocatalyst. Redox enzymes, also named oxidoreductases, typically use cofactors to facilitate the transfer of electrons. While there are numerous redox enzymes known, there is a limited set of redox cofactors available in nature used by these enzymes. Some redox cofactors are rather limited in the type of redox reaction that they can support. For example, nicotinamide cofactors (NAD+ or NADP+) solely enable oxidation and reduction reactions by the transfer of a hydride. There are also cofactors that are used for different types of redox reactions. For example, flavin cofactors (FAD or FMN) facilitate oxidation by hydride transfer but can also support electron transfer, oxygenations, halogenations and other types of reactions. All redox enzymes belong Figure 1. Structural formulas of some cofactors: vitamin B12, prenylated to the EC (Enzyme Commission) class I. According to the EC classification, FMN, FAD, and heme. oxidoreductases can be subclassified into 22 different subclasses, dependent on the type of reaction and substrate(s). A more general classification of the most common 9 10 8 1 Oxidases and peroxidases and their roles in biocatalysis Chapter I

1.1 Introduction redox enzymes is shown in Table 1. Redox enzymes have found numerous 1.1.1 Background applications which include: textile and food processing, synthesis and modification 1.1.1.1 Redox enzymes of polymers, oxidative degradation of pollutants, (asymmetric) synthesis of fine In redox reactions, electron transfer takes place from one reactant to another. In chemicals and pharmaceuticals, and as part of biosensors for a variety of analytical [3,4] synthetic chemistry, these reactions often require harsh conditions (e.g. elevated and clinical applications. temperatures and high pressure) and proceed with low chemo-, regio- and enantioselectivity. A large number of enzymes on the other hand are capable of performing redox reactions with an astonishing high degree of selectivity. These biological catalysts typically require cofactors for catalysis because the building blocks of proteins, amino acids, are not well suited for redox chemistry. Only in some rare cases, redox enzymes perform catalysis without any cofactor [1,2]. Not only redox enzymes, but also many other enzymes rely on cofactors: it is estimated that about half of all enzymes depend on one or more cofactors. Cofactors come in many flavors, ranging from metal ions to highly complex molecules such as vitamin B12 and prenylated FMN (Figure 1). They enable enzymes to catalyze a plethora of reactions that would simply be impossible without a cofactor. The importance of cofactors in nature can also be deduced from the dedicated, complex and energy-consuming biosynthetic pathways of many cofactors. Cofactors can be classified into two cofactor types: prosthetic groups and coenzymes. Prosthetic groups are cofactors that are tightly, sometimes even covalently, bound. A well-known example is the heme cofactor bound in hemoglobin. Coenzymes are cofactors that are loosely bound to the target enzymes and which dissociate upon being converted, to be used by another coenzyme- dependent enzyme. Well-known examples are the NAD(P)+-dependent alcohol dehydrogenases. When considering enzymes as biocatalysts, a dependency on coenzymes presents a challenge as it will require an approach that will recycle the coenzyme. This is not the case when dealing with enzymes that contain a prosthetic group which can be regarded as an integral part of the biocatalyst. Redox enzymes, also named oxidoreductases, typically use cofactors to facilitate the transfer of electrons. While there are numerous redox enzymes known, there is a limited set of redox cofactors available in nature used by these enzymes. Some redox cofactors are rather limited in the type of redox reaction that they can support. For example, nicotinamide cofactors (NAD+ or NADP+) solely enable oxidation and reduction reactions by the transfer of a hydride. There are also cofactors that are used for different types of redox reactions. For example, flavin cofactors (FAD or FMN) facilitate oxidation by hydride transfer but can also support electron transfer, oxygenations, halogenations and other types of reactions. All redox enzymes belong Figure 1. Structural formulas of some cofactors: vitamin B12, prenylated to the EC (Enzyme Commission) class I. According to the EC classification, FMN, FAD, and heme. oxidoreductases can be subclassified into 22 different subclasses, dependent on the type of reaction and substrate(s). A more general classification of the most common 9 10 9 1 Oxidases and peroxidases and their roles in biocatalysis Chapter I

Table 1. Classification of the major redox enzyme classes. EC Subclass Function Typical cofactors 1.1.X.X dehydrogenases/reductases nicotinamide 1.X.3.X oxidases flavin, copper 1.11.1.X peroxidases heme 1.14.X.X oxygenases iron, heme, flavin 1.11.2.X peroxygenases heme 1.11.1.6 catalases heme 1.1.2 Oxidases In this thesis, research is presented in which oxidases and peroxidases were studied as biocatalysts. Oxidases are enzymes that utilize molecular oxygen to catalyze oxidations and yield water or hydrogen peroxide as a by-product.[5,6] An equation – showing how oxidases operate is shown below. Substrate + O2 → Product + H2O2 or H2O 8α 8α Some known examples of oxidases that are exploited as biocatalysts are: glucose oxidase, cholesterol oxidase, and NAD(P)H oxidase. Oxidases contain a prosthetic group that is required for catalysis which is typically a flavin or copper cofactor. The flavin-containing oxidases have been classified into six different classes: the Glucose Methanol Choline (GMC)-type oxidases, the Vanillyl Alcohol Oxidase (VAO)-type oxidases, the amine oxidases, the sulfhydryl oxidases, the acyl CoA oxidase-type oxidases and the 2-hydroxyacid oxidases.[7] These different classes are based on sequence and structural similarities. 1.1.1.2 GMC-type oxidases The first type of flavin-containing oxidases we will discuss are the GMC-type oxidases. GMC-type oxidases can oxidize primary alcohols to their corresponding aldehydes. Their activity is not restricted to primary alcohols as (mutant) enzymes have also been reported to catalyze the conversion of secondary alcohols to ketones.[8] They have also been reported to be able to convert thiols to their corresponding thioaldehydes.[9] GMC-type oxidases can thus be considered to be somewhat promiscuous. Yet, they seem to be specialized in catalyzing alcohols. The general mechanism by which GMC-type oxidases operate is by a direct hydride transfer from the substrate to the N5 of the flavin cofactor. This is made easier by proton abstraction by a conserved active-site histidine. The hydride transfer and proton abstraction either happen at the same time following a concerted mechanism or the hydride transfer occurs after proton abstraction following a stepwise mechanism.[10–12] A famous example of a GMC-type oxidase is glucose oxidase, which is perhaps the most widely applied redox enzyme. This fungal enzyme is efficient in oxidizing glucose into gluconic acid and has been extensively explored 11 12 10 1 Oxidases and peroxidases and their roles in biocatalysis Chapter I

Table 1. Classification of the major redox enzyme classes. EC Subclass Function Typical cofactors 1.1.X.X dehydrogenases/reductases nicotinamide 1.X.3.X oxidases flavin, copper 1.11.1.X peroxidases heme 1.14.X.X oxygenases iron, heme, flavin 1.11.2.X peroxygenases heme 1.11.1.6 catalases heme 1.1.2 Oxidases In this thesis, research is presented in which oxidases and peroxidases were studied as biocatalysts. Oxidases are enzymes that utilize molecular oxygen to catalyze oxidations and yield water or hydrogen peroxide as a by-product.[5,6] An equation – showing how oxidases operate is shown below. Substrate + O2 → Product + H2O2 or H2O 8α 8α Some known examples of oxidases that are exploited as biocatalysts are: glucose oxidase, cholesterol oxidase, and NAD(P)H oxidase. Oxidases contain a prosthetic group that is required for catalysis which is typically a flavin or copper cofactor. The flavin-containing oxidases have been classified into six different classes: the Glucose Methanol Choline (GMC)-type oxidases, the Vanillyl Alcohol Oxidase (VAO)-type oxidases, the amine oxidases, the sulfhydryl oxidases, the acyl CoA oxidase-type oxidases and the 2-hydroxyacid oxidases.[7] These different classes are based on sequence and structural similarities. 1.1.1.2 GMC-type oxidases The first type of flavin-containing oxidases we will discuss are the GMC-type oxidases. GMC-type oxidases can oxidize primary alcohols to their corresponding aldehydes. Their activity is not restricted to primary alcohols as (mutant) enzymes have also been reported to catalyze the conversion of secondary alcohols to ketones.[8] They have also been reported to be able to convert thiols to their corresponding thioaldehydes.[9] GMC-type oxidases can thus be considered to be somewhat promiscuous. Yet, they seem to be specialized in catalyzing alcohols. The general mechanism by which GMC-type oxidases operate is by a direct hydride transfer from the substrate to the N5 of the flavin cofactor. This is made easier by proton abstraction by a conserved active-site histidine. The hydride transfer and proton abstraction either happen at the same time following a concerted mechanism or the hydride transfer occurs after proton abstraction following a stepwise mechanism.[10–12] A famous example of a GMC-type oxidase is glucose oxidase, which is perhaps the most widely applied redox enzyme. This fungal enzyme is efficient in oxidizing glucose into gluconic acid and has been extensively explored 11 12 11 1 Oxidases and peroxidases and their roles in biocatalysis Chapter I

– → → αhelices, a middle domain made up of a β terminal domain of α αβ α β

13 14 12 1 Oxidases and peroxidases and their roles in biocatalysis Chapter I

– → → αhelices, a middle domain made up of a β terminal domain of α αβ α β

13 14 13 1 Oxidases and peroxidases and their roles in biocatalysis Chapter I

–

15 16 14 1 Oxidases and peroxidases and their roles in biocatalysis Chapter I

–

15 16 15 1

Oxidases and peroxidases and their roles in biocatalysis 16 Oxidasesand peroxidases and their rolesbiocatalysisin 15

Family Substrates Cofactor Cofactor binding Examples

–

Type Substrate Cofactor Examples

→

→ →

Compound II + AH•

→

2AH•

state) + H state) + AH•

Chapter I

1 Oxidases and peroxidases and their roles in biocatalysis Chapter I

→ → Compound II + AH• → state) + AH• + H – → 2AH• 15 18 17 1 Oxidases and peroxidases and their roles in biocatalysis Chapter I

so no seuene o stutua eatonsp t an o te peous aateed peodases e most eent assaton o peodases poposed tat peodase enmes a unde one o ou oups ese ou eme peodase supeames ae te peodaseataase peodaseooenase peodaseote dsmutase and peodasepeoenase supeames ese ou supeames de n oea od ate ste atetue and enmat attes oa te est aateed and most de apped peodase s oseads peodase Hoseads peodase H) s a pant enme o onsdeae nteest as een son o te past entues e ommon used H s n at a mtue o peodase sooms soated om oseads In seen dstnt soenmes o oseads ee soated– In 2 H soenmes ee soated om tee ommea pepaatons o oseads usn soeet ousn2 In most pepaatons oee H CA s te most pomnent soom H ontans an on potopopn I eme oato e pesene o te eme β oato endes te enme a ons oo and maes t eas to eauate ts put usn te enetsa ) aue e aue s dened te ato eteen te asoanes at nm oet and) to tat at 2 nm poten and) It as een son tat H s e dut to podue usn eteooous osts en onsden eomnant epesson o H seea eatues soud e taen nto onsdeaton In H CA ou dsude des ae omed eteen Cs2 Cs Cs2 and Cs22 e poten poduton soud e paaeed t poduton and nopoaton o te eme oato and nopoaton o a aum on uae osaton o H tends to mae ts eomnant poduton dut e poduton o eomnant H n Escherichia coli tpa esuts n omaton o nuson odes e tee ae epots tat am soue epesson n E. coli tese metods seem dut to e epodued o ead to mnute amounts o soue poten eent te use o east Pichia pastoris as een son to e moe eete n podun soue and ate H As mentoned aoe eent anote peodase am as een dsoeed te soaed tpe peodases ue to te att o te st desed peodase on des te name edeoon eodase as oned e dependent β te st epoted as soated om a unus ate studes eeaed tat s ae peaent n atea enomes euene anass studes ae ed to poposn tee ou o sometmes een e deent suasses Aodn to te eoase dataase s a nto ou oups A to ) e n assatons an on tee oups te td and out oups om one oup Cass A s ae eoned te pesene o a atseeton mot n te poten 19 20 18 1 Oxidases and peroxidases and their roles in biocatalysis Chapter I so no seuene o stutua eatonsp t an o te peous aateed peodases e most eent assaton o peodases poposed tat peodase enmes a unde one o ou oups ese ou eme peodase supeames ae te peodaseataase peodaseooenase peodaseote dsmutase and peodasepeoenase supeames ese ou supeames de n oea od ate ste atetue and enmat attes oa te est aateed and most de apped peodase s oseads peodase Hoseads peodase H) s a pant enme o onsdeae nteest as een son o te past entues e ommon used H s n at a mtue o peodase sooms soated om oseads In seen dstnt soenmes o oseads ee soated– In 2 H soenmes ee soated om tee ommea pepaatons o oseads usn soeet ousn2 In most pepaatons oee H CA s te most pomnent soom H ontans an on potopopn I eme oato e pesene o te eme β oato endes te enme a ons oo and maes t eas to eauate ts put usn te enetsa ) aue e aue s dened te ato eteen te asoanes at nm oet and) to tat at 2 nm poten and) It as een son tat H s e dut to podue usn eteooous osts en onsden eomnant epesson o H seea eatues soud e taen nto onsdeaton In H CA ou dsude des ae omed eteen Cs2 Cs Cs2 and Cs22 e poten poduton soud e paaeed t poduton and nopoaton o te eme oato and nopoaton o a aum on uae osaton o H tends to mae ts eomnant poduton dut e poduton o eomnant H n Escherichia coli tpa esuts n omaton o nuson odes e tee ae epots tat am soue epesson n E. coli tese metods seem dut to e epodued o ead to mnute amounts o soue poten eent te use o east Pichia pastoris as een son to e moe eete n podun soue and ate H As mentoned aoe eent anote peodase am as een dsoeed te soaed tpe peodases ue to te att o te st desed peodase on des te name edeoon eodase as oned e dependent β te st epoted as soated om a unus ate studes eeaed tat s ae peaent n atea enomes euene anass studes ae ed to poposn tee ou o sometmes een e deent suasses Aodn to te eoase dataase s a nto ou oups A to ) e n assatons an on tee oups te td and out oups om one oup Cass A s ae eoned te pesene o a atseeton mot n te poten 19 20 19 1 Oxidases and peroxidases and their roles in biocatalysis Chapter I

Chem. Biol. 2014 21 – Tetrahedron 2003 59 – Curr. Opin. Biotechnol. 10 – 1999 Ind. Biotechnol. 2005 1 – Advances in Enzymology and Related Areas of Molecular Biology 2011 Fundamentals of Enzymology (Third Edition) 2001 Appl. Microbiol. Biotechnol. 2013 97, a ββ linkage) in high yield and ChemBioChem 2012 13 – Angew. Chem. Int. Ed. Engl. 2014 53 – Cellulomonas bogoriensis Cbo FEBS J. 2013 280 – Biochemistry 2012 51 – Biochemistry 2010 49 – Biomol. Concepts 2014 5 – Cbo and the enzyme’s activity was tested using several dyes as substrates. The enzyme Appl. Environ. Microbiol. 2001 67 – Trends Biochem. Sci. 23 – 1998 Arch. Biochem. Biophys. 2008 474 – Structure 1997 5, – FEBS J. 2008 275 – Molecules 2018 23

21 22 20 1 Oxidases and peroxidases and their roles in biocatalysis Chapter I

Chem. Biol. 2014 21 – Tetrahedron 2003 59 – Curr. Opin. Biotechnol. 10 – 1999 Ind. Biotechnol. 2005 1 – Advances in Enzymology and Related Areas of Molecular Biology 2011 Fundamentals of Enzymology (Third Edition) 2001 Appl. Microbiol. Biotechnol. 2013 97, a ββ linkage) in high yield and ChemBioChem 2012 13 – Angew. Chem. Int. Ed. Engl. 2014 53 – Cellulomonas bogoriensis Cbo FEBS J. 2013 280 – Biochemistry 2012 51 – Biochemistry 2010 49 – Biomol. Concepts 2014 5 – Cbo and the enzyme’s activity was tested using several dyes as substrates. The enzyme Appl. Environ. Microbiol. 2001 67 – – Trends Biochem. Sci. 1998 23 Arch. Biochem. Biophys. 2008 474 – Structure 1997 5, – FEBS J. 2008 275 – Molecules 2018 23

21 22 21 1 Oxidases and peroxidases and their roles in biocatalysis Chapter I

Eur. J. Biochem. 1995 234 – Nat. Commun. 2015 6 – A. Winkler, A. Łyskowski, S. Riedl, M. Puhl, T. M. Kutchan, P. Nat. Chem. Biol. 2008 4 – PLoS One 2019 14 – ChemBioChem 2010 11 – ACS Symp. Ser. 1998 – J. Biol. Chem. 1999 274, Curr. Opin. Chem. Biol. 1999 3 – Structure 1999 7 Biotechnol. Adv. 2006 – EMBO J. 2003 22 – Enzyme Res. 2011 2011 Arch. Biochem. Biophys. 2010 493 – Phytochem. Biomol. Concepts 2011 2 – Rev. 2004 3 – Curr. Opin. Struct. Biol. 1992 2 – 2013 Appl. Environ. Microbiol. 1999 65 – Appl. Microbiol. Biotechnol. 2011 91 – J. Biol. Chem. 1966 241 – J. Biol. Chem. 1967 242 – Biochemistry 2005 44 – J. Biol. Chem. 1968 Biochem. 243 – Biophys. Res. Commun. 2011 405 – 1971 – FEBS Lett. 1994 351 – Plant Physiol. 1977 60 – Mol. Genet. Metab. 2000 71 – FEBS Lett. 1976 72 – 1998 13 – Pharm. Bioprocess. 2013 1 – J. Exp. J. Biosci. Bioeng. 2018 126 – Med. 1998 188 – Chem. Rev. 1996 96 – J. Biotechnol. 2016 233 – Arch. Biochem. Biophys. 2015 574 – JBIC J. Biol. Inorg. Chem. Adv. Synth. Catal. 2011 353 – 1997 2 – Biochemistry 1998 37 – 23 24 22 1 Oxidases and peroxidases and their roles in biocatalysis Chapter I

Bacterial enzymes involved in lignin degradation

Gonzalo de Gonzalo, Dana I. Colpa, Mohamed Habib and Marco W. Fraaije J. Biotechnol. 2016 236

J. Biotechnol. 2016 236

25 Bacterial enzymes involved in lignin degradation Chapter II Bacterial enzymes involved in lignin degradation Chapter II

2 – – – – – – ough the β ough the β ββ, β4, and β ββ, β4, and β – –

29 28 29 28 26 Bacterial enzymes involved in lignin degradation Chapter II Bacterial enzymes involved in lignin degradation Chapter II

2 – – – – – – ough the β ough the β ββ, β4, and β ββ, β4, and β – –

29 28 29 28 27 Bacterial enzymes involved in lignin degradation Chapter II Bacterial enzymes involved in lignin degradation Chapter II

well be the enyme that was described long before the first fungal y was described. able . ccurrence of ys in bacterial genomes. y performing a 2 analysis of the predicted proteomes, homologs of nown ys were identified. Organism – DyP type A B C Escherichia coli Thermobifida fusca et al. reported on a thorough study on lignin decomposition by “soil Rhodococcus jostii microorganisms”, which presumable were bacteri – Streptomyces viridosporus strain Streptomyces coelicolor Amycolatopsis sp. i Pseudomonas sp. train p ys hae a protomer weight of around – a and arious oligomeric states hae been obsered . h ey belong to the peroxidase chlorite dismutase superfamily of proteins and contain a noncoalently bound heme b cofactor. ys show a dimeric ferredoxinlie fold consisting of a fourstranded anti parallel βsheet surrounded by αhelices. y type peroxidases contain a highly consered motif and a consered proximal histidine, which acts– as the fifth ligand of the heme iron. et, while ys are structurally unrelated to the common fungal

peroxidases, they exhibit similar catalytic properties wit h haing similar redox potentials and reactiities. urthermore, some of the bacterial ys are secreted ia the at secretion machinery which adds to the analogy with the secreted fungal peroxidases. ased on seuence characteristics, ough the ysβ hae been diided in four classes in the – βeroxiaseβ, β4, anddatabase β . roteins belonging to classes – are mainly found in bacteria, while class ys are extracellular fungal representaties . lass ys typically hae a atsignal seuence and are therefore secreted. n contrast, the y protein seuences of class and ys do not disclose any secretion signal peptides, suggesting that they are intracellular enymes. he nterro database currently contains y seuences. pproximately thirty of these – enymes hae been isolated and characteried,. ys are mainly actie at acidic p and show a ery broad substrate profile, including seeral classes of synthetic bacterial ‘lignin peroxidase’ was described from Streptomyces viridosporus dyes, monophenolic compounds, veratryl alcohol, βcarotenes, n and lignin model compounds, but their physiological substrates still remain unnown. y – peroxidases can also catalyse interesting synthetic reactions such as enantioselectie Streptomyces sulfoxidations, heme deferrochelatations and een carbonyl olefination 29 30 31 28 28 Bacterial enzymes involved in lignin degradation Chapter II Bacterial enzymes involved in lignin degradation Chapter II

well be the enyme that was described long before the first fungal y was described. able . ccurrence of ys in bacterial genomes. y performing a analysis of the predicted proteomes, homologs of nown ys were identified. 2 Organism – DyP type A B C Escherichia coli Thermobifida fusca et al. reported on a thorough study on lignin decomposition by “soil Rhodococcus jostii microorganisms”, which presumable were bacteri – Streptomyces viridosporus strain Streptomyces coelicolor Amycolatopsis sp. i Pseudomonas sp. train p ys hae a protomer weight of around – a and arious oligomeric states hae been obsered . h ey belong to the peroxidase chlorite dismutase superfamily of proteins and contain a noncoalently bound heme b cofactor. ys show a dimeric ferredoxinlie fold consisting of a fourstranded anti parallel βsheet surrounded by αhelices. y type peroxidases contain a highly consered motif and a consered proximal histidine, which acts– as the fifth ligand of the heme iron. et, while ys are structurally unrelated to the common fungal

peroxidases, they exhibit similar catalytic properties wit h haing similar redox potentials and reactiities. urthermore, some of the bacterial ys are secreted ia the at secretion machinery which adds to the analogy with the secreted fungal peroxidases. ased on seuence characteristics, ough the ysβ hae been diided in four classes in the – βeroxiaseβ, β4, anddatabase β . roteins belonging to classes – are mainly found in bacteria, while class ys are extracellular fungal representaties . lass ys typically hae a atsignal seuence and are therefore secreted. n contrast, the y protein seuences of class and ys do not disclose any secretion signal peptides, suggesting that they are intracellular enymes. he nterro database currently contains y seuences. pproximately thirty of these – enymes hae been isolated and characteried,. ys are mainly actie at acidic p and show a ery broad substrate profile, including seeral classes of synthetic bacterial ‘lignin peroxidase’ was described from Streptomyces viridosporus dyes, monophenolic compounds, veratryl alcohol, βcarotenes, n and lignin model compounds, but their physiological substrates still remain unnown. y – peroxidases can also catalyse interesting synthetic reactions such as enantioselectie Streptomyces sulfoxidations, heme deferrochelatations and een carbonyl olefination 29 30 31 28 29 Bacterial enzymes involved in lignin degradation Chapter II Bacterial enzymes involved in lignin degradation Chapter II

2 β β – – Bs Bacillus subtilis Pp Pseudomonas putida Svi Saccharomonospora viridis Tfu Thermobifida fusca Svi Tfu Tc Thermomonospora curvata Amycolatopsis – β Tfu

Tfu β Tfu ough the β ββ, β4, and β Tc Rhodococcus jostii Tfu β ion of the Cα Cβ linkages of the model substrate and subsequent DyP Tc β β Bs – β Tc β

Bs 29 32 33 28 30 Bacterial enzymes involved in lignin degradation Chapter II Bacterial enzymes involved in lignin degradation Chapter II

2 β β – – Bs Bacillus subtilis Pp Pseudomonas putida Svi Saccharomonospora viridis Tfu Thermobifida fusca Svi Tfu Tc Thermomonospora curvata Amycolatopsis – β Tfu

Tfu β Tfu ough the β ββ, β4, and β Tc Rhodococcus jostii Tfu β ion of the Cα Cβ linkages of the model substrate and subsequent DyP Tc β β Bs – β Tc β

Bs 29 32 33 28 31 Bacterial enzymes involved in lignin degradation Chapter II Bacterial enzymes involved in lignin degradation Chapter II

β Rhodococcus jostii DyPB cleaves the CαCβ linkage of the phenolic lignin dimer guaiacylglycerolβ through the breakage of the CαCβ bond. Thus Bs – 2 – Tfu Pseudomonas fluorescence Svi – kcat − – the Δ dypB Rhodococcus jostii –

– Bl Mt Anabaena ough the β ββ, β4, and β Pseudomonas putida kcat − kcatKM – Pleurotus eryngii Phanerochaete chrysosporium Pseudomonas fluorescence Escherichia coli 29 34 35 28 32 Bacterial enzymes involved in lignin degradation Chapter II Bacterial enzymes involved in lignin degradation Chapter II

– Bl Mt Anabaena ough the β ββ, β4, and β Pseudomonas putida kcat − kcatKM – Pleurotus eryngii Phanerochaete chrysosporium Pseudomonas fluorescence Escherichia coli 29 34 35 28 33 Bacterial enzymes involved in lignin degradation Chapter II Bacterial enzymes involved in lignin degradation Chapter II

2 – – (TEMPO) and 2,2′ – S. coelicolor S. violaceusniger S. griseus ough the β ββ, β4, and β (2,2′ –

E. coli Eucalyptus globulus 29 36 37 28 34 Bacterial enzymes involved in lignin degradation Chapter II Bacterial enzymes involved in lignin degradation Chapter II

2 – – (TEMPO) and 2,2′ – S. coelicolor S. violaceusniger S. griseus ough the β ββ, β4, and β (2,2′ –

E. coli Eucalyptus globulus 29 36 37 28 35 Bacterial enzymes involved in lignin degradation Chapter II Bacterial enzymes involved in lignin degradation Chapter II

sae steiliation method his amneatie ateim was ale to deade linin and ie staw he seened Pantoea ananatis d enome eealed that this miooanism has the otential to ode inta and etaellla laases o 2 tatie laaseenodin enes wee disoeed and stdied om seene analses it was onlded that a had the hihest homolo to tial ateial laases hs eominant a was haa teied as ein a dimei enme with hih atiit at low – ales and a modeate themostailit In vitro linin deadation a in the esene o as mediato eslted in onesion ate h he omed odts ontained seeal low molela weiht aomati omonds sh as enediaoaldehde eneneoanoi aid – and henol aases om Bacillus seies ae in eneal toleant to hih temeates and alaline onditions whih maes them sitale o linin deadation Streptomyces S. coelicolor S. lividans S. notnatel most o these laases ae intaellla whih omliates lae sale viridosporus Amycolatopsis E. coli odtion eentl an etaellla laase was otained om Bacillus tequilensis – an ateim isolated om a ae mill elent he otimm temeate – o this laase was – while it een etains at o its atiit at he laase showed an otimal atiit at esides ein an etaellla enme its stailit mae laase a sel ioatalst to e eloited on indstial sale aase odtion was otimied sin a medim ontainin n and e slates as well as − ethanol laase– was emloed in the ioleahin o sotwood l esltin in a edtion o in the aa

nme and a ine ase in ihtness ddition o hdoenotiaole as mediato at low onentation m led to a the imoement in its eomane eeal ateial stains otained om soils o a iodiesit ih ainoest in e S. coelicolor hae een tested in the oidationough the o β his eslted in the isolation o two βBacillusβ, β stains4, and Bacillusβ atrophaeus stain and Bacillus pumilus stain oth miooanisms ehiit intaellla and etaellla laase atiities themoe at linin and the linin model omond aialleolβ aial ethe wee sessll deaded oth stains his sests that these stains hao inteestin laases – laase om Bacillus pumilus ot disoeed enome minin has een sessll loned and oeeessed in E. coli his enme showed a hih themostailit with a maimm atiit at ot was aale o oidiin seeal henoli omonds showin otimal ales at netal to alaline whih maes it an inteestin ioatalst o the iotehnoloial aliations In addition to the “small” two Thermus thermophilius is a themohili ateia whih odes an intaellla laase thlaase his ateial laase was sessll eessed in P antoea ananatis E. coli 29 38 39 28 36 Bacterial enzymes involved in lignin degradation Chapter II Bacterial enzymes involved in lignin degradation Chapter II

sae steiliation method his amneatie ateim was ale to deade linin and ie staw he seened Pantoea ananatis d enome eealed that this miooanism has the otential to ode inta and etaellla laases o tatie laaseenodin enes wee disoeed and stdied om seene 2 analses it was onlded that a had the hihest homolo to tial ateial laases hs eominant a was haa teied as ein a dimei enme with hih atiit at low – ales and a modeate themostailit In vitro linin deadation a in the esene o as mediato eslted in onesion ate h he omed odts ontained seeal low molela weiht aomati omonds sh as enediaoaldehde eneneoanoi aid – and henol aases om Bacillus seies ae in eneal toleant to hih temeates and alaline onditions whih maes them sitale o linin deadation Streptomyces S. coelicolor S. lividans S. notnatel most o these laases ae intaellla whih omliates lae sale viridosporus Amycolatopsis E. coli odtion eentl an etaellla laase was otained om Bacillus tequilensis – an ateim isolated om a ae mill elent he otimm temeate – o this laase was – while it een etains at o its atiit at he laase showed an otimal atiit at esides ein an etaellla enme its stailit mae laase a sel ioatalst to e eloited on indstial sale aase odtion was otimied sin a medim ontainin n and e slates as well as − ethanol laase– was emloed in the ioleahin o sotwood l esltin in a edtion o in the aa

nme and a ine ase in ihtness ddition o hdoenotiaole as mediato at low onentation m led to a the imoement in its eomane eeal ateial stains otained om soils o a iodiesit ih ainoest in e S. coelicolor hae een tested in the oidationough the o β his eslted in the isolation o two βBacillusβ, β stains4, and Bacillusβ atrophaeus stain and Bacillus pumilus stain oth miooanisms ehiit intaellla and etaellla laase atiities themoe at linin and the linin model omond aialleolβ aial ethe wee sessll deaded oth stains his sests that these stains hao inteestin laases – laase om Bacillus pumilus ot disoeed enome minin has een sessll loned and oeeessed in E. coli his enme showed a hih themostailit with a maimm atiit at ot was aale o oidiin seeal henoli omonds showin otimal ales at netal to alaline whih maes it an inteestin ioatalst o the iotehnoloial aliations In addition to the “small” two Thermus thermophilius is a themohili ateia whih odes an intaellla P antoea ananatis laase thlaase his ateial laase was sessll eessed in E. coli 29 38 39 28 37 Bacterial enzymes involved in lignin degradation Chapter II Bacterial enzymes involved in lignin degradation Chapter II

finall ig oiies glutathione an releases the final rout igure o other glutathione reutases fro Sphingobium s strain ig an ig are also ale to atale the sae reation as ig ut these enes are 2 atie on the oosite enantioer of the aronl ooun fore the oiation of guaialglerol β guaial ith ig eentl the stereoreferene of ig has een stuie together ith to noel glutathione lases ig fro – Novosphingobium s an ig fr o Thiobacillus denitrificans in the enati egraation of a lignin oel sustrate employing an enzymatic cascade of βetherases an glutathione lases he teste enzymes showed β enantioreferene his enantioreferene an e inreas e – or een reerse utagenesis 8 s these ene sstes are inherent intraellular as is an intraellular etaolite the are not inole in the isolated from γ initial egraation of lignin et the a la a role in the egraation of the sall lignin oligoers fore in the first stes of lignin egraation an a eelo as useful ioatalsts in the fiel of lignoellulose utiliation – igure egraation of guaialglerol β guaial Sphingobium s LigD catalyzes the alcohol oxidation of the lignin substrate Cα; (2) LigF catalyzes the incorporation of glutathione at the Cβ position, and finally (3) LigG oxidizes glutathione leaing to the final rout Pseudomonas stutzeri Pseudomonas putida ough the β ββ, β4, and β igure 5. Multienzymatic degradation of the biphenyl lignin derivative 2,2′ dependent β ihro3,3′ietho5,5′iaroihenl hingoonas auioilis In the ‘80 s the first glutathione dependent β our enes are inole ig atales the eethlation of the catalyzing the reductive cleavage of β etho grou ring fission is atale the iogenase ig The first detailed studies were reported for the βO4 aryl hrolsis is atale ig an onersion of aroanilli ai into system from the α Sphingobium– anilli ai is erfore to earolases ig an ig composed of three separate proteins; LigD (aCα (a β he role of sueroie isutases in aterial lignin oifiation E. coli enzyme system cleaves the β eentl to aterial anganeseeenent sueroie isutases ns β ere isoere to hae lignin egraing atiit ueroie isutases tiall atale the isroortionation of sueroie anion raial into oleular ogen the Cα of lignin substrate from the alcohol to the corresponding ketone; (2) LigF an hrogen eroie an la a e role in ellular rotetion against oiatie cleaves the intermediate with the attachment of glutathione at the Cβ position, and stress ueroie isutases are in general intraellular enes ut soe 29 40 41 28 38 Bacterial enzymes involved in lignin degradation Chapter II Bacterial enzymes involved in lignin degradation Chapter II

finall ig oiies glutathione an releases the final rout igure o other glutathione reutases fro Sphingobium s strain ig an ig are also ale to atale the sae reation as ig ut these enes are atie on the oosite enantioer of the aronl ooun fore the 2 oiation of guaialglerol β guaial ith ig eentl the stereoreferene of ig has een stuie together ith to noel glutathione lases ig fro – Novosphingobium s an ig fr o Thiobacillus denitrificans in the enati egraation of a lignin oel sustrate employing an enzymatic cascade of βetherases an glutathione lases he teste enzymes showed β enantioreferene his enantioreferene an e inreas e – or een reerse utagenesis 8 s these ene sstes are inherent intraellular as is an intraellular etaolite the are not inole in the isolated from γ initial egraation of lignin et the a la a role in the egraation of the sall lignin oligoers fore in the first stes of lignin egraation an a eelo as useful ioatalsts in the fiel of lignoellulose utiliation – igure egraation of guaialglerol β guaial Sphingobium s LigD catalyzes the alcohol oxidation of the lignin substrate Cα; (2) LigF catalyzes the incorporation of glutathione at the Cβ position, and finally (3) LigG oxidizes glutathione leaing to the final rout Pseudomonas stutzeri Pseudomonas putida ough the β ββ, β4, and β igure 5. Multienzymatic degradation of the biphenyl lignin derivative 2,2′ dependent β ihro3,3′ietho5,5′iaroihenl hingoonas auioilis In the ‘80 s the first glutathione dependent β our enes are inole ig atales the eethlation of the catalyzing the reductive cleavage of β etho grou ring fission is atale the iogenase ig The first detailed studies were reported for the βO4 aryl hrolsis is atale ig an onersion of aroanilli ai into system from the α Sphingobium– anilli ai is erfore to earolases ig an ig composed of three separate proteins; LigD (aCα (a β he role of sueroie isutases in aterial lignin oifiation E. coli enzyme system cleaves the β eentl to aterial anganeseeenent sueroie isutases ns β ere isoere to hae lignin egraing atiit ueroie isutases tiall atale the isroortionation of sueroie anion raial into oleular ogen the Cα of lignin substrate from the alcohol to the corresponding ketone; (2) LigF an hrogen eroie an la a e role in ellular rotetion against oiatie cleaves the intermediate with the attachment of glutathione at the Cβ position, and stress ueroie isutases are in general intraellular enes ut soe 29 40 41 28 39 Bacterial enzymes involved in lignin degradation Chapter II Bacterial enzymes involved in lignin degradation Chapter II

eales of etraellular atiit hae een reorte eentl to etraellular etherase as ientifie Sphingomonas paucimobilis as foun to haror ns fro Sphingobacterium s n an n ere ientifie another interesting egraation atha relate to lignin egraation hih an artiall urifie Interestingl oth enes are ale to erfor the includes a dioxygenase. A biphenyl compound (2,2′ihro3,3′ietho5,5′ 2 egraation of rganosol an raft lignin as ell as ifferent lignin oel iaroihenl as ientifie as a groth sustrate for Sphingomonas paucimobilis sustrates into seeral oouns ll the routs fore resulte fro arlCα 8 our ifferent tes of enes are inole in the initial stes of and CαCβ bond oxidative cleavage reactions as well as from Oeethlation egraing this ihenl igig earolases ig a hrolase atiit he lignin oiation reatiit of these ns an e assigne to the ig an iron eenent – eethlase an ig an etraiol iogenase igure foration of a hrol raial ith a high oiant effiien It reains to e he eethlation of the etho grou taes lae ig he rout fro estalishe hether suh sueroie isutases are roising aniates for this leaage ste is the sustrate for the iogenase ig hih leas to a ring iotehnologial aliation in the area of lignoellulose egraation an hether fission rout ieling aroanilli ai an aro – the are tial aterial ligninolti enes hroentaienoi ai he rout aroanilli ai is onerte into anilli ai in a roess atal e to earolase enes ig an atalaseeroiases are assoiate ith lignoellulose egraation ig hese finings suggest that iogenases reresents another tool use eentl a roteoi aroah a atalaseeroiase o as ientifie ateria to assist in lignin egraation as a heeontaining ene serete Amycolatopsis s i hen inuate ith lignoellulosi aterial o roie further eiene for its inoleent in utloo lignin egraation the ene as reoinantl roue an urifie o ateria o not ossess the regular eroiases that fungi elo for lignin as foun to e ale to onert a henoli lignin oel ooun hile a egraation his a e ue to intrinsi iffiulties in eressing these rather ethlate eriatie as not a sustrate ith eeriental onfiration that this ole roteins that are tiall gloslate ontain seeral isulhie ons atalase ero iase ats on a ligninlie ooun a role of o in lignin an inororate seeral aliu ions an a hee ofator oling an roessing oifiation see feasile uture researh ill tell hether suh etraellular a reuire seial onitions that are not oatile ith the aterial ahiner aterial atalase eroiases are freuentl use ateria to oif lignin for rotein roution In line ith this it is orth noting that –ost of the attets to roue eroiases fro the lant eroiase suerfail faile et the

aterial iogenases a la a role in lignin egraation te eroiases are soehat less ole onerning their rotein struture no In 0 a euliar aterial ene as esrie that is oose of a iogenase aliu ining sites gloslation or isulhie ons neee an are ie oain an a ligninining oain80 gain this ene as isolate fro a srea aong ateria eoinant eression of arious s in oli tiall Streptomyces isolate an as foun to e assoiate ith a ooas hih las its iele high leels of eression hih is in shar ontrast ith the eression of eggs in oo herefore it as antiiate that the isolate ateriu oul ehiit fungal eroiases8 his aes these aterial eroiases interesting targets for lant ioass egraation aailities Inee a etaile harateriation reeale ough the β ene eeloent he aterial laases also see to e suite for large sale a suite of serete enes inole in hrolti an oiatie atta of ββ, β4, and β reoinant ene roution reent etaile stu reeale that lignoellulose s art of the ientifiation of serete roteins the 8 otiiing eression onitions arious aterial laases an e roue in E. rotein eerge ase on the rotein seuene it aeare to e the result of a coli8 fusion of a intraiol iogenase an a arohrate ining oule he rotein also ontains an terinal attransloation signal etie hih onfirs its hile the nuer of non aterial eroiases an laases that a e inole etraellular loation eoinant eression of the ene in E. coli as in lignin egraation has gron rearal in the last to eaes there a still – suessful an alloe the eluiation of the rstal struture of the iogenase e unisoere ene tes aroun that la a role in aterial lignin egraation oain urtherore it oul e onfire that the ene as atie as eeral ne aniates hae surfae in the last fe ears ie sura urtherore iogenase ith seeral atehol eriaties Intriguingl it as isoere that one lass of enes that has een oerlooe are the enes that nee to roie the arohrate ining oule islae affinit toars sntheti lignin the aterial eroiases the reuire hrogen eroie uh oiases hae een olers ientifie for the fungal eroiases an siilarl it is eete that ateria serete oiases et hat te of oiases an their resetie sustrates hae still to e he inoleent of a iogenase in a aterial egraation atha of lignin estalishe related compound is not unprecedented. One of the bacteria from which a β 29 42 43 28 40 Bacterial enzymes involved in lignin degradation Chapter II Bacterial enzymes involved in lignin degradation Chapter II

eales of etraellular atiit hae een reorte eentl to etraellular etherase as ientifie Sphingomonas paucimobilis as foun to haror ns fro Sphingobacterium s n an n ere ientifie another interesting egraation atha relate to lignin egraation hih an artiall urifie Interestingl oth enes are ale to erfor the includes a dioxygenase. A biphenyl compound (2,2′ihro3,3′ietho5,5′ egraation of rganosol an raft lignin as ell as ifferent lignin oel iaroihenl as ientifie as a groth sustrate for Sphingomonas paucimobilis 2 sustrates into seeral oouns ll the routs fore resulte fro arlCα 8 our ifferent tes of enes are inole in the initial stes of and CαCβ bond oxidative cleavage reactions as well as from Oeethlation egraing this ihenl igig earolases ig a hrolase atiit he lignin oiation reatiit of these ns an e assigne to the ig an iron eenent – eethlase an ig an etraiol iogenase igure foration of a hrol raial ith a high oiant effiien It reains to e he eethlation of the etho grou taes lae ig he rout fro estalishe hether suh sueroie isutases are roising aniates for this leaage ste is the sustrate for the iogenase ig hih leas to a ring iotehnologial aliation in the area of lignoellulose egraation an hether fission rout ieling aroanilli ai an aro – the are tial aterial ligninolti enes hroentaienoi ai he rout aroanilli ai is onerte into anilli ai in a roess atal e to earolase enes ig an atalaseeroiases are assoiate ith lignoellulose egraation ig hese finings suggest that iogenases reresents another tool use eentl a roteoi aroah a atalaseeroiase o as ientifie ateria to assist in lignin egraation as a heeontaining ene serete Amycolatopsis s i hen inuate ith lignoellulosi aterial o roie further eiene for its inoleent in utloo lignin egraation the ene as reoinantl roue an urifie o ateria o not ossess the regular eroiases that fungi elo for lignin as foun to e ale to onert a henoli lignin oel ooun hile a egraation his a e ue to intrinsi iffiulties in eressing these rather ethlate eriatie as not a sustrate ith eeriental onfiration that this ole roteins that are tiall gloslate ontain seeral isulhie ons atalase ero iase ats on a ligninlie ooun a role of o in lignin an inororate seeral aliu ions an a hee ofator oling an roessing oifiation see feasile uture researh ill tell hether suh etraellular a reuire seial onitions that are not oatile ith the aterial ahiner aterial atalase eroiases are freuentl use ateria to oif lignin for rotein roution In line ith this it is orth noting that –ost of the attets to roue eroiases fro the lant eroiase suerfail faile et the

aterial iogenases a la a role in lignin egraation te eroiases are soehat less ole onerning their rotein struture no In 0 a euliar aterial ene as esrie that is oose of a iogenase aliu ining sites gloslation or isulhie ons neee an are ie oain an a ligninining oain80 gain this ene as isolate fro a srea aong ateria eoinant eression of arious s in oli tiall Streptomyces isolate an as foun to e assoiate ith a ooas hih las its iele high leels of eression hih is in shar ontrast ith the eression of eggs in oo herefore it as antiiate that the isolate ateriu oul ehiit fungal eroiases8 his aes these aterial eroiases interesting targets for lant ioass egraation aailities Inee a etaile harateriation reeale ough the β ene eeloent he aterial laases also see to e suite for large sale a suite of serete enes inole in hrolti an oiatie atta of ββ, β4, and β reoinant ene roution reent etaile stu reeale that lignoellulose s art of the ientifiation of serete roteins the 8 otiiing eression onitions arious aterial laases an e roue in E. rotein eerge ase on the rotein seuene it aeare to e the result of a coli8 fusion of a intraiol iogenase an a arohrate ining oule he rotein also ontains an terinal attransloation signal etie hih onfirs its hile the nuer of non aterial eroiases an laases that a e inole etraellular loation eoinant eression of the ene in E. coli as in lignin egraation has gron rearal in the last to eaes there a still – suessful an alloe the eluiation of the rstal struture of the iogenase e unisoere ene tes aroun that la a role in aterial lignin egraation oain urtherore it oul e onfire that the ene as atie as eeral ne aniates hae surfae in the last fe ears ie sura urtherore iogenase ith seeral atehol eriaties Intriguingl it as isoere that one lass of enes that has een oerlooe are the enes that nee to roie the arohrate ining oule islae affinit toars sntheti lignin the aterial eroiases the reuire hrogen eroie uh oiases hae een olers ientifie for the fungal eroiases an siilarl it is eete that ateria serete oiases et hat te of oiases an their resetie sustrates hae still to e he inoleent of a iogenase in a aterial egraation atha of lignin estalishe related compound is not unprecedented. One of the bacteria from which a β 29 42 43 28 41 Bacterial enzymes involved in lignin degradation Chapter II Bacterial enzymes involved in lignin degradation Chapter II

It is interesting to note that onl reentl also for the egraation of the ellulose Sci. 2016 113 art of lant ioass oiatie enes are reognie as aor laers in the Bioengineered 2016 7 – egraation roess he oerontaining soalle lti olsaharie 2 onoogenases s hae een foun to e essential in the atta of the rather inert arts of ellulose an other olsaharies he reent fining of the Fungal Genet. Biol. isin grou that the s seuestere the reuire eletrons fro the lignin 2014 72 – art of lant ioass suggests a strong lin eteen the egraation of the ifferent – Tetrahedron 8 2002 ioass oieties It ill e interesting to see hether aterial s an e 58 – line to other reo enes serete ateria an hether aterial lignin egraation is intertine ith lant olsaharie egraation Arch. Biochem. Biophys. 1986 251 – – ith the realiation of the aterial ailit to oif the lignin art of lant ioass ioheial stuies on aterial lignin egraation athas an their resetie enes has een reitalise ith ne aroahes to ientif ne Environ. Microbiol. 2013 15 – ligninegraing ateria8 in oination ith etaile genoi roteoi an ioheial stuies the ientities an roles of aterial ligninolti enes ill e unoere in the oing ears nolegeents his or as suorte the grauate rogra sntheti iolog for aane etaoli engineering roet nuer 00000 he etherlans onalo e onalo an aal rogra thans I for ersonal – funing ohae ai reeie funing fro the ultural ffairs an issions etor inistr of igher uation gt eferenes Science 2012 336 – sgher ashir Ial Chem. Eng. Res. Des. 92 – 2014 8 Soil Sci. 1930 30 – ough the β agausas eha i hanra hen ais aison ion ilna eller angan ββ, β 4, and β asar aler shalinsi usan an Science 2014 344 8 Genome Announc. aaguhi asunua gino ono Curr. Opin. Biotechnol. 2013 1 2016 42 0– – ottlie elar Bacteriol. Rev. 1951 15 J. Am. Chem. Soc. 2011 133 – anhole eets orreel alh oeran Plant Physiol. Curr. Opin. Struct. Biol. 1992 2 –

2010 153 8 –0 Appl. elai oliati I ann in s ariaslani Microbiol. Biotechnol. 2010 86 – in a aei ress 2013 –8 Cell. Mol. Life Sci. 2009 66 – elsen iihele eters oe Proc. Natl. Acad. 29 44 45 28 42 Bacterial enzymes involved in lignin degradation Chapter II Bacterial enzymes involved in lignin degradation Chapter II

It is interesting to note that onl reentl also for the egraation of the ellulose Sci. 2016 113 art of lant ioass oiatie enes are reognie as aor laers in the Bioengineered 2016 7 – egraation roess he oerontaining soalle lti olsaharie onoogenases s hae een foun to e essential in the atta of the 2 rather inert arts of ellulose an other olsaharies he reent fining of the Fungal Genet. Biol. isin grou that the s seuestere the reuire eletrons fro the lignin 2014 72 – art of lant ioass suggests a strong lin eteen the egraation of the ifferent – Tetrahedron 8 2002 ioass oieties It ill e interesting to see hether aterial s an e 58 – line to other reo enes serete ateria an hether aterial lignin egraation is intertine ith lant olsaharie egraation Arch. Biochem. Biophys. 1986 251 – – ith the realiation of the aterial ailit to oif the lignin art of lant ioass ioheial stuies on aterial lignin egraation athas an their resetie enes has een reitalise ith ne aroahes to ientif ne Environ. Microbiol. 2013 15 – ligninegraing ateria8 in oination ith etaile genoi roteoi an ioheial stuies the ientities an roles of aterial ligninolti enes ill e unoere in the oing ears nolegeents his or as suorte the grauate rogra sntheti iolog for aane etaoli engineering roet nuer 00000 he etherlans onalo e onalo an aal rogra thans I for ersonal – funing ohae ai reeie funing fro the ultural ffairs an issions etor inistr of igher uation gt eferenes Science 2012 336 – sgher ashir Ial Chem. Eng. Res. Des. 92 – 2014 8 Soil Sci. 1930 30 – ough the β agausas eha i hanra hen ais aison ion ilna eller angan ββ, β 4, and β asar aler shalinsi usan an Science 2014 344 8 Genome Announc. aaguhi asunua gino ono Curr. Opin. Biotechnol. 2013 1 2016 42 0– – ottlie elar Bacteriol. Rev. 1951 15 J. Am. Chem. Soc. 2011 133 – anhole eets orreel alh oeran Plant Physiol. Curr. Opin. Struct. Biol. 1992 2 –

2010 153 8 –0 Appl. elai oliati I ann in s ariaslani Microbiol. Biotechnol. 2010 86 – in a aei ress 2013 –8 Cell. Mol. Life Sci. 2009 66 – elsen iihele eters oe Proc. Natl. Acad. 29 44 45 28 43 Bacterial enzymes involved in lignin degradation Chapter II Bacterial enzymes involved in lignin degradation Chapter II

J. Ind. Microbiol. Biotechnol. 2014 41 – N. Lončar, D. I. Colpa, M. W. Fraaije, Tetrahedron 2016 72 – Arch. Biochem. Biophys. 2015 574 – Arch. Biochem. 2 Arch. Biochem. Biophys. 2015 574 – Biophys. 2016 594 – Appl. Environ. Microbiol. 1999 65 – – Biochemistry 2011 50 – J. Biol. Chem. 2007 282 – Biochem. J. 1986 236 – Appl. Environ. Microbiol. 1988 – 54 – Arch. Biochem. Biophys. 2015 574 – J. Biotechnol. FEBS Lett. 2012 586 – 1990 13 – Can. J. Microbiol. 1998 44 – ACS Chem. Biol. 2013 8 – Arch. Biochem. Biophys. Biochemistry 2015 574 – 1998 37 – Appl. Microbiol. Biotechnol. 2013 97 – J. Mol. Biol. 2005– 354 – Nucleic Acids Res. 2013 41 Appl. Environ. Microbiol. 2009 75 – Proc. Natl. Acad. Sci. U. S. A. 2009 106 – Nat. Struct. Mol. Biol. 2008 15 – ough the β ChemCatChem 2016 8 – ββ, β4, and β J. Biol. Chem. 2014 289 – Arch. Biochem. Biophys. 2015 574 – Trends Biotechnol. 2006 24 – Sci. Rep. 2015 5 Trends Biotechnol. Appl. Microbiol. Biotechnol. – 2011 29 – 2014 98 – Enzyme Res. 2011 2011 PLoS One 2014 9 Biotechnol. Bioeng. 2004 86 – J. Biol. Chem. 2015 290 – Appl. Environ. Microbiol. 1995 61 – ACS Chem. Biol. 2012 7 – 29 46 47 28 44 Bacterial enzymes involved in lignin degradation Chapter II Bacterial enzymes involved in lignin degradation Chapter II

J. Ind. Microbiol. Biotechnol. 2014 41 – N. Lončar, D. I. Colpa, M. W. Fraaije, Tetrahedron 2016 72 – Arch. Biochem. Biophys. 2015 574 – Arch. Biochem. 2 Arch. Biochem. Biophys. 2015 574 – Biophys. 2016 594 – Appl. Environ. Microbiol. 1999 65 – – Biochemistry 2011 50 – J. Biol. Chem. 2007 282 – Biochem. J. 1986 236 – Appl. Environ. Microbiol. 1988 – 54 – Arch. Biochem. Biophys. 2015 574 – J. Biotechnol. FEBS Lett. 2012 586 – 1990 13 – Can. J. Microbiol. 1998 44 – ACS Chem. Biol. 2013 8 – Arch. Biochem. Biophys. Biochemistry 2015 574 – 1998 37 – Appl. Microbiol. Biotechnol. 2013 97 – J. Mol. Biol. 2005– 354 – Nucleic Acids Res. 2013 41 Appl. Environ. Microbiol. 2009 75 – Proc. Natl. Acad. Sci. U. S. A. 2009 106 – Nat. Struct. Mol. Biol. 2008 15 – ough the β ChemCatChem 2016 8 – ββ, β4, and β J. Biol. Chem. 2014 289 – Arch. Biochem. Biophys. 2015 574 – Trends Biotechnol. 2006 24 – Sci. Rep. 2015 5 Trends Biotechnol. Appl. Microbiol. Biotechnol. – 2011 29 – 2014 98 – Enzyme Res. 2011 2011 PLoS One 2014 9 Biotechnol. Bioeng. 2004 86 – J. Biol. Chem. 2015 290 – Appl. Environ. Microbiol. 1995 61 – ACS Chem. Biol. 2012 7 – 29 46 47 28 45 Bacterial enzymes involved in lignin degradation Chapter II Bacterial enzymes involved in lignin degradation Chapter II

Trends Biotechnol. 2000 18 – FEBS Lett. 1989 249 – Cell. Mol. Life Sci. 2015 72 – 2 Appl. Environ. Environ. Sci. Process. Impacts 2015 17 – Microbiol. 2009 75 – FEBS Lett. 1990 267 – – Biosci. Biotechnol. Adv. Microbiol. 2014, 4 Biochem. 2011 75 – Green Chem. – 2015 17 – Biochemistry 2014 53 – Appl. Environ. Microbiol. 2014 80 – ACS Chem. Biol. 2015 10 – J. Biol. Chem. 2013 288 – Int. Microbiol. 2009 12 – J. Basic Microbiol. 2009 BioResources 2011, 6 49 Suppl 1 Sci. Rep. 2015 5 Lett. Appl. Microbiol. 2014 58 – – Biotechnol. Lett. 2015 37 – Sci. Rep. 2015 5 3 Biotech é á 2015 5 – í í ACS Sustain. Chem. Eng. 2016 4 – ough the β Biotechnol. Bioeng. 2013 110 – ββ, β4, and β BMC Biotechnol. 2011 11 Extremophiles 2005 9 – Biotechnol. Lett. 2012 34 – – Biotechnol. Lett. 2003 25 – Bioresour. Technol. 2008 99 – Proc. Natl. Acad. Sci. U. S. A. 2014 111 – 29 48 49 28 46 Bacterial enzymes involved in lignin degradation Chapter II Bacterial enzymes involved in lignin degradation Chapter II

Trends Biotechnol. 2000 18 – FEBS Lett. 1989 249 – Cell. Mol. Life Sci. 2015 72 – Appl. Environ. 2 Environ. Sci. Process. Impacts 2015 17 – Microbiol. 2009 75 – FEBS Lett. 1990 267 – – Biosci. Biotechnol. Adv. Microbiol. 2014, 4 Biochem. 2011 75 – Green Chem. – 2015 17 – Biochemistry 2014 53 – Appl. Environ. Microbiol. 2014 80 – ACS Chem. Biol. 2015 10 – J. Biol. Chem. 2013 288 – Int. Microbiol. 2009 12 – J. Basic Microbiol. 2009 BioResources 2011, 6 49 Suppl 1 Sci. Rep. 2015 5 Lett. Appl. Microbiol. 2014 58 – – Biotechnol. Lett. 2015 37 – Sci. Rep. 2015 5 3 Biotech é á 2015 5 – í í ACS Sustain. Chem. Eng. 2016 4 – ough the β Biotechnol. Bioeng. 2013 110 – ββ, β4, and β BMC Biotechnol. 2011 11 Extremophiles 2005 9 – Biotechnol. Lett. 2012 34 – – Biotechnol. Lett. 2003 25 – Bioresour. Technol. 2008 99 – Proc. Natl. Acad. Sci. U. S. A. 2014 111 – 29 48 49 28 47

iaai e a e eaai ii ie i a iaeeiae aae e e ‐

ae ai ee e ikola Lončar, Milos Trajkovic, a a aaie

ei ii a eae iaaia i a e e eai i a iaeeiae aae ee e i ee i iai i ee iae a a eiae ii ie aeia‐ a e‐ e aae eai ae aaae e aii e iae e ie a a e eie ee ‐a ea e e e eie i e e eiae e ai i a i ii ee iae a a a ae aeae ie a ae ai a a iee e e a e e ei ii a e a ee aa i i‐ e eae a iae‐ ‐ ‐ i e ei ii e eai a iie i e aiee e ie ie ie ie ii ie a ae a ai e ae ie e eeai chromatography revealed the formation of relatively small lignin oligomers (≈1000 a i ieia eeea‐ ie a eee ea aei eae e aai i e eae a e‐ aerial contained α 4/β 4, β 4, β β, β iae a ieii i ee eae iiae a e iaaia 3 e aeia e eee aa‐ ii‐ ‐ ie‐ ‐ ‐ ‐ ee‐ a ee i ae ie ie e eaii ae a ee i ae e ie a eae i ae ia ii e eee‐ e eee e eee a aae a aie iaai‐ aa e eaai ii ie aeia a eia aae eia ‐ A Biocatalytic One Pot Approach for the Preparation of Lignin Oligomers i ae i ae

Using an Oxidase/Peroxidase Cascade M. H. M. Habib, P. J. Deuss, N. Lončar, M. Trajkovic, M. W. Fraaije, Adv. Synth. Catal. 2017 359 – Enzyme System

Mohamed Habib, Peter J. Deuss, Nikola Loncar> Milos Trajkovic, and Marco W. Fraaije

iaai e a e eaai ii ie i a iaeeiae aae e e ‐

ae ai ee e ikola Lončar, Milos Trajkovic, a a aaie

ei ii a eae iaaia i a e e eai i a iaeeiae aae ee e i ee i iai i ee iae a a eiae ii ie aeia‐ a e‐ e aae eai ae aaae e aii e iae e ie a a e eie ee ‐a ea e e e eie i e e eiae e ai i a i ii ee iae a a a ae aeae ie a ae ai a a iee e e a e e ei ii a e a ee aa i i‐ e eae a iae‐ ‐ ‐ i e ei ii e eai a iie i e aiee e ie ie ie ie ii ie a ae a ai e ae ie e eeai chromatography revealed the formation of relatively small lignin oligomers (≈1000 a i ieia eeea‐ ie a eee ea aei eae e aai i e eae a e‐ aerial contained α 4/β 4, β 4, β β, β iae a ieii i ee eae iiae a e iaaia e aeia e eee aa‐ ii‐ ‐ ie‐ ‐ ‐ ‐ ee‐ a ee i ae ie ie e eaii ae a ee i ae e ie a eae i ae ia ii e eee‐ e eee e eee a aae a aie iaai‐ aa e eaai ii ie aeia a eia aae eia ‐

i ae i ae

M. H. M. Habib, P. J. Deuss, N. Lončar, M. Trajkovic, M. W. Fraaije, Adv. Synth. Catal. 2017 359 –

49 A biocatalytic one-pot approach for the synthesis of lignin-like oligomers Chapter III

– ‐ – ‐ ‐ linked via among others, ‐β 4, β 5, β β, β 1, α 5′ linkages, of which β ‐ – ‐ ‐ ‐ ‐‐ ‐ ‐ ‐ ‐ ‐ 3 ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐

– ‐

53 54 50 A biocatalytic one-pot approach for the synthesis of lignin-like oligomers Chapter III

– ‐ – ‐ ‐ linked via among others, ‐β 4, β 5, β β, β 1, α 5′ linkages, of which β ‐ – ‐ ‐ ‐ ‐‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ 3 ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐

– ‐

53 54 51 A biocatalytic one-pot approach for the synthesis of lignin-like oligomers Chapter III

‐ ‐ ‐ 3 ‐ ‐ Svi ‐ Svi

Svi 2 ‐ ‐ 55 56 52 A biocatalytic one-pot approach for the synthesis of lignin-like oligomers Chapter III

‐ ‐ ‐ 3 ‐ ‐ Svi ‐ Svi

Svi 2 ‐ ‐ 55 56 53 A biocatalytic one-pot approach for the synthesis of lignin-like oligomers Chapter III

‐ ‐ ‐ 3 ‐ ‐ to an eugenol (1) unit through a β ‐ ‐ ‐ ‐ Svi ‐ ‐ ‐ ‐ Svi ‐ ‐ ‐ ‐ ‐ ‐ – ‐ ‐ Svi 1 vide infra ‐ ‐ 2

57 58 54 A biocatalytic one-pot approach for the synthesis of lignin-like oligomers Chapter III

‐ ‐ ‐ 3 ‐ ‐ to an eugenol (1) unit through a β ‐ ‐ ‐ ‐ Svi ‐ ‐ ‐ ‐ Svi ‐ ‐ ‐ ‐ ‐ ‐ – ‐ ‐ Svi 1 vide infra ‐ ‐ 2

57 58 55 A biocatalytic one-pot approach for the synthesis of lignin-like oligomers Chapter III

1 clearly signals belonging to lignin α β 4, β β, β 5′ could be ‐ ‐ ‐ ‐ ‐ ‐ ‐ 3 4 ≈33 α 4/β 4, β 4, β 5, β β link 5 ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ synthesized lignin has 48.7% α 4/β 4 and β – – – β ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ The‐ amount of β β linkages reaches 13.2% in the synthetic lignin oligomers. This ‐ – ‐ ‐ in situ

59 60 56 A biocatalytic one-pot approach for the synthesis of lignin-like oligomers Chapter III

59 60 57 A biocatalytic one-pot approach for the synthesis of lignin-like oligomers Chapter III

found that only β 5′ and β ‐ aforementioned linkages,‐ ‐ ‐ α 4, ‐ β β linkages and a dibenzodioxocin unit (see ‐ ‐ ‐ ‐ ‐ ‐ cycle was ≈290 3 ‐ ‐ ‐ ‐ ‐ ‐ d6 ‐ ‐‐ – ‐ 1 Svi 10β cells in 500 ‐ ‐ Svi ‐ 61 62 58 A biocatalytic one-pot approach for the synthesis of lignin-like oligomers Chapter III

found that only β 5′ and β ‐ aforementioned linkages,‐ ‐ ‐ α 4, ‐ β β linkages and a dibenzodioxocin unit (see ‐ ‐ ‐ ‐ ‐ ‐ cycle was ≈290 3 ‐ ‐ ‐ ‐ ‐ ‐ d6 ‐ ‐‐ – ‐ 1 Svi 10β cells in 500 ‐ ‐ Svi ‐ 61 62 59 A biocatalytic one-pot approach for the synthesis of lignin-like oligomers Chapter III

Svi ‐ ‐ Svi ‐ ‐ − − ‐ ‐ ‐ 3 ‐ Svi μL volume of the diluted sample was ‐ ‐ ‐ μ ‐ ‐ Svi – – – − ‐ and stored‐ at −20 ‐ ‐ ‐ Svi ‐ − − Svi ϵ − − μ ≈ 44,000 ϵ ‐ ‐ ‐

63 64 60 A biocatalytic one-pot approach for the synthesis of lignin-like oligomers Chapter III

63 64 61 A biocatalytic one-pot approach for the synthesis of lignin-like oligomers Chapter III

3 – – – ‐ − ‐ μ μm MIXED − ‐ ‐ ‐ μm solvent filter from Waters (Milford, MA, USA) under ‐ d6 ‐ ‐ ‐ ‐ μ Annu. Rev. Plant Biol. 2003 54 – ‐ 2013 Curr. Opin. Biotechnol. 2016 42 – ‐ 65 66 62 A biocatalytic one-pot approach for the synthesis of lignin-like oligomers Chapter III

– 3 – – ‐ − ‐ μ μm MIXED − ‐ ‐ ‐ μm solvent filter from Waters (Milford, MA, USA) under ‐ d6 ‐ ‐ ‐ ‐ μ Annu. Rev. Plant Biol. 2003 54 – ‐ 2013 Curr. Opin. Biotechnol. 2016 42 – ‐ 65 66 63 A biocatalytic one-pot approach for the synthesis of lignin-like oligomers Chapter III

aie eei 2001 – 2013 e aa ea 1993 i a e a i ie i 2004 – 2014 e eee 1965 2016 e a – 2012 – 2016 – a aaie a i 2014 2016 – – 3 2014 – a aaa aiee 2003 – aie iiai ae iaa ieie 2016 – 2013 – ee ae ie ie a ie ae ae e 2009 – 2016 – aeie a aie a ii 1998 e ai i ae aa – e a 2005 – 1987 N. Lončar, D. I. Colpa, M. W. Fraaije, 2016 – –

1994 – 2008 – 1989 – 2015 – 2016 – éá í í 2016 – 2007 – 2016 – 2015 67 68 64 A biocatalytic one-pot approach for the synthesis of lignin-like oligomers Chapter III

1994 – 2008 – 1989 – 2015 – 2016 – éá í í 2016 – 2007 – 2016 – 2015 67 68 65

The Biocatalytic Synthesis of Syringaresinol from 2,6-Dimethoxy-4- allylphenol in One-Pot Using a Tailored Oxidase/Peroxidase System 2018 Mohamed Habib, Milos Trajkovic and Marco W. Fraaije

2018

67 The biocatalytic synthesis of syringaresinol using an oxidase/peroxidase system Chapter IV The biocatalytic synthesis of syringaresinol using an oxidase/peroxidase system Chapter IV

– – – – hol (2) units linked via a β–β linkage. The lignan is known to display hol (2) units linked via a β–β linkage. The lignan is known to display 4 73 72 73 72 68 The biocatalytic synthesis of syringaresinol using an oxidase/peroxidase system Chapter IV The biocatalytic synthesis of syringaresinol using an oxidase/peroxidase system Chapter IV

– – – – hol (2) units linked via a β–β linkage. The lignan is known to display hol (2) units linked via a β–β linkage. The lignan is known to display 4 73 72 73 72 69 The biocatalytic synthesis of syringaresinol using an oxidase/peroxidase system Chapter IV The biocatalytic synthesis of syringaresinol using an oxidase/peroxidase system Chapter IV

sinapyl alcohol within 22 h when 5 μM of the enzyme was added to a solution – – alcohol monomers linked to each other through a β –β linkage. When EUGO I427A (5 μM) and HRP (0.65 μM) were added to a reaction containing 10 mM 2,6 hol (2) units linked via a β–β linkage. The lignan is known to display 4 – = 0.46 μM). This mutant enzyme displays a more than – = 0.77 μM). The other two – – 0.34 μM, I427G: = 0.39 μM). Yet, the I427G mutant suffered – = 0.39 μM). The steady – allylphenol as a substrate apart from the β–β linkages from syringaresinol. A signal for a β en modified on the α however, the exact nature of this β formation of β–β dimers over typical β allylphenol into sinapyl alcohol using EUGO I427A. When 0.5 μM of EUGO 73 74 75 72 70 The biocatalytic synthesis of syringaresinol using an oxidase/peroxidase system Chapter IV The biocatalytic synthesis of syringaresinol using an oxidase/peroxidase system Chapter IV

sinapyl alcohol within 22 h when 5 μM of the enzyme was added to a solution – – alcohol monomers linked to each other through a β –β linkage. When EUGO I427A (5 μM) and HRP (0.65 μM) were added to a reaction containing 10 mM 2,6 hol (2) units linked via a β–β linkage. The lignan is known to display 4 – = 0.46 μM). This mutant enzyme displays a more than – = 0.77 μM). The other two – – 0.34 μM, I427G: = 0.39 μM). Yet, the I427G mutant suffered – = 0.39 μM). The steady – allylphenol as a substrate apart from the β–β linkages from syringaresinol. A signal for a β en modified on the α however, the exact nature of this β formation of β–β dimers over typical β allylphenol into sinapyl alcohol using EUGO I427A. When 0.5 μM of EUGO 73 74 75 72 71 The biocatalytic synthesis of syringaresinol using an oxidase/peroxidase system Chapter IV The biocatalytic synthesis of syringaresinol using an oxidase/peroxidase system Chapter IV

β cells were grown in 5 mL Luria – – isolated lignan. In addition to the syringaresinol, ≈80 mg of insoluble product was hol (2) units linked via a β–β linkage. The lignan is known to display 4 molecular weight (MW ≈ 44,000 Da). For determination of steady state kinetic ɛ 73 76 77 72 72 The biocatalytic synthesis of syringaresinol using an oxidase/peroxidase system Chapter IV The biocatalytic synthesis of syringaresinol using an oxidase/peroxidase system Chapter IV

β cells were grown in 5 mL Luria – – isolated lignan. In addition to the syringaresinol, ≈80 mg of insoluble product was hol (2) units linked via a β–β linkage. The lignan is known to display 4 molecular weight (MW ≈ 44,000 Da). For determination of steady state kinetic ɛ 73 76 77 72 73 The biocatalytic synthesis of syringaresinol using an oxidase/peroxidase system Chapter IV The biocatalytic synthesis of syringaresinol using an oxidase/peroxidase system Chapter IV

– – hol (2) units linked via a β–β linkage. The lignan is known to display 4 – 2014 2018 – 2015 D. I. Colpa, N. Lončar, M. Schmidt, M. W. Fraaije, 2017 2017 J. Deuss, N. Lončar, M. Trajkovic, M. W. Fraaije, – 2017 1996 – 2016 – А. А. Semenov, A. G. Enikeev, V. B. Khobrakova, Y. G. Razuvayeva, S. М. 2013 – 73 78 79 72 74 The biocatalytic synthesis of syringaresinol using an oxidase/peroxidase system Chapter IV The biocatalytic synthesis of syringaresinol using an oxidase/peroxidase system Chapter IV

– – hol (2) units linked via a β–β linkage. The lignan is known to display 4 – 2014 2018 – 2015 D. I. Colpa, N. Lončar, M. Schmidt, M. W. Fraaije, 2017 2017 J. Deuss, N. Lončar, M. Trajkovic, M. W. Fraaije, 2017 – 1996 – 2016 – А. А. Semenov, A. G. Enikeev, V. B. Khobrakova, Y. G. Razuvayeva, S. М. 2013 – 73 78 79 72 75 The biocatalytic synthesis of syringaresinol using an oxidase/peroxidase system Chapter IV

2012 – 2017 – 2017 – 2016 – 2007 – 4

73 80 76 The biocatalytic synthesis of syringaresinol using an oxidase/peroxidase system Chapter IV

2012 – 2017 – 2017 – 2016 – 2007 –

73 80

Characterization of a New DyP-Peroxidase from the Alkaliphilic Cellulomonad, Cellulomonas bogoriensis 2019

Mohamed Habib, Henriëtte J. Rozeboom and Marco W. Fraaije

2019

79 Characterization of a New DyP-Peroxidase from Cellulomonas bogoriensis Chapter V Characterization of a New DyP-Peroxidase from Cellulomonas bogoriensis Chapter V

5 − − 85 84 85 84 80 Characterization of a New DyP-Peroxidase from Cellulomonas bogoriensis Chapter V Characterization of a New DyP-Peroxidase from Cellulomonas bogoriensis Chapter V

5 − − 85 84 85 84 81 Characterization of a New DyP-Peroxidase from Cellulomonas bogoriensis Chapter V Characterization of a New DyP-Peroxidase from Cellulomonas bogoriensis Chapter V

w t C b o D yP

0 .8 4 0 7 K P i 5 0 m M , p H 7 2 8 0 1 m M H 2 O2 0 .6 e 1 m M D ith io n ite c

n a 4 3 2 b r 0 .4 o

b A 0 .2 5 0 3 5 58 6 3 1 0 .0 0 0 0 0 0 0 0 0 0 0 5 0 5 0 5 0 5 0 3 3 4 4 5 5 6 6 7 W av e le n g th (n m ) ( a) kobs at pH 4 (s −1) max Conc. Dye Type Dye E201D TfuDyP (nm) (µM) wt CboDyP Hemin CboDyP b E20 1 DC b o D y P 0 .8 2 8 0 K P i 5 0 m M , p H 7 5 1 m M H 2 O 2 0 .6 3,4’,4’’,4’’’ e 4 0 7

c 1 m M D ith io n ite

a b r 0 .4

b 4 3 2 A 0 .2 5 0 3 5 5 8 6 3 1 0 .0 0 0 0 0 0 0 0 0 0 0 5 0 5 0 5 0 5 0 5 0 2 3 3 4 4 5 5 6 6 7 W a v e le n g th (n m ) (b ) μ b μ − 85 86 87 84 82 Characterization of a New DyP-Peroxidase from Cellulomonas bogoriensis Chapter V Characterization of a New DyP-Peroxidase from Cellulomonas bogoriensis Chapter V

w t C b o D yP

0 .8 4 0 7 K P i 5 0 m M , p H 7 2 8 0 1 m M H 2 O2 0 .6 e 1 m M D ith io n ite c

n a 4 3 2 b r 0 .4 o s

b A 0 .2 5 0 3 5 58 6 3 1 0 .0 0 0 0 0 0 0 0 0 0 0 5 0 5 0 5 0 5 0 3 3 4 4 5 5 6 6 7 W av e le n g th (n m ) ( a) kobs at pH 4 (s −1) max Conc. Dye Type Dye E201D TfuDyP (nm) (µM) wt CboDyP Hemin CboDyP b E20 1 DC b o D y P 0 .8 2 8 0 K P i 5 0 m M , p H 7 1 m M H 2 O 2 5 0 .6 3,4’,4’’,4’’’ e 4 0 7 c 1 m M D ith io n ite

n a b r 0 .4 o

b 4 3 2 A 0 .2 5 0 3 5 5 8 6 3 1 0 .0 0 0 0 0 0 0 0 0 0 0 5 0 5 0 5 0 5 0 5 0 2 3 3 4 4 5 5 6 6 7 W a v e le n g th (n m ) (b ) μ b μ − 85 86 87 84 83 Characterization of a New DyP-Peroxidase from Cellulomonas bogoriensis Chapter V Characterization of a New DyP-Peroxidase from Cellulomonas bogoriensis Chapter V

β β α − − μ 3 μ 3 4 −− 4 −− – – μ , 33 μ μ , , , , , , , , 4 β α α 3 – – μ – , , ε δ 5 , , 4 3 4 – , moved about 0.8 Å toward Glu201 by the “bulkier” Thr330, instead of Cys, Ser or , 4 , , , 43 , , 4 33 4, 3 4, 3 4, 3 − 4, 3 85 88 89 84 84 Characterization of a New DyP-Peroxidase from Cellulomonas bogoriensis Chapter V Characterization of a New DyP-Peroxidase from Cellulomonas bogoriensis Chapter V

β β α − − μ 3 μ 3 4 −− 4 −− – – μ , 33 μ μ , , , , , , , , 4 β α α 3 – – μ – , , ε δ , 5 , 4 3 4 – , moved about 0.8 Å toward Glu201 by the “bulkier” Thr330, instead of Cys, Ser or , 4 , , , 43 , , 4 33 4, 3 4, 3 4, 3 − 4, 3 85 88 89 84 85 Characterization of a New DyP-Peroxidase from Cellulomonas bogoriensis Chapter V Characterization of a New DyP-Peroxidase from Cellulomonas bogoriensis Chapter V

the tunnel ore whih is narrower in y 0.8 Å omared to y and findin was for aetonitrile and butanone where no meltin temerature was t both 1.0 Å. This hannel rovides aess of the hydroen eroide to the reorded at 20 vv solvent onentration as the enyme must have already been heme 13. denatured uon addin the solvent before the eeriment ommened. The meltin temeratures of the enyme with and without the solvent an be seen in iure S11. .3 aterials and ethods .3.1 Chemials and eaents 1 was suli ed from ros ranis ittsburh, , S. sa restrition enyme was sulied from ew nland iolabs swih, , S. T liase and T liase buffer were sulied from Thermofisher Sientifi altham, , S. ll media omonents and amiillin antibioti were from isher Sientifi hemials ittsburh, , S. S rotease enyme was rovided by GCC Groninen, The etherlands. nstantlueT rotein stain was urhased from edeon San ieo, C, S. The iare Sin inire kit was urhased from iaen ilden, Germany. .3.2 nyme Clonin y was odon otimied for lonin in and ordered with sa overhans from Thermofisher Sientifi as GenertT StrinsT. The enyme was then loned usin the Goldenate ssembly i rotool into a vetor harborin a hista at the terminus followed by a S rotein. The histaSy vetor was then transformed into ew nland iolabs 10β ometent ells and rown overniht at 3 C on luria bertani lates sulied with 0 μ m−1 amiillin. The y 201 mutant, y 5 22 mutant and y 1 mutant were onstruted usin the rimers mentioned in Table S1 usin the uikhane mutaenesis tehniue.The reation miture ontained fu ltra otstart C aster i 1, n lasmid temlate, 0. μ of the rimer miture and omleted to 30 μ usin illi water. The rotool for the C was C for min initial denaturation, 33 yles of C

iure 2. a The y dimer showin the heme and iron brown in the ative denaturation for 30 s, 1 C annealin for y and C annealin for y site buried in between the alha helies and the beta sheets and b the ative site and y for 30 s, and an etension time of 3 min at 2 C. The final etension of y showin the heme and iron brown and the 201 residue hinderin the time was 1 min at 2 C. fter the C was omleted, n was added to eah aessibility to the ative site c the σweihted 2mo eletron density usin of the C reations to remove the non mutated methylated lasmid and leave only hases from the final model is ontoured at 1. σ level. Contours more than 2.2 Å the mutated lones. The samles were then treated by heatin at 80 C for 10 min from any of the dislayed atoms have been removed for larity. o water or other to denature the n. inally, the C reation roduts were transformed into liands are observed near the heme ofator. 10β ells and inubated on lates ontainin 0 μ m−1 amiillin, at 3 C overniht. Colonies were then iked and used to inoulate m broth .2. Thermal Stability of y ontainin 0 μ m −1 amiillin, overniht. The overniht ulture was then y was found to melt at a temerature of . 0.2 C as determined by urified for the neessary l asmids usin the iaen re Sin inire kit the Thermofluor tehniue in 10 m otassium hoshate buffer, . o adopting the manufacturer’s protocol. Finally, the purified plasmid was sent to inrease in meltin temerature of the enyme was reorded for all the different GTC bersber, Germany for seuenin. solvents tested at vv. The meltin temerature droed for all the solvents − tested when shiftin from to 20 vv onentration. The most sinifiant 85 90 91 84 86 Characterization of a New DyP-Peroxidase from Cellulomonas bogoriensis Chapter V Characterization of a New DyP-Peroxidase from Cellulomonas bogoriensis Chapter V the tunnel ore whih is narrower in y 0.8 Å omared to y and findin was for aetonitrile and butanone where no meltin temerature was t both 1.0 Å. This hannel rovides aess of the hydroen eroide to the reorded at 20 vv solvent onentration as the enyme must have already been heme 13. denatured uon addin the solvent before the eeriment ommened. The meltin temeratures of the enyme with and without the solvent an be seen in iure S11. .3 aterials and ethods .3.1 Chemials and eaents 1 was suli ed from ros ranis ittsburh, , S. sa restrition enyme was sulied from ew nland iolabs swih, , S. T liase and T liase buffer were sulied from Thermofisher Sientifi altham, , S. ll media omonents and amiillin antibioti were from isher Sientifi hemials ittsburh, , S. S rotease enyme was rovided by GCC Groninen, The etherlands. nstantlueT rotein stain was urhased from edeon San ieo, C, S. The iare Sin inire kit was urhased from iaen ilden, Germany. .3.2 nyme Clonin y was odon otimied for lonin in and ordered with sa overhans from Thermofisher Sientifi as GenertT StrinsT. The enyme was then loned usin the Goldenate ssembly i rotool into a vetor harborin a hista at the terminus followed by a S rotein. The histaSy vetor was then transformed into ew nland iolabs 10β ometent ells and rown overniht at 3 C on luria bertani lates sulied with 0 μ m−1 amiillin. The y 201 mutant, y 22 mutant and y 1 mutant were onstruted usin the rimers 5 mentioned in Table S1 usin the uikhane mutaenesis tehniue.The reation miture ontained fu ltra otstart C aster i 1, n lasmid temlate, 0. μ of the rimer miture and omleted to 30 μ usin illi water. The rotool for the C was C for min initial denaturation, 33 yles of C

.. nyme pression and urification lasmids from each of the three wild type enyme s and three mutant enymes were transformed into β cells and inoculated onto agar plates containing μg m− ampicillin. n oernight pre culture m in roth was inoculated using one of the isolated colonies from each type of clone and the medium supplemented with μ g m − ampicillin. he p reculture was used to inoculate a μ m terrific roth medium and ampicillin was added to gie μg m− as μ a final concentration in the medium. he culture was grown at until an μ optical density of . was reached at which an amount euialent to gie a concentration of . arainose was added to induce protein epression. he flas was then transferred to an incuator operating at and rpm. fter h, the cultures were harested y centrifugation at for min at ecman oulter, anti centrifuge, . rotor. he pellet was disrupted y sonication s on, s off for min at amplitude using a onics ira ell proe sonicator. he sonicated cells were centrifuged for min at , rpm to separate the cell deris from the cellfree etract. he cell free etract was filtered using hatman F . memrane syringe filters to remoe remaining deris. μ he cell free etract was added to graity flow columns paced with m i μ sepharose Fast flow resin after first euilirating the resin using column olumes of uffer . he ound protein was washed using column olumes of uffer followed y column olumes of uffer . he protein was finally eluted using uffer until all the protein was remoed from the column. he protein was − − then desalted using an cono ac esalting repaced raity Flow olumn ioad using uffer . he compositions of uffers – are found in upplementary aterials, ale . h e desalted fraction was isualied y running μ μ 5 an gel followed y staining using nstantlue rotein tain to assess purity and sie of the eluted protein. .. etermination of nyme oncentration

he enyme concentration of each of the si ariant s was determined ased on the predicted molar etinction coefficient at nm using the rotparam tool the etinction coefficients at nm are y . m− cm−, y . m− cm−, and for y . m− cm−. .. etermination of the ctiity of y with ifferent yes spectrum scan was done for each of eactie lue , isperse lue , and ndigotetrasulfonate using μ of dye concentration to calculate the etinction μ coefficient at the waelength that gae the highest asorance. his was repeated μ for cid ed and copper phthalocyanine dyes ut at μ. hen to a plastic μ cuette, μ of citrate uffer was added followed y hydrogen peroide to gie a final concentration of μ. he dye was then added to a final concentration of μ or μ depending on the dye used see ale . he enyme was finally − added to gie a concentration of n and the reaction followed oer s at the 85 92 93 84 88 Characterization of a New DyP-Peroxidase from Cellulomonas bogoriensis Chapter V Characterization of a New DyP-Peroxidase from Cellulomonas bogoriensis Chapter V

he enyme concentration of each of the si ariant s was determined ased on the predicted molar etinction coefficient at nm using the rotparam tool the etinction coefficients at nm are y . m− cm−, y . m− cm−, and for y . m− cm−. .. etermination of the ctiity of y with ifferent yes spectrum scan was done for each of eactie lue , isperse lue , and ndigotetrasulfonate using μ of dye concentration to calculate the etinction μ coefficient at the waelength that gae the highest asorance. his was repeated μ for cid ed and copper phthalocyanine dyes ut at μ. hen to a plastic μ cuette, μ of citrate uffer was added followed y hydrogen peroide to gie a final concentration of μ. he dye was then added to a final concentration of μ or μ depending on the dye used see ale . he enyme was finally − added to gie a concentration of n and the reaction followed oer s at the 85 92 93 84 89 Characterization of a New DyP-Peroxidase from Cellulomonas bogoriensis Chapter V Characterization of a New DyP-Peroxidase from Cellulomonas bogoriensis Chapter V

− α − 5 α – We would like to thank Dana I. Colpa and Nikola Lončar for fruitful discussions. − 85 94 95 84 90 Characterization of a New DyP-Peroxidase from Cellulomonas bogoriensis Chapter V Characterization of a New DyP-Peroxidase from Cellulomonas bogoriensis Chapter V

− α − 5 α – We would like to thank Dana I. Colpa and Nikola Lončar for fruitful discussions. − 85 94 95 84 91 Characterization of a New DyP-Peroxidase from Cellulomonas bogoriensis Chapter V Characterization of a New DyP-Peroxidase from Cellulomonas bogoriensis Chapter V

– 2017 – 2005 – 2018 2011 – – 2016 2012 – – 2016 – N. Lončar, D.I. Colpa, M.W. Fraaije. Exploring the Biocatalytic Potential 2013 – 2016 – 5 2011 – 2014 – 2007 2016 – 2010 – 1968 , 2016 – – − 2011 – 2015 – 85 96 97 84 92 Characterization of a New DyP-Peroxidase from Cellulomonas bogoriensis Chapter V Characterization of a New DyP-Peroxidase from Cellulomonas bogoriensis Chapter V

– 2017 – 2005 – 2018 2011 – – 2016 2012 – – 2016 – N. Lončar, D.I. Colpa, M.W. Fraaije. Exploring the Biocatalytic Potential 2013 – 2016 – 5 2011 – 2014 – 2007 2016 – 2010 – 1968 , 2016 – – − 2011 – 2015 – 85 96 97 84 93 Characterization of a New DyP-Peroxidase from Cellulomonas bogoriensis Chapter V

2010 – 2014 –

2010 – 2012 2006 – 5

85 98 94 Characterization of a New DyP-Peroxidase from Cellulomonas bogoriensis Chapter V

2010 – 2014 –

2010 – 2012 2006 –

85 98

Expression and characterization of

a resurrected DyP-type peroxidase

Maria Laura Mascotti, Mohamed H. Habib, and Marco W. Fraaije

97 Expression and characterization of a resurrected DyP-type peroxidase Chapter VI Chapter VI Expression and characterization of a resurrected DyP-type peroxidase called consensus method to enerate stale proteins. hateer the reason may e it appears that can e effectie in eneratin roust ariants of a particular called consensus method to enerate stale proteins. hateer the reason may e enzyme family. it appears that can e effectie in eneratin roust ariants of a particular enzyme family. n the contet of our study it is orth notin that in recent years has een performed on funal peroidases that elon to the peroidase catalase n the contet of our study it is orth notin that in recent years has een superfamily hich harors the ersatile peroidases linin peroidases and performed on funal peroidases that elon to the peroidase catalase mananese peroidases. n an attempt to resurrect etinct peroidases from the end superfamily hich harors the ersatile peroidases linin peroidases and of the aroniferous period yuso ernandez and coorers decided to select mananese peroidases. n an attempt to resurrect etinct peroidases from the end enes from seuenced enomes and to perform upon them. his era of the aroniferous period yuso ernandez and coorers decided to select as chosen as it as the period here the first lininolytic enzymes incl. enes from seuenced enomes and to perform upon them. his era peroidases must hae appeared . hese enzymes ould oriinate from olyporales as chosen as it as the period here the first lininolytic enzymes incl. species. he first linin deradin peroidase is supposedly a n peroidase peroidases must hae appear ed. hese enzymes ould oriinate from olyporales oidizin phenolic linin that accordin to the study ould not chane until species. he first linin deradin peroidase is supposedly a n peroidase the appearance of an eposed tryptophan oidizin nonphenolic linin to yield a oidizin phenolic linin that accordin to the study ould not chane until ersatile peroidase. ater another eolution ary eent resulted in the loss of the the appearance of an eposed tryptophan oidizin nonphenolic linin to yield a n indin site eneratin the first linin peroidase that eoled to the etant ersatile peroidase. ater another eolutionary eent resulted in the loss of the form y improin catalytic efficiency. fter the appearance of the eposed catalytic n indin site enerat in the first linin peroidase that eol ed to the etant tryptophandeterminants an of increase enzyme infunction staility is atreferred acidic pto as as “horizontal seen leadin approach”. to an increase in formdeterminants y improin of enzyme catalytic function efficiency. is referred fter theto as appearance “horizontal of approach”. the eposed catalytic the oidizin poer of these enzymes. ecently it as shon that the eolution tryptophan an increase in staility at acidic p as seen leadin to an increase in of funal peroidases inoled in linin deradation paralleled the eolution of the oidizin poer of these enzymes. ecently it as shon that the eolution oody plants. hese studies reealed details on ho peroidases of this of funal peroidases inoled in linin deradation paralleled the eolution of particular peroidase family hae eoled to fulfill their current tass. oody plants. hese studies reealed details on ho peroidases of this particular peroidase family hae eoled to fulfill their current tass. n this chapter e descrie an study of the family of y type peroidases ys. y this e aimed at eneratin a roust y. ys ere first discoered n this chapter e descrie an study of the family of y type peroidases y im and hoda here they identified a ene from ec that ys. y this e aimed at eneratin a roust y. ys ere first discoered encodes for a noel type of peroidase and named it y. ys hae a y im and hoda here they identified a ene from ec that characteristic feature of ein ale to decolorize arious dyes such as encodes for a no el type of peroidase and named it y. ys hae a anthrauinone s and azo dyes hence their name ye decolorizin eroidases. characteristic feature of ein ale to decolorize arious dyes such as ys do not share seuence homoloy ith the typical plant or animal peroidases anthrauinone s and azo dyes hence their name ye decolorizin eroidases. that elon to the peroidasecatalase superfamily. ys form a distinct peroidase ys do not share seuence homoloy ith the t ypical plant or animal peroidases 6 family that is part of the peroidase chlorite dismutase superfamily . ys are that elon to the peroidase catalase superfamily. ys form a distinct peroidase found in funi ut are much more aundant in act eria– . hey hae een family that is part of the peroidase chlorite dismutase superfamily . ys are classified into four roups and . hile class and ys are mainly found in funi ut are much more aundant in act eria –. hey hae een from acterial oriin class ys are mainly found in funi. he type ys classified into four roups and . hile class and ys are mainly cluster more closely ith class ys and also display similar enzyme actiity from acterial oriin class ys are mainly found in funi. he type ys features. fe eamples of acteria that contain ys are cluster more closely ith class ys and also display similar enzyme actiity sp. and features. fe eamples of acteria that contain ys are . n funi ys can e found in sp. and and ehrens descried the epression . n funi ys can e found in of funal ys usin a coldshoc inducile epression system from . his and ehrens descried the epression of funal ys usin a coldshoc inducile epression system from . his 103 102 103 102 98 Expression and characterization of a resurrected DyP-type peroxidase Chapter VI Chapter VI Expression and characterization of a resurrected DyP-type peroxidase called consensus method to enerate stale proteins. hateer the reason may e it appears that can e effectie in eneratin roust ariants of a particular called consensus method to enerate stale proteins. hateer the reason may e enzyme family. it appears that can e effectie in eneratin roust ariants of a particular enzyme family. n the contet of our study it is orth notin that in recent years has een performed on funal peroidases that elon to the peroidase catalase n the contet of our study it is orth notin that in recent years has een superfamily hich harors the ersatile peroidases linin peroidases and performed on funal peroidases that elon to the peroidase catalase mananese peroidases. n an attempt to resurrect etinct peroidases from the end superfamily hich harors the ersatile peroidases linin peroidases and of the aroniferous period yuso ernandez and coorers decided to select mananese peroidases. n an attempt to resurrect etinct peroidases from the end enes from seuenced enomes and to perform upon them. his era of the aroniferous period yuso ernandez and coorers decided to select as chosen as it as the period here the first lininolytic enzymes incl. enes from seuenced enomes and to perform upon them. his era peroidases must hae appeared . hese enzymes ould oriinate from olyporales as chosen as it as the period here the first lininolytic enzymes incl. species. he first linin deradin peroidase is supposedly a n peroidase peroidases must hae appear ed. hese enzymes ould oriinate from olyporales oidizin phenolic linin that accordin to the study ould not chane until species. he first linin deradin peroidase is supposedly a n peroidase the appearance of an eposed tryptophan oidizin nonphenolic linin to yield a oidizin phenolic linin that accordin to the study ould not chane until ersatile peroidase. ater another eolution ary eent resulted in the loss of the the appearance of an eposed tryptophan oidizin nonphenolic linin to yield a n indin site eneratin the first linin peroidase that eoled to the etant ersatile peroidase. ater another eolutionary eent resulted in the loss of the form y improin catalytic efficiency. fter the appearance of the eposed catalytic n indin site enerat in the first linin peroidase that eol ed to the etant tryptophandeterminants an of increase enzyme infunction staility is atreferred acidic pto as as “horizontal seen leadin approach”. to an increase in determinantsform y improin of enzyme catalytic function efficiency. is referred fter theto as appearance “horizontal of approach”. the eposed catalytic the oidizin poer of these enzymes. ecently it as shon that the eolution tryptophan an increase in staility at acidic p as seen leadin to an increase in of funal peroidases inoled in linin deradation paralleled the eolution of the oidizin poer of these enzymes. ecently it as shon that the eolution oody plants. hese studies reealed details on ho peroidases of this of funal peroidases inoled in linin deradation paralleled the eolution of particular peroidase family hae eoled to fulfill their current tass. oody plants. hese studies reealed details on ho peroidases of this particular peroidase family hae eoled to fulfill their current tass. n this chapter e descrie an study of the family of y type peroidases ys. y this e aimed at eneratin a roust y. ys ere first discoered n this chapter e descrie an study of the family of y type peroidases y im and hoda here they identified a ene from ec that ys. y this e aimed at eneratin a roust y. ys ere first discoered encodes for a noel type of peroidase and named it y. ys hae a y im and hoda here they identified a ene from ec that characteristic feature of ein ale to decolorize arious dyes such as encodes for a no el type of peroidase and named it y. ys hae a anthrauinone s and azo dyes hence their name ye decolorizin eroidases. characteristic feature of ein ale to decolorize arious dyes such as ys do not share seuence homoloy ith the typical plant or animal peroidases anthrauinone s and azo dyes hence their name ye decolorizin eroidases. that elon to the peroidasecatalase superfamily. ys form a distinct peroidase ys do not share seuence homoloy ith the t ypical plant or animal peroidases family that is part of the peroidase chlorite dismutase superfamily . ys are that elon to the peroidase catalase superfamily. ys form a distinct peroidase 6 found in funi ut are much more aundant in act eria– . hey hae een family that is part of the peroidase chlorite dismutase superfamily . ys are classified into four roups and . hile class and ys are mainly found in funi ut are much more aundant in act eria –. hey hae een from acterial oriin class ys are mainly found in funi. he type ys classified into four roups and . hile class and ys are mainly cluster more closely ith class ys and also display similar enzyme actiity from acterial oriin class ys are mainly found in funi. he type ys features. fe eamples of acteria that contain ys are cluster more closely ith class ys and also display similar enzyme actiity sp. and features. fe eamples of acteria that contain ys are . n funi ys can e found in sp. and and ehrens descried the epression . n funi ys can e found in of funal ys usin a coldshoc inducile epression system from . his and ehrens descried the epression of funal ys usin a coldshoc inducile epression system from . his 103 102 103 102 99 Expression and characterization of a resurrected DyP-type peroxidase Chapter VI Chapter VI Expression and characterization of a resurrected DyP-type peroxidase called consensus method to enerate stale proteins. hateer the reason may e it appears that can e effectie in eneratin roust ariants of a particular enzyme family. n the contet of our study it is orth notin that in recent years has een NEB 10β competent cells (New performed on funal peroidases that elon to the peroidase catalase superfamily hich harors the ersatile peroidases linin peroidases and mananese peroidases. n an attempt to resurrect etinct peroidases from the end of the aroniferous period yuso ernandez and co orers decidedβ to select enes from seuenced enomes and to perform upon them. his era as chosen as it as the period here the first lininolytic enzymes incl. peroidases must hae appeared. hese enzymes ould oriinate from olyporales species. he first linin deradin peroidase is supposedly a n peroidase oidizin phenolic linin that accordin to the study ould not chane until the appearance of an eposed tryptophan oidizin nonphenolic linin to yield a ersatile peroidase. ater another eolutionary eent resulted in the loss of the n indin site eneratin the first linin peroidase that eoled to the etant formThe Interpro y improin family catalytic IPR006314 efficiency. corresponding fter the appearanceto DyPs (≈14000 of the eposed sequences) catalytic plus determinants of enzyme function is referred to as “horizontal approach”. tryptophan an increase in staility at acidic p as seen leadin to an increase in the oidizin poer of these enzymes. ecently it as shon that the eolution of funal peroidases inoled in linin deradation paralleled the eolution of oody plants. hese studies reealed details on ho peroidases of this particular peroidase family hae eoled to fulfill their current tass. n this chapter e descrie an study of the family of y type peroidases ys . y this e aimed at eneratin a roust y. ys ere first discoered y im and hoda here they identified a ene from ec that encodes for a noel type of peroidase and named it y. ys hae a characteristic feature of ein ale to decolorize arious dyes such as anthrauinone s and azo dyes hence their name yedecolorizin eroidases. ys do not share seuence homoloy ith the typical plant or animal peroidases that elon to the peroidase catalase superfamily. ys form a distinct peroidase 6 family that is part of the peroidasechlorite dismutase superfamily. ys are found in funi ut are much more aundant in act eria–. hey hae een classified into four roups and . hile class and ys are mainly from acterial oriin class ys are mainly found in funi. he type ys cluster more closely ith class ys and also display similar en zyme actiity features. fe eamples of acteria that contain ys are sp. and . n funi ys can e found in and ehrens descried the epression of funal ys usin a coldshoc inducile epression system from . his 103 104 105 102 100 Expression and characterization of a resurrected DyP-type peroxidase Chapter VI Chapter VI Expression and characterization of a resurrected DyP-type peroxidase called consensus method to enerate stale proteins. hateer the reason may e it appears that can e effectie in eneratin roust ariants of a particular enzyme family. n the contet of our study it is orth notin that in recent years has een NEB 10β competent cells (New performed on funal peroidases that elon to the peroidase catalase superfamily hich harors the ersatile peroidases linin peroidases and mananese peroidases. n an attempt to resurrect etinct peroidases from the end of the aroniferous period yuso ernandez and co orers decidedβ to select enes from seuenced enomes and to perform upon them. his era as chosen as it as the period here the first lininolytic enzymes incl. peroidases must hae appeared. hese enzymes ould oriinate from olyporales species. he first linin deradin peroidase is supposedly a n peroidase oidizin phenolic linin that accordin to the study ould not chane until the appearance of an eposed tryptophan oidizin nonphenolic linin to yield a ersatile peroidase. ater another eolutionary eent resulted in the loss of the n indin site eneratin the first linin peroidase that eoled to the etant formThe Interpro y improin family catalytic IPR006314 efficiency. corresponding fter the appearanceto DyPs (≈14000 of the eposed sequences) catalytic plus determinants of enzyme function is referred to as “horizontal approach”. tryptophan an increase in staility at acidic p as seen leadin to an increase in the oidizin poer of these enzymes. ecently it as shon that the eolution of funal peroidases inoled in linin deradation paralleled the eolution of oody plants. hese studies reealed details on ho peroidases of this particular peroidase family hae eoled to fulfill their current tass. n this chapter e descrie an study of the family of y type peroidases ys . y this e aimed at eneratin a roust y. ys ere first discoered y im and hoda here they identified a ene from ec that encodes for a noel type of peroidase and named it y. ys hae a characteristic feature of ein ale to decolorize arious dyes such as anthrauinone s and azo dyes hence their name yedecolorizin eroidases. ys do not share seuence homoloy ith the typical plant or animal peroidases that elon to the peroidase catalase superfamily. ys form a distinct peroidase family that is part of the peroidasechlorite dismutase superfamily. ys are 6 found in funi ut are much more aundant in act eria–. hey hae een classified into four roups and . hile class and ys are mainly from acterial oriin class ys are mainly found in funi. he type ys cluster more closely ith class ys and also display similar en zyme actiity features. fe eamples of acteria that contain ys are sp. and . n funi ys can e found in and ehrens descried the epression of funal ys usin a coldshoc inducile epression system from . his 103 104 105 102 101 Expression and characterization of a resurrected DyP-type peroxidase Chapter VI Chapter VI Expression and characterization of a resurrected DyP-type peroxidase called consensus method to enerate stale proteins. hateer the reason may e it appears that can e effectie in eneratin roust ariants of a particular enzyme family. n the contet of our study it is orth notin that in recent years has een performed on funal peroidases that elon to the peroidase catalase superfamily hich harors the ersatile peroidases linin peroidases and mananese peroidases. n an attempt to resurrect etinct peroidases from the end of the aroniferous period yusoernandez and coorers decided to select enes from seuenced enomes and to perform upon them. his era as chosen as it as the period here the first lininolytic enzymes incl. peroidases must hae appeared. hese enzymes ould oriinate from olyporales species. he first linin deradin peroidase is supposedly a n peroidase oidizin phenolic linin that accordin to the study ould not chane until the appearance of an eposed tryptophan oidizin nonphenolic linin to yield a ersatile peroidase. ater another eolutionary eent resulted in the loss of the n indin site enerat in the first linin peroidase that eol ed to the etant form y improin catalytic efficiency. fter the appearance of the eposed catalytic tryptophan an increase in staility at acidic p as seen leadin to an increase in determinants of enzyme function is referred to as “horizontal approach”. the oidizin poer of these enzymes. ecently it as shon that the eolution of funal peroidases inoled in linin deradation paralleled the eolution of oody plants. hese studies reealed details on ho peroidases of this particular peroidase family hae eoled to fulfill their current tass. n this chapter e descrie an study of the family of ytype peroidases ys . y this e aimed at eneratin a roust y. ys ere first discoered y im and hoda here they identified a ene from ec that encodes for a noel type of peroidase and named it y. ys hae a characteristic feature of ein ale to decolorize arious dyes such as anthrauinone s and azo malonate dyes hence formation their atname 270 yenm (εdecolorizin eroidases. ys do not share seuence homoloy ith the t ypical plant or animal peroidases that elon to the peroidase catalase superfamily. ys form a distinct peroidase 6 family that is part of the peroidase chlorite dismutase superfamily . ys are found in funi ut are much more aundant in acteria–. hey hae een classified into four roups and . hile class and ys are mainly from acterial oriin class ys are mainly found in funi. he type ys cluster more closely ith class ys and also display similar enzyme actiity features. fe eamples of acteria that contain ys are sp. and . n funi ys can e found in and ehrens descried the epression of funal ys usin a cold shoc inducile epression system from . his 103 106 107 102 102 Expression and characterization of a resurrected DyP-type peroxidase Chapter VI Chapter VI Expression and characterization of a resurrected DyP-type peroxidase called consensus method to enerate stale proteins. hateer the reason may e it appears that can e effectie in eneratin roust ariants of a particular enzyme family. n the contet of our study it is orth notin that in recent years has een performed on funal peroidases that elon to the peroidase catalase superfamily hich harors the ersatile peroidases linin peroidases and mananese peroidases. n an attempt to resurrect etinct peroidases from the end of the aroniferous period yusoernandez and coorers decided to select enes from seuenced enomes and to perform upon them. his era as chosen as it as the period here the first lininolytic enzymes incl. peroidases must hae appeared. hese enzymes ould oriinate from olyporales species. he first linin deradin peroidase is supposedly a n peroidase oidizin phenolic linin that accordin to the study ould not chane until the appearance of an eposed tryptophan oidizin nonphenolic linin to yield a ersatile peroidase. ater another eolutionary eent resulted in the loss of the n indin site enerat in the first linin peroidase that eol ed to the etant form y improin catalytic efficiency. fter the appearance of the eposed catalytic tryptophan an increase in staility at acidic p as seen leadin to an increase in determinants of enzyme function is referred to as “horizontal approach”. the oidizin poer of these enzymes. ecently it as shon that the eolution of funal peroidases inoled in linin deradation paralleled the eolution of oody plants. hese studies reealed details on ho peroidases of this particular peroidase family hae eoled to fulfill their current tass. n this chapter e descrie an study of the family of ytype peroidases ys . y this e aimed at eneratin a roust y. ys ere first discoered y im and hoda here they identified a ene from ec that encodes for a noel type of peroidase and named it y. ys hae a characteristic feature of ein ale to decolorize arious dyes such as anthrauinone s and azo malonate dyes hence formation their atname 270 yenm (εdecolorizin eroidases. ys do not share seuence homoloy ith the t ypical plant or animal peroidases that elon to the peroidase catalase superfamily. ys form a distinct peroidase family that is part of the peroidase chlorite dismutase superfamily . ys are 6 found in funi ut are much more aundant in acteria–. hey hae een classified into four roups and . hile class and ys are mainly from acterial oriin class ys are mainly found in funi. he type ys cluster more closely ith class ys and also display similar enzyme actiity features. fe eamples of acteria that contain ys are sp. and . n funi ys can e found in and ehrens descried the epression of funal ys usin a cold shoc inducile epression system from . his 103 106 107 102 103 Expression and characterization of a resurrected DyP-type peroxidase Chapter VI Chapter VI Expression and characterization of a resurrected DyP-type peroxidase called consensus method to enerate stale proteins. hateer the reason may e it appears that can e effectie in eneratin roust ariants of a particular enzyme family. n the contet of our study it is orth notin that in recent years has een performed on funal peroidases that elon to the peroidasecatalase superfamily hich harors the ersatile peroidases linin peroidases and mananese peroidases. n an attempt to resurrect etinct peroidases from the end of the aroniferous period yusoernandez and coorers decided to select enes from seuenced enomes and to perform upon them. his era as chosen as it as the period here the first lininolytic enzymes incl. peroidases must hae appeared. hese enzymes ould oriinate from olyporales species. he first linin deradin peroidase is supposedly a n peroidase oidizin phenolic linin that accordin to the study ould not chane until the appearance of an eposed tryptophan oidizin nonphenolic linin to yield a ersatile peroidase. ater another eolutionary eent resulted in the loss of the n indin site eneratin the first linin peroidase that eoled to the etant form y improin catalytic efficiency. fter the appearance of the eposed catalytic tryptophan an increase in staility at acidic p as seen leadin to an increase in determinants of enzyme function is referred to as “horizontal approach”. the oidizin poer of these enzymes. ecently it as shon that the eolution of funal peroidases inoled in linin deradation paralleled the eolution of oody plants. hese studies reealed details on ho peroidases of this particular peroidase family hae eoled to fulfill their current tass. n this chapter e descrie an study of the family of y type peroidases ys . y this e aimed at eneratin a roust y. ys ere first discoered y im and hoda here they identified a ene from ec that encodes for a noel type of peroidase and named it y. ys hae a characteristic feature of ein ale to decolorize arious dyes such as anthrauinone s and azo dyes hence their name yedecolorizin eroidases. ys do not share seuence homoloy ith the typical plant or animal peroidases that elon to the peroidasecatalase superfamily. ys form a distinct peroidase 6 family that is part of the peroidase chlorite dismutase superfamily . ys are found in funi ut are much more aundant in act eria –. hey hae een classified into four roups and . hile class and ys are mainly from acterial oriin class ys are mainly found in funi. he type ys cluster more closely ith class ys and also display similar enzyme actiity features . fe eamples of acteria that contain ys are sp. and . n funi ys can e found in and ehrens descried the epression of funal ys usin a coldshoc inducile epression system from . his 103 108 109 102 104 Expression and characterization of a resurrected DyP-type peroxidase Chapter VI Chapter VI Expression and characterization of a resurrected DyP-type peroxidase called consensus method to enerate stale proteins. hateer the reason may e it appears that can e effectie in eneratin roust ariants of a particular enzyme family. n the contet of our study it is orth notin that in recent years has een performed on funal peroidases that elon to the peroidasecatalase superfamily hich harors the ersatile peroidases linin peroidases and mananese peroidases. n an attempt to resurrect etinct peroidases from the end of the aroniferous period yusoernandez and coorers decided to select enes from seuenced enomes and to perform upon them. his era as chosen as it as the period here the first lininolytic enzymes incl. peroidases must hae appeared. hese enzymes ould oriinate from olyporales species. he first linin deradin peroidase is supposedly a n peroidase oidizin phenolic linin that accordin to the study ould not chane until the appearance of an eposed tryptophan oidizin nonphenolic linin to yield a ersatile peroidase. ater another eolutionary eent resulted in the loss of the n indin site eneratin the first linin peroidase that eoled to the etant form y improin catalytic efficiency. fter the appearance of the eposed catalytic tryptophan an increase in staility at acidic p as seen leadin to an increase in determinants of enzyme function is referred to as “horizontal approach”. the oidizin poer of these enzymes. ecently it as shon that the eolution of funal peroidases inoled in linin deradation paralleled the eolution of oody plants. hese studies reealed details on ho peroidases of this particular peroidase family hae eoled to fulfill their current tass. n this chapter e descrie an study of the family of y type peroidases ys . y this e aimed at eneratin a roust y. ys ere first discoered y im and hoda here they identified a ene from ec that encodes for a noel type of peroidase and named it y. ys hae a characteristic feature of ein ale to decolorize arious dyes such as anthrauinone s and azo dyes hence their name yedecolorizin eroidases. ys do not share seuence homoloy ith the typical plant or animal peroidases that elon to the peroidasecatalase superfamily. ys form a distinct peroidase family that is part of the peroidase chlorite dismutase superfamily . ys are 6 found in funi ut are much more aundant in act eria –. hey hae een classified into four roups and . hile class and ys are mainly from acterial oriin class ys are mainly found in funi. he type ys cluster more closely ith class ys and also display similar enzyme actiity features . fe eamples of acteria that contain ys are sp. and . n funi ys can e found in and ehrens descried the epression of funal ys usin a coldshoc inducile epression system from . his 103 108 109 102 105 Expression and characterization of a resurrected DyP-type peroxidase Chapter VI Chapter VI Expression and characterization of a resurrected DyP-type peroxidase called consensus method to enerate stale proteins. hateer the reason may e it appears that can e effectie in eneratin roust ariants of a particular enzyme family. n the contet of our study it is orth notin that in recent years has een performed on funal peroidases that elon to the peroidasecatalase superfamily hich harors the ersatile peroidases linin peroidases and mananese peroidases. n an attempt to resurrect etinct peroidases from the end of the aroniferous period yusoernandez and coorers decided to select enes from seuenced enomes and to perform upon them. his era as chosen as it as the period here the first lininolytic enzymes incl. peroidases must hae appeared. hese enzymes ould oriinate from olyporales species. he first linin deradin peroidase is supposedly a n peroidase oidizin phenolic linin that accordin to the study ould not chane until the appearance of an eposed tryptophan oidizin nonphenolic linin to yield a ersatile peroidase. ater another eolutionary eent resulted in the loss of the n indin site eneratin the first linin peroidase that eoled to the etant form y improin catalytic efficiency. fter the appearance of the eposed catalytic tryptophan an increase in staility at acidic p as seen leadin to an increase in determinants of enzyme function is referred to as “horizontal approach”. the oidizin poer of these enzymes. ecently it as shon that the eolution of funal peroidases inoled in linin deradation paralleled the eolution of oody plants. hese studies reealed details on ho peroidases of this particular peroidase family hae eoled to fulfill their current tass. n this chapter e descrie an study of the family of ytype peroidases ys. y this e aimed at eneratin a roust y. ys ere first discoered y im and hoda here they identified a ene from ec that encodes for a no el type of peroidase and named it y. ys hae a characteristic feature of ein ale to decolorize arious dyes such as anthrauinone s and azo dyes hence their name ye decolorizin eroidases. y s do not share seuence homoloy ith the t ypical plant or animal peroidases that elon to the peroidase catalase superfamily. ys form a distinct peroidase 6 family that is part of the p eroidase chlorite dismutase superfamily . ys are found in funi ut are much more aundant in acteria–. hey hae een classified into four roups and . hile class and ys are mainly from acterial oriin class ys are mainly found in funi. he type ys cluster more closely ith class ys and also display similar enzyme actiity features. fe eamples of acteria that contain ys are sp. and . n funi ys can e found in and ehrens descried the epression of funal ys usin a cold shoc inducile epression system from . his 103 110 111 102 106 Expression and characterization of a resurrected DyP-type peroxidase Chapter VI Chapter VI Expression and characterization of a resurrected DyP-type peroxidase called consensus method to enerate stale proteins. hateer the reason may e it appears that can e effectie in eneratin roust ariants of a particular enzyme family. n the contet of our study it is orth notin that in recent years has een performed on funal peroidases that elon to the peroidasecatalase superfamily hich harors the ersatile peroidases linin peroidases and mananese peroidases. n an attempt to resurrect etinct peroidases from the end of the aroniferous period yusoernandez and coorers decided to select enes from seuenced enomes and to perform upon them. his era as chosen as it as the period here the first lininolytic enzymes incl. peroidases must hae appeared. hese enzymes ould oriinate from olyporales species. he first linin deradin peroidase is supposedly a n peroidase oidizin phenolic linin that accordin to the study ould not chane until the appearance of an eposed tryptophan oidizin nonphenolic linin to yield a ersatile peroidase. ater another eolutionary eent resulted in the loss of the n indin site eneratin the first linin peroidase that eoled to the etant form y improin catalytic efficiency. fter the appearance of the eposed catalytic tryptophan an increase in staility at acidic p as seen leadin to an increase in determinants of enzyme function is referred to as “horizontal approach”. the oidizin poer of these enzymes. ecently it as shon that the eolution of funal peroidases inoled in linin deradation paralleled the eolution of oody plants. hese studies reealed details on ho peroidases of this particular peroidase family hae eoled to fulfill their current tass. n this chapter e descrie an study of the family of ytype peroidases ys. y this e aimed at eneratin a roust y. ys ere first discoered y im and hoda here they identified a ene from ec that encodes for a no el type of peroidase and named it y. ys hae a characteristic feature of ein ale to decolorize arious dyes such as anthrauinone s and azo dyes hence their name ye decolorizin eroidases. y s do not share seuence homoloy ith the typical plant or animal peroidases that elon to the peroidase catalase superfamily. ys form a distinct peroidase family that is part of the p eroidase chlorite dismutase superfamily . ys are 6 found in funi ut are much more aundant in acteria–. hey hae een classified into four roups and . hile class and ys are mainly from acterial oriin class ys are mainly found in funi. he type ys cluster more closely ith class ys and also display similar enzyme actiity features. fe eamples of acteria that contain ys are sp. and . n funi ys can e found in and ehrens descried the epression of funal ys usin a cold shoc inducile epression system from . his 103 110 111 102 107 Expression and characterization of a resurrected DyP-type peroxidase Chapter VI Chapter VI Expression and characterization of a resurrected DyP-type peroxidase called consensus method to enerate stale proteins. hateer the reason may e it appears that can e effectie in eneratin roust ariants of a particular enzyme family. n the contet of our study it is orth notin that in recent years has een performed on funal peroidases that elon to the peroidasecatalase superfamily hich harors the ersatile peroidases linin peroidases and mananese peroidases. n an attempt to resurrect etinct peroidases from the end of the aroniferous period yusoernandez and coorers decided to select enes from seuenced enomes and to perform upon them. his era as chosen as it as the period here the first lininolytic enzymes incl. peroidases must hae appeared. hese enzymes ould oriinate from olyporales → species. he first linin deradin peroidase is supposedly a n peroidase → Compound II + AH• oidizin phenolic linin that accordin to the study ould not chane until the appearance of an eposed tryptophan oidizin nonphenolic linin to yield a → state) + AH• + H ersatile peroidase. ater another eolutionary eent resulted in the loss of the n indin site eneratin the first linin peroidase that eol ed to the etant form y improin catalytic efficiency. fter the appearance of the eposed catalytic → O + 2AH• tryptophan an increase in staility at acidic p as seen leadin to an increase in determinants of enzyme function is referred to as “horizontal approach”. the oidizin poer of these enzymes. ecently it as shon that the eolution of funal peroidases inoled in linin deradation paralleled the eolution of oody plants. hese studies reealed details on ho peroidases of this particular peroidase family hae eoled to fulfill their current tass. 0 .4 n this chapter e descrie an study of the family of ytype peroidases 0 .3 ys. y this e aimed at eneratin a roust y. ys ere first discoered e c

y im and hoda here they identified a ene from ec that a b encodes for a noel type of peroidase and named it y. ys hae a r 0 .2 o

characteristic feature of ein ale to decolorize arious dyes such as b

y im and hoda here they identified a ene from ec that a b encodes for a noel type of peroidase and named it y. ys hae a r 0 .2 o

characteristic feature of ein ale to decolorize arious dyes such as b

A anthrauinones and azo dyes hence their name yedecolorizin eroidases. 0 .1 ys do not share seuence homoloy ith the typical plant or animal peroidases that elon to the peroidasecatalase superfamily. ys form a distinct peroidase 0 .0 family that is part of the peroidasechlorite dismutase superfamily. ys are 3 0 0 4 0 0 5 0 0 6 0 0 7 0 0 6 found in funi ut are much more aundant in acteria–. hey hae een W a v e le n g th (n m ) classified into four roups and . hile class and ys are mainly from acterial oriin class ys are mainly found in funi. he type ys cluster more closely ith class ys and also display similar enzyme actiity features. fe eamples of acteria that contain ys are sp. and . n funi ys can e found in and ehrens descried the epression of funal ys usin a coldshoc inducile epression system from . his 103 112 113 102 109 Expression and characterization of a resurrected DyP-type peroxidase Chapter VI Chapter VI Expression and characterization of a resurrected DyP-type peroxidase called consensus method to enerate stale proteins. hateer the reason may e iteonstuted appears that s aecan atata e effectie ompetent in eneratin Inteestn roust ariants aso mananese of a particular as ae e aes enten onstants o te eaton o An and enzymeound to family. e aepted as sustate e aesenten net onstants o ea AnAt t A H2O2 and n 2+ o A H2O2 and n2+ ae son n ae Catat ates o te n the contet of our study it is orth notin that in recent years has een ustate nme s ) ) s ) to s on and A ae sma e te aues o AnAt performed on funal peroidases that elon to the peroidasecatalase ae someat e 2 od) sos a o s and a An 2 superfamily hich harors the ersatile peroidases linin peroidases and o 2 en att s tested usn A as a sustate And t A mananese peroidases. n an attempt to resurrect etinct peroidases from the end as a sustate te as epoted to e s ) Cea te AnAt 2 aofnesta the aroniferous s dspa period oe attesyusoernandez ut te and coorers decided s aso to pat select o enes from seuenced enomes and to perform upon them. his era An te oup III Cass s see ue 2) not a desendant o oup I as chosen as it as the period here the first lininolytic enzymes incl. see ue 2) Inteestn te atat ates and aues ound o te anesta AnAt 2 2 peroidasess ae ose must to haete aues appear otaneded. hese enzymes ould en oriinate from o olyporales A as species. he first linin deradin peroidase is supposedly a n peroidase An used as a sustate s te on desendant o oema dataoidizin ae aaae phenolic e linin atat that atesaccordin o te to to the s study on nould2+ ae not sma chane as untile AnAt 2 the appearance of an eposed tryptophan oidizin nonphenolic linin to yield a as te aues Aan te ae aso tn te ane o te att peous ersatile peroidase. ater another eolution ary eent resulted in the loss of the An 2 noted te etant enme e antes o ot s o te des H2O2 a n indin site eneratin the first linin peroidase that eoled to the etant ee ute t aues n te o ane Aso o doen peode AnAt 2 form y improin catalytic efficiency. fter the appearance of the eposed catalytic eate o aues ee ound ndatn tat te esueted s peom tryptophan an increase in staility at acidic p as seen leadin to an increase in determinants of enzymeAn function is referred to as “horizontal approach”. e as peodases on A antaunone and ao des 2+ n the oidizin poer of these enzymes. ecently it as shon that the eolution AnAt of funal peroidases inoled in linin deradation paralleled the eolution of oody plants. hese studies reealed details on ho peroidases of this particular peroidase family hae eoled to fulfill their current tass. a etemned usn A as a sustate Optma pH and tempeatue n this chapter e descrie an study of the family of ytype peroidases ys. y this e aimed at eneratin a roust y. ys ere first discoered e optma pH o att o An and te AnAt ee ound to y im and hoda here they identified a ene from ec that e at a pH o ue ) s s sma to peous aateed s o encodes for a noel type of peroidase and named it y. ys hae a optma att as een eoded at o pH aues characteristic feature of ein ale to decolorize arious dyes such as anthrauinones and azo dyes hence their name yedecolorizin eroidases. ys do not share seuence homoloy ith the typical plant or animal peroidases that elon to the peroidasecatalase superfamily. ys form a distinct peroidase 6 family that is part of the peroidasechlorite dismutase superfamily. ys are found in funi ut are much more aundant in acteria–. hey hae een classified into four roups and . hile class and ys are mainly from acterial oriin class ys are mainly found in funi. he type ys cluster more closely ith class ys and also display similar enzyme actiity features. fe eamples of acteria that contain ys are sp. and . n funi ys can e found in and ehrens descried the epression of funal ys usin a coldshoc inducile epression system from . his 103 114 115 102 110 Expression and characterization of a resurrected DyP-type peroxidase Chapter VI Chapter VI Expression and characterization of a resurrected DyP-type peroxidase called consensus method to enerate stale proteins. hateer the reason may e iteonstuted appears that s aecan atata e effectie ompetent in eneratin Inteestn roust ariants aso mananese of a particular as ae e aes enten onstants o te eaton o An and enzymeound to family. e aepted as sustate e aesenten net onstants o ea AnAt t A H2O2 and n 2+ o A H2O2 and n2+ ae son n ae Catat ates o te n the contet of our study it is orth notin that in recent years has een ustate nme s ) ) s ) to s on and A ae sma e te aues o AnAt performed on funal peroidases that elon to the peroidasecatalase ae someat e 2 od) sos a o s and a An 2 superfamily hich harors the ersatile peroidases linin peroidases and o 2 en att s tested usn A as a sustate And t A mananese peroidases. n an attempt to resurrect etinct peroidases from the end as a sustate te as epoted to e s ) Cea te AnAt 2 aofnesta the aroniferous s dspa period oe attesyusoernandez ut te and coorers decided s aso to pat select o enes from seuenced enomes and to perform upon them. his era An te oup III Cass s see ue 2) not a desendant o oup I as chosen as it as the period here the first lininolytic enzymes incl. see ue 2) Inteestn te atat ates and aues ound o te anesta AnAt 2 2 peroidasess ae ose must to haete aues appear otaneded. hese enzymes ould en oriinate from o olyporales A as species. he first linin deradin peroidase is supposedly a n peroidase An used as a sustate s te on desendant o oema dataoidizin ae aaae phenolic e linin atat that atesaccordin o te to to the s study on nould2+ ae not sma chane as untile AnAt 2 the appearance of an eposed tryptophan oidizin nonphenolic linin to yield a as te aues Aan te ae aso tn te ane o te att peous ersatile peroidase. ater another eolution ary eent resulted in the loss of the An 2 noted te etant enme e antes o ot s o te des H2O2 a n indin site eneratin the first linin peroidase that eoled to the etant ee ute t aues n te o ane Aso o doen peode AnAt 2 form y improin catalytic efficiency. fter the appearance of the eposed catalytic eate o aues ee ound ndatn tat te esueted s peom tryptophan an increase in staility at acidic p as seen leadin to an increase in determinants of enzymeAn function is referred to as “horizontal approach”. e as peodases on A antaunone and ao des 2+ n the oidizin poer of these enzymes. ecently it as shon that the eolution AnAt of funal peroidases inoled in linin deradation paralleled the eolution of oody plants. hese studies reealed details on ho peroidases of this particular peroidase family hae eoled to fulfill their current tass. a etemned usn A as a sustate Optma pH and tempeatue n this chapter e descrie an study of the family of ytype peroidases ys. y this e aimed at eneratin a roust y. ys ere first discoered e optma pH o att o An and te AnAt ee ound to y im and hoda here they identified a ene from ec that e at a pH o ue ) s s sma to peous aateed s o encodes for a noel type of peroidase and named it y. ys hae a optma att as een eoded at o pH aues characteristic feature of ein ale to decolorize arious dyes such as anthrauinones and azo dyes hence their name yedecolorizin eroidases. ys do not share seuence homoloy ith the typical plant or animal peroidases that elon to the peroidasecatalase superfamily. ys form a distinct peroidase family that is part of the peroidasechlorite dismutase superfamily. ys are 6 found in funi ut are much more aundant in acteria–. hey hae een classified into four roups and . hile class and ys are mainly from acterial oriin class ys are mainly found in funi. he type ys cluster more closely ith class ys and also display similar enzyme actiity features. fe eamples of acteria that contain ys are sp. and . n funi ys can e found in and ehrens descried the epression of funal ys usin a coldshoc inducile epression system from . his 103 114 115 102 111 Expression and characterization of a resurrected DyP-type peroxidase Chapter VI Chapter VI Expression and characterization of a resurrected DyP-type peroxidase called consensus method to enerate stale proteins. hateer the reason may e it appears that can e effectie in eneratin roust ariants of a particular thermostablty relects the condtons at hch the DyP as unctonn tll the enzyme family. p H 3 reconstructed DyPs are nterestn enzymes as the etant DyPs derved rom t have hardly been studed nly or one o these DyPs some more detaled normaton n the contet of our study it is orth notin that in recent years p H 4has een An c AltD yP 7 4 s avalable he DyP rom has been shon to be an nterestn and p H 5 performed on funal peroidases that elon to the peroidasecatalase unue bocatalyst or the roducton o nootatone uture studes ll p H 6 superfamily hich harors the ersatile peroidases linin peroidases and reveal hether the ncDyP can also be used or selectve odatons o p H 7 mananese peroidases. n an attempt to resurrect etinct peroidases from the end terenods of the aroniferous period yusoernandez and coorers decidedp H 8 to select enesAn c D yfromP 7 4 seuenced enomes and to perform upon them. his era y resurrectn t o alternatve ancestral DyPs or the same node n the as chosen as it as the period here the first lininolytic enzymes incl. hyloenetc tree to catalytcally cometent DyPs ere created he to DyPs peroidases must hae appeared. hese enzymes ould oriinate from olyporales der by substtutons hle they dslay very smlar roertes nly hen species. he first0 linin deradin2 peroidase4 is supposedly6 a n peroidase loon at the netc arameters o sever al dye substrates some derences ere oidizin phenolic linin that accordin to the study ould not chane until ound or the dyes manly derences n values ere observed s most o the the appearance of an eposed tryptophan- 1 oidizin nonphenolic linin to yield a k o b s (s ) mutatons seem to be on the surace o the roten derences n substrate anty ersatile peroidase. ater another eolutionary eent resulted in the loss of the may be elaned by alternatve docn stes or these dyes on the enzymes DyP n indin site eneratin the first linin peroidase that eoled to the etant catalyzed dye odatons are suosed to occur throuh lon rane electron transer form y improin catalytic efficiency. fter the appearance of the eposed catalytic odaton uon bndn o the substrate on the surace o the roten nother tryptophan an increase in staility at acidic p as seen leadin to an increase in nterestndeterminants ndn of enzyme s the functionobserved is oretreferred band to asat “horizontala relatvely hhapproach”. avelenth the oidizin poer of these enzymes. ecently it as shon that the eolution nm DyPs and other hemorotens usually have ths oret band at around nm of funal peroidases inoled in linin deradation paralleled the eolution of hs ea at nm may hnt to a ormer role o these ancestral DyPs andor may oody plants. hese studies reealed details on ho peroidases of this suest that these DyPs may need to be actvated or ull actvt y ost etant unal particular peroidase family hae eoled to fulfill their current tass. DyPs dslay much hher actvtes hen comared th the ncDyP and n this chapter e descrie an study of the family of ytype peroidases ltncDyP hs may be due to only a small ercentae o DyP that s ys. y this e aimed at eneratin a roust y. ys ere first discoered catalytcally cometent et the DyP rom dslayed smlar moderate y im and hoda here they identified a ene from ec that actvtes hch may suest that DyPs o ths subrou have relatvely lo encodes for a noel type of peroidase and named it y. ys hae a actvtes nortunately no heme absorbance sectrum or has been characteristic feature of ein ale to decolorize arious dyes such as reorted hch ould reveal hether unal DyPs rom these subrou alays anthrauinones and azo dyes hence their name yedecolorizin eroidases. dslay a devant heme abs orbance sectrum ys do not share seuence homoloy ith the typical plant or animal peroidases eerences that elon to the peroidasecatalase superfamily. ys form a distinct peroidase family that is part of the peroidasechlorite dismutase superfamily. ys are 6 yusoernndez artnez uzDueas found in funi ut are much more aundant in acteria–. hey hae een 2017 – classified into four roups and . hile class and ys are mainly from acterial oriin class ys are mainly found in funi. he type ys erl enhard 2016 cluster more closely ith class ys and also display similar enzyme actiity features. fe eamples of acteria that contain ys are ochber hornton – 2017 sp. and . n funi ys can e found in D berles 2007 and ehrens descried the epression of funal ys usin a cold shoc inducile epression system from . his ucerandl Pauln 1965 – that the resurrected DyPs are not very ‘old’ enzymes and the observed moderate 103 ehmann Pasamontes assen yss 2000 – 116 117 102 112 Expression and characterization of a resurrected DyP-type peroxidase Chapter VI Chapter VI Expression and characterization of a resurrected DyP-type peroxidase called consensus method to enerate stale proteins. hateer the reason may e it appears that can e effectie in eneratin roust ariants of a particular thermostablty relects the condtons at hch the DyP as unctonn tll the enzyme family. p H 3 reconstructed DyPs are nterestn enzymes as the etant DyPs derved rom t have hardly been studed nly or one o these DyPs some more detaled normaton n the contet of our study it is orth notin that in recent years p H 4has een An c AltD yP 7 4 s avalable he DyP rom has been shon to be an nterestn and p H 5 performed on funal peroidases that elon to the peroidasecatalase unue bocatalyst or the roducton o nootatone uture studes ll p H 6 superfamily hich harors the ersatile peroidases linin peroidases and reveal hether the ncDyP can also be used or selectve odatons o p H 7 mananese peroidases. n an attempt to resurrect etinct peroidases from the end terenods of the aroniferous period yusoernandez and coorers decidedp H 8 to select enesAn c D yfromP 7 4 seuenced enomes and to perform upon them. his era y resurrectn t o alternatve ancestral DyPs or the same node n the as chosen as it as the period here the first lininolytic enzymes incl. hyloenetc tree to catalytcally cometent DyPs ere created he to DyPs peroidases must hae appeared. hese enzymes ould oriinate from olyporales der by substtutons hle they dslay very smlar roertes nly hen species. he first0 linin deradin2 peroidase4 is supposedly6 a n peroidase loon at the netc arameters o sever al dye substrates some derences ere oidizin phenolic linin that accordin to the study ould not chane until ound or the dyes manly derences n values ere observed s most o the the appearance of an eposed tryptophan- 1 oidizin nonphenolic linin to yield a k o b s (s ) mutatons seem to be on the surace o the roten derences n substrate anty ersatile peroidase. ater another eolutionary eent resulted in the loss of the may be elaned by alternatve docn stes or these dyes on the enzymes DyP n indin site eneratin the first linin peroidase that eoled to the etant catalyzed dye odatons are suosed to occur throuh lon rane electron transer form y improin catalytic efficiency. fter the appearance of the eposed catalytic odaton uon bndn o the substrate on the surace o the roten nother tryptophan an increase in staility at acidic p as seen leadin to an increase in nterestndeterminants ndn of enzyme s the functionobserved is oretreferred band to asat “horizontala relatvely hhapproach”. avelenth the oidizin poer of these enzymes. ecently it as shon that the eolution nm DyPs and other hemorotens usually have ths oret band at around nm of funal peroidases inoled in linin deradation paralleled the eolution of hs ea at nm may hnt to a ormer role o these ancestral DyPs andor may oody plants. hese studies reealed details on ho peroidases of this suest that these DyPs may need to be actvated or ull actvt y ost etant unal particular peroidase family hae eoled to fulfill their current tass. DyPs dslay much hher actvtes hen comared th the ncDyP and n this chapter e descrie an study of the family of ytype peroidases ltncDyP hs may be due to only a small ercentae o DyP that s ys. y this e aimed at eneratin a roust y. ys ere first discoered catalytcally cometent et the DyP rom dslayed smlar moderate y im and hoda here they identified a ene from ec that actvtes hch may suest that DyPs o ths subrou have relatvely lo encodes for a noel type of peroidase and named it y. ys hae a actvtes nortunately no heme absorbance sectrum or has been characteristic feature of ein ale to decolorize arious dyes such as reorted hch ould reveal hether unal DyPs rom these subrou alays anthrauinones and azo dyes hence their name yedecolorizin eroidases. dslay a devant heme abs orbance sectrum ys do not share seuence homoloy ith the typical plant or animal peroidases eerences that elon to the peroidasecatalase superfamily. ys form a distinct peroidase family that is part of the peroidasechlorite dismutase superfamily. ys are yusoernndez artnez uzDueas 6 found in funi ut are much more aundant in acteria–. hey hae een 2017 – classified into four roups and . hile class and ys are mainly from acterial oriin class ys are mainly found in funi. he type ys erl enhard 2016 cluster more closely ith class ys and also display similar enzyme actiity features. fe eamples of acteria that contain ys are ochber hornton – 2017 sp. and . n funi ys can e found in D berles 2007 and ehrens descried the epression of funal ys usin a cold shoc inducile epression system from . his ucerandl Pauln 1965 – that the resurrected DyPs are not very ‘old’ enzymes and the observed moderate 103 ehmann Pasamontes assen yss 2000 – 116 117 102 113 Expression and characterization of a resurrected DyP-type peroxidase Chapter VI Chapter VI Expression and characterization of a resurrected DyP-type peroxidase called consensus method to enerate stale proteins. hateer the reason may e it appears that heeler can e meffectie arusee in eneratin roustarms ariants of a particular 2009 – enzyme2016 family. – n the contet amocy of our study obauer it is orth chaner notin that asselhuberin recent years coluss has een 2010 – performedoud on funal Prer peroidases P urtmuller that elon bner to the peroidase catalase superfamily2015 hich –harors the ersatile peroidases linin peroidases and 2016 – mananese peroidases. n an attempt to resurrect etinct peroidases from the end yusoernndez uzDueas artnez of the aroniferous period yusoernandez and coorers decided to select 2018 – enes from seuenced enomes and to perform upon them. his era as chosen as it as the period here the first lininolytic enzymes incl. 2016 – yusoernndez encoret utrrez uzDueas peroidases must hae appeared. hese enzymes ould oriinate from olyporales artnez 2019 2018 species. he first linin deradin peroidase is supposedly a n peroidase oidizin phenolic m linin hoda that accordin to the 1999 study ould not– chane until the appearance of an eposed tryptophan oidizin nonphenolic linin to yield a ersatile peroidase. uano ater another eolution2009 ary eent– resulted in the loss of the 2011 – n indin site eneratin the first linin peroidase that eoled to the etant form y improin ers catalytic Pecyna efficiency. ellner fter the orrch appearance orn of the eposed teen catalytic 2014 tryptophanorchter an increase llrch in staility at acidic p as seen 2013 leadin to an– increase in determinants – of enzyme function is referred to as “horizontal approach”. the oidizin poer of these enzymes. ecently it as shon that the eolution of funal N. peroidasesLončar, D. I.inoled Colpa, M. in W.linin Fraaije, deradation paralleled 2016 the eolution– of oody plants. hese studies reealed details on ho peroidases of this 2008 – ahmanour D u 2015 – particular peroidase family hae eoled to fulfill their current tass. abb ozeboom raae 2019 2018 n this chapter e descrie an study of the family of ytype peroidases ys. y this e aimed at eneratin a roust y. ys ere first discoered van loos D orres Pazmo nter raae y im and hoda here they identified a ene from ec that 2010 – encodes for a noel type of peroidase and named it y. ys hae a – 2018 characteristic ronfeature of ein han ale to decolorize arious 2014 dyes – such as anthrauinones and azo dyes hence their name yedecolorizin eroidases. y s do not ubeta share seuence rshna homoloy aoorith the tPypical ozbal plant orD animal cullan peroidases 2018 – that elonelrod to the peroidase D ller catalaseP bdube superfamily. mbn ys form stahova a distinct et peroidaseal 6 family that2007 is part of– the peroidasechlorite dismutase superfamily. ys are 2016 – found in funi ut are much more aundant in acteria–. hey hae een classified into four ola roups ame and . ashmoto hile class shda and ys shaa are mainly 2018 from acterialhbata oriin aa class ys are mainly found2009 in funi.– he type ys cluster more closely ith class ys and also display similar enzyme actiity u Du an D hu uan e u u features. fe eamples of acteria that contain ys are 2012 2011 – sp. and . n funi ys can e found in m shaa ra hoda 1995 – and ehrens descried the epression of funal ys usin a coldshoc inducile epression system from . his Puhse zeda a eorre nter orn 103 118 119 102 114 Expression and characterization of a resurrected DyP-type peroxidase Chapter VI Chapter VI Expression and characterization of a resurrected DyP-type peroxidase called consensus method to enerate stale proteins. hateer the reason may e it appears that heeler can e meffectie arusee in eneratin roustarms ariants of a particular 2009 – enzyme2016 family. – n the contet amocy of our study obauer it is orth chaner notin that asselhuberin recent years coluss has een 2010 – performedoud on funal Prer peroidases P urtmuller that elon bner to the peroidase catalase superfamily2015 hich –harors the ersatile peroidases linin peroidases and 2016 – mananese peroidases. n an attempt to resurrect etinct peroidases from the end yusoernndez uzDueas artnez of the aroniferous period yusoernandez and coorers decided to select 2018 – enes from seuenced enomes and to perform upon them. his era as chosen as it as the period here the first lininolytic enzymes incl. 2016 – yusoernndez encoret utrrez uzDueas peroidases must hae appeared. hese enzymes ould oriinate from olyporales artnez 2019 2018 species. he first linin deradin peroidase is supposedly a n peroidase oidizin phenolic m linin hoda that accordin to the 1999 study ould not– chane until the appearance of an eposed tryptophan oidizin nonphenolic linin to yield a ersatile peroidase. uano ater another eolution2009 ary eent– resulted in the loss of the 2011 – n indin site eneratin the first linin peroidase that eoled to the etant form y improin ers catalytic Pecyna efficiency. ellner fter the orrch appearance orn of the eposed teen catalytic 2014 tryptophanorchter an increase llrch in staility at acidic p as seen 2013 leadin to an– increase in determinants – of enzyme function is referred to as “horizontal approach”. the oidizin poer of these enzymes. ecently it as shon that the eolution of funal N. peroidasesLončar, D. I.inoled Colpa, M. in W.linin Fraaije, deradation paralleled 2016 the eolution– of oody plants. hese studies reealed details on ho peroidases of this 2008 – ahmanour D u 2015 – particular peroidase family hae eoled to fulfill their current tass. abb ozeboom raae 2019 2018 n this chapter e descrie an study of the family of ytype peroidases ys. y this e aimed at eneratin a roust y. ys ere first discoered van loos D orres Pazmo nter raae y im and hoda here they identified a ene from ec that 2010 – encodes for a noel type of peroidase and named it y. ys hae a – 2018 characteristic ronfeature of ein han ale to decolorize arious 2014 dyes – such as anthrauinones and azo dyes hence their name yedecolorizin eroidases. y s do not ubeta share seuence rshna homoloy aoorith the tPypical ozbal plant orD animal cullan peroidases 2018 – that elonelrod to the peroidase D ller catalaseP bdube superfamily. mbn ys form stahova a distinct et peroidaseal family that2007 is part of– the peroidasechlorite dismutase superfamily. ys are 2016 – 6 found in funi ut are much more aundant in acteria–. hey hae een classified into four ola roups ame and . ashmoto hile class shda and ys shaa are mainly 2018 from acterialhbata oriin aa class ys are mainly found2009 in funi.– he type ys cluster more closely ith class ys and also display similar enzyme actiity u Du an D hu uan e u u features. fe eamples of acteria that contain ys are 2012 2011 – sp. and . n funi ys can e found in m shaa ra hoda 1995 – and ehrens descried the epression of funal ys usin a coldshoc inducile epression system from . his Puhse zeda a eorre nter orn 103 118 119 102 115 Summary, Conclusion and Future Outlook

Summary, Conclusion and Future Outlook Summary, Conclusion and Future Outlook Summary, Conclusion and Future Outlook inae eebin natua inin he oube action a ao anazed and a ound to contain to die and a oube tetae hate eot on a biocatatic ethod o the nthei o inaeino inaeino i a coound that ha both edicina a e a indutia ue he deeoed biocatatic cacade tat ith diethoxaheno and aain the ue o eueno oxidae and hoeadih eoxidae a biocatat he tuctue o eueno oxidae a tudied to dein utant enze hich oud hae oe actiit on diethoxaheno than the id te enze he to aino acid that ee conideed o utation ee a and e hee eidue ae coe to the ethox ou o the ubtate o hich oe ace needed to be ceated he utant a ound to be the bet utant out o the deined utant b exhibitin ioed inetic aaete ina acoho a ceated o diethoxaheno uin the eueno oxidae utant ina acoho i a eatie exenie coound heea it diethoxecuo i eatie chea ddition o hoeadih eoxidae to the eaction ieded inaeino a ao oduct he exeient a eeated on a cae and oduced inaeino in ied hi ne deeoed biocatatic cacade eaction oide an ea ocedue to oduce inaeino o chea tatin ateia diethoxaheno ate and dioxen ne te eoxidae i eoted in chate he eoxidae i naed ate the oani in hich it a identiied he β enze a identiied hen doin a each in the databae uin the β euence o a a ue he euence ha a at ina euence thouh hich it caiie a a a he enze doe not hae the conentiona oti the otheie coneed aatate ha been eaced b a utaate hi a thouht to cone noe chaacteitic to thi enze n act ith oin at aaine condition it a hoed that oud be actie at eatie hih a ound to act ie a coon eoxidae ith a otiu o actiit at aound uin eactie bue a a ubtate he i ectu o ecobinant hoed that the otein a u oaded ith hee he eect o addin dithionite and hdoen eoxide a ao tudied and hoed that the enze behae ie a tica te eoxidae he etin teeatue o a ound to be 5 he actiit o the id te enze a coaed to that o the utant are: β 4, α 4, β β, β 5’ and the dibenzodioxocin unit in hich the actie ite aatate a etoed nteetin the aatate are: β 4, α 4, β β, β 5’ and the dibenzodioxocin unit utant hoed an aot od ioeent in actiit hen coaed ith the id te enze ina the cta tuctue o a oed eeain detai on it actie ite achitectue nceta euection 121 121 122 118 Summary, Conclusion and Future Outlook Summary, Conclusion and Future Outlook Summary, Conclusion and Future Outlook inae eebin natua inin he oube action a ao anazed and a ound to contain to die and a oube tetae hate eot on a biocatatic ethod o the nthei o inaeino inaeino i a coound that ha both edicina a e a indutia ue he deeoed biocatatic cacade tat ith diethoxaheno and aain the ue o eueno oxidae and hoeadih eoxidae a biocatat he tuctue o eueno oxidae a tudied to dein utant enze hich oud hae oe actiit on diethoxaheno than the id te enze he to aino acid that ee conideed o utation ee a and e hee eidue ae coe to the ethox ou o the ubtate o hich oe ace needed to be ceated he utant a ound to be the bet utant out o the deined utant b exhibitin ioed inetic aaete ina acoho a ceated o diethoxaheno uin the eueno oxidae utant ina acoho i a eatie exenie coound heea it diethoxecuo i eatie chea ddition o hoeadih eoxidae to the eaction ieded inaeino a ao oduct he exeient a eeated on a cae and oduced inaeino in ied hi ne deeoed biocatatic cacade eaction oide an ea ocedue to oduce inaeino o chea tatin ateia diethoxaheno ate and dioxen ne te eoxidae i eoted in chate he eoxidae i naed ate the oani in hich it a identiied he β enze a identiied hen doin a each in the databae uin the β euence o a a ue he euence ha a at ina euence thouh hich it caiie a a a he enze doe not hae the conentiona oti the otheie coneed aatate ha been eaced b a utaate hi a thouht to cone noe chaacteitic to thi enze n act ith oin at aaine condition it a hoed that oud be actie at eatie hih a ound to act ie a coon eoxidae ith a otiu o actiit at aound uin eactie bue a a ubtate he i ectu o ecobinant hoed that the otein a u oaded ith hee he eect o addin dithionite and hdoen eoxide a ao tudied and hoed that the enze behae ie a tica te eoxidae he etin teeatue o a ound to be 5 he actiit o the id te enze a coaed to that o the utant are: β 4, α 4, β β, β 5’ and the dibenzodioxocin unit in hich the actie ite aatate a etoed nteetin the aatate are: β 4, α 4, β β, β 5’ and the dibenzodioxocin unit utant hoed an aot od ioeent in actiit hen coaed ith the id te enze ina the cta tuctue o a oed eeain detai on it actie ite achitectue nceta euection 121 121 122 119 Summary, Conclusion and Future Outlook Summary, Conclusion and Future Outlook Summary, Conclusion and Future Outlook n chate the econtuction euection and chaacteization o an anceta aaine o actiit ith hain the tuctue o aaiabe utant i decibed euence ee ubected to eea iteatie ibaie coud be deined o the eneation o aiant actie at oe neuta ocee o eoin edundancie eainent buidin a hoenetic tee and nothe aeain eectie i to exoe the oibiit o utatin anua inection to ied a eiabe dataet o euence te caeu anai uch that it oud unction in uoxidation and hdoxation eaction and hence o the euence anceta euence econtuction a eoed on the behae a a eoxenae o the euected anceta coud ee a bai ina hoenetic tee ode a eected o anceta euection noin o enze enineein eot that to o it ineae ae o eiou chaacteized enze o aiant o eeence thi anceto nc and nct ee exeed and thoouh chaacteized he actiit o nc and nct a deteined uin iueido eu endeboch eee eea dieent de eactie bue eactie bue eactie bac 5 2019 555 and iect bue he ichaei enten inetic contant ee deteined o ie i ai hou uan the de hdoen eoxide and n 2017 – utue eectie uiaa aauchi oita aeda ihibe he ucce o oducin inin oioe tatin o eueno uin on to 1996 – enze in a oneot ahion deontate the oe o cacade biocatai t anie oande auua ene nad ao oide ea acce to ininie oioe uin dieent ixtue o iadi eauand aaue ucot ai aheno the aount o ethox ou in the eneated ininie ateia can 2017 – be tuned hu inin o dieent ant ouce can be iiced nthetic inin oe can ee a auabe eeach ode coound to inetiate inin aoization o exae to tud thei deadation b ethod uch a oi and ozonoi he boad ane o inae ound in the enzentheized β inin oide a eect ode o thoe tudin inin deadation hi ininie ateia a ao be ued a additie in ateia uch a ahat o atic t ha been hon that uch coound can be ued a an ahat binde odiie xcet o inin the cobination o eueno oxidae and a eoxidae can ao be ued to ntheize inaeino in a oneot ahion inaeino i a coound that can be extacted o ioated o natua ouce ant oee uch ethod ae tediou he abiit to oduce thi coound in the ab ith uch a hih ied and uit tatin o a chea ecuo ee a a ood bai to deeo the oduction o thi coound on a ae cae inaeino ha been hon to be bioactie and a be ued o otection aaint atic cance utue o coud ao inoe tetin inaeino a cooe inaeino ha attactie eatue o thi a i it can be eaded a a dio and ii it contain a ciued biuan oiet hich ae it tuctue iid t a are: β4, α4, ββ, β 5’ and the dibenzodioxocin unit be a candidate o ue a ubtitute o biheno in indutia oe ot eiou chaacteized dia actiit at athe o aue 5 he ootunit to hae a oeatin at neuta to iht aaine oud be e attactie hi i the cae eecia hen uin an oxidae and a eoxidae toethe in oneot eaction xidae hae a eeence o a neuta to iht 121 123 124 120 Summary, Conclusion and Future Outlook Summary, Conclusion and Future Outlook Summary, Conclusion and Future Outlook n chate the econtuction euection and chaacteization o an anceta aaine o actiit ith hain the tuctue o aaiabe utant i decibed euence ee ubected to eea iteatie ibaie coud be deined o the eneation o aiant actie at oe neuta ocee o eoin edundancie eainent buidin a hoenetic tee and nothe aeain eectie i to exoe the oibiit o utatin anua inection to ied a eiabe dataet o euence te caeu anai uch that it oud unction in uoxidation and hdoxation eaction and hence o the euence anceta euence econtuction a eoed on the behae a a eoxenae o the euected anceta coud ee a bai ina hoenetic tee ode a eected o anceta euection noin o enze enineein eot that to o it ineae ae o eiou chaacteized enze o aiant o eeence thi anceto nc and nct ee exeed and thoouh chaacteized he actiit o nc and nct a deteined uin iueido eu endeboch eee eea dieent de eactie bue eactie bue eactie bac 5 2019 555 and iect bue he ichaei enten inetic contant ee deteined o ie i ai hou uan the de hdoen eoxide and n 2017 – utue eectie uiaa aauchi oita aeda ihibe he ucce o oducin inin oioe tatin o eueno uin on to 1996 – enze in a oneot ahion deontate the oe o cacade biocatai t anie oande auua ene nad ao oide ea acce to ininie oioe uin dieent ixtue o iadi eauand aaue ucot ai aheno the aount o ethox ou in the eneated ininie ateia can 2017 – be tuned hu inin o dieent ant ouce can be iiced nthetic inin oe can ee a auabe eeach ode coound to inetiate inin aoization o exae to tud thei deadation b ethod uch a oi and ozonoi he boad ane o inae ound in the enzentheized β inin oide a eect ode o thoe tudin inin deadation hi ininie ateia a ao be ued a additie in ateia uch a ahat o atic t ha been hon that uch coound can be ued a an ahat binde odiie xcet o inin the cobination o eueno oxidae and a eoxidae can ao be ued to ntheize inaeino in a oneot ahion inaeino i a coound that can be extacted o ioated o natua ouce ant oee uch ethod ae tediou he abiit to oduce thi coound in the ab ith uch a hih ied and uit tatin o a chea ecuo ee a a ood bai to deeo the oduction o thi coound on a ae cae inaeino ha been hon to be bioactie and a be ued o otection aaint atic cance utue o coud ao inoe tetin inaeino a cooe inaeino ha attactie eatue o thi a i it can be eaded a a dio and ii it contain a ciued biuan oiet hich ae it tuctue iid t a are: β4, α4, ββ, β 5’ and the dibenzodioxocin unit be a candidate o ue a ubtitute o biheno in indutia oe ot eiou chaacteized dia actiit at athe o aue 5 he ootunit to hae a oeatin at neuta to iht aaine oud be e attactie hi i the cae eecia hen uin an oxidae and a eoxidae toethe in oneot eaction xidae hae a eeence o a neuta to iht 121 123 124 121 Nederlandse Samenvatting

Translated by Jeroen Drenth

Nederlandse Samenvatting Nederlandse Samenvatting

en obenst an 5 onoosbaa inineacti mateiaa ed beeit met een scaa conesie. et onoosbaa oduct bee ae ieoo enoemde cemisce bindinen te beatten. De oosbae actie beatte tee sooten dimeen en een oosbaa tetamee. n oodstu odt een bioatatisce metode besoen oo et sntetiseen an sinaesino. inaesino is een sto die zoe medisce as industie toeassinen ent. De ontiede bioatatisce cascade stat met dimetoxaeno en de eede enoemde combinatie an eueno oxidase en oseadis eoxidase as atasatoen. idens deze studie ed ezoct naa manieen om de snteseoute te otimaiseen. Daaom ed de stuctuu an eueno oxidase bestudeed oo et onteen an enzmmutanten die mee – actiiteit zouden unnen etonen met dimetoxaeno dan et – idte enzm. ee aminozuuesiduen eden uiteozen as mutatie taets namei a en e. Deze esiduen zitten dict bi de metoxoe an et substaat aa mee uimte oo moest oden eceed. e mutatie bee de ootste toename in atatisce actiiteit te ebben an ae esceende mutanten. De conesie an dimetoxaeno eataseed doo de eueno oxidase aiant esuteede in de sntese an sina acoo. Dit oduct is eatie duu tei de beinsto uist i oedoo is. oeoein an oseadis eoxidase aan deze eactie esuteede in de omin an sinaesino as oododuct. en scaa conesie a obenst. Deze nieue biosntetisce oute is een maeie ocedue om sinaesino te maen uit oedoe statmateiaen namei ate dimetoxaeno en moecuai zuusto. en nieu Dte eoxidase odt besceen in oodstu . Deze nieue eoxidase daat de naam D naa et oanisme aauit et esoeed ed . et enzm ed edentiiceed bi een seac in de database aabi de seuentie an D as eeentie ed ebuit. De D eet een at secetiesinaaseuentie aadoo et as een ass D odt ecassiiceed. Dit enzm beat niet de conentionee Dseuentie D in dit enzm is de nomaa econseeede asataat D eanen doo een utamaat . iedoo ed esecueed dat dit enzm niet eede etoonde eienscaen zou unnen ebben. eoot ed dat dit enzm actie zou zin o eatie oe aaden aanezien oed oeit onde basisce omstandieden oe . ctiiteitmetinen met eactie bue as substaat ieten ecte zien dat D etzede otimum eet as andee Dte eoxidases namei een otimum ond . et sectum β–– α–– β–β –5’ en de dibenzodioxocine unit. De massa an ecombinant eoduceed D iet zien dat et enzm oedi eaden – as met eem de benodide coacto. oeoein an ditioniet en atestoeoxide a etzede eect as bi andee Dte eoxidases. et

127 128 124 Nederlandse Samenvatting Nederlandse Samenvatting

127 128 125 Nederlandse Samenvatting Nederlandse Samenvatting

scinbae smetunt an dit enzm bee 5 te zin. De atatisce actiiteit an et idte enzme en dat an een mutant aabi de utamaat as eanen doo de nomaa econseeede asataat eden met eaa eeeen. ot ote eassin bee de actiiteit an de mutant tien ee oe te zin. o de istastuctuu an D ed oeeded ee ons inzict a in de acitectuu an et actiee centum an et enzm. ncestae D esuectie n oodstu odt de econstuctie esuectie en aateisatie an een ancestae oooudeie D besceen. D seuenties eden ondeoen aan esciende iteatiee ocessen oo et eideen an zic eaende seuenties eainment et bouen an een oenetisce stamboom en andmatie insectie om zo een betoubae dataset an seuenties te – ien. a naueuie anase an deze seuenties ed een ancestae seuentieeconstuctie ondenomen ebuimaende an de oenetisce stamboom. noount ed eseecteed oo ancestae esuectie omdat tee etainen anuit dit unt eaateiseede D eoxidases beatten. ee aianten an deze oooude Da en Db eden tot exessie ebact en extensie eaateiseed. De atatisce actiiteit an deze tee rale DyP’s enzmen ed beaad met tee esciende eustoen en dit a de icaeisenten constanten oo beide eustoen en oo atestoeoxide. oeomstesectie 2019 De successen die eboet zin met de oductie an inineoiomeen anuit eueno – met beu an maa tee enzmen in n ot – aten de act an 2017 cascade bioatase zien. Deze metode biedt oo maei toean tot inine – actie oiomeen oo een ote esceideneid aan toeassinen. et et aien an esciende aenoen an de oeeeeid metoxoeen in et 1996 – inineactie oduct oden aestemd. iedoo an de ininestuctuu an esciende antensooten oden naebootst. Deze sntetisce ininemodeen unnen oden toeeast in ondezoeen naa inine aoisatie 2017 zoas et bestudeen an esciende deadatieocessen as ose en – ozonose. De ote aiteit aan esciende sooten cemisce bindinen in enzmesntetiseede modeen maen deze ebindinen ideae modeen oo abaastudies. ede zouden deze inineactie mateiaen oo unnen oden ebuit as additieen in mateiaen as asat en unststoen. De combinatie an eueno oxidase en oseadis eoxidase in n ot is niet aeen escit oo de sntese an ininemodeen maa oo oode sntese an sinaesino. inaesino an oden extaeed uit anten maa deze

129 130 126 Nederlandse Samenvatting Nederlandse Samenvatting

129 130 127 Curriculum vitae Acknowledgements

Curriculum vitae Acknowledgements ae a as r ya a raa rl e raae ver the course of this PhD there were a lot of people of whom had an influence from the Faculty of Pharmacy at Cairo University after getting his Bachelor’s degree either directly or indirectly in helping me achieve my degree. First of all would ay e e e r as a ea asssa a e Deare lie to than my Mom, Dad, brother (Kareem), Nahla, and Yusuf (my rly a ly a e aly Paray ar ersy e nephew) for the continuous mental and physical support during my PhD years. his Master’s degree from the same faculty in July 2014. His thesis dissertation was would also lie to than the rest of my family from both my Mom and my Dad’s titled “Study on oxidoreductases re y aera slae r e ya side for their generous support during my visits to Egypt in the form of family environment”. He started his PhD in the Molecular Enzymology group under the gatherings. sers Pr ar raae aary s r se e se ases a erases r e r alale eal rs s would also lie to than Marco for giving me the opportunity to do my PhD in r as e y e lral e a sss er e sry his la. thin am lucy to have Marco as my PhD supervisor as he has a talent er a y in directing his students to do research in a friendly environment. He has a way of nowing exactly what you need without asing and has a cheerful attitude in List of Publications directing you in the la. Marco also always eeps his door open welcoming any inuiries you may have. gain would lie to than Marco for this opportunity e al D la a raae 2016 and wish him all the est in his future experiences. – would lie to than Dick my second supervisor for the uestions he raised during H. M. Habib, P. J. Deuss, N. Lončar, M. Trajkovic, M. W. Fraaije, my wor discussions. His comments enriched the discussion and made me loo 2017 – deeper into my wor to find answers to his uestions. a ra raae – 2018 would lie to than Peter Deuss for his collaoration in my first experimental a e raae 2019 paper. His contriution really added depth to the paper and we managed to pulish it in a reputale ournal.

Of course I couldn’t have finished this PhD without the help and support of my colleagues and friends in the la. would lie to first egin with thaning the residents of ffice 114.0104. hen first came to roningen was welcomed y Hemant, Alex and Elisa in the office. always had chats with Hemant aout different topics lie aout our cultural acgrounds and how they differ from those acgrounds found in Europe. Hemant also always provided me with different suggestions when had some prolems in my experiments. e also enoyed playing suash together on several occasions. Alex provided me with help and advice in my first days in roningen. recall he told me where can uy my first ie in roningen. e also discussed how to improve poster designs using doe llustrator. Elisa started her wor one month efore came to roningen. So we were more or less the same with getting to now our surroundings. Elisa had a very cheerful attitude and we had some nice outings together. She always helped whenever needed in the la and was a good friend to have oth inside and outside of the la. hen came the new residents of office 104 Milos, Alejandro, Qinglong and Yiming. Milos is a very helpful person. He always provided assistance whenever needed whether that e with the 131 132 128 Curriculum vitae Acknowledgements

M or other machines e also helped in analyin data whenever it was too oer the years. We occasionally had ick chats reardin lie topics in eneral and comple and I needed to discuss the data with someone Milos was always there e they ere pleasant. also had some publications toether Alejandro always had this calm nature and friendly attitude e is a ood listener to problems you may face inside or outside n room there as Antonija, Gautier, Peter, Nina, Arne, Marzena and . old like to thank or her stron and spportie character. the lab and offers supportive suestions Qinglong is a very responsible man e Yapei Antonija is a ood person to tal to when you are facin some troubles as he will provide Antonija is a ood person to talk to or conselin. he alays reets yo ith a elcomin attitde. is a ery cheerl and n character to be arond. e constructive support inally Yiming is our newest member in the office Gautier alays played some nice and happy sons inside the lab. e also has eperience Yiming is a very cheerful and funny character e always reets you with a smile and has a happy lauh I would lie to than the office members for providin sin some o the more adanced eipment in the lab and that is ery helpl. breafast on so many different occasions hat was a nice esture Peter is a ery nice character and elcomes yo ith a arm smile. Yapei is a ery hard orker and old alays ask or adice on orkrelated matters. as happy I will then move to room I will start by thanin Elvira Romero for her to anser her estions hen cold. Nina as a ood riend to hae. he as a continuous help and support she provided inside the lab with her etensive nice person to talk to pls she as a hard orker in the lab and elcomed any eperience she has ained over the years Elvira was a ood person to tal to estion may hae had happily. old like to thank Arne or bein a ood riend whenever I had a problem in the lab but also outside of the lab Elvira listened very een ater he let or rop. We enoyed nice times toether especially the days e well and provided solutions to many problems I would lie to than her for all the ent to his place or a barbee. old also like to thank Marzena or the nice support she provided over the years at roninen Max was a ood friend to have times e had toether. in the lab with his impressive wor dedication and helpful character especially in n room there as molecular bioloy wor Max assisted me in doin some protein mutaenesis wor Nikola, Nikkolas, Christiaan, Estela, Hugo and for one of my publications e lied to teach the mechanism of how everythin Hesam. ikola old like to thank or instrctin me in orkin ith the T wors and that is a ood attitude to help better understand what it is you are doin machines in the lab and the P as ell. Nikola has etensie eperience in orkin ith these eipment. alays had constrctie criticism on some Max also lied to eep a ood worlife balance and oranied a lot of suash Nikola volleyball and basetball ames e also too the intiative on oraniin some o the eperiments as condctin in the lab and alays ae sestions on ho barbeues and many parties at his place I would lie to wish him all the best in his to improe my ork. am thankl to Nikola or his sestions in the lab as that improed my ork reslts. also had a leadin character and pshed or new postdoc position hen comes Caterina Caterina is a cheerful and lovin Nikola character he always reets people with a happy smile and offers help whenever hain barbees and other actiities otside o the lab. Him and Max alays needed he is also very fair in her attitude with other people I would lie to than steered the rop otside o the lab to try to be inoled in dierent actiities. is a ery cheerl y. e is a ood riend to hae inside and otside o her for oraniin the trip to chiermonnioo hat was fun Sam was a nice friend Nikkolas to have in the lab e wored hard and was a nice person to tal to In my earlier the lab. e alays makes sarcastic remarks and is a ery nny indiidal. is a ery ood lab technician. e alays tries to help ot in iin broken days Suzan was still here Suzan and I shared moments of bein the only Christiaan microbioloists in the lab and we had happy discussions over that I would lie to machinery or other adets inside the lab. e proides a lot o assistance hen than her for all the ood times we had toether both in and out of the lab e needed inside the lab. Estela is a nice riend. enoy chattin ith Estela abot amily and other isses oer lnch. is a ery practical character. e is alays went out with her family to one of the festivals in roninen Andrea Fabara is a Hugo ood friend to have e first met when she was doin her masters proect with tryin to maimie the beneit rom a sinle eperiment in the lab. enoy the chats e hae oer coee breaks. old like to ish a speedy recoery. hope Nina. Then she came back to do her PhD in Marco’s lab. We had some happy talks Hesam he ill be better soon and come back to the lab. toether I would lie to also than Dana for our collaboration in worin with DyPs Dana is a very oranied and very dedicated individual he helped me a lot n room there is Elvira Bombino, Heijn, Eduardo, Carlos and Simone. when I started worin with DyP enymes Dana has a very calm and nice character old like to thank Elvira Bombino or her happy attitde een thoh she is and she helped me in stressful times by talin in a calm and entle manner I am alays complainin abot somethin. talk to Elvira hen ant to cheer p as very happy I had the chance to wor with Dana before she moved to another roup she alays has somethin nny or ood to say. Heijn and Carlos I didn’t have for doin her postdoc I would lie to than Misun for her nice attitude and help lenthy talks ith bt they are alays hard orkin and bsy doin comptational ork. The chats had ith Heijn abot birdatchin and the places e isited 133 134 130 cnowledements Acknowledgements

ee ve nie Eduardo I aa ened tain t and havin nh tethe e fascinating. I enoyed the time she was here in roningen and the times we went i a nie en and i aa n t e and Simone i a ve eeted en out together and wish we can collaorate together in the future in different proects. ith a eaant ie Simone heed e e the ne the and i aa ein an etin I a have eadin the the ig thank you goes to iaa and iia for the times we went to see a movie intent together in the cinema or ust having casual chats every now and then. I would also like to thank the students I supervised whilst doing my h. Jeroen, Fabiola, Eleonora, Friso, Cyntia, Thai and Brenda in ie s was a very good student ut a it unlucky with his results. I am sure he will do good I d ie t than Jeroen hi eaant and nie attitde Fabiola a he wherever he decides to go for his future steps. also did well in her Bachelor’s nie attitde and tn haate that i etie needed in the a Eleonora proect. I enoyed supervising during her work in the la. I also wish her the a a a eaant en t have in the a Friso I d ie t than a the nie tie e had tethe e ened in t the inea evea tie t ee very est in her net step. i did well and was very proactive in his work. e e te vie e a hted at hi ae evea tie and e didn’t need much guidance to get around in the lab. I wish all three of my students all the est in their future endeavours. ened ain ita and inin a e n tethe thee Brenda I ened and had the eae in ith in a e a eeient Brenda i a nn I would like to thank my friends ha ass a saha for their constant haate t e and and ha a ve ha and d ah I aa ened tain support and good lunches movies and footall matches we went to see together. I t Cyntia hen he a din he h in the e e t ta at an would also like to thank my neighor ha for all the footall matches we dieent et I d ie t a than t Thai a e the ht tie watched together and the outings we went to together. e t t n eah the e a had a ht aatin in ne Thai’s ae I a ate t have nn Thai I would like to also thank my colleagues and friends from work at gypt for their constant support throughout my years in the etherlands. They always gave me a I would also like to thank everyone from the Roelfe’s group for the happy times we push to work harder and produce more results. I am grateful for having this support haed tethe it a the i the Ivana, Manu, Lilliana, from home. Sambika Cora and Nathalie I d ie t ena than a the n and nie tie e had tethe th at niveit and tide the niveit he i Thank you… ee aa hee and ee ve tive individa t e and I d Mohamed a ie t than Dowine he ind attitde I d a ie t than Matt and Rachel the nie tie e had tethe I aina had e hat ith Shreyans which I liked very much. The new generation of Roelfe’s students namely Linda, Claudia and Reuben a deeve t e thaned I d eeia ie t than Linda for her support and our frequent chats together. I can’t thank Linda enh he ntant t and d ie t ih he a the et ith he h Ginny a a n t e and Clemens deeve t e thaned hi nttive itii din diin and ein a d iend e e t ta at nient t tethe and di hi viit t t and advente he had ve thee I d ie t than Rudy the d tie in hienni and in the ee dinne e had tethe e the viit e had in ninen et a i iat Filippo, Mathias and Laura ae e thee viit I ened the tie I had tethe ith Filippo e had e nie hat tethe Mathias i a ve n en t e ith e ent t tethe t ath a ta ae in the t a ne e a ent t evea the tie Laura I d ie t than ein a aat in the ina hate the thei e eeiene ith aneta eene enttin i ite 135 136 132 nedeent s