Enzymatic functionalization and degradation of natural and synthetic polymers

Von der Fakultät für Mathematik, Informatik und Naturwissenschaften der RWTH Aachen University zur Erlangung des akademischen Grades einer Doktorin der Naturwissenschaften genehmigte Dissertation

vorgelegt von

Shohana Subrin Islam

M. Sc. Biotechnologie

aus

Dhaka, Bangladesch

Berichter: Univ. -Prof. Dr. Ulrich Schwaneberg

Univ. -Prof. Dr. Lothar Elling

Tag der mündlichen Prüfung: 23.01.2019

Diese Dissertation ist auf den Internetseiten der Universitätsbibliothek verfügbar.

To my mom & my sister-the two persons in the world who always stand by me

Table of content

Table of content

Table of content ______v Publications and patents ______ix Abstract ______xi 1. General introduction ______1 1.1 Enzymatic functionalization of (bio)polymers ______1

1.2 Enzymatic degradation of polymers ______3

1.3 Protein engineering ______5 1.3.1 Directed evolution of ______6 1.3.2 KnowVolution – Directed Evolution 2.0 ______9

1.4 Aims of the dissertation ______11

2. Engineering of an aryl toward sulfation of saccharides ______13 2.1 Declaration ______13

2.2 State of the art ______13 2.2.1 Sulfation in nature ______13 2.2.2 Sulfation of glycosaminoglycans______14 2.2.3 Chemical sulfation of (poly)saccharides ______15 2.2.4 Classes of ______16 2.2.5 Aryl sulfotransferases from Desulfitobacterium hafniense ______19 2.2.6 Directed evolution of sulfotransferases ______20 2.2.7 Objectives ______21

2.3 Material and methods ______22 2.3.1 Chemicals ______22 2.3.2 Cloning of astA and astB ______22 2.3.3 Construction of astB SeSaM library ______23 2.3.4 Site-saturation and site-directed mutagenesis ______25 2.3.5 Cultivation of libraries in 96-well microtiter plates ______25 2.3.6 pNPS based screening system for 96-well microtiter plates ______26 2.3.7 Production in shaking flasks ______26 2.3.8 Purification of ASTB-WT and variants ______27 2.3.9 Kinetic characterization of ASTB-WT and variants ______27 2.3.10 Evaluation of pH activity profile and pH stability ASTB-WT and variants ______27 2.3.11 Determination of organic solvent resistance of ASTB-WT and variants ______28 2.3.12 Determination of thermostability of ASTB-WT and variants ______28 2.3.13 Computational analysis of ASTB homology model ______28

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2.3.14 Synthesis of (mono)sulfated GlcNAc-linker-tBoc ______29 2.3.15 Analytical and semi-preparative HPLC ______29 2.3.16 Mass spectrometry (MS) ______30 2.3.17 Nuclear magnetic resonance (NMR) ______31

2.4 Results ______32 2.4.1 Selection of an aryl sulfotransferase ______32 2.4.2 Development of a continuous pNPS based screening system ______33 2.4.3 Directed evolution of ASTB toward GlcNAc ______35 2.4.3.1 Generation and screening of astB SeSaM library ______36 2.4.3.2 Screening of ASTB SeSaM library ______38 2.4.3.3 Kinetic characterization of ASTB-WT and variants for GlcNAc ______39 2.4.4 KnowVolution campaign of ASTB towards cellobiose ______40 2.4.4.1 Phases of KnowVolution ______40 2.4.4.2 Production and purification of ASTB-WT and selected variants ______44 2.4.4.3 Kinetic characterization of ASTB-WT and improved variants ______45 2.4.4.4 Determination of sulfated cellobiose monosulfate ______46 2.4.4.5 ASTB Activity toward mono-, di-, and oligosaccharides ______47 2.4.4.6 Sulfation of GlcNAc-linker-tBoc by ASTB-WT and improved variants ______48 2.4.4.7 pH activity and pH stability ASTB-WT and improved variants ______52 2.4.4.8 Organic co-solvent activity of ASTB-WT and improved variants ______53 2.4.4.9 Thermostability of ASTB-WT and improved variants ______55

2.5 Discussion ______56

3. Engineering of a cutinase toward degradation of synthetic polymers ______61 3.1 Declaration ______61

3.2 State of the art ______61 3.2.1 Plastics and their applications ______61 3.2.2 Environmental pollution by microplastic ______62 3.2.2.1 Polyethylene terephthalate (PET) ______64 3.2.2.2 Polyurethane (PUR) ______65 3.2.3 Biodegradation of PET and PUR ______66 3.2.4 Cutinases from Thermomonospora curvata (Tcur0390/Tcur1278) ______67 3.2.5 Directed polymer degradation by binding domains ______69 3.2.6 Objectives ______72

3.3 Material and methods ______73 3.3.1 Chemicals ______73 3.3.2 Use of eGFP-anchor fusion protein for binding tests ______73 3.3.3 Construction of cutinase-anchor fusion protein ______74 3.3.4 Production of Tcur1278-WT and Tcur1278-anchors in Pichia pastoris ______74 3.3.5 Determination of cutinase activity via pNPB assay ______75 3.3.6 Evaluation of polyester-PUR degradation ______75 vi

Table of content

3.3.7 Evaluation of PET-film degradation ______75 3.3.8 Dynamic light scattering (DLS) ______76 3.3.9 Field emission scanning electron microscopy (FE-SEM) ______76

3.4 Results ______77 3.4.1 Selection of anchors for binding to polyester-PUR and PET ______77 3.4.2 Cultivation of Tcur1278-WT and Tcur1278-anchors in Pichia pastoris ______78 3.4.3 Determination of polyester-PUR degradation ______79 3.4.4 Specific activity of Tcur1278-WT and Tcur1278-TA2 ______80 3.4.5 Quantification of polyester-PUR degradation ______81 3.4.5.1 MTP-based turbidity assay ______81 3.4.5.2 DLS of polyester-PUR particles ______84 3.4.5.3 FE-SEM analysis of polyester-PUR degradation ______85 3.4.6 Broadening the targeted degradation by anchor peptides to PET ______86

3.5 Discussion ______87

4. Summary and conclusion ______93 5. References ______97 6. Appendix ______109 6.1 List of abbreviation ______109

6.2 List of DNA and protein sequences ______111

6.3 List of oligonucleotides ______118

6.4 List of computational data ______121

6.5 List of analytical data ______123

6.6 List of figures ______126

6.7 List of tables ______128

7. Declaration ______129 8. Author contribution ______131 9. Acknowledgment ______133 10. Curriculum vitae ______135

vii

Publications and patents

Publications and patents

Parts of this thesis have been published:

Islam S, Mate DM, Martínez R, Jakob F, Schwaneberg U. 2018. A robust protocol for directed aryl sulfotransferase evolution towards the carbohydrate building block GlcNAc. Biotechnology and Bioengineering, 115:1106-1115. Islam S, Laaf D, Infanzón B, Palentová H, Davari MD, Jakob F, Křen V, Elling L, Schwaneberg U. 2018. KnowVolution campaign of an aryl sulfotransferase increases activity toward cellobiose. Chemistry – A European Journal, 24(64), 17117-17124. Islam S*, Weber L*, Jakob F, Schwaneberg U. 2018. Targeting microplastic in the void of diluted suspension. Environment International, 123, 428-435 (*shared first authorship).

Publications in collaboration:

Ji Y, Islam S, Mertens A, Sauer S, Jakob F, Schwaneberg U. 2018. Ditected aryl sulfotransferase evolution toward improved sulfation stoichiometry on the example of catechols. Applied Microbiology and Biotechnology, DOI: 10.1007/s00253-019-09688-0. Ji Y*, Islam S*, Dhoke G, Davari MD, Schwaneberg U. 2019. Regioselective O-Sulfation of high value chemicals by a reengineered aryl sulfotransferase. In preparation (*shared first authorship).

Parts of this thesis were filed for patent application:

Schwaneberg U, Sözer S, Mertens A, Davari MD, Jakob F, Islam S, Weber L. 2018. Fusion Peptides or Proteins, their Use, and Systems and Kits based thereupon, for the Separation and/or Detection of Plastics, particularly of Microplastics. EP18178726.8.

Mussmann N, Wieland S, Degering C, Zimmermann W, Wei R, Jakob F, Islam S, Schwaneberg U, Haarmann T, Lorenz P, Rachinger M, Schreiter M, Schwerdtfeger R. 2018 Mittel erhaltend rekombinante Polyesterase. DE 102018210605.3.

Mussmann N, Wieland S, Degering C, Zimmermann W, Wei R, Jakob F, Islam S, Schwaneberg U, Weber L, Rübsam R, Eidener J, Haarmann T, Lorenz P, Schwerdtfeger R. 2018. Mittel erhaltend Polyesterase I. DE 102018210608.8.

ix

Abstract

Abstract

Enzymes are well-known as efficient catalysts for chemical reactions under mild conditions (ambient temperature, low pressure) with high chemo-, regio-, and enantioselectivity. Properties such as specific activity, stability, and resistance can be improved using protein engineering to tailor enzymes. This work reports the engineering of two different classes of enzymes ( and ) toward efficient functionalization of biopolymers and depolymerization of synthetic polymers, respectively.

New functionalities can be introduced to polysaccharides by applying chemical and enzymatic technologies. Glycosaminoglycan (GAG) is one of the most important classes of functional polysaccharides found in nature. Natural GAGs are mainly isolated from animal sources leading to mass animal farming. Alternative to natural GAGs, chemically sulfated polysaccharide (e.g., cellulose) can mimic the functionalities of GAGs including anti- coagulant and anti-viral activity. Enzymatic sulfation by sulfotransferases in contrast to chemical sulfation offers a greener alternative to introduce groups to the building blocks of polysaccharides in aqueous solution, at ambient temperature, and with high chemo- and regioselectivity. Bacterial aryl sulfotransferases (ASTs) are interesting biocatalyst due to a broad acceptor range and cost-effective sulfate donors. Interestingly, ASTs are able to sulfate small saccharides (e.g., glucose) slowly. Therefore, tailoring of ASTs by protein engineering methods is needed to increase the activity of these enzymes for effective sulfation of saccharides, and later polysaccharides.

The aryl sulfotransferase B (ASTB) from Desulfitobacterium hafniense was advanced toward a synthetically attractive sulfation agent for saccharides. A directed evolution protocol was developed and validated for ASTB. The well-known para-nitrophenylsulfate (pNPS) quantification system was extended to a continuous and robust screening system in 96-well MTP format with a true coefficient of variation below 15%. Firstly, a random mutagenesis library of ASTB was screened to identify variants with improved sulfation activity toward the GAG-building block N-acetylglucosamine (GlcNAc). The best-identified variant ASTB-V1 (Val579Asp) showed an up to 3.4-fold increased specific activity (U/mg) toward GlcNAc in comparison to ASTB-WT. Secondly, ASTB was further subjected to a knowledge-gaining directed evolution campaign (KnowVolution) toward the cellulose building block cellobiose. The final recombination variant ASTB-M5 (Leu446Pro/Val579Lys) resulted in a 7.6‐fold increase in specific activity (6.15 U/mg) toward cellobiose. Computational analysis discovered an important role of Leu446Pro in the substrate‐binding and suggested Val579Lys as a distal substitution. Mass spectrometry (MS) revealed a monosulfation of GlcNAc as well as cellobiose. Structure elucidation by nuclear magnetic resonance (NMR) confirmed the partial regioselective sulfation of GlcNAc‐linker‐tBoc at rare positions (C‐3 or C‐4), whereas C‐6 preferred is by

xi

Abstract chemical methods. These findings represent the first successful directed evolution campaign followed by KnowVolution of an AST for regioselective sulfation of saccharides.

The abundance of microplastic in the landfill and marine environment causes harm to the living organisms and in long run to human race. A natural degradation of such polymer requires from 50 to 100 years or even more depending on the size and type. Other than the most commonly used synthetic polymers as plastics (e.g., polyethylene and polypropylene), polyethylene terephthalate (PET) and polyurethane (PUR) can undergo biodegradation by hydrolytic enzymes. For instance, cutinases are reported to degrade both PET and PUR. A challenging task for the enzymes is to target the microplastic particles for depolymerization under natural circumstances. Hydrolytic enzymes (e.g., cellulases and depolymerases) for natural polymers (e.g., cellulose and polyhydroxyalkanoate) appear with a so-called substrate-binding domain. The substrate-binding domain binds specifically to the polymeric material to bring the catalytic domain of the enzymes to its substrate. Inspired by nature’s solution, enzymes without binding domain can be fused to material-binding peptides (e.g., hydrophobins, cellulose binding domains, and anchor peptides), which bind strongly to polymers such as PET and PUR.

Synthetic polymer degradation by a cutinase was accelerated using anchor peptides as adhesion promoters. Two anchor peptides, liquid chromatography peak I (LCI) and Tachystatin A2 (TA2) were fused to the bacterial cutinase Tcur1278 to accelerate the degradation of polyester-PUR nanoparticle and PET films. The half-life of polyester-PUR nanoparticles was reduced from 41.8 h to 6.2 h (6.7-fold) in a diluted suspension (0.057% w/v) by Tcur1278-TA2. Dynamic light scattering (DLS) revealed the smaller size of degraded nanoparticles (from 0.33 nm down to 0.08 nm) by Tcur1278-TA2 compared to Tcur1278-WT. Field emission scanning electron microscopy (FE-SEM) enabled the comparison of the degraded polyester-PUR nanoparticle layer by Tcur1278 with and without TA2. Furthermore, the principle of targeting synthetic polymer by anchor peptide was applied to PET. A 16-fold accelerated PET-foil degradation was documented by Tcur1278-TA2 compared to Tcur1278-WT. This study reports the first proof of principle of targeted and accelerated degradation by the usage of anchor peptides as adhesion promoters fused to a synthetic polymer degrading .

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General introduction

1. General introduction

1.1 Enzymatic functionalization of (bio)polymers

Enzymatic functionalization of biopolymers is abundant in nature. Biopolymers such as DNA, proteins, lipids, and carbohydrates are reversibly or irreversibly modified for instance by addition of chemical functional groups and complex molecules (sugars and polypeptides)[1]. Methylation of DNA plays a key role in the epigenetic regulation of gene expression, in which enzymes like methyltransferases introduce methyl group to DNA nucleotides (cytosine and adenine)[2]. Apart from DNA functionalization, a variety of post- translational modification and functionalization of proteins are known for diverse biological functions and regulation mechanisms[1, 3]. Building blocks of proteins (amino acids) are enzymatically functionalized during the post-translational functionalization[3]. The most prominent reversible covalent modifications of proteins are shown in Figure 1. Phosphorylation and dephosphorylation are carried out by kinases and phosphatases that are often involved in activation and inactivation of enzymes[4]. Typically, amino acids containing hydroxyl groups such as serine, threonine, and tyrosine undergo phosphorylation and dephosphorylation[4a]. Another important protein functionalization is acetylation by acetyltransferases on serine and lysine residues, which regulates e.g., DNA recognition, protein-protein interaction, and protein stability[5]. Methylation/demethylation by methyltransferases/demethylases of lysine residues in protein like histones is strongly involved in the activation and repression of gene transcription[6]. One of the best-characterized histone code is the methylation of lysine[7]. Further enzymatic functionalization of proteins by introducing chemical groups includes acylation, hydroxylation, and sulaftion[3]. Moreover, protein can be enzymatically ubiquitinated and sumoylated, whereas proteins like ubiquitin and SUMO (small ubiquitin-related modifier) are ligated by the act of a to the target protein[8]. Ubiquitination and sumoylation play roles in a wide range of cellular processes e.g., apoptosis, DNA repair, signal transduction, nucleocytoplasmic signaling, regulation of gene expression, and many more[9]. Glycosylation is one kind of co- and post-translational modification that occurs not only in proteins but also in lipids[10]. In such kind of modifications, glycosyltransferases are responsible to attach glycans (glycosyl donor e.g.,

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General introduction activated nucleotide sugars) by their enzymatic activity to proteins or lipids (glycosyl acceptor)[11]. N- (glycans are attached to a nitrogen atom of asparagine or arginine residues) and O-linked glycosylation (glycans are attached to the hydroxyl oxygen atom of serine, threonine, tyrosine, hydroxylysine, or hydroxyproline residues) are commonly needed for protein folding, protein regulation, cell-cell interaction, immune regulation, etc.[10].

Specific enzymes in cellular catabolism and metabolism functionalize small and large sugar molecules. For instance, small sugar like glucose undergoes phosphorylation at C-6 by hexokinase or glucokinase as it enters a living cell[12]. Thus, glucose receives a highly negatively charged functional group, so that it is not easily diffused out of the cell. The phosphorylated sugar, glucose 6-phosphate represents the starting molecule of two major metabolic pathways; glycolysis and pentose phosphate pathway[13]. Furthermore, the dietary disaccharide sucrose (β-D-fructofuranosyl α-D-glucopyranoside) is transported into the cell with a phosphate group at C-6 (glucosyl moiety) introduced by

Figure 1. Post-translation functionalization and modification. Only some of the most prominent reversible PTMs are in category attachment of chemical groups and complex molecules are shown.

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General introduction the phosphoenolpyruvate-dependent sugar phosphotransferase system[14]. Finally, sucrose 6-phosphate is converted to lactic acid in the Embden–Meyerhof glycolytic pathway[14]. Acetylation is another key enzymatic functionalization of sugar building blocks, such as amino sugar glucosamine. During the hexosamine biosynthetic pathway, glucosamine 6-phosphate is acetylated by acetyltransferase and finally ends up as UDP- N-acetylglucosamine (UDP-GlcNAc), which one of the building blocks in polysaccharides like chitin[15] and glycosaminoglycans (GAGs)[16].

Sulfation (often termed as sulfurylation) is among the enzymatic sugar functionalization one of the crucial sugar modification. Enzymes called PAPS-dependent sulfotransferases are responsible to introduce sulfate groups regioselectively to the sugar moieties[17]. Sulfated sugars are highly negatively charged macromolecules that naturally appear in seaweeds and animals[18]. Particularly, most of the plant polysaccharides e.g., fucans, ulvans, agargans, and carrageenans are present in seaweeds like brown algae[18-19]. Fucans are for instance highly sulfated complex structures with L-fucose building blocks, which is found in brown algae as well as marine invertebrates[20]. Furthermore, ulvans occur in green seaweeds, which is mainly made of sulfated L-rhamnose units at 3’ OH-groups that is linked to xylose or uronic acids[21]. Carrageenans isolated from red algae are on the other hand structured linear polysaccharides with sulfated or non-sulfated repeating units of L- and/or D-galactose and 3,6-anhydro-D- or L-galactose[22]. Another class of pharmaceutically important mostly sulfated polysaccharides is glycosaminoglycans (GAGs). GAG such as heparin is a highly sulfated polysaccharide, which is clinically used as an anticoagulant[23].

1.2 Enzymatic degradation of polymers

Biopolymers such as cellulose, hemicellulose, and lignin are abundant in nature as lignocellulosic biomass[24]. These biopolymers built huge networks by covalent and non- covalent crosslinking[24]. Among them, cellulose is the most abundant and simple linear homopolymer, which consists of D-glucose units linked by β-1,4 glycosidic bonds to grow as cellobiose building blocks[25]. Degradation of cellulose is generally performed in nature by cellulases (endoglucanases/exoglucanases), which hydrolyze internal β-1,4 glycosidic bonds or set saccharide units free from the end of the chain. Cellulases are typically made

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General introduction of two domains: catalytic domain and cellulose binding domain (CBD; in general CBM: carbohydrate binding domain)[26]. CBM binds to the carbohydrate polymer and thereby locally bring the catalytic domain to it to initiate the degradation[26-27]. Hemicellulose (heteropolymer) consists of D-xylose, D-mannose, D-galactose, D-glucose, L-arabinose, 4-O-methyl-glucuronic, D-galacturonic, and D-glucuronic acids, whereas the sugar units are linked by β-1,4 and branched β-1,3-glycosidic bonds[24]. Due to a high xylan content in hemicellulose, xylanases play the main role in depolymerization of hemicellulose[28]. Microorganisms produce biodegradable polyesters e.g., polyhydroxyalkanoates (PHA) under abnormal conditions, which serve as energy storage source[25]. Polyhydroxybutyrate (PHB) and polyhydroxyvalerate (PHV) are used as biodegradable polyester for instance in the medical and pharmaceutical industries (e.g., suture thread, implant, drug release)[29]. Biological degradation of such polyesters is performed by PHA depolymerases. Similar to cellulases, PHA depolymerases act on the polymer PHA via a substrate-binding domain and the catalytic domain performs the depolymerization[30].

Synthetic aliphatic, aromatic and mixed polymers are widely used plastics and end up as plastic waste in the environment[31]. The degradation of plastic is difficult due to their inertness and durability[32]. Nevertheless, depolymerization of some synthetic polymers by microbes and their metabolic enzymes seems to be a potential tool for greener plastic degradation. Microbial plastic degradation takes place in sequential steps (Figure 2): 1) bio-fragmentation (hydrolytic cleavage of polymers by enzymes), 2) assimilation

(uptake of oligomers by microbes), and 3) mineralization (production of CO2, H2O, CH4, and other metabolites)[25]. Mostly extracellular depolymerases are involved in the enzymatic breakdown of synthetic polymers[25]. Polyesters and polyurethanes are susceptible to microbial/enzymatic attack due to their accessible ester and urethane bonds[29]. Microbes assimilate the degradation products as carbon and nitrogen source[33]. Aliphatic polyesters such as polylactic acid (PLA), polycaprolactone (PCL) undergo easily hydrolysis by fungal or bacterial enzymes like esterases, in which the ester bonds within the polymer are cleaved[25]. Aromatic-aliphatic polyesters e.g., polyethylene terephthalate (thermoplastic polymers) are also reported to be degraded by like esterase[34], lipases[35], and cutinases[36]. Thermoset polymers like polyurethane can

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General introduction

be degraded by PUR esterases[37], polyamidases[38], and cutinases[39]. Biodegradation of synthetic polymers is cheap, environmentally friendly, and thereby very much appreciated by the society[29]. Though, the slow rate of microbial and enzymatical degradation makes it challenging.

Figure 2. Microbial degradation of synthetic polymer. Microbes produces extracellular enzymes (depolymerases with or without substrate binding domain) that target synthetic polymer for degradation (bio-fragmentation). Thereby, free monomers, dimers, and oligomers are utilized by microbes as energy source (assimilation). And finally, oxidized metabolites (CO2, H2O, and CH4) are produced (mineralization). Adapted from Pathak and Navneet, 2017.

1.3 Protein engineering

Nature has been evolving proteins for over billions of years to adapt them to environmental changes[40]. Even though proteins and their functions are highly complex, nature is able to engineer their behavior, functionality, and even structure[41]. Humans are also employing evolutionary approaches for thousands of years for plant and animal breeding purposes to achieve desirable characteristics[40]. Protein engineering is a highly attractive field since three to four decades because of the growing demand of enzymes as

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General introduction biocatalysts. While chemical catalysts are already well-established tools and delivered products of daily use without any help of biocatalysts for over 200 years, biocatalysts are perhaps making their way rapidly to serve as an alternative due to a bunch of advantages[42]. Dissimilar to chemical catalysts, biocatalysts show selectivity (regio-, chemo-, and enantioselectivity), biodegradability, sustainability, and function in aqueous media without high temperature and pressure[42-43]. Enzymes have been already part of large number of industrial success stories including hydrolases in detergent industries, amylases in food industries, phytases in feed industries, laccases textile industries, and more[42-43]. Despite all these great features, enzymes often face some crucial limitations to be applied for industrial purposes. Among them, low activity, low stability (temperature, pH, solvent), short lifetime, and low turnover number are the main drawbacks making many interesting enzymes unsuitable for chemical synthesis[42]. Keeping nature’s evolutionary performance in mind, protein engineers have been designing and tailoring existing proteins to overcome the aforementioned limitations by using laboratory evolution methods[41]. Successfully evolved enzymes for non-biological conditions (e.g., pH, temperature, and solvent resistance) find their applications in the synthesis of bulk chemicals as well as pharmaceutical precursors[44]. The method of enzyme engineering can vary depending on the targeted enzyme property. There are generally two directions of protein engineering: i) rational design and ii) directed evolution[45].

1.3.1 Directed evolution of enzymes

Directed evolution of enzymes has emerged as a powerful and versatile algorithm for tailoring enzymes properties to industrial demands as well as understanding the structure-function relationships[43b, 46]. This principle was first mentioned by Spiegelman in 1965-1967 by performing “evolution in a tube” complementing Darwinian evolution with some remarkable simplicity. Directed evolution i) targets rather individual molecules within an organism (microevolution) rather than a whole organism (macroevolution) as existing in nature; ii) aims targeted, not blindly by chance; iii) does not require thousands and millions of years, but happens rapidly[47]. Such a laboratory experiment consists of three key steps, the gene encoding a protein of interest is randomized by mutagenesis methods, applying an appropriate screening and selection strategy mutants are identified

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General introduction

from the pool of mutant libraries showing an improved property, and finally the genes encoding improved variants are isolated[48] (Figure 3). Iterative cycles of these three steps accumulate beneficial mutations in improved variants, thus they are desirably evolved towards new functions and properties[40, 49]. In the past two decades, enzyme engineering via directed evolution has been successfully applied to tailor various enzyme properties counting activity[50], regioselectivity[51], thermostability[52], enantioselectivity[53], organic solvent stability[54], and pH stability[55]. Prof. Dr. Frances Arnold received the Nobel prize in chemistry for her pioneer work on “directed evolution of enzymes” in 2018[56].

Selection of the appropriate method to generate diversity plays an important role in a directed evolution experiment[44]. Typically, PCR-based random mutagenesis methods (e.g., error-prone PCR[45], SeSaM[57], CASTing[58], cepPCR[59]) and DNA recombination methods (e.g., StEP[47], DNA shuffling[60], PTRec[61]) are widely used for randomization of the parent gene of interest in Step I. DNA recombination methods allow recombination of genes often derived from the same family that are preselected by nature for their function[62]. Oppositely, random mutagenesis approaches integrate mutations in one

Figure 3. General scheme of a directed evolution campaign. Three steps are performed iteratively in directed evolution: diversity generation is performed on DNA level, followed by screening of generated variants on the protein level for a target property, and finally, isolation and sequence analysis of improved variant, which is the starting point of the next cycle. The figure was adapted from Güven et al. 2010.

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General introduction gene of interest to adapt proteins further to process conditions or to elucidate structure- function relationships. Also, synthetic chemistry-based methods (e.g., randomization of codons) and whole cell approaches (e.g., mutator strains, mutator plasmids) exist[62]. Random mutagenesis libraries commonly consist of 1,000 to 10,000 clones that need to be screened in step II of a directed evolution approach[44]. Robust and reliable enzyme production and thus high throughput screening system is necessary to perform a successful evolution campaign[44]. Depending on the library size, gene expression system, and reaction, screening can be performed with the ascending order of throughput in agar plates, microtiter plates (MTP; 96-, 384-well format), cells as microreactors, whole cell, and in vitro compartmentalization[44]. Agar plate screening systems are considered as low to medium throughput and mostly suitable as pre-screening since they are rather qualitative than quantitative methods. The most popular screening system is MTP format due to the availability of standardized equipment, flexibility, and ability to quantify enzyme activity, which makes it medium to high throughput category of screening[44]. MTP screening is always dependent on the chromogenic or fluorogenic reaction compounds limiting screening of enzymes converting or producing no colored fluorescent compounds. Screening in high to ultra-high throughput manner can be achieved by microencapsulation (water/oil/water microemulsions) of whole cells or in vitro compartmentalization in combination with flow cytometry[63]. The later approach is very useful since this approach is time-saving by removing all the cell growth steps and allows library translation without loss of any gene diversity and screening of toxic proteins[64]. Since the diversity generated is astronomical (e.g., a small protein with 100 amino acid can theoretically yield 20100 = ~10130 variants) and most of the enzymes range from 180- 600 amino acids, it is impossible to screen all the protein sequence space with existing methods[41, 43b, 44]. However, identification of improved variants in step III of the campaign is usually possible by screening a library comprising 1,000–2,000 variants as almost 50% of the positions contribute to the improved property[65]. Commonly 3-5 variants with improved properties are identified after each round of evolution. The “best” variant serves as a template to start the iterative cycles of the subsequent round of evolution. In average, identified variants comprise after each round one or more amino acid substitution depending on the choice of the mutagenesis method[44].

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General introduction

1.3.2 KnowVolution – Directed Evolution 2.0

As aforementioned, directed evolution has been serving over the past few decades as a very popular and powerful tool. In a typical directed enzyme evolution campaign, often ≥ 4 rounds are necessary to end up with variants exhibiting the improved targeted property. Thus, the traditional approach is very time consuming (more than a year) and requires a huge screening effort. Moreover, in each round of evolution mutations are accumulated, whereas all of them might not necessarily influence the targeted property. Typically, the “best” variant after several rounds of directed evolution ends up with 6 to 12 substitutions[66]. Protein engineers are moving toward combined and knowledge gaining evolution approaches to overcome some of the mentioned drawbacks. The ideal next-generation directed evolution should require less time, less experimental effort, and be able to gain knowledge about the structure-function relationship on a molecular level. Nowadays, a comprehensive protein design with combined methods is of growing interest, which brings the desired properties for ideal directed enzyme evolution with it. Important examples in this field are: i) ProSAR (protein sequence activity relationship analysis based strategy)[60], ii) MORPHING (mutagenic organized recombination process by homologous in vivo grouping)[67], and iii) KnowVolution (knowledge gaining directed evolution)[68].

KnowVolution is one of the newest concept reported in the literature, which consists of four phases[68] (Figure 4). The phase I (identification) requiring approximately 2 weeks starts with a traditional directed evolution experiment, in which gene diversity is generated by random mutagenesis and/or DNA shuffling. Depending on the gene size and the chosen mutagenesis method, typically 20 beneficial variants are identified. Around 12 positions should be considered to be involved in the improved property, whereas these often are found clustered in a few protein regions and can occur several times. Site- saturation mutagenesis of potentially beneficial positions is performed in 2-3 weeks in phase II (determination). This phase allows the determination of positions contributing to the improvement since often 50% of the identified positions in phase I are not beneficial. Sequencing of 16 beneficial clones per positions is suggested to gain already insights about the type of amino acid substitutions. In phase II, 4-6 amino positions with known substitutions contributing to the improvement are expected. Phase III (selection) of

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General introduction

KnowVolution deals with the computer-assisted structural analysis of determined positions and substitutions in phase II. The visual inspection of the protein structure or a homology model allows a deeper knowledge in a few days, whether the positions are in close proximity and how they can interact with each other. Furthermore, the selection phase enables to answer crucial questions to make a decision for the final phase including the positions to be recombined, the positions to be grouped, and the substitutions to be generated. Finally, the phase IV (recombination) materializes in 1-2 weeks the suggestions made in phase III by recombining amino acid positions by multi-site saturation (3 to 5 positions) or one by one recombination of most beneficial substitutions. Site-saturation (phase II) and recombination (phase III) of selected positions achieve improvements which are equivalent or even better than those obtained in several rounds of standard directed evolution experiments. KnowVolution campaigns can be performed in iterative manner as well if further improvements are desired[68]. KnowVolution success stories are well documented in the literature for e.g., glucose oxidase[69], alkaline protease[70], phytase[55, 71], cellulase[72], and recently hyaluronic acid synthase[73].

Figure 4. Four phases of KnowVolution. A KnowVolution campaign comprises four defined phases: i) identification of potential beneficial positions by applying directed evolution, ii) determination of beneficial substitutions by individual site saturation, iii) selection of amino acid substitutions for recombination by visual inspection via computational analysis, and iv) recombination of selected amino acid substitutions by multiple site saturation or iterative recombination. Iterative cycles of KnowVolution is possible depending on the desired improvement of target property. Adapted from Cheng et al. 2015.

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Aims of the dissertation

1.4 Aims of the dissertation

GAGs are very important functional polysaccharides in all kingdoms of life and are of high pathophysiological interest. Since the isolation of natural GAGs is mainly limited to animal sources, in vitro synthesis of GAGs and/or GAG-like molecules is sustainable for a longer run. Enzymatic sulfation of GAG building blocks (e.g., GlcNAc, GalNAc) or GAG mimetics (e.g., glucose, cellobiose) might represent the first step of an alternative synthesis. The first aim of this dissertation, therefore, is to design a novel aryl sulfotransferase for effective and selective sulfation of polysaccharide units. To achieve this, the aryl sulfotransferase B (ASTB) was re-engineered to improve its specific activity toward polysaccharide building blocks. A robust and continuous screening system was developed and validated in one round of directed evolution using the monosaccharide, GlcNAc. A KnowVolution campaign furthermore improved the activity of ASTB toward a disaccharide (cellobiose). First structural and functional insights from computational analysis were gained to understand beneficial amino acid substitutions. Final ASTB variants were also characterized. To benchmark ASTB as a suitable biocatalyst for sulfation, a product (GlcNAc-linker-tBoc monosulfate) was synthesized by ASTB wild-type and its improved variants to determine conversion, yield, and purity. Finally, structural elucidation by NMR was performed to determine the position of sulfation. This work will open a new catalytic route to regioselectively sulfate saccharides for in vitro synthesis of GAGs/GAG mimetics.

The dramatic increase of plastic usage in the last 70 years generated an astronomical amount of plastic waste. Particularly, plastic debris produced by extensive degradation (e.g., microplastic, nanoplastic) pollute coastal and marine habitats constantly and thereby end up in the food chain. Furthermore, humans are directly or indirectly responsible for releasing microplastic into the environment through many cosmetic products. Sustainable plastic management strategies to avoid and minimize microplastic contaminations are of high importance to ensure a supply of healthy food and water. Biodegradation (microbial and/or enzymatic) is one sustainable and green way to decompose microplastic. Hydrolytic enzymes (e.g., cutinases, esterases, and amidases) are known to degrade synthetic polymers such as PET and PUR. However, the degradation happens often very slowly due to the lack of enzyme-polymer adjacency. To address this, as the second aim of this dissertation, enzymes were designed for effective and faster

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Aims of the dissertation degradation of synthetic polymers. Fusion proteins made of a polymer degrading enzyme (cutinase) and a polymer adhesion promoter (anchor peptide) were designed for controlled and effective degradation. Two reported anchor peptides (LCI and TA2) were analyzed for the binding ability to selected synthetic polymers (polyester-PUR and PET). The anchor peptides were then fused to the target cutinase, Tcur1278. Degradation of polyester-PUR nanoparticles by Tcur1278 with and without fused anchor peptide were evaluated through degradation kinetics and nanoparticle half-life. Methods such as DLS and FE-SEM were further applied to achieve a closer look into the degraded nanoparticles in solution and as a solid particle layer. Finally, the developed platform was expanded toward another synthetic polymer PET (PET-film). Weight loss of PET-films after the treatment with Tcur1278 wild-type and Tcur1278-anchor peptide fusion were determined to evaluate their degradation performance.

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2. Engineering of an aryl sulfotransferase toward sulfation of saccharides

2.1 Declaration

Parts of this chapter has been published:

Islam S, Mate DM, Martínez R, Jakob F, Schwaneberg U. 2018. A robust protocol for directed aryl sulfotransferase evolution towards the carbohydrate building block GlcNAc. Biotechnology and Bioengineering, 115:1106-1115[74] Islam S, Laaf D, Infanzón B, Palentová H, Davari MD, Jakob F, Křen V, Elling L, Schwaneberg U. 2018. KnowVolution campaign of an aryl sulfotransferase increases activity toward cellobiose. Chemistry – A European Journal, 24(64), 17117-17124[75]

2.2 State of the art

2.2.1 Sulfation in nature

Sulfation often termed as sulfurylation (should not be confused with sulfonation) is a

- covalent functionalization process of a molecule, in which a sulfate group (OSO3 ) from a donor molecule is attached to an acceptor molecule[17]. Sulfation can take place at O- or N-atom, therefore, called O- and N-sulfation, respectively. Such a functionalization of a molecule provides various physicochemical properties to it, e.g., anionic charge, size, degradability, and solubility[76]. Sulfation is a prominent biological event in all kingdoms of life[77]. Nature is a very old specialist in using sulfoconjugation on endogenous and exogenous molecules for detoxification and cellular response mechanisms[78]. Especially in the case of xenobiotics and small endogenous molecules (e.g., neurotransmitter, drugs, and steroids), sulfation takes place in a variety of organisms. Sulfation is defined to be crucial for the detoxification pathway since the toxic molecules become more water- soluble and get easier excreted through the kidneys and bile[79]. Nevertheless, sulfation can be also involved in the metabolic activation of carcinogenic and mutagenic pathways[80]. Biological processes such as viral entry into cells, leukocyte adhesion, molecular signaling, and anticoagulation get activated based upon sulfoconjugation events. A large enzyme class called sulfotransferases (STs) are responsible for sulfation in biological systems[17].

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2.2.2 Sulfation of glycosaminoglycans

A special class of sulfated polysaccharides is well-known as GAGs, which is mostly abundant on the animal cell surface and extracellular matrices (ECM)[81]. GAGs are very important biomolecules since these display various kind of pharmacological activities including anticoagulant, antiviral, and antithrombotic activities[82]. They find their functions both in the structural organization of ECM and regulation of biological activities of e.g., growth factors, cytokines, morphogens, and enzymes[83]. A variety of biological functions of GAGs depend on their specific interactions with proteins, which is commonly mediated by the negatively charged groups (e.g., sulfate groups) in GAGs and positively charged residues in respective interacting proteins[84]. Generally, GAGs are linear and highly sulfated polysaccharides that are composed of disaccharide repeating units of an amino sugar (N-acetylated or N-sulfated glucosamine or N-acetylgalactosamine) and a uronic acid (glucuronic acid or iduronic acid) or galactose[85]. Five main types of GAGs are synthesized in biological pathways (Figure 5 and Table 1). Among them, heparin/heparan sulfate (HS), chondroitin sulfate (CS), dermatan sulfate (DS), and keratan sulfate (KS) are naturally sulfated. Though, hyaluronan (HA) is the only GAG which is not sulfated in nature. The sulfation of GAG happens in a very specific manner by enzyme class called PAPS-dependent sulfotransferase, which sacrifices a sulfate group of the sulfate donor (3’-phosphoadenosine 5’-phosphosulfate) to sulfate the building blocks of GAGs[17].

Figure 5. General structure of glycosaminoglycans. Building blocks of heparin/heparan sulfate (HS), chondroitin sulfate (CS), dermatan sulfate (DS), keratan sulfate (KS), and hyaluronan (HA) are shown. Figure is taken from Gemma et al. 2008.

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Table 1. GAGs with their location and functions. Data collected from Wong et al. 2004, Gemma et al. 2008, Lindahl et al. 2015, DeAngelis et al. 2013.

GAGs Location Function

Angiogenesis, blood coagulation, Heparin/Heparan sulfate Cell surface, extracellular growth factor binding, and (HS) matrix activation

Chondroitin sulfate Cell surface, brain, cartilage Cartilage development, neuronal (CS) functions, lymphocyte binding, T-cell response

Dermatan sulfate Skin, central nervous system Carcinogenesis, wound repair, (DS) and fibrosis

Keratan sulfate Cartilage, cornea Corneal transparency (KS)

Hyaluronan Extra-, peri-cellular matrix Elastoviscosity of liquid (HA) connective tissues, control of tissue hydration and water transport, cell detachment, mitosis, migration, tumor development and metastasis, and inflammation

2.2.3 Chemical sulfation of (poly)saccharides

Chemical sulfation is a widely discussed topic in polysaccharide chemistry. Chemically sulfated cellulose was reported to be highly soluble[87], anticoagulant[88], and antiviral[89]. Moreover, sulfated cellulose undergoes better enzymatic degradation compared to natural cellulose due to improved solubility of cellulose sulfate in water[87, 90]. These novel properties are of high interest for pharmaceutical and biotechnological application of cellulose[91].

Chemical processes can perform sulfation mainly in two ways: homogeneous sulfation

[92] (e.g., N2O4/DMF/SO3, [Bmim]Cl/DMF/ClSO3H, DMF/NH2SO3H) or heterogeneous

[93] sulfation (e.g., propanol/H2SO4, pyridine/ClSO3H) . Regioselective sulfation of polysaccharides by chemical means is a challenging task without protection/deprotection chemistry and the C-6 position is targeted preferably for sulfation (preference: C-6 > C-2 >(>>) C-3)[18, 94] Regioselective sulfation at C-3 is rarely reported in literature, but it is

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[93b] possible by employing cellulose-2,6-trifluoroacetate in a SO3/pyridine/DMF system . In case of chemical sulfation of GAGs (e.g., dermatan sulfate and chondroitin sulfate), the C- 6 position in GalNAc (amino sugar in the disaccharide GAG building block) show dominant

sulfation (tri- or tetrabutylammonium salts in SO3∙DMF) compared to other targetable sites (negligible sulfation at C-4 in GalNAc and C-3 in GlcA/L-IdoA)[95]. Moreover, chemical sulfation of naturally non-sulfated GAGs (e.g., hyaluronic acid[96] and heparosan[97]) revealed the C-6 position as preferred sulfation target in contrast to C-4.

2.2.4 Classes of sulfotransferases

Sulfotransferases catalyze the transfer of a sulfate group from a donor to an acceptor molecule[17]. They can be classified depending on their donor molecule (PAPS-dependent and PAPS-independent), heritage (eukaryotic and prokaryotic) and location (cytosolic and membrane-associated). Generally, sulfotransferases are distinguished according to their cellular location, which is mainly eukaryotic sulfotransferases. Human sulfotransferases are very well-described in the literature[79-80, 98]. Especially, human cytosolic sulfotransferases (SULT1, SULT2, and SULT4 families) have been subjected to deeper studies including crystal structures over the few decades[99]. The SULT3 family has been discovered in mouse and rabbit so far sulfating only amino groups[100]. There is only little known about the SULT5 family, of which only one member was isolated from mice[101]. Cytosolic SULTs are mainly responsible for the metabolism of xenobiotics and small endogenous substrates such as steroids, bile acids, and neurotransmitters[79]. Cytosolic

Figure 6. General protein structure of cytosolic PAPS-dependent ST and catalyzed reaction. A) Structure of Human SULT1A1 in homodimeric form (PDB: 2D06). Monomers are indicated with different colors (blue and cyan). B) A PAPS-dependent sulfotransferase transfers sulfate group from donor PAPS to an acceptor carrying an alcohol group. The main product of the reaction is a sulfated acceptor molecule, while PAP is the by-product. Figure 6B taken from Wong et al. 2004.

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SULTs have a broad substrate spectrum including phenol, catecholamines, hydroxysteroids, estrogens, aryl hydroxylamines, and thyroid hormones[17, 79]. 3’- phosphoadenosine 5’-phosphosulfate (PAPS) is the universal sulfate donor in the reactions catalyzed by cytosolic SULTs[102]. The general reaction scheme is shown in Figure 6B, which requires an acceptor (e.g., R-OH) and PAPS as a donor. The reaction products

- are sulfated acceptor (R-OSO3 ) and the desulfated 3’-phosphoadenosine-5’-phosphate (PAP). The SULTs are typically globular proteins with a single α/β domain forming a five- stranded parallel β-sheets surrounded by α-helices (Figure 6A). The β-sheets forms the PAPS- and the core catalytic residues[99a].

The other large class of STs is called membrane-associated STs located in the Golgi apparatus of the cell and are involved in crucial biological processes by sulfating larger biomolecules, such as carbohydrates and proteins[79, 98c]. This class uses the universal donor PAPS and as acceptor sugar moieties as well as protein/peptides (tyrosine provides the acceptor OH-group)[17, 76]. Membrane-associated STs are highly specific to their acceptor molecules other than cytosolic SULTs. Sulfated carbohydrates and proteins act in various extracellular recognition processes[76].

Sulfotransferases can also be classified according to their donor molecules. Since the eukaryotic STs (cytosolic SULTs and membrane-associated STs) use PAPS as the universal donor, these are termed as PAPS-dependent STs. There are other sulfotransferases that do not accept PAPS as a donor, but phenolic donors such as para-nitrophenylsulfate, 4- acetylphenylsulfate, 4-methylumbelliferylsulfate[77]. PAPS-independent STs are found in prokaryotes that are mostly located in the periplasm of δ- and ε-proteobacteria and called aryl(sulfate) sulfotransferases (ASTs/ASSTs). ASTs are responsible for sulfation in commensal intestinal bacteria accepting a very promiscuous type of aromatic sulfuric esters as donors and phenols as acceptors[77]. ASTs were isolated from different bacterial origins in human, rat, and mouse intestinal flora i.e., human intestinal bacterium Eubacterium A-44[103], rat intestinal bacterium Klebsiella K-36[104], and mouse intestinal bacterium Haemophilus K-12[105]. Furthermore, PAPS-independent ASTs were isolated and characterized derived from Enterobacter amnigenus AR-37[106], Citrobacter freundii[107], Salmonella typhimurium[108], and uropathogenic Escherichia coli CFT073[109]. Interestingly, among all the E. coli strains, ASST is upregulated only in the uropathogenic

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organisms suggesting that ASST might play a role in urinary tract bacterial infections[110]. ASST from Escherichia coli CFT073 is one of the most deeply studied PAPS-independent ASST elucidating the genomic context, molecular protein structure, reaction mechanism, and architecture[77, 109, 111].

The uropathogenic E. coli strain CFT073 contain the gene cluster encoding ASST and two disulfide bond forming proteins (DsbL and DsbI) as a tricistronic operon on the genome. DsbL and DslBI are homologous to DslA/DslB dithiol oxidase system, that is only found in the uropathogenic strains[111]. The specific DsbL/DsbI system catalyzes the single disulfide bond formation in each monomer of the homodimeric ASST[109]. Three crystal structures of ASST from E. coli CFT073 were reported ( (PDB) accession number: 3ELQ, 3ETS, and 3ETT)[111] describing ASST as a homodimeric protein (Figure 7A) with a small N-terminal domain (residues 1–116) contributing in a seven-stranded β-propeller fold and a large C-terminal domain (residues 117–571). The active site is formed in the cleft of the center of the propeller[109]. Malojcić et al. suggested a possible reaction mechanism that follows the ping-pong bi-bi mechanism (Figure 7B). Ping-pong bi-bi reaction mechanism is based on a two-substrate and two-product system. Bacterial ASSTs

Figure 7. Protein structure of ASST from uropathogenic E. coli and its reaction mechanism. A) ASST in its homodimeric state (PDB: 3ETS). B) Reaction mechanism of ASST following ping-pong- bi-bi mechanism. In the first step the atom of p-nitrophenyl sulfate (sulfate donor) is nucleophilically attacked by the catalytic histidine (H436), which results in a covalent sulfohistidine intermediate and p-nitrophenolate. Dissociation of p-nitrophenolate and binding of the phenolic acceptor molecule occurs during the second step, followed by the nucleophilic attack from phenol on the sulfur group in the intermediate. Finally, sulfated phenol is released afterwards from the active site. Figure 7B is taken from Malojcic et al. 2008.

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Engineering of an aryl sulfotransferase toward sulfation of saccharides follow this kind of multi-substrate reaction system, in which a histidine has the catalytic activity similar to eukaryotic sulfotransferases. The catalytic reaction of ASST from E. coli follows two steps. During the first step, the sulfate donor molecule (e.g., para- nitrophenylsulfate (pNPS)) binds into the active site and the histidine residue at position 436 initiates a nucleophilic attack on the S-atom of the donor that the enzyme. After the release of the co-product (p-nitrophenylate), the second step begins with the binding of the acceptor substrate (e.g., phenol). The sulfate group from the covalent sulfohistidine intermediate is then transferred to the acceptor and dissociation of the final product that is sulfated by the enzyme follows[111].

2.2.5 Aryl sulfotransferases from Desulfitobacterium hafniense

Desulfitobacterium hafniense is a gram-positive dehalorespiring bacterium and belongs to the Clostridia class that dechlorinates halogenated compounds under anaerobic conditions by dehalorespiration[112]. Two different aryl sulfotransferases (ASTA: GeneBank accession – KTE90238 and ASTB: GeneBank accession – WP_015942610) from D. hafniense were recently isolated. These aryl sulfotransferases from Firmicutes (class I) share relatively less sequence similarity with other ASSTs from Proteobacteria (class II). Van der Horst and co-workers reported ASTA that consists of 628 amino acids (molecular weight ~74.4 kDa) and interestingly shows activity not only towards phenolic substrates but also with aliphatic alcohols (e.g., butanol, pentanol, octanol, glycerol, and D-glucose). The sulfation of aliphatic acceptors by an aryl sulfotransferase was unexpected since these enzymes are known for sulfating phenolic acceptors[77]. This finding opens new routes to sulfate non-phenolic molecules including carbohydrate building blocks[74]. In past six years, ASTA was reported by different groups showing its outstanding performance as a biocatalyst sulfating steroids[113], flavonoids[114], and as recently reported, large molecules like lignin[115]. Moreover, substrate engineering was reported to increase the sulfation activity of ASTA toward small carbohydrates (e.g., β-D-glucopyranose, β-D-N- acetylglucosamine) by covalently introducing an aromatic group (phenyl or p- nitrophenyl)[116]. Another aryl sulfotransferase (ASTB) from D. hafniense (626 amino acids long) can be found in NCBI deposited as ArsR family transcriptional regulator. ASTA and ASTB share 46% protein sequence identity (Figure 8).

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Figure 8. Sequence alignment between ASTA and ASTB from Desulfitobacterium hafniense. Identical amino acids are highlighted by green color. The active site residue His350 is marked by a red box.

2.2.6 Directed evolution of sulfotransferases

PAPS-dependent sulfotransferases have been targeted for directed evolution campaigns to improve substrate specificity[117] and thermostability[99b]. In these campaigns, PAPS was used as the sulfate donor with colorimetric (p-nitrophenol; pNP) or fluorogenic (3-cyano- 7-hydroxycoumarin; 3CyC) acceptor molecules. The screening systems using pNP and 3CyC were successfully employed in three directed evolution studies. In all three campaigns, human PAPS-dependent cytosolic sulfotransferases (SULT1A1 or SULT1E1) were targeted[99b, 117-118]. For instance, 600 clones of SULTE1 generated by targeted mutagenesis were screened with the fluorometric 3CyC assay in MTP-format. The most beneficial SULTE1 variants showed an up to 10.6°C enhanced thermal resistance[117].

In the case of PAPS-independent STs (e.g., bacterial ASTs), no successful directed evolution campaign has been reported. Nevertheless, in one publication a directed evolution attempt for increased uridine sulfation was reported for a bacterial sulfotransferase LipB[119]. LipB was derived from Streptomyces sp. SANK 60405, which is

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Engineering of an aryl sulfotransferase toward sulfation of saccharides responsible for sulfation of the nucleoside antibiotic liposidomycin B-I and other acceptors. The random LipB mutagenesis library was generated by error-prone PCR (epPCR). A colorimetric pNPS-based endpoint assay in 96-well microtiter plate (MTP)- format was used to screen 200 variants resulting in no LipB variant with improved uridine sulfation[119].

2.2.7 Objectives

The main goal of this chapter was to tailor a bacterial aryl sulfotransferase to improve its specific activity towards small saccharides (Figure 9). The objectives can be divided into three parts:

i) Establishment of a robust screening system for selected enzyme and acceptor molecules ii) Validation of the screening system in a round of directed evolution toward a monosaccharide acceptor (GlcNAc) iii) A full KnowVolution campaign of an AST toward a disaccharide acceptor (cellobiose)

Figure 9. Engineering of aryl sulfotransferase B (ASTB) for higher sulfation activity toward saccharides. Illustrated is ASTB as dimer and the saccharides targeted for sulfation are indicated as rectangles

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2.3 Material and methods

Material and methods for this chapter were adapted from Islam et al. 2018[74-75].

2.3.1 Chemicals

All chemicals used in this study were of analytical grade or higher quality and were purchased from AppliChem (Darmstadt, Germany), Sigma Aldrich Chemie (Taufkrichen, Germany) or VWR International (Darmstadt, Germany) unless specified. The restriction enzymes and were purchased from New England Biolabs (Frankfurt am Main, Germany) while DNA polymerases were prepared in-house. The SeSaM kit for library generation including buffers, primers, nucleotides, and polymerases was obtained from SeSaM Biotech GmbH (Aachen, Germany).

2.3.2 Cloning of astA and astB

The wildtype astA (GeneBank accession number: KTE90238) and astB (GeneBank accession number: WP_015942610) gene from D. hafniense were received from evoxx GmbH (Monheim, Germany) as E. coli codon optimized synthetic gene including NdeI and SacI restriction sites for cloning. After double digestion with NdeI and SacI the target gene was sub-cloned into pET22b(+) applying T4 DNA ligase. The resulting plasmids were transformed into chemically competent E. coli BL21 Gold (DE3) (Agilent Technologies, Santa Clara, USA). For later purification of the target protein, Strep-tag II (Ser-Ala-Trp- Ser- His-Pro-Gln-Phe-Glu-Lys)[120] was attached at the C-terminal end of respective genes by using 2-step PCR protocol (Table 2). The PCRs were performed in 200 µL PCR tubes (VWR, Darmstadt, Germany) using a thermal cycler gradient PCR machine (Mastercycler® pro, Eppendorf, Hamburg, Germany). Sequencing of all sub-cloned genes at Eurofins MWG Operons (Ebersberg, Germany) confirmed correct cloning and insertion of the Strep-tag II.

Table 2. Two-step PCR program. Step 1 Temperature [°C] Time [sec] Cycle [x] Initial denaturation 98 60 1 Denaturation 98 30 Annealing 55 30 5 Elongation 72 240 Step 2 Denaturation 98 30 Annealing 55 30 20 Elongation 72 240 Final elongation 72 600 1

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2.3.3 Construction of astB SeSaM library

A random mutagenesis library of astB[74] was generated using sequence saturation mutagenesis (SeSaM-Tv-classic[121]). The SeSaM-library was generated in four steps: 1) generation of a DNA-fragment pool with random size distribution; 2) enzymatic fragment elongation with universal bases; 3) full-length gene synthesis; and 4) universal base replacement[57].

Preliminary step: Generation of dsDNA template for Step 1 and Step 3 For step 1 and step 3 template-generation, a 50 µL PCR-mixture contained: 1x PhuS Buffer; (New England Biolabs); 0.2 mM of each dNTP; 0.5 µM of each primer (SeSaM_F1 and R3 for Step 1 forward template-DNA, SeSaM_R3 and F1 for Step 1 reverse template-DNA, SeSaM_R3 and F1_up Step 3 forward template-DNA and SeSaM_F1 and R3_dn for Step 3 reverse template-DNA; Table 12); 0.5 U PhuS polymerase (homemade), and 40 ng/kb plasmid template DNA. The following PCR protocol was employed: 98°C for 30 s (1 cycle); 98°C for 15 s, 65°C for 30 s, 72°C for 70 s (18 cycles); 72°C for 5 min (1 cycle). The PCR products were digested with DpnI (20 U, 37°C, 3 h) and purified (Nucleospin Extract II kit from Macherey Nagel, Düren, Germany).

Step 1: Generation of ssDNA fragment pool with a random size distribution The generated ASTB Step 1 template-DNA was amplified by PCR in presence of dATPαS and dGTPαS to randomly incorporate chemically cleavable phosphorothioated bonds. PCR solutions (50 µL) contained: 1x Taq Pol buffer, 0.5 µM of each primer (Bio_SeSaM_F and R3 for Step 1 forward template DNA and Bio_SeSaM_R and F1 for Step 1 reverse template- DNA; Table 12), 5 U Taq DNA polymerase, and 360 ng/kb of the SeSaM template-DNA (Step 1 forward and reverse template DNA). According to the desired library, the following nucleotide combinations were used for A and G libraries: 0.2 mM of each dATP, dGTP, dTTP and dCTP. The following dATPαS and dGTPαS concentrations were used: A-forward library: 35%, G-forward library: 25%, A-reverse library: 30%, A-reverse library: 25%. For both setups, the same PCR protocol was followed: 94°C for 2 min (1 cycle); 94°C for 30 s, 65°C for 30 s, 72°C for 70 s (19 cycles); 72°C for 3 min (1 cycle). PCR products were purified

(Nucleo Extract II kit with NT buffer) and eluted (92 µL ddH2O). The phosphorothiodiester bonds were cleaved (30 min, 70°C) in SeSaM-Taq DNA polymerase buffer (1x),

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supplemented with iodine (20 mM in EtOH). Cleaved biotinylated DNA fragments were isolated by magnetic streptavidin beads (M-PVA SAV1, Chemagen, Baesweiler, Germany) and ssDNA was purified employing the NTC binding buffer (Nucleospin Extract II kit).

Step 2: Enzymatic fragment elongation with universal bases Single-stranded DNA fragments generated in Step 1 were elongated at the 3´-OH groups via TdT (terminal deoxynucleotidyl transferase) by the incorporation of dPTP (37°C, 120 min) and terminated by heat inactivation (30 min, 75°C). Reaction mixtures (25 µL) contained: 1x TdT buffer, 0.25 mM CoCl2, ~1 pmol of ssDNA fragment (from Step 1), 5 pmol dPTP, and 10 U TdT. The ssDNA was purified using NucleoSpin Gel and PCR Clean- up kit with NTC buffer (Macherey Nagel, Düren, Germany).

Step 3: Full-length gene synthesis Full-length astB genes were generated using dPTPαS elongated DNA fragments (from step 2) as primers and step 3 dsDNA as templates (generated during the preliminary step). The PCRs (50 µL) contained: 80 ng of ssDNA from step 2, 80 ng of dsDNA step 3 DNA template, 1x Super Taq buffer, 0.2 mM of each dNTPs, and 1 U 3D1 polymerase. PCR protocolwas employed as follows: 94°C for 2 min (1 cycle); 94°C for 30 s, 52°C for 30 s, 72°C for 2 min 10 s (29 cycles). PCR products were purified using NucleoSpin Gel and PCR Clean-up kit (Macharey Nagel, Düren, Germany).

Step 4: Universal base replacement Universal bases were replaced by standard nucleotides during PCR amplification. The PCR solutions (50 µL) contained: 0.5 U SeSaM Taq polymerase TM, 0.5 µM of each primer (SeSaM_F and SeSaM_R, Primer sequence in Appendix, Table 12), 50 ng full-length DNA from Step 3, 0.2 mM of each dNTP, 1x SeSaM Taq DNA polymerase buffer. The following PCR protocol was employed: 94°C for 2 min (1 cycle); 94°C for 30 s, 60°C for 30 s, 72°C for 1 min 40 s (15 cycles); 72°C for 3 min (1 cycle). The amplified PCR product was run on 0.8% agarose gel and gel purified with NucleoSpin Gel and PCR Clean-up kit and NT buffer (Macherey Nagel, Düren, Germany). The final astB-SeSaM library was achieved by combining 200 ng of each library, which was introduced into pET22b(+) by phosphorothioate-based ligase-independent gene cloning (PLICing)[122].

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2.3.4 Site-saturation and site-directed mutagenesis

Individual site-saturation using NNK codon at amino acid positions 111, 144, 446, 579, 608, 615 and 624 in ASTB was performed by 2-step PCR according to Table 2. Recombination of beneficial substitutions (L446P, V579K, G608D, and S615G) was also accomplished following the same procedure using individual single mutants as templates. The PCRs were performed in 200 µL PCR tubes (VWR, Darmstadt, Germany) using a thermal cycler gradient PCR machine (Mastercycler® pro, Eppendorf, Hamburg, Germany). All PCR products were subjected to DpnI digestion (37°C, 18 h) followed by enzyme inactivation (60°C, 15 min) and DNA purification using PCR purification kit (Macherey-Nagel, Düren, Germany). Purified PCR products (50-100 ng) were transformed into chemically competent E. coli BL21 Gold (DE3) (Agilent Technologies, Santa Clara, USA) for expression and screening.

2.3.5 Cultivation of libraries in 96-well microtiter plates

Colonies of respective libraries (SeSaM or SSM) were transferred from LB agar plates into 96-well MTP (PS-F-bottom, Greiner Bio-One, Frickenhausen, Germany) containing 150 µL/well LB media (10 g L-1 tryptone, 5 g L-1 yeast extract, 10 g L-1 NaCl) supplemented with ampicillin (100 µg/mL). After cultivation (37°C, 900 rpm, 70% humidity, 24 h) in an MTP shaker (Multitron II, Infors GmbH, Einsbach, Germany), 80 µL glycerol (50% (w/w)) was added and the plates were stored at - 80°C as master plates. For expression, pre- cultures were inoculated using MTP replicator (96 pins) and cultivated (37°C, 900 rpm, 70% humidity, 24 h). TB medium (medium (80%): 10 g L-1 tryptone, 5 g L-1 yeast extract,

-1 -1 -1 4 g L glycerol; buffer (20%): 2.31 g L KH2PO4 and 12.54 g L K2HPO4) with a volume of 140 µL (supplemented with 100 µg/mL ampicillin) was transferred into microtiter plates (PS-V-bottom, Greiner Bio-One, Frickenhausen, Germany) and each well was inoculated using 10 µL of pre-culture. After initial growth (37°C, 900 rpm, 70% humidity, 2.5 h), expression was induced by adding IPTG (100 mM) to a final concentration of 0.1 mM and the cultures were cultivated for protein production overnight (30°C, 900 rpm, 70% humidity, 18 h) in an MTP shaker (Multitron II, Infors GmbH). Cells were harvested by using a centrifuge (4°C, 2900 x g, 10 min; 5810 R, Eppendorf, Hamburg, Germany) and stored at - 20°C for 1 h. Cell pellets were thawed and resuspended in 150 µL lysozyme

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Engineering of an aryl sulfotransferase toward sulfation of saccharides solution (1.5 mg/mL in Tris-HCl, 100 mM, pH 9.0) and incubated in an MTP shaker (37°C, 900 rpm, 70% humidity, 1 h; Multitron II, Infors GmbH). Subsequently, the MTPs were centrifuged (4°C, 2,900 x g, 30 min) and the ASTB containing clarified cell lysate was used for screening assay.

2.3.6 pNPS based screening system for 96-well microtiter plates

A 96-well MTP based screening system was established. After cultivation and cell lysis, 10 µL AST containing clarified cell lysate was transferred into a fresh MTP (PS-F-bottom, Greiner Bio-One) containing in total 5 µL reaction buffer (100 mM Tris-HCl, pH 9.0) and 80 µL of 350 mM mono- or disaccharides dissolved in the reaction buffer (final concentration 280 mM). The reaction mixture was incubated (800 rpm, 5 min, RT) in an MTP shaking device (TiMix2, Edmund Bühler GmbH, Hechingen, Germany). Reactions were started by supplementing 5 µL of pNPS (10 mM) to a final concentration of 0.5 mM.

-1 -1 The release of pNP was kinetically detected at 405 nm (εpNP=14.7 mM cm ) in an MTP reader (cycles: 30, kinetic interval: 20 s, RT, Time: 10 min; TECAN Sunrise, Männerdorf, Switzerland). One unit (1 U) of ASTB activity is defined by the required amount of enzyme for the formation of 1 µmol pNP per minute (Equation 1).

푼 ∆푬 ∙ 푽 푻풐풕풂풍 풂풄풕풊풗풊풕풚 ( ) = 푻 풎푳 ∈ퟒퟎퟓ 풏풎∙ 풅푴푻푷 ∙ 푽풍풚풔풂풕풆

Equation 1. The volumetric activity of ASTB using pNPS Assay. ΔE: calculated slope of initial linear part of the kinetic curve in absorbance per min, VLysate: lysate volume (10 µL), VT: total reaction -1 -1 volume (100 µL), Ԑ405 nm: molar extinction coefficient of pNP (14.7 mM cm ), dMTP represents the diameter of a well of MTP with 100 µL working volume in 96-well, flat bottom, 0.2855 cm).

2.3.7 Production in shaking flasks

E. coli BL21 Gold (DE3) cells containing pET22b(+)-ASTB-WT or ASTB variants were inoculated to an OD600 of 0.05 by using an overnight-culture in 200 mL TB medium in a 1 L

Erlenmeyer flask. The cultures were grown until an OD600 of 0.6-0.8 was achieved (37°C, 200 rpm, 2-3 h). By the addition of 0.1 mM IPTG, expression was induced, and the cultures were grown overnight (30°C, 200 rpm, 18 h). For production in 2 L Erlenmeyer flask, 400 mL culture volume was used. To avoid the production of inclusion bodies in 400 mL cultures, the expression was performed at a lower temperature (20°C, 200 rpm, 48 h).

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Cells were harvested (4°C, 11,279 x g, 15 min; Sorvall, ThermoFischer Scientific, Darmstadt, Germany) and pellets were stored at - 80°C until further use.

2.3.8 Purification of ASTB-WT and variants

Frozen pellets (1 g cell in 5 mL buffer) were resuspended in NP buffer (7.68 g L-1

-1 NaH2PO4.2H2O (50 mM) and 17.54 g L NaCl (300 mM); pH 8.0 adjusted with NaOH) and incubated on ice. Lysozyme (1.5 mg/mL) was added prior to cell disruption via sonication on ice (5 min, 30 s pulse, 15 s pause, 70% amplitude) using ultrasonicator (Vibra-Cell™ Ultrasonic Liquid Processor, VCX 130, Sonics & Materials, Inc., Newtown, CT, USA). The cell lysate was centrifuged (4°C, 11,279 x g, 45 min), the supernatant was filtered (0.8 µm filter unit) and used for purification. ASTB-WT and variants were purified using Strep- Tactin column (Strep-Tactin Superflow Plus Cartridge-5 mL, Qiagen, Hilden, Germany) by gravity flow according to manufacturer’s protocol. For elution of the bound Strep-tag II

-1 proteins, a buffer including desthiobiotin was used (7.68 g L NaH2PO4.2H2O (50 mM), 17.54 g L-1 NaCl (300 mM), and 0.5 g L-1 desthiobiotin (2.5 mM); pH 8.0 adjusted with NaOH). Eluted proteins were concentrated using 15 mL centrifugal filters (Amicon® Ultra, cut off 30 kDa, Merck Chemicals GmbH, Darmstadt, Germany). Subsequently, the buffer was exchanged with Tris-HCl (100 mM, pH 9.0) in the same centrifugal filters. Purified proteins were stored at 4°C for several months.

2.3.9 Kinetic characterization of ASTB-WT and variants

Only purified enzymes were used for determination of kinetic parameters. Purified ASTB- WT, -V1, -M1, -M2, and -M5 were diluted in Tris-HCl buffer (100 mM, pH 9.0) to a final concentration of 0.06 to 0.12 µM in MTP. Kinetic constants of ASTB-WT and ASTB-M1, - M2, and -M5 were determined by measuring the initial rates at various resorcinol concentrations (0 to 6 mM) with 0.06 µM enzyme and at various GlcNAc, glucose and cellobiose concentrations (0 to 295 mM) with 0.12 µM enzyme. All the reactions were performed in six replicates with 0.5 mM pNPS as fixed donor concentration and performed as described for the screening system (Chapter 2.3.6.)

2.3.10 Evaluation of pH activity profile and pH stability ASTB-WT and variants

The pH profile of ASTB-WT and ASTB variants (ASTB-M1, -M2, -M5) were determined

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using Briton and Robinson buffer (200 mM H3BO3, 200 mM H3PO4, and 200 mM CH3COOH, adjustment of the desired pH with 0.2 M NaOH; working concentration: 100 mM, pH: 5.0 – 12.0). Donor and acceptor of the pNPS based assay were prepared in Briton and Robinson buffer at different pH values. The reactions were carried out in 8 replicates with 0.12 µM enzyme and as described in Chapter 2.3.6. Highest activity at respective pH value was considered as 100%.

For determination of pH stability, purified ASTB-WT and ASTB variants (ASTB-M1, -M2, - M5) were incubated in Briton and Robinson buffer (100 mM, pH 5.0 – 12.0) for up to 6 h. Samples were taken after 0, 1, 2, 4, and 6 h and supplemented into the reaction to perform ASTB activity assay (Chapter 2.3.6). Activity was set as 100% at all tested pH values (pH 6.0 – 11.0) at 0 h.

2.3.11 Determination of organic solvent resistance of ASTB-WT and variants

The organic solvent resistance profiles of ASTB-WT and ASTB-M1, -M2, -M5 were determined using 0 – 50% (v/v) dimethyl sulfoxide (DMSO), methanol, ethanol, acetone, and acetonitrile and 0 – 20% tetrahydrofuran (THF). Reactions were performed in 100 µL using 122 mM cellobiose, 0.5 mM pNPS and 0.12 µM enzyme in a Tris-HCl (100 mM, pH 9.0) buffer system. Enzyme activity was set as 100% (buffer activity), when no organic solvent was present in the reaction.

2.3.12 Determination of thermostability of ASTB-WT and variants

Purified ASTB and variants (ASTB-M1, -M2, -M5) in Tris-HCl buffer (100 mM, pH 9.0) were incubated in 200 µL PCR tubes (VWR, Darmstadt, Germany) at varied temperatures (gradient from 23°C to 67.1°C; 10 min) in a thermal cycler (Mastercycler® pro, Eppendorf, Hamburg, Germany). The samples were subsequently cooled down on ice (referred as 4°C) for 5 min. The residual ASTB activity was measured using the pNPS based screening assay (Chapter 2.3.6).

2.3.13 Computational analysis of ASTB homology model

Molecular modelling was performed in a collaboration with Dr. B. Infanzón and Dr. M. D. Davari (Institute of Biotechnology, RWTH Aachen University). The 3D structure of ASTB- WT was modelled by using I-TASSER server[123], which is a hierarchical protein structure

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Engineering of an aryl sulfotransferase toward sulfation of saccharides modelling approach based on the secondary structure enhanced Profile-Profile threading Alignment (PPA)[124]. The best model was selected based on the C-score (confident score). The model was further verified for protein geometry by Structural Analysis and Verification Server (SAVES) online tools (i.e. Verify 3D, PROCHECK, ERRAT[125], and ProSA[126]). To check the stability of the model, molecular dynamics (MD) simulations were performed and structural stability of the generated model in water was estimated by calculation of the root mean square deviation (RMSD) from the initial structure in MD simulations performed for 100 ns in 0.9 % (0.15 M) and 7.5 % (1.3 M) NaCl. In order to generate the homodimer of the ASTB, the protein-protein docking was performed by using SymmDock Server[127]. The dimeric structure used in calculations was assembled by expanding the crystal symmetry. PDBePISA web server[128] was used to identify the most stable dimer interface in the ASTB.

2.3.14 Synthesis of (mono)sulfated GlcNAc-linker-tBoc

Enzymatic sulfation of GlcNAc-linker-tBoc was performed at ambient temperature under continuous stirring in Tris-HCl buffer (100 mM, pH 9.0) in 2 mL scale (in 5 mL glass vials; ND18, VWR International GmbH, Darmstadt, Germany) with ASTB-WT and selected variants. Each reaction contained 80 µmol GlcNAc-linker-tBoc and 40 µM purified enzyme. Reactions were initiated with 16 µmol pNPS and after every 24 h further 16 µmol was added until 72 h. Proteins from the enzymatic reactions were separated by using 2 mL centrifugal filters (Amicon® Ultra, Merck Chemicals GmbH, Darmstadt, Germany) with a molecular weight cut-off of 30 kDa.

2.3.15 Analytical and semi-preparative HPLC

Analytical HPLC measurements were performed on an HPLC system (Nexera X2, Shimadzu Deutschland GmbH, Duisburg, Germany) using a Multospher-APS-HP-5 μm HILIC column (250 x 4.6 mm; CS Chromatographie, Langerwehe, Germany). Proteins from the biotransformation reactions were separated by using 0.5 mL centrifugal filters (Amicon® Ultra, Merck Chemicals GmbH, Darmstadt, Germany) with a cut off of 10 kDa. Samples were diluted 40 times with a defined mobile phase (75% acetonitrile, 5 mM ammonium acetate, pH 4.3) and 10 – 40 µL was injected onto the column mentioned. Isocratic separation of the samples was obtained over a time course of 10-20 min and at a flow

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Engineering of an aryl sulfotransferase toward sulfation of saccharides rate of 0.5 – 1 mL/min. The UV absorbance of GlcNAc-linker-tBoc and sulfated GlcNAc- linker-tBoc were monitored at 254 nm. A calibration curve of GlcNAc-linker-tBoc within a concentration range between 0.005 and 2.0 mM was used to calculate the obtained product concentrations.

Preparative HPLC purifications were performed in a collaboration with Dr. D. Laaf and Prof. Dr. L. Elling (Laboratory for Biomaterials, Institute of Biotechnology and Helmholtz- Institute for Biomedical Engineering, RWTH Aachen University). An HPLC system from Knauer (pump 1000, UV detector 2600, Berlin, Germany) using a Multospher-APS-HP- 5 μm HILIC column (250 x 10 mm; CS Chromatographie, Langerwehe, Germany) was employed. Samples were diluted ~ 4 times in the mobile phase (75 % (v/v) acetonitrile, 5 mM (w/v) ammonium acetate, pH 4.3) and applied via an injection valve. Isocratic separation of the analytes was obtained (15 min; flow rate of 3.5 mL/min). The UV absorbance of GlcNAc-linker-tBoc and sulfated GlcNAc-linker-tBoc was monitored at 254 nm. Fractions were manually collected and analyzed via analytical HPLC (column: Multospher-APS-HP-5 μm; 250 x 4.6 mm; CS Chromatographie); mobile phase: 75 % (v/v) acetonitrile dissolved in 5 mM (w/v) ammonium acetate, pH 4.3; flow rate: 1 ml/min; time course: 10 min). Fractions containing the sulfated product were pooled and evaporated (60°C, 200 - 300 mbar, rotary evaporator, vacuum pump CVC2, Vauubrand GmbH, Moenchengladbach, Germany). After evaporation, 8.5 mL deionized water and 1.5 mL HEPES buffer (1 M, pH 7.0) were supplemented to the 5 mL product for neutralization. In total, 15 mL sample was used to isolate the final product by solid-phase extraction using Sep-Pak® C18 1/3/6cc Vac Cartridges (Waters, Eschborn, Germany) according to the manufacturer’s protocol. Finally, 3-6 mL sample containing highly pure product was recovered and evaporated (45°C, Eppendorf Concentrator Plus, Hamburg, Germany) to obtain ~3.5-5 mg product, which was verified by analytical HPLC.

2.3.16 Mass spectrometry (MS)

MS analysis was performed in a collaboration with Dr. D. Laaf and Prof. L. Elling (Laboratory for Biomaterials, Institute of Biotechnology and Helmholtz-Institute for Biomedical Engineering, RWTH Aachen University). Purified sulfated GlcNAc-linker-tBoc was used to perform electrospray ionization mass spectrometry (ESI-MS). Analysis of pure

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Engineering of an aryl sulfotransferase toward sulfation of saccharides substance (0.1-0.2 mM) was realized by usage of Multospher 120 RP 18 HP-3μm (60 x 2 mm; CS-Chromatographie), a flow rate of 0.2 mL/min, a mobile phase of acetonitrile/water (50:50) and defined conditions (needle voltage = 4 kV, temperature = 400°C, cone voltage = 100 V, negative mode; Finnigan Surveyor MSQ Plus, ThermoFischer Scientific, Darmstadt, Germany). Mass/charge-ratio (m/z ratio) was considered for detection of target molecules.

2.3.17 Nuclear magnetic resonance (NMR)

All NMR data were obtained in a collaboration with Dr. H. Pelantová and Prof. Dr. V. Křen (Institute of Microbiology, Czech Academy of Sciences, Prague, Czech Republic). NMR spectra were acquired on a Bruker AVANCE III 700 MHz spectrometer (700.13 MHz for 1H and 176.05 MHz for 13C) and a Bruker AVANCE III 600 MHz spectrometer (600.23 MHz for

1 13 H and 150.93 MHz for C) in D2O (99.98% D, ARMAR Chemicals, Döttingen, Switzerland) at 303 K. Proton spectra were referenced to residual signal of water (σH: 4.508 ppm); carbon chemical shifts to the signal of acetone (σc: 30.50 ppm). NMR experiments: COSY, HSQC, HSQC-TOCSY, and 1d-TOCSY were performed using standard manufacturers’ software. 1H NMR and 13C NMR spectra were zero filled to fourfold data points and multiplied by window function before Fourier transformation. The two-parameter double-exponential Lorentz-Gauss function was applied for 1H to improve resolution and line broadening (1 Hz) was applied to get better 13C signal-to-noise ratio.

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2.4 Results

The results are divided into 4 parts as follows: i) selection of the appropriate AST for evolution studies among ASTA and ASTB, ii) development of pNPS-based screening system for small sugars, iii) directed evolution of selected AST toward a monosaccharide and iv) KnowVolution campaign of selected AST for a disaccharide.

2.4.1 Selection of an aryl sulfotransferase

The enzyme selection criterion was based on the performance of the enzymes in presence of target acceptors, saccharides. Two aryl sulfotransferases (ASTA and ASTB) were successfully produced in E. coli BL21 Gold (DE3). Their activity in clarified crude cell extracts was evaluated by pNPS-based assay toward a phenolic acceptor (e.g., resorcinol), monosaccharides (e.g., GlcNAc and glucose) and a disaccharide (e.g., cellobiose). As shown in Figure 10, the volumetric activity of ASTB is significantly higher for all tested acceptors compared to ASTA. ASTB showed 4.3-fold and 5.3-fold higher activity toward the evaluated monosaccharides, GlcNAc and glucose, respectively. Furthermore, a 5.7 times better performance of ASTB than ASTA was recorded for the disaccharide acceptor cellobiose. Overall, for saccharides, ASTB seems to be the better candidate to start evolution studies with. In addition to the targeted sugar acceptors, the enzymes were evaluated for a phenolic acceptor, resorcinol, which belongs to the natural acceptor class of ASTs. Similar to the saccharides, ASTB showed a 3.2-fold higher volumetric activity

Figure 10. Comparison of activity between ASTA and ASTB. A) The volumetric activity of ASTA and ASTB is shown in U/L for tested monosaccharides GlcNAc and glucose, and disaccharide, cellobiose. B) volumetric activity of ASTA and ASTB is shown in U/L for phenolic acceptor, resorcinol.

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Engineering of an aryl sulfotransferase toward sulfation of saccharides in comparison to ASTA toward resorcinol. Taken together, ASTB was selected as the target enzyme for the directed evolution and KnowVolution campaigns in this study.

2.4.2 Development of a continuous pNPS based screening system

The well-known pNPS assay for sulfotransferases was advanced and optimized for sulfation of small saccharides. N-acetylglucosamine (GlcNAc) as monosaccharide and cellobiose as disaccharide were selected as the target acceptor molecules for the assay. The principle of the screening assay is shown in Figure 11A for GlcNAc and in Figure 11B for cellobiose. Both reactions follow the same concept using pNPS as donor molecule, which is desulfated by the act of active ASTB to form the chromogenic by-product pNP (yellow color detectable at 405 nm). The sulfate group is ideally transferred to the target molecule GlcNAc or cellobiose, whereas the sulfated saccharide is not visible in the detection system. The ultimate detection of the sulfated product must be followed by HPLC and MS detection methods.

Step by step optimization of screening parameters is obligatory for the development of robust screening systems in directed enzyme evolution. The main optimized parameters for pNPS assay were the employed volume of cell-free extract as well as donor (pNPS) and acceptor (GlcNAc and cellobiose) concentrations. Influence of varied volumes of clarified free extract containing ASTB-WT in the pNPS assay was analyzed and a linear range throughout the tested volume (0–15 μl) was recorded (Figure 12A). A volume of 10 µL was selected for screening toward GlcNAc and cellobiose that corresponds to a concentration

Figure 11. Targeted reactions for establishment of pNPS-based screening system. Sulfation of A) GlcNAc and B) cellobiose using pNPS as donor molecule by the act of ASTB. In both cases a monosulfated product is released and as by-product pNP is formed.

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of ~0.12 µM ASTB for both acceptors (Figure 12B). The linear detection range of the pNPS screening system was determined by varying the donor concentration (0–1mM pNPS) and keeping a constant acceptor concentration (150 mM GlcNAc and cellobiose; Figure 12C). A linearity between ASTB activity and varied pNPS concentrations was only observed between 0.0 and 0.5 mM pNPS. A surprising observation was made for both acceptors at pNPS concentration higher than 0.5 mM showing an inhibitory effect on ASTB (Figure 12C). Hence, 0.5 mM was chosen as a suitable pNPS concentration for the screening system. The linear detection range for acceptors was determined between 25 and 300 mM for cellobiose and between 25 and 325 mM for GlcNAc at a pNPS concentration of 0.5 mM (Figure 12D). Finally, 280 mM was selected for both acceptors. Furthermore, a

Figure 12. Assay parameters of pNPS assay using GlcNAc and cellobiose as acceptors. A) Linear correlation between ASTB total activity [U] with 280 mM GlcNAc (circles) or cellobiose (squares) and 0.5 µM pNPS and volume of cell free extract. B) Volumetric activity [U/L] of ASTB at different enzyme concentrations. Reactions were carried out using 0-0.5 µM of purified ASTB-WT, 280 mM cellobiose and 0.5 mM pNPS. C) Effect of pNPS on ASTB activity. Reactions were performed using 0-1.0 mM pNPS, 150 mM GlcNAc or cellobiose and 10 µL cell-free extract. D) Calibration curve for GlcNAc and cellobiose between 0 and 325 mM using 0.5 mM pNPS and 10 µL cell free extract. All reported values and respective standard deviations are obtained from six replicates.

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low background activity (<0.05 AU/h for GlcNAc (Figure 13A) and <0.1 AU/h for cellobiose (Figure 13B)) was determined.

Applying the optimized conditions (0.5 mM pNPS, 280 mM GlcNAc/cellobiose, and 10 μl cell-free extract), the key parameter coefficient of variation (CV) of the pNPS assay was obtained. The apparent CV was determined to be 11.7% and 8.9% for GlcNAc and cellobiose, respectively by a screening of 96 identical ASTB wild-type clones (using a 96- well MTP) on a pET22b(+) vector in E. coli BL21 Gold (DE3) cells. For the determination of true CV, 96 identical clones harboring the pET22b(+) empty vector were analyzed. Finally, the true CV was determined to be 14.3% GlcNAc and 10.5% cellobiose after subtracting the empty vector background from the apparent CV (Figure 13B). Taken together, the established pNPS assay exhibits a CV below 15%, which was demonstrated elsewhere to represent a reliable and robust screening system[72, 129].

Figure 13. Coefficient of variation of pNPS assay with GlcNAc and cellobiose as acceptors. Activity of ASTB-WT towards A) GlcNAc and B) cellobiose in 96-well format plotted in descending order. The apparent coefficient of variation () represents activity values without subtracting the background of empty vector, and the true coefficient of variation () was calculated after EV background () subtraction.

2.4.3 Directed evolution of ASTB toward GlcNAc

One round of directed evolution was performed to improve ASTB’s specific activity toward GlcNAc. Randomization of astB was achieved by applying SeSaM. Screening of the generated ASTB library was performed using established pNPS based screening system. Best-identified variant showing increased sulfation activity toward GlcNAc was characterized.

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2.4.3.1 Generation and screening of astB SeSaM library

The diversity of astB was generated by applying SeSaM-Tv-classic[121b]. Electrophoretic analyses of individual SeSaM steps for astB library generation are shown in Figure 14. Preliminary step I was performed for template generation for step 1 and step 3 (Figure 14A). The following dATPαS and dGTPαS concentrations were selected according to the fragmentation pattern: A forward library-35% (Figure 14B), G forward library-25% (Figure 14C), A reverse library-30% (Figure 14D), and G reverse library-25% (Figure 14E). The selected concentrations were used in step 1 for construction of both SeSaM A and G

Figure 14. Generation of astB SeSaM library. A) Preliminary step I- PCR amplification to generate template for step 1 and step 3 (expected band size: 2.1 kb). 1: 1 kb DNA ladder, 2: Step 1 forward library template, 3: Step 1 reverse library template, 4: Step 3 reverse library template, 3: Step 3 forward library template. B) – E) Determination of optimal phosphorothioate percentage using different concentrations of dATPαS or dGTPαS (1: 1 kb DNA ladder, 2: 0%, 3: 10%, 4: 15%, 5: 20%, 6: 25%, 7: 30%, 8: 35% and 9: 40%) for A_forward library (B), G_forward library (C), A_reverse library (D), G_reverse library (E). F) Elongation of FITC-labeled primer by TdT. 1: In positive control each band represents an addition of one universal base to FITC-labeled primer by TdT and 2: negative control (only FITC-labeled primer without TdT reaction) and G) Final A and G libraries. 1: 1 kb DNA ladder, 2: A forward library, 3: G forward library, 4: A reverse library, and 5: G reverse library (Islam et al. 2018).

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Engineering of an aryl sulfotransferase toward sulfation of saccharides libraries. In step 2, the terminal deoxynucleotidyl transferase (TdT) catalyzed the incorporation of universal bases (dPTαP) at the 3’-OH of the single stranded library fragments. The quality control for the addition of dPTαP was persued by analyzing the number of incorporated P nucleotides at the 5’-FITC labeled oligonucleotide, which was visualized on a 36% acrylamide gel using a phosphorimager (Figure 13F). The final A- forward, G-forward, A-reverse and G-reverse libraries were obtained by amplification (Figure 14G). ASTB-SeSaM library was achieved by combining 200 ng of each library, which was introduced into pET22b(+) by phosphorothioate-based ligase-independent gene cloning (PLICing)[122]. The used primers are listed in the list of oligonucleotides (Table 12). The PLICing product was transformed into chemically competent E. coli BL21 Gold (DE3) for expression. The quality of the generated SeSaM library was verified by analyzing sequences of 12 improved mutants. Sequencing revealed 62.5% transitions (Ts) and 37.5% transversions (Tv) (see Table 3) indicating a Ts/Tv ratio of 1.7, which is comparable to the reported value for the SeSaM-Tv-classic protocol (Ts/Tv: 1.8) [121a]. The percentage of WT occurrence was 17% among 12 sequenced mutants. As sequencing analysis matched the requirements of a good mutant library, astB SeSaM library was used for the subsequent screening.

Table 3. Sequencing analysis of mutants from astB SeSaM library. Twelve SeSaM variants (SeSaM1-12) are listed with individual nucleotide and amino acid substitutions as well as number of transitions and transversions. Nucleotide Amino acid Transitions Transversion Mutant substitutions substitutions [Ts] [Tv] SeSaM1 TC L446P 1 0 SeSaM2 AC, AT E111D, T144S 0 2 SeSaM3 TC, TA V579D, V624M 1 1 SeSaM4 GA, AT G608D, S615C 1 1 SeSaM5 TA, CT, GAa L225Q, S629F 1 1 SeSaM6 TC, TC V242A, F383S 2 0 SeSaM7 - - 0 0 SeSaM8 TA V579D 0 1 SeSaM9 TC L628P 1 0 SeSaM10 AG N277S 1 0 SeSaM11 GA A252T 1 0 SeSaM12 TC S629P 1 0 aSilent mutation

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2.4.3.2 Screening of ASTB SeSaM library

In order to validate the established pNPS screening system, the generated ASTB-SeSaM library was expressed in E. coli BL21 Gold (DE3) and screened toward GlcNAc. Screening of 1,760 SeSaM mutants for improved ASTB activity toward GlcNAc yielded 20 ASTB variants with increased activity in lysed crude extracts (>1.5-fold compared to ASTB-WT). The selected 20 ASTB variants were rescreened in 8 replicates each to identify the 5 most promising variants. The volumetric activities of the 5 selected variants (SeSaM2, 3, 4, 8, and 10) in clarified crude extracts are shown in Figure 15. The variant SeSaM8 (ASTB- V579D; blue bar in Figure 15) yielded an up to 3.2-fold increased activity for GlcNAc (ratio variant/WT). Sequencing analysis revealed that the variants carried one or two amino acid substitutions (see Table 3). Interestingly, the amino acid exchange from valine to aspartic acid at position 579 was identified in two improved variants (SeSaM3 and SeSaM8). The “best” variant SeSaM8 is designated as ASTB-V1 in the following chapter.

Figure 15. ASTB variants for GlcNAc sulfation. Volumetric activity of ASTB-WT and the five SeSaM variants (SeSaM2, 3, 4, 8, and 10) toward GlcNAc in clarified cell-free extract employing the continuous pNPS screening system. GlcNAc (280 mM) as acceptor; pNPS (0.5 mM) as donor was used. The reported initial activity values and error bars correspond to average values of eight replicates. EV (empty vector) represents the background activity.

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2.4.3.3 Kinetic characterization of ASTB-WT and variants for GlcNAc

ASTB-WT and ASTB-V1 were purified to perform the final characterization. The kinetic characterization of the purified ASTB-WT and the “best” variant ASTB-V1 was performed by comparing initial activities applying the established pNPS screening system for GlcNAc. A comparison of activity values in crude lysates and of purified ASTB variants will clarify whether the pNPS screening system is suitable to identify ASTB variants with improved initial activities. Kinetic constants were not obtained since the increase of specific activity in respect to the GlcNAc concentrations (0-280 mM) remained linear up to the solubility limit of GlcNAc (~50 mg/mL in water; Figure 16). Nevertheless, the increase in specific activity of ASTB-V1 (V579D) over a varied GlcNAc concentration range was clearly shown, which validates the pNPS screening system for directed ASTB evolution. Obtained improvements in specific activities range between 2.7 to 3.4-fold depending on employed GlcNAc concentrations (75 mM: 3.4-fold; 25 mM 2.7-fold; ASTB-V1). Under screening

Figure 16. Determination of initial activities of ASTB-WT and ASTB-V1 with GlcNAc. Purified ASTB-WT (green) and ASTB-V1 (blue) were employed using the pNPS-screening system. Specific activities in U/mg are plotted against varied GlcNAc acceptor concentrations. (0 to 280 mM). Measurements were performed in 8 replicates.

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Engineering of an aryl sulfotransferase toward sulfation of saccharides conditions (280 mM) a 2.9-times increased specific activity was accomplished (ASTB-V1: 0.84 U/mg vs. ASTB-WT: 0.29 U/mg).

2.4.4 KnowVolution campaign of ASTB toward cellobiose

A KnowVolution approach was pursued to improve ASTB’s specific activity towards a disaccharide, cellobiose. All four phases of KnowVolution are explained in detail below.

2.4.4.1 Phases of KnowVolution

Phase I (Identification)

KnowVolution campaign was initiated with the ASTB-SeSaM library, which was used to perform directed ASTB evolution for GlcNAc. The screening was performed in MTPs, wherein one plate contained 86 ASTB variants and 8 controls (4 clones of ASTB-WT and 4 clones of EV). After screening of 1,760 clones, 9 variants were selected with ≥ 1.5-fold

Figure 17. Determination of the volumetric activity (U/L) of ASTB-WT and selected variants in the re-screening phase. Volumetric activity of ASTB-WT and the five SeSaM variants (SeSaM1-4, 7, 8, 10, 11, and 12) toward cellobiose in clarified cell-free extract employing the continuous pNPS screening system. Cellobiose (280 mM) as acceptor; pNPS (0.5 mM) as donor was used. The reported initial activity values and error bars correspond to average values of eight replicates. EV (empty vector) represents the background activity.

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Engineering of an aryl sulfotransferase toward sulfation of saccharides improvement and were re-screened in 2 MTPs in 8 replicates each. Figure 17 shows the obtained results for all re-screened variants. Among them four improved variants (SeSaM1: 2.3-fold, SeSaM2: 1.9-fold, SeSaM3: 2.0-fold, and SeSaM4: 2.5-fold; shown as blue bars in Figure 17) were identified. Sequencing results of SeSaM variants revealed either single or double amino acid substitutions were incorporated (Table 3). In total, 7 beneficial positions (E111, T144, L446, V579, G608, S615, and V624) were identified for phase II.

Phase II (Determination)

All 7 beneficial positions (111, 144, 446, 579, 608, 615, and 624) were saturated individually by site-saturation mutagenesis (SSM). For each SSM library, 188 clones were screened. SSM libraries of 111, 144, and 624 did not yield any improved variant. Only 4 (446, 579, 608 and 615) out of 7 positions showed an actual effect on the ASTB activity for cellobiose. SSM 446, SSM 579, SSM 608, and SSM 615 yielded 9, 13, 4 and 4 improved variants, respectively. Table 4 contains the number of mutants for each position and the frequency of each substitution, which were sequenced (only improved variants were sequenced). The best-improved variant from each SSM library were determined: ASTB- M1: 2.6-fold, ASTB-M2: 4.5-fold, ASTB-M3: 2.7-fold, and ASTB-M4: 2.3-fold.

Table 4. Beneficial positions and substitutions with their effect on enzyme properties. RBold and underlined letters indicate the best substitutions. Position # sequenced Substitutions (# occurance) Improved property E111 - - no effect T144 - - no effect L446 9 P (4), S (2), K (1), A (1), V (1) activity towards cellobiose V579 13 N (3), L (2), P (2), R (2), G (2), D (1), activity towards cellobiose K (1) G608 3 E (1), A (1), D (1) soluble expression S615 4 G (3), E (1) soluble expression V624 - - no effect

Phase III (Selection)

A structural model of ASTB (Figure 18) was generated by homology modelling based on the known ASST protein structure (PDB: 3ETS). The multiple sequence alignment between ASTB and 250 similar sequences with high similarity by PSI-BLAST search[130]

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Engineering of an aryl sulfotransferase toward sulfation of saccharides demonstrated high conservation of H220, H283, R301, and H350 (see WebLogo[131] representation in Figure 50). The catalytic residue H350 (undergoes transient covalent sulfation during the catalytic cycle) was further confirmed by substituting histidine by alanine residue leading to no active ASTB (Figure 51). The structural stability of the ASTB model in water was estimated by calculation of the root mean square deviation (RMSD; Figure 52). Though ASTB forms a dimer in the native state and the typical ß-propeller structure similar to ASST, the flexible C-terminal region is very different compared to ASST. In order to identify, whether the flexible C-terminal part (506-628) really play a role on the ASTB activity, a truncated version of ASTB was generated (ASTB-506stop). Interestingly, there was no ASTB activity determined for ASTB-506stop, which confirms the necessity of the C-terminal region for activity (Figure 51). The average of root mean square fluctuation (RMSF) per residue confirmed C-terminal region as highly flexible (Figure 52). Four beneficial positions (446, 579, 608, and 615) were visualized using the proposed structural model of ASTB-WT (Figure 18A) to localize the beneficial positions. Position L446 is localized in the proximity to the catalytic histidine (H350) and positions V579, G608, and S615 are situated in the loop of the C-terminal variable region (Figure 18B).

Figure 18. ASTB structural model. A) The dimeric structural model of ASTB is shown. Monomers are highlighted in light and dark cyan. Catalytic histidine (H350) and key residues (H220, H283, and R301) forming together the active site are shown in red and blue sticks, respectively; (B) Monomer of ASTB highlighting the residues shown as green ball represent positions that were substituted (L446P and V579K) in variant ASTB-M5. (Adapted from Islam et al. 2018).

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Phase IV (Recombination)

The “best” amino acid substitutions for each beneficial position (L446P, V579K, S608D, and S615G) were recombined to study the synergistic effect of combined substitutions on ASTB activity. The recombination was performed one by one, whereas variants with double substitutions were generated by site-directed mutagenesis (ASTB-M5: L446P/V579K, ASTB-M6: L446P/S615G, ASTB-M7: V579K/G608D, ASTB-M8: V579K/S615G, and ASTB-M9: G608D/S615G). It was found that only two substitutions (L446P and V579K) improved activity toward cellobiose and two other substitutions (G608D and S615G) contributed to higher soluble expression (Figure 19A). The SDS-PAGE analysis of clarified cell lysate of all variants in Figure 19B confirmed higher protein content for those, which contain one of the two responsible substitutions for soluble expression or both (G608D or/and S615G). The aim of the KnowVolution campaign was the improvement of ASTB’s specific activity toward cellobiose. Therefore, further recombination of all four positions was not necessary. ASTB-M5 (L446P/V579K) was identified as the final variant of KnowVolution, wherein both substitutions contribute only to the increased cellobiose activity exhibiting a synergistic effect with an improvement of 7.3-fold.

Figure 19. Activity and SDS-PAGE analysis of the final single and recombination ASTB variants. Volumetric activity of ASTB-WT and the nine KnowVolution variants (ASTB-M1 to ASTB-M9) toward cellobiose in clarified cell-free extract employing the continuous pNPS screening system. Cellobiose 280 mM as acceptor; pNPS 0.5 mM as donor was used. The reported initial activity values and error bars correspond to average values of eight replicates. (B) SDS-PAGE analysis of ASTB-WT and KnowVolution variants (ASTB-M1 to ASTB-M9). Crude preparations (clarified lysates after lysozyme treatment) were analyzed (expected protein size: ~72 kDa). EV: empty vector, M: protein marker.

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2.4.4.2 Production and purification of ASTB-WT and selected variants

ASTB-WT, single variants ASTB-M1 and ASTB-M2, and the final recombination variant ASTB-M5 were produced in flasks to purify the enzymes. The analysis of all collected fractions of Strep II tag purification was performed via SDS-PAGE. Typically, no ASTB was detected in flow-through fractions because most of the target protein is bound to the Strep-Tactin column as expected. In the subsequent washing steps, a little amount of desired ASTB is washed off. Finally, most of the bound protein was eluted in the elution phase, which was highly pure. The final washing step carried a very little amount of desired protein. The highly pure elution fractions were collected, pooled, and dialyzed against the working buffer (100 mM Tris-HCl, pH 9.0) and analyzed via SDS-PAGE (Figure 20A). Typically, from 5-8 g cell pellet (wet cell mass) around 50-100 mg purified protein was yielded. Furthermore, ASTB-WT and ASTB variants were analyzed by native-PAGE, which proved ASTB’s appearance as a dimeric protein under natural conditions (Figure 20B).

Figure 20. SDS-PAGE and Native-PAGE analysis of ASTB and ASTB variants. A) SDS-PAGE analysis of purified ASTB-WT (lane 1), ASTB-M1 (lane 2), ASTB-M2 (lane 3), and ASTB-M5 (lane 5). Lane 1 and 6 show the protein ladder. Expected size: ~ 72 kDa (monomer) B) Native-PAGE analysis of cell- free extract containing ASTB-WT (lane 1), ASTB-M1 (lane 2), ASTB-M2 (lane 3), and ASTB-M5 (lane 5). Lane 1 show the protein ladder. Expected size: ~ 144 kDa (dimer).

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2.4.4.3 Kinetic characterization of ASTB-WT and improved variants

The kinetic characterization of the purified ASTB-WT and the three variants (ASTB-M1, -M2 and -M3) was performed with four acceptor molecules; resorcinol, GlcNAc, glucose, and the KnowVolution target acceptor cellobiose. The only phenolic acceptor was resorcinol, which belongs to the natural acceptor class (phenols) of ASTB. The typical Michaelis-Menten kinetics was obtained for ASTB-WT as well as three final ASTB variants with resorcinol as acceptor (Figure 21A). Table 5 summarizes the kinetic constants for

ASTB-WT, ASTB-M1, ASTB-M2, and ASTB-M5. The KM and Vmax values were increased for all three variants compared to ASTB-WT toward resorcinol, in which Vmax increased up to 2.6-times for ASTB-M5 in contrast to ASTB-WT. Detailed mechanistic and kinetic constants were not performed for sugars (GlcNAc, glucose, and cellobiose) since the activity increase

Figure 21. Reaction kinetics of ASTB-WT and final variants with different acceptors. Initial activities of purified ASTB-WT and three final variants (ASTB-M1, ASTB-M2, and ASTB-M5) were determined after 10 min conversion employing the pNPS-screening system. Specific activities in U/mg are plotted against varied concentrations of A) resorcinol (0 to 5 mM), B) GlcNAc (0 to 295 mM), C) glucose (0 to 1280 mM), and D) cellobiose (0 to 295 mM).

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Engineering of an aryl sulfotransferase toward sulfation of saccharides in respect to the employed concentration range of GlcNAc (0-300 mM: Figure 21B), glucose (0-1280 mM; Figure 21C), and cellobiose (0-295 mM; Figure 21D) did not follow any kinetic model. Higher concentrations for GlcNAc and cellobiose were not experimentally possible because of their solubility limit. Nevertheless, specific activities were determined by comparing initial activities with the optimized pNPS screening system (Figure 21). For all variants, the highest improvement in specific activity for the KnowVolution target acceptor cellobiose was observed at 75 mM (ASTB-M1: 3.4-fold, ASTB-M2: 3.5-fold; the synergistic effect in the recombination variant, ASTB-M5: 7.6-fold). Under screening conditions (280 mM) following folds of improvement regarding specific activity were found: ASTB-M1: 2.3-fold, ASTB-M2: 3.3-fold, and ASTB-M5: 5.8-fold. Comparison between wild type and variants revealed 2.2-, 3.0-, and 5.4-fold improved specific activity at 295 mM cellobiose (highest employed concentration) with ASTB-M1, ASTB-M2, and ASTB-M5, respectively (Table 5 summarizes the specific activities in U/mg).

Table 5. Kinetic constants (KM and Vmax) of ASTB-WT, ASTB-M1, ASTB-M2, and ASTB-M5 with resorcinol, GlcNAc, glucose, and cellobiose as acceptors.

Resorcinol GlcNAc Glucose Cellobiose

1 1 1 KM Vmax Vmax Vmax Vmax Enzyme [mM] [U/mg] [U/mg] [U/mg] [U/mg] ASTB-WT 0.74 ± 0.1 102 ± 8 0.28 ± 0.01 2.1 ± 0.2 1.1 ± 0.04 ASTB-M1 1.3 ± 0.2 119 ± 9 0.30 ± 0.01 2.1 ± 0.1 2.5 ± 0.2 ASTB-M2 2.0 ± 0.3 136 ± 11 0.62 ± 0.01 4.6 ± 0.4 3.4 ± 0.1 ASTB-M5 2.8 ± 0.5 262 ± 25 0.58 ± 0.01 5.8 ± 0.2 6.2 ± 0.1 1Highest measured specific activity under experimental conditions

2.4.4.4 Determination of sulfated cellobiose monosulfate

Enzymatic sulfation of cellobiose by ASTB-WT and the final variant ASTB-M5 was confirmed by electronspray ionization mass spectrometric (ESI-MS). Theoretical mass of cellobiose monosulfate is 422.07, which is not identified in the negative control without enzyme (Figure 22A). A product with an m/z ratio ([M-H]-) of 421 was identified in the conversions performed with ASTB-WT (Figure 22B) and ASTB-M5 (Figure 22C) for 24 h, which correlates with the theoretical mass of a monosulfated a cellobiose.

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Figure 22. Electronspray ionization mass spectrometric analysis of enzymatically sulfated cellobiose. A) cellobiose without any enzymatic modification, B) enzymatic sulfation of cellobiose by ASTB-WT, and (C) ASTB-M5. Theoretical mass of cellobiose monosulfate is 422.07 and expected m/z ratio in negative mode was 421.07. Conversions were performed for 24 h.

2.4.4.5 ASTB Activity toward mono-, di-, and oligosaccharides

All generated final ASTB variants for GlcNAc and cellobiose were investigated with a variety of small sugar molecules including mono-, di-, and trisaccharides (Table 6). For this set of experiments, the established pNPS based screening system was used in combination with clarified cell lysates. As a general trend, ASTB-M2 (V579K) and ASTB-M5 (L446P/V579K) were improved toward all tested saccharides. Interestingly, the single amino acid substitution L446P in ASTB-M2 increased the activity toward glucose and not toward its C-4 epimer galactose. Accordingly, L446P contributed to activity improvement toward cellobiose (two D-glucose units linked by 1,4 β-glycosidic bond) but not toward lactose (D-galactose and D-glucose linked by 1,4 β-glycosidic bond). Moreover, L446P did not improve the activity toward maltose (two D-glucose units linked by 1,6 β-glycosidic bond) indicating the role of the structural difference of sugars on the substitution.

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Table 6. Improvement of ASTB variant activity toward saccharides compared to ASTB-WT. V/WT indicates the ratio of volumetric activity between variant and WT.

ASTB-M1 ASTB-M2 ASTB-M5 Sugar type Sugar acceptors [V/WT] [V/WT] [V/WT] GlcNAc 1.6 ± 0.2 3.6 ± 0.5 2.9 ± 0.7 Glucose 2.1 ± 0.5 5.3 ± 0.8 6.1 ± 0.5 Galactose 0.9 ± 0.2 2.5 ± 0.2 2.7 ± 0.8 Monosaccharide Fructose 1.4 ± 0.3 2.4 ± 0.3 2.4 ± 0.5 Mannose 1.5 ± 0.1 1.8 ± 0.1 1.8 ± 0.5 Ribose 1.1 ± 0.0 2.1 ± 0.2 1.9 ± 0.3 Cellobiose 2.6 ± 0.5 4.5 ± 0.5 7.3 ± 0.5 Saccharose 1.2 ± 0.2 2.0 ± 0.2 2.5 ± 0.2 Disaccharide Lactose 1.3 ± 0.1 1.9 ± 0.2 2.7 ± 0.4 Maltose 1.0 ± 0.1 4.9 ± 0.7 2.6 ± 0.4 Trisaccharide Raffinose 2.4 ± 0.1 2.5 ± 0.3 2.7 ± 0.3

2.4.4.6 Sulfation of GlcNAc-linker-tBoc by ASTB-WT and improved variants

A tBoc (tert-butyloxycarbonyl) protecting group labeled GlcNAc-amino linker molecule (GlcNAc-linker-tBoc[132]) was subjected to perform sulfation with ASTB-WT and variants. This modified GlcNAc was selected due to its easy detection with a UV-detector (254 nm) in the HPLC system. The enzymatic sulfation of GlcNAc-linker-tBoc in presence of pNPS as a donor by ASTB is represented in Figure 23. The “best” variant from directed evolution toward GlcNAc (ASTB-V1) and the “best” variants from KnowVolution campaign toward

Figure 23. Targeted reaction for HPLC analysis of GlcNAc sulfation. Sulfation of GlcNAc-linker- tBoc using pNPS as donor molecule by the act of ASTB. A monosulfated product and a by-product pNP are formed. cellobiose (ASTB-M1, ASTB-M2, and ASTB-M5) were analyzed for product formation. Figure 24 shows the product formation by ASTB-WT and all the aforementioned variants over 50 h, which were performed in analytical scale (80 µmol GlcNAc-linker-tBoc, 80 µM pNPS, and 40 µM purified enzyme) and analyzed by HPLC. The product formation by ASTB- M2, ASTB-M5, and ASTB-V1 is higher compared to the ASTB-WT at every time point. All three variants performed sulfation in similar rate (~2.0-fold higher product formation).

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Interestingly, the only variant ASTB-M1 did not perform better than ASTB-WT indicating that the amino acid substitution L446P does not play a role in the improvement of GlcNAc- linker-tBoc sulfation. These results also indicate that the substitution V579D is better than V579K for the sulfation of GlcNAc-linker-tBoc.

Product characterization was performed with the “best” variant (ASTB-V1) identified in the directed evolution round (toward GlcNAc) and the final variant (ASTB-M5) generated in the KnowVolution campaign (toward cellobiose). The reaction was further optimized in order to isolate the formed product by ASTB-WT, ASTB-M5, and ASTB-V1. The donor molecule pNPS inhibits the reaction as already found out while establishing the screening system (Figure 12C). Therefore, the product formation (monosulfated GlcNAc-linker-tBoc) was enhanced by feeding pNPS (16 µmol after every 24 h to 80 µmol GlcNAc-linker-tBoc) slowly to the reaction catalyzed by ASTB-WT (see an example of synthesis in Figure 25A).

Figure 24. Sulfation of GlcNAc-linker-tBoc by ASTB variants over time. Figure shows ASTB-WT (green), ASTB-V1 (grey), ASTB-M1 (blue), ASTB-M2 (cyan), and ASTB-M5 (red). Reactions (80 µmol GlcNAc-linker-tBoc, 16 µM pNPS, and 40 µM purified enzyme) were performed in analytical scale and analyzed by HPLC (75 % (v/v) acetonitrile dissolved in 5 mM (w/v) ammonium acetate, pH 4.3; 1 mL/min; 10 min).

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Product formation was boosted for ASTB-WT by feeding pNPS from 10 µmol to 27 µmol in a duration of 72 h, which corresponds to a conversion of 33.8% GlcNAc-linker-tBoc into its monosulfated product (Table 7). A conversion of 67.3% (2-fold) and 87.1% (2.7-fold) was achieved by ASTB-M5 and ASTB-V1, respectively (Table 7). The products (Figure 26) were isolated and purified using semi-preparative HPLC for determination and structure elucidation by NMR. Figure 26 represents the HPLC as well as MS chromatogram of ASTB- WT, ASTB-M5, and ASTB-V1. ESI-MS fragmentation confirmed a monosulfation of GlcNAc- linker-tBoc (m/z ratio of [M-H]- = 500.7-500.8) for all enzymatic conversions. 1H- and 13C- NMR (Figure 53, Figure 54, Figure 55) confirmed the attachment of a sulfate group either at position C-3 or C-4 for ASTB-WT, ASTB-M5, and ASTB-V1. The sulfated products 3-O- sulfo-GlcNAc-linker-tBoc (Figure 25B) and 4-O-sulfo-GlcNAc-linker-tBoc (Figure 25C) were found as a mixture in a synthesis ratio of 55-60% and 40-45%, respectively (for NMR-data see Table 13 (3-O-sulfo-GlcNAc-linker-tBoc) and Table 14 (4-O-sulfo-GlcNAc-linker-tBoc)).

Figure 25. Separation of reaction components by analytical HPLC and structures of novel sulfated compounds. A) HPLC was performed with a defined mobile phase (75% acetonitrile, 5 mM ammonium acetate, pH 4.3), at 1.0 ml/min for 10 min. Retention time of pNP at 3.203 min, GlcNAc-linker-tBoc at 4.064 min, pNPS at 4.372 min, GlcNAc-linker-tBoc sulfate at 6.424 min. Chemical structure of B) 3-O-sulfo-GlcNAc-linker-tBoc and C) 4-O-sulfo-GlcNAc-linker-tBoc.

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Table 7. Semi-preparative synthesis of sulfated GlcNAc-linker-tBoc by ASTB-WT, ASTB-M5, and ASTB-V1.

Enzyme GlcNAc-linker-tBoc pNPS1 Conversion2 Final yield3

[µM] [µmol] [µmol] [µmol] [mg]

ASTB-WT 40 80 16+16+16=48 27.0 (33.8%) 3.5 (8.6%)

ASTB-M5 40 80 16+16+16=48 53.9 (67.3%) 4.2 (10.5%)

ASTB-V1 40 80 16+16+16=48 69.7 (87.1%) 5.0 (12.5%)

Figure 26. HPLC and MS analysis of purified sulfated product. HPLC analysis of semi-purified product for A) ASTB-WT, C) ASTB-M5, and E) ASTB-V1 are shown on the left side. Following peaks were identified: ~3.5 min non-modified GlcNAc-linker-tBoc, 5.9-6.1 min monosulfated GlcNAc- linker-tBoc (mobile phase: 75 % (v/v) acetonitrile dissolved in 5 mM (w/v) ammonium acetate, pH 4.3; flow rate: 1 ml/min; time course: 10 min); ESI-MS-spectra of novel sulfated compounds by B) ASTB-WT, D) ASTB-M5, and F) ASTB-V1. Theoretical m/z of monosulfated GlcNAc-linker-tBoc is 500.1 ([M-H]-).

116 µmol pNPS was fed into reactions every 24 h. 2Conversion in µmol was determined by analytical HPLC measurements (before purification). Conversion in % gives the percentage of sulfated product compared to the initial amount of substrate. 3Final yield after complete purification. Yield in % represents the product yield relative to initial substrate (calculated by analytical HPLC measurements after purification). Page 51 of 135

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2.4.4.7 pH activity and pH stability ASTB-WT and improved variants

The Britton and Robinson buffer system was employed to study the pH activity and stability due to its broad buffering range (pH 2.0 – 12.0). As depicted in Figure 27, ASTB- WT, as well as the variants (ASTB-M1, -M2 and -M5), show a bell-shaped pH activity profile at a pH range of 5.0 to 12.0. The trend of the pH activity profiles is very similar between the wildtype and the variants. Neither a shift nor a dramatical change in the residual activities was observed. The pH optimum of all enzymes lies clearly at pH 10.0.

The pH stability of ASTB-WT and the final ASTB variants was examined at a pH range of 6.0 to 11.0 for a duration of 6 h. The relative activities of the enzymes did not vary much between pH 6.0 and pH 9.0 (80 – 100%). Though, all enzymes showed an optimum at pH 10.0 (Figure 27), the stability of them decreases significantly at pH 10 over time (Figure 28A). ASTB-WT is the most unstable enzyme at pH 10 losing about 80% of its initial

Figure 27. pH activity profile of ASTB-WT and variants within a pH range of 5.0-12.0. (A) pH activity profiles of ASTB-WT (green), ASTB-M1 (blue), ASTB-M2 (cyan), and ASTB-M5 (red). Activities were measured in 100 mM Britton and Robinson buffer at different pH values (pH 5.0- 12.0) using cellobiose as acceptor. ASTB activity was considered as 100 % at pH 10.0. All plotted mean values and standard errors were calculated from four replicates.

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activity, while ASTB-M5 seems to be the most stable enzyme at this pH value keeping 60% of its residual activity (Figure 28B).

Figure 28. pH stability of ASTB-WT and variants after 6 h. A) pH stability profile of ASTB-WT (green), ASTB-M1 (blue), ASTB-M2 (cyan), and ASTB-M5 (red) between pH 6 and 12 after 6 h of incubation. B) pH stability at pH 10.0. Enzymes were incubated in 100 mM Britton and Robinson buffer at pH 10.0, and residual activity was measured at 0, 1, 2, 4, and 6 h using pNPS assay. Experiments were performed in four replicates for each point.

2.4.4.8 Organic co-solvent activity of ASTB-WT and improved variants

The resistance of ASTB-WT and final variants were investigated with six different water- miscible organic co-solvents (DMSO, methanol, ethanol, tetrahydrofuran, acetone, and acetonitrile; Figure 29). Among them, ASTB-WT showed better resistance in the presence of the tested alcohols (methanol: Figure 29B and ethanol: Figure 29C) as co-solvent. Especially, in presence of 5-25% ethanol, a dramatic increase of residual ASTB-WT activity up to 140% was observed, whereas the ASTB-variants lost 50% of their activity already at 5% ethanol. Interestingly, the resistance profiles of the single variant ASTB-M1 shifted toward higher DMSO and acetone concentrations. As shown for DMSO (Figure 29A) and acetone (Figure 29E), ASTB-M1 can tolerate higher co-solvent concentration compared to the ASTB-WT and other variants. ASTB-M1 shows a 2-fold increased tolerance at 40% DMSO and 2.5-fold at 5% acetone (>1.5-fold throughout the tested acetone concentrations). Furthermore, ASTB-M1 showed slightly higher tolerance as well with all tested tetrahydrofuran and acetonitrile concentrations as co-solvents.

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Figure 29. Organic co-solvent resistance of ASTB-WT and variants. Relative activities of purified ASTB-WT (green), ASTB-M1 (blue), ASTB-M2 (cyan), and ASTB-M5 (red) as a function of organic co-solvent concentration (0-50 % (v/v)). A) DMSO, B) methanol, C) ethanol, D) tetrahydrofuran, E) acetone, and F) acetonitrile. Relative activity is the ratio of specific activity in the presence of organic co-solvent to that in the absence of organic co-solvent (0 %; reaction performed in buffer). Experiments were performed in four replicates for each point.

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2.4.4.9 Thermostability of ASTB-WT and improved variants

The temperature stability of ASTB-WT and KnowVolution variants (ASTB-M1, -M2, and -M5) was determined in a temperature range between 4°C and 67.1°C after 10 min incubation at respective temperature (Figure 30). ASTB-WT and ASTB-M2 behave similarly throughout the measured temperature range. The biggest change was observed for ASTB-M1 and ASTB-M5, wherein ASTB-M1 is more and ASTB-M5 less stable than the wild- type between 4°C and 35°C. For instance, at 35°C ASTB-M1 lost only 10% of its initial activity and all other variants including ASTB-WT lost about 30%. The melting temperature of all enzymes was obtained to be very similar, which lies between 48-50°C. All enzymes did not show any residual activity at >65°C. Nevertheless, at 60°C the final KnowVolution variant, ASTB-M5 retained 25% of its residual activity and ASTB-WT only 10%.

Figure 30. Temperature stability of ASTB-WT and variants between 4 and 67.1°C. Stability profiles of ASTB-WT (green), ASTB-M1 (blue), ASTB-M2 (cyan), and ASTB-M5 (red) are illustrated. Activities were measured using pNPS assay and cellobiose as acceptor. The highest ASTB activity measured was considered as 100 %. The dotted line across the figure represents T50, at which only 50 % of the enzyme activity is measured. All plotted mean values and standard errors were calculated from four replicates.

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2.5 Discussion

Carbohydrates like GAGs are valuable sulfated and non-sulfated (only hyaluronan) pharmaceutical compounds, which are mainly isolated in industrial scale from animal sources (e.g., pig, bovine, and shark)[16] and produced through fermentation of suitable microbial strains[133]. Chemical synthesis these molecules is a very challenging task due to the long chain lengths and complex patterns of functionalization (mostly sulfation) and epimerization. Oligosaccharides with more than five sugar units are often difficult requiring protection/deprotection chemistry and thereby many synthesis steps and finally suffering from low final yields[86]. The biggest challenging issue is the regio- and chemoselective sulfation of hydroxyl groups by chemical processes[78a]. In vitro (chemo)enzymatic synthesis of GAGs is alternatively an attractive and well-studied field in glycobiotechnology[86]. Specialized enzymes in such processes can address the selectivity issue. Reports of enzymatic and chemoenzymatic synthesis of keratan sulfate oligosaccharides employed human PAPS-dependent sulfotransferases[134], chemical sulfation[135] for the sulfation steps, and glycosyltransferases for the elongation of the oligosaccharide chains. However, the enzymes used in the studies are not applicable for industrial purpose due to their expensive dependency (PAPS: ~92 M €/kg; Sigma- Aldrich). PAPS-independent sulfotransferases from prokaryotes (e.g., aryl sulfotransferases) are of high synthetic importance due to their promiscuous substrate scope (phenolic: antibiotics, steroids, and flavonoids; non-phenolic: D-glucose, glycerol, 1-butanol, 2-phenylethanol, and cyclohexanol) reported in the recent studies[114-116, 136]. Three crucial properties of PAPS-independent sulfotransferases make them attractive biocatalysts in (poly)saccharide functionalization: 1) sulfation of non-phenolic hydroxyl groups, 2) PAPS-independency, and 3) production in conventional bacterial expression systems. Nature designed these enzymes for a variety of aromatic sulfuric acid esters and phenols as sulfate donors and acceptors[77]. Therefore, these enzymes are needed to be re-engineered especially in terms of activity towards non-phenolic acceptors like small and larger sugar molecules for application in glycobiotechnology.

The aryl sulfotransferase ASTB from D. hafniense was targeted in this study to engineer for a model monosaccharide (GlcNAc) and a disaccharide (cellobiose). The hexosamine

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GlcNAc appears as a building block of several GAGs (such as heparan sulfate, heparin, and keratin sulfate) and is in most of the cases sulfated[137]. On the other hand, cellobiose is the building block of the most abundant polymer on Earth, cellulose and chemically sulfated cellulose is pharmaceutically valuable[88-89].

A robust and reliable screening system is key to every successful protein engineering study that allows tailoring of enzyme properties to application demands[44]. Sulfotransferase properties to address often comprise enzyme activity[118], substrate specificity[117], and substrate inhibition[99b]. In this case, the specific activity was the target ASTB property to improve toward the aforementioned acceptors. Sulfotransferase screening system based on pNPS is often applied, since this allows a colorimetric detection of product formation (here by-product pNP)[78b, 103, 138]. The pNPS screening system was expanded and validated in 96-well MTP format for the first time for ASTB as enzyme and GlcNAc and cellobiose as acceptor substrate. Parameters of the screening system including the volume of the cell- free extract, donor, and acceptor concentration were systematically optimized for GlcNAc and cellobiose (Figure 12). Finally, a true CV of 14.3% and 10.5% were determined for pNPS screening system applying GlcNAc and cellobiose as acceptors, respectively (Figure 13). Successfully employed MTP-based screening systems in directed evolution campaigns with a true CV below 15% are reported (e.g., cellulase[72], esterase[129b], and epoxygenase[129a]). The continuous pNPS screening system was successfully validated in a directed evolution round and subsequently used for a KnowVolution campaign of ASTB (Figure 31).

A random mutagenesis library of astB was generated by applying SeSaM method, which was used for both directed evolution and KnowVolution phase I. The selection of an appropriate technique to generate gene diversity is an important decision to start a smart directed evolution campaign. SeSaM with its advantageous features over regular random mutagenesis methods has already been well-established and applied in various successful enzyme evolution studies[72, 139]. To date, there is no report of directed sulfotransferase evolution, in which SeSaM was applied to construct libraries. SeSaM identifies itself by its transversion enrichment and subsequent mutations, which generates chemically more diverse substitutions than other PCR-based methods[57, 140]. Overall, 35% of 1,760 SeSaM

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Engineering of an aryl sulfotransferase toward sulfation of saccharides variants showed sulfotransferase activity in the generated ASTB library (an active variant was defined by the 10 % of retained WT-activity). The library possesses a Ts/Tv ratio of 1.7, which is in close agreement with the reported value for a SeSaM-Tv-classic (Ts/Tv: 1.8) as mutagenesis method[121a]. One round of directed evolution of ASTB yielded the best variant ASTB-V1 (V579D) with an up to 3.4-fold enhanced specific activity toward GlcNAc as target acceptor and 2.4-fold increased product formation (GlcNAc-linker-tBoc). Thereby, the successful validation of the developed pNPS screening system was demonstrated.

The engineering of ASTB was further expanded to a full KnowVolution campaign while using cellobiose as target acceptor. This strategy was selected to avoid several rounds of directed evolution without gaining any deeper molecular knowledge. In total 3,067 were screened in the entire campaign toward cellobiose activity. In the ASTB KnowVolution study, in total 7 potential positions were identified for improving activity toward cellobiose in phase I. Saturation of those positions yielded 4 beneficial positions (L446, V579, G608, and S615) showing a positive effect on the activity. Accordingly, 3 non- beneficial positions (42.5% of identified positions) were eliminated in the early stage of

Figure 31. Overview of KnowVolution campaign of ASTB toward cellobiose. One round of directed evolution yielded seven potentially beneficial positions (E111, T144, L446, V579, G608, S615, and V624; positions in italic came from double substituted variants) in phase I. In phase II, individual site-saturation mutagenesis (NNK codon) of all seven positions were performed. Four positions yielded higher activities and the substitutions (L446P, V579K, G608D, and S615G) were selected for recombination based on an ASTB homology model (phase III). Finally, in phase IV recombination was performed to generate double substituted variants (site-directed mutagenesis). Adapted from Islam et al. 2018.

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Engineering of an aryl sulfotransferase toward sulfation of saccharides the campaign. Reports show 50% of the potentially identified substitutions in an evolution round do not contribute to the actual improvement[68]. Computational analysis (phase III) revealed that the positions are located relatively far from each other so that they were not grouped and individual recombination was performed. One by one recombination in phase IV revealed amino acid positions contribute to activity (446 and 579) as well as soluble expression (608 and 615). Furthermore, recombination of them showed cooperative effect while combining amino acid substitutions for improved activity (L446P and V579K) with each other. In every phase, ASTB activity was improved for cellobiose from 2.5-fold (phase I) to 4.5-fold (phase II) and finally to 7.3-fold (phase IV). All these results prove the effectivity of a KnowVolution campaign achieving the final ASTB variant (ASTB-M5) with two amino acid substitutions, in which both contribute individually and the recombination synergistically to the activity of ASTB. Positions L446 and V579 were never reported for any aryl sulfotransferase before. Interestingly, L446P plays a role in only in sulfation of glucose and glucose-based oligosaccharides (e.g., cellobiose, maltose). Structural analysis of ASTB confirmed that 446 is located in the close proximity of the active site (Figure 18B). The flexibility of the entrance channel loop to the binding pocket might be increased by the substitution of leucine to proline, which might facilitate enough space for disaccharides. On the other hand, V579K seems to have a common positive influence on the ASTB activity towards all tested mono-, di- and trisaccharides (Table 6). V579K represents a distal substitution sitting in an extended loop in the C-terminal region and not being in contact with the dimer interface (Figure 18). High flexibility of C-terminal loop including position 579 was ensured by the calculation of root mean square fluctuation (RMSF) per residue of ASTB (Figure 52). Therefore, V579K might change the protein conformational motion leading to an improved sulfation activity towards GlcNAc as well as cellobiose[141].

Structure elucidation of the sulfated GlcNAc-linker-tBoc disclosed the selectivity of ASTB. ASTB-WT and its variants (ASTB-V1 and ASTB-M5) are able to only monosulfate GlcNAc- linker-tBoc, which was confirmed by MS analysis (Figure 26). NMR data finally confirmed a partial regioselectivity at C-3 (40-45%) and C-4 position (55-60%) when sulfation takes place by ASTB (Figure 55). In most of the applied chemical cellulose sulfation approaches, the regioselectivity is C-6 > C-2 >(>>) C-3[18, 94]. To date, there was no report of C-3/C-4

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Engineering of an aryl sulfotransferase toward sulfation of saccharides sulfation of GlcNAc or similar saccharides by any AST. Therefore, ASTB and its variants represent an enzymatic route to sulfate mono- and oligosaccharides at rare hydroxyl groups. The influence of the best substitutions identified for ASTB activity was investigated for the following enzyme properties; 1) pH profile, 2) pH-stability, 3) thermostability, and 4) organic co-solvent activity. Experiments regarding pH profile (pH 2.0 – 12.0) revealed a bell-shaped profile for ASTB-M1, ASTB-M2, and ASTB-M5 similar to ASTB-WT without any shift in pH optimum at pH 10 (Figure 27). Interestingly, the variants, ASTB-M1 (L446P) and ASTB-M5 (L446P/V579K) showed higher pH stability over 6 h at the optimum pH compared to ASTB-WT (Figure 28). These findings indicate that L446P/V579K influences both activity and pH stability and ASTB can be further improved for pH stability. On the other hand, the best amino acid substitutions found in KnowVolution did not change the melting temperature of ASTB variants in comparison to the ASTB-WT (Figure 30). Nevertheless, ASTB-M1 showed relatively higher temperature stability between 30° and 40°C than ASTB-WT, whereas the recombination variant ASTB- M5 was less stable compared to ASTB-WT. This experimental finding showed again that ASTB can be evolved for further properties other than activity. In the presence of organic co-solvents such as DMSO and acetone, variants like ASTB-M1 and ASTB-M5 showed higher residual activity than ASTB-WT (Figure 29). This opens further possibilities to tailor ASTB for organic solvents, which might be needed for substrate solubilization in biocatalysis.

Taken together, ASTB is a very attractive and novel biocatalytic tool for chemo- /regioselective sulfation of saccharides at rare positions (C-3/C-4). In this study, a robust continuous pNPS-based screening system for ASTB was developed for GlcNAc and cellobiose, which can be very likely expanded to other pharmaceutically interesting sugars and/or substrate classes (for instance, antibiotics[78b, 142], steroids[113, 143], and flavonoids[114b]). It was demonstrated that ASTB can be re-engineered by using directed evolution followed by KnowVolution to improve its specific activity towards non-natural acceptors like saccharides. Two key positions (446 and 579) for improvement of activity were identified, which were never reported before for any AST. Further protein engineering studies of ASTB might improve the activity toward larger polysaccharides like cellulose and chitin and provide fully regioselective sulfation only at C-3 or C-4.

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Engineering of a cutinase toward degradation of synthetic polymes

3. Engineering of a cutinase toward degradation of synthetic polymers

3.1 Declaration

Parts of this chapter have been published as:

Islam S*, Apitius L*, Jakob F, Schwaneberg U. 2019. Targeting microplastic in the void of diluted suspension. Environment international, 123, 428-435 (*shared first authorship)[144].

Parts of this chapter has been filled for patent applications:

Schwaneberg U, Sözer S, Mertens A, Davari MD, Jakob F, Islam S, Weber L. 2018. Fusion Peptides or Proteins, their Use, and Systems and Kits based thereupon, for the Separation and/or Detection of Plastics, particularly of Microplastics. EP18178726.8.

Mussmann N, Wieland S, Degering C, Zimmermann W, Wei R, Jakob F, Islam S, Schwaneberg U, Haarmann T, Lorenz P, Rachinger M, Schreiter M, Schwerdtfeger R. 2018 Mittel erhaltend rekombinante Polyesterase. DE 102018210605.3.

Mussmann N, Wieland S, Degering C, Zimmermann W, Wei R, Jakob F, Islam S, Schwaneberg U, Weber L, Rübsam R, Eidener J, Haarmann T, Lorenz P, Schwerdtfeger R. 2018. Mittel erhaltend Polyesterase I. DE 102018210608.8.

3.2 State of the art

3.2.1 Plastics and their applications

Nowadays, the use of plastic is abundant in every household. From household items to industrial applications, the use of plastic has increased dramatically since the middle of the 20th century[145]. More than 330 million tonnes of plastic is produced in 2016 worldwide and one-third of it is used in packaging[146]. An overview of plastic production between 1950 and 2016 is depicted in Figure 32. The word “Plastics” came from a Greek word plastikos that means “capable of being shaped or molded”. Plastics typically made of synthetic polymers of petrochemical origin are indeed malleable or flexible and can be molded in any shape[147]. The specific properties of plastics e.g., versatility, light- weightiness, durability, cost-effectivity, robustness, and resistance are the main reasons behind the high demand for plastics[147-148]. Table 8 shows the common synthetic polymers with their various applications.

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Plastics can be classified into two main groups: thermoplastics and thermoset plastics[149]. Thermoplastics (e.g., polyolefins such as polypropylene and polyethylene) are generated by breaking the double bond in the original olefin by additional polymerization to form new carbon-carbon bonds, the carbon-chain polymers. Thermoplastics make around 92%

Figure 32. Worldwide manufactured plastics from 1950 to 2016 and European plastic demand by the synthetic polymers in 2016. A) Data includes thermoplastics, polyurethanes, and other plastics (thermosets, adhesives, coatings, and sealants). Does not include PET, PA, PP, and polyacrylic-fibers. B) Numbers are shown in percentage. Adapted from PlasticsEurope, 2017.

of used plastics globally. They can be repeatedly molded by heating and cooling and are considerably non-biodegradable[32]. Thermoset plastics, for instance, polyester and polyurethane are produced by condensation of a carboxylic acid and an alcohol or amine to form polyester or polyamide[150]. Unlike thermoplastics, thermoset plastics cannot be reshaped after solidifying[149] and are potentially susceptible to biodegradation[32]. Only 8% of the worldwide used plastics are derived from thermoset plastics[32].

3.2.2 Environmental pollution by microplastic

Carpenter and his colleagues belonged to one of the first groups, who drew attention to the pollution caused by plastic debris in the coastal areas[151]. Since then the accumulation of used plastic debris in the environment increased dramatically as the consumption of plastic rises day by day[152]. Over the past few decades, conventional materials for packaging e.g., glass, paper or metal are replaced by cheap plastic materials[148]. Once plastic waste of all sizes is discarded in nature, it gets washed away and enters the marine environment [145]. The overall disposed plastic can be distinguished according to their sizes: megaplastic (>100 mm), macroplastic (>20 mm), mesoplastic (5-20 mm), and microplastic (<5 mm). Plastic waste can undergo several modes of degradation processes

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Engineering of a cutinase toward degradation of synthetic polymes such as photo-, bio-, thermooxidative degradation, and wave action[152-153]. Unfortunately, all kind of mentioned degradation processes are very slow and thus plastic debris survive the marine environment for an extremely long time period[154]. The pollution caused by plastic debris is ubiquitous and is a threat to the marine biota as well as the human being[31]. Even if the use of plastics stopped immediately, the waste will persist for centuries in the environment[152].

Table 8. Most widely used synthetic polymers and their applications. Information gathered from Zheng et al. 2005, Andrady 2011, PlasticsEurope 2016.

Synthetic polymer Abbreviation Application Low-density polyethylene LDPE Films, packaging High-density polyethylene HDPE Milk and juice jugs, toys, pipes Polyethylene terephthalate PET Beverage bottles, tubes, films Polyester PE Fibers, textiles Polypropylene PP Rope, bottle caps, automotive parts Polyamide PA Fibers, tubes, netting, traps Polystyrene PS Tanks, containers, compact-discs (CD) Polyvinyl chloride PVC Pipes, frames, floor covering Polyurethane PUR Coating, insulation, paints, tires Polylactic acid PLA Coatings Polymethyl methacrylate PMMA Contact lenses, plexiglas

A recent concern is the accumulation of smaller plastic fragments (well-known as microplastics) in the marine habitats, which cannot be visualized by naked-eye. Microplastics can end up in the oceans mainly in two ways: 1) direct introduction by human beings through drainage and 2) natural breakdown and fragmentation of macro- and mesoplastics[148]. Microplastics are typically used for manufacturing cosmetic and pharmaceutical products (e.g., scrubbers, facial cleansers, toothpaste) and air-blasting technologies[31, 156]. Thus, the wastewater containing microplastics gets directly introduced into the sewage system after daily use[31]. Moreover, synthetic fibers from washing clothes are also one of the major microplastic contaminants in the sewage water ending up in the marine ecosystem. Reports showed that a single load of laundry from domestic washing machines can set >1900 fibers free in the wastewater[153]. Although the

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Engineering of a cutinase toward degradation of synthetic polymes use of microplastics is intrinsically tied to the daily household, the disposal of it plays a threat to the environment, especially the marine biota[157]. Since microplastics are highly hydrophobic materials and possess a high surface area to volume ratio, these are highly susceptible to act as adsorbent or reservoir of toxic chemicals so-called “persistent organic pollutants (POPs)” found in the environment[147, 158]. Due to their smaller size, microplastics and/or contaminated micro-debris can be ingested by a variety of organisms from invertebrates to higher mammalian organisms[157b]. The first reports of microplastic ingestion showed invertebrates (e.g., amphipods, lugworms, and barnacles) to ingest microplastics within a few days[156]. Furthermore, the most frequently studied invertebrate is a mussel type (Mytilus edulis), which was reported to ingest microplastic even within 12 h in the experimental phase[159]. Microplastic ingestion is also well-spread among the marine animals including seabirds[160], turtles[161], fishes[162], and sperm whales[163]. The ingested non-degradable microplastics by marine organisms can block their intestinal tract, inhibit secretion of gastric enzymes, affect steroid hormone levels, delay in ovulation, and lead to infertility[157c]. Thus, the microplastics get accumulated in the marine food web, which has become an open threat to the marine ecosystem over the few decades[31, 153].

The human population as mentioned above get exposed to microplastics by their daily life consumables. For instance, microparticles in the toothpaste can unintentionally be swallowed and finally can enter and get adsorbed in the gastronomical tract[164]. Evidence exists that the high microplastic contaminants in seafood are a major threat to food safety of human[165]. Additionally, alternate ingestion of microplastic by a human can provoke transposition of chromosomes resulting in diseases such as infertility, breast cancer, skin damage, and obesity[31]. Potential human health risks by microplastic pollution are not analyzed in detail yet, which demands more studies on the harmful effect of microplastic on a human in the future[31, 166].

3.2.2.1 Polyethylene terephthalate (PET)

Polyethylene terephthalate (PET) is one of the most commonly used thermoplastic polymer of polyester family globally. In Europe, PET was 7.4% of all commonly manufactured plastic in the year 2016[146]. The most well-known application of PET is

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Engineering of a cutinase toward degradation of synthetic polymes packaging for instance, of beverage bottles. PET can appear as a transparent (amorphous) and as an opaque or white colored (semi-crystalline) material depending on processing and application[167]. The monomer of PET can be synthesized in two ways: 1) esterification between terephthalic acid and ethylene glycol (water as a byproduct), or 2) transesterification reaction between ethylene glycol and dimethyl terephthalate (methanol as a byproduct)[168]. Subsequently, after esterification/transesterification, the polymerization is performed via polycondensation reaction of the monomers, wherein ethylene glycol is released as a byproduct that is recycled within PET production[167]. Figure 33 shows a common chemical structure of PET.

Figure 33. Chemical structure of polyethylene terephthalate (PET). PET is synthesized by the transesterification between ethylene glycol and dimethyl terephthalate. “n “states for the number of monomeric repeating units.

3.2.2.2 Polyurethane (PUR)

Polyurethane (PUR) is a versatile class of synthetic polymers that find their applications in industrial and medical fields. Products made of polyurethanes are very diverse such as coatings, fibers, tires, paints, adhesives, sponges, insulation, and many more[32, 169]. Among all the European manufactured plastics, PUR was about 7.5% in 2016 (Figure 32B). PUR is typically mixed polymers composed of polyisocyanates and polyol containing isocyanate and hydroxyl groups, respectively. The reaction between isocyanate (N=C=O) and hydroxyl groups (OH) ends up as a urethane bond, which appears as a repeating unit in the final PUR polymer[170]. The used polyisocyanates for PUR synthesis are mainly diisocyanates (e.g., 2,4-tolylene diisocyanate, 4,4’-diphenylmethane diisocyanate, 1,3- xylylene diisocyanate). On the other hand, two types of polyols are commonly used, namely poly(ester)-type (e.g., poly(butylene adipate), poly(ethylene-butylene adipate), polycaprolactone) and poly(ether)-type (e.g., poly(oxytetramethylene) glycol, poly(oxypropylene) glycol, poly(oxypropylene)-poly(oxyethylene) glycol). As for a linking agent, for instance, 1,4-butanediol, ethylene glycol, and glycerol are employed[171]. PUR chains are not only made of carbon atoms but rather of heteroatoms including oxygen,

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Engineering of a cutinase toward degradation of synthetic polymes carbon, and nitrogen[172]. Figure 34 shows the simplified formula of linear PUR. Depending on the changes and variations if the polyhydroxyl and polyfunctional nitrogen compounds, different classes of PUR can be produced[170b]. Furthermore, variations in the number of substitutions and the linker between and within branch chains manufacture linear to branched and flexible to rigid PUR[173].

Figure 34. Chemical structure of polyurethane (PUR). R1 and R2 indicate the side chains of diols and diisocyanate, respectively. n states for the number of repeating units.

3.2.3 Biodegradation of PET and PUR

Degradation of synthetic polymers can occur due to changes in polymer properties and functionalities induced by any physical, chemical, and biological reactions. Degraded polymers exhibit changes in their material properties, for instance, cracking, erosion, discoloration, phase separation, or delamination[174]. Mainly three types of degradation are prominent: photo, thermo-oxidative, and biological degradation[29]. Biodegradation of polymers especially synthetic polymers due to plastic pollution is often termed in combination with waste management[29, 32, 175]. Compared to photo and thermal degradation processes, biodegradation does not require heat, UV-light or high-energy radiation and is environmentally friendly[29]. Biodegradation is leaded mainly by living organisms, which can break down organic substances. Conventional petrochemical polymers are typically not degradable by the microbial attack or take decades to be degraded[175b]. But some synthetic plastics such as polyester polyurethanes, polyethylene with starch blend are biodegradable[32].

PET and especially modified PET are reported to undergo degradation by enzymatic processes. Modified PET contains comonomers like polybutylene adipate/terephthalate (PBAT) and polytetramethylene adipate/terephthalate (PTMAT), which exhibit ester, ether, and amide bonds. These functional linkages are vulnerable to enzymatic hydrolysis. Synthetic polyesters containing aromatic components as for PET require high reaction temperatures (glass transition temperature of PET is approximately 75°C) for

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Engineering of a cutinase toward degradation of synthetic polymes hydrolysis[176]. Therefore, thermophilic microorganisms producing thermostable hydrolases (cutinases, esterase, and lipases) are capable of degrading aromatic polyesters, which is well documented in the literature[36, 177]. Mostly the thermophilic actinomycetes from Thermobifida species such as Thermobifida fusca[178], Thermobifida cellulosilytica[178a], and Thermobifida alba[34, 179] produce PET-hydrolyzing enzymes. Moreover, filamentous fungi Fusarium oxysporum and Fusarium solani have been reported to grow on medium containing PET fibers and to degrade it[180]. Most recently reported bacterium called Ideonella sakaiensis 201-F6 as well can degrade and assimilate PET[181].

Degradation of PUR is classified into three categories 1) fungal, 2) bacterial, and 3) enzymatic degradation[169, 182]. PUR-degradation by fungi is mainly reported for the polyester-PUR, in which fungal enzymes (esterase and urethanase) hydrolyze ester and urethane bonds. Four fungal species, Curvularia senegalensis, Fusarium solani, Aureobasidium pullulans, and Cladosporium sp. were shown to degrade polyester- PUR[183]. As an example, an extracellular polyurethanase (PUase) from Curvularia senegalensis displaying high PUR degrading activity (based on the ester linkage cleavage) was reported[183]. Bacterial PUR degradation is reported for a variety of bacterial strains such as Corynebacterium sp.[184], Pseudomonas aeruginosa[184], Comamonas acidovorans[185], Acinetobacter calcoaceticus[186], and Bacillus subtilis[187] that utilize polyester-PUR solid cubes or PUR paint as carbon, nitrogen and energy source to grow. Two types of PUases are isolated and characterized so far, for instance, Comamonas acidovorans TB-35 produces an extracellular (PueA/B) and a membrane-bound esterase (PudA), whereas PudA catalyzes the main polyester-PUR degradation[37, 188]. Akutsu and coworkers reported a two-step degradation process: PudA binds by hydrophobic adsorption onto the insoluble polyester-PUR and subsequently, the hydrolysis of polyester-PUR takes place by the act of its esterase activity[37].

3.2.4 Cutinases from Thermomonospora curvata (Tcur0390/Tcur1278)

The enzyme class cutinase was originally discovered in plant fungi (e.g., Fusarium solani pisi) for hydrolysis of the ester bonds of the plant polymer cutin, which is composed of hydroxylated C-16- and C-18-fatty acids linked via ester bonds. Cutinases (EC 3.1.1.74)

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Engineering of a cutinase toward degradation of synthetic polymes function as serine esterases and are a member of the α/β hydrolase superfamily. Cutinases have a of Ser–His–Asp, whereas the catalytic residue serine is exposed to solvent[177a]. Unlike lipases, cutinases lack the typical lid structure and do not need any interfacial activation. Furthermore, dissimilar to esterases, cutinases can hydrolyze lipid substrates[177a]. Apart from plant fungi, a variety of bacterial cutinases were isolated and characterized such as Thermobifida fusca[189], Thermobifida alba[34], Thermobifida cellulosilytica[190], Thermomonospora fusca[191], and Thermomonospora curvata[192]. All mentioned thermophilic actinomycetes produce synthetic polyester hydrolyzing cutinases[36, 177a, d]. Hydrolysis of aliphatic polyesters e.g., PCL[189, 193] as well as aliphatic-aromatic co-polyesters such as PET[178-179, 192, 194] and PTT[195] have been well documented.

In this study, the focus was on a cutinase called Tcur1278 originated from Thermomonospora curvata, which is a facultative aerobic thermophilic actinomycete isolated from composts containing plant materials. The strain T. curvata DSM 43183 has an optimal growth at 50°C[196] and at a wide range of pH (7.5 to 11.0)[197]. Recently, two cutinases from T. curvata DSM 43183 (Tcur1278 and Tcur0390) were characterized for PET[192] and polyester-PUR[39] degradation. Tcur1278 functions optimally against typical

Figure 35. Structural model of the cutinase Tcur1278. The model was generated by applying YASARA (structure version 13.1.25). The catalytic triade consists of serine (Ser), histindine (His), and aspartic acid (Asp) residues (highlighted in red).

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model esterase substrate p-nitrophenyl butyrate (pNPB) at 60°C and at pH 8.5. Tcur1278 retains more than 80% and the initial activity after incubation for 60 min at 55°C[192]. Figure 35 shows a structural model of Tcur1278 generated using YASARA Structure version 17.4.17[124] and the catalytic triad (Ser-His-Asp) of Tcur1278 (Figure 35).

3.2.5 Directed polymer degradation by binding domains

Enzymes generally work in aqueous phases and catalyze the reaction of water-soluble substrates. The performance of enzymes can be affected in the presence of water- insoluble substrates. Polymer-degrading hydrolases show interesting characteristics with their water-insoluble substrates (e.g., natural and synthetic polymers), which allow the enzymes to bind the surface of the insoluble substrate[198]. Adsorption and desorption of enzymes onto polymer surfaces during hydrolysis is accomplished by the so-called binding domains/modules[27]. The best example found in nature is the reaction mechanism of cellulases. The biodegradation of the natural polymer cellulose is conducted by cellulases, which are comprised of a cellulose binding domain (CBD) and a catalytic domain (see Figure 36). CBD is separated from the catalytic domain by a flexible linker. The role of the CBD is to bind cellulose and thereby locally bring the catalytic domain to it to initiate the

Figure 36. Schematic presentation of cellulose degradation by cellulase. Cellulose binding domain (blue) binds to cellulose polymer (green), which is connected via a linker domain (red) to the catalytic domain(orange). The catalytic domain is responsible for the degradation of cellulose chains into building blocks.

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Engineering of a cutinase toward degradation of synthetic polymes degradation[26-27]. Similar behavior was also found among poly(hydroxyalkanoate) (PHA) depolymerase that degrades the insoluble polyester PHA[199]. Similar to cellulases and PHA depolymerase, the membrane-bound polyurethanase PudA was considered to possess a hydrophobic-PU-surface binding domain and a catalytic domain[37]. Fusion of surface binding domains to hydrolases for instance cutinases is reported to stimulate PET hydrolsysis[200]. For example, Ribitsch and coworkers fused cutinase (Thc_Cut1 from Thermomyces cellullosylitica) covalently to small cysteine-rich fungal proteins called hydrohobins[200a], which can naturally adsorb to hydrophobic surfaces[201]. Cutinase- hydrophobin fusion proteins were capable to enhance PET hydrolysis up to 16-times than that of the free cutinase[200a]. Additionally, Thc_Cut1 was covalently fused to carbohydrate binding module (CBM)[200c] and polyhydroxyalkanoate binding module (PBM)[178a], wherein the hydrolysis of PET was stimulated by the introduction of these binding modules as well.

Apart from the afromentioned binding modules (hydrophobins, CBMs, and PBMs), polymer-binding peptides (PBPs) or anchor peptides can be used to bind to several classes of materials such as PP[202], PS[203], metal[204], platinum[205]. Fusion of anchor peptides to enzymes to achieve enzyme immobilization onto the polymeric surface has been documented. For instance, a glutathione S-transferase was fused to PS-binding peptides (PS19 and PS23) for directed immobilization on polystyrene substrates[206]. Nature evolved peptides to interact with biological polymers such as phospholipids (e.g., bacterial cell membranes) over millions of years. One class of short water-soluble peptides are called anti-microbial peptides (AMPs) due to their defensive properties against a variety of microbes. AMPs attach to the biological membrane by electrostatic or hydrophobic interaction to form barrels or pores and thus disrupt the microbial membrane[207]. Binding of AMPs such as cecropin-A, LCI, and Tachystatin A2 to a synthetic ABA block co-polymer (PIB1000–PEG6000–PIB1000), PP, and PS was demonstrated in several reports[202, 208].

AMPs are found in all kingdoms of life and vary in length (usually from 10 to 100 amino acids), secondary structure, charge, amino acid composition, and hydrophobicity[209]. Based on their secondary structure, AMPs can be classified into four kinds: 1) α-helix, 2) β-sheet, 3) mixed (α-helix and β-sheet), and 4) unstructured (Figure 37). In this thesis, the anchor peptide Tachystatin A2 (TA2) was employed as a binding module to accelerate

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PET and polyester-PUR degradation by a cutinase. TA2 is a positively charged and β-sheeted AMP derived from horseshoe crab hemocyte (Limulus polyphemus) and shows a broad spectrum of antimicrobial activity against Gram-negative and Gram-positive bacteria and fungi[210]. Recently, TA2 was demonstrated to bind the synthetic polymer, PS[208b].

Figure 37. Different types of AMPs. A) α-helical cecropin A (PDB: 2LA2), B) β-sheeted LCI (PDB: 2B9K), C) mixed structured defensin 5 (PDB: 2KSK), and D) unstructured SMAP-29 (PDB: 1FRY).

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3.2.6 Objectives

The main objective of this part of the thesis was to establish a directed synthetic polymer degradation by the fusion of adhesion promotors (anchor peptides) to a degrading enzyme (Figure 38). The objectives can be divided into four parts:

i) Selection of anchor peptides for binding to polyester-PUR nanoparticles and PET-based textiles ii) Generation and production of fusion proteins of cutinase (Tcur1278-WT and selected anchor peptides (LCI/TA2) iii) Evaluation of targeted polyester-PUR degradation by Tcur1278-WT and Tcur1278-LCI/TA2 iv) Expanding the anchor peptide technology to PET-film degradation

Figure 38. Targeted microplastic degradation in high diulted suspension by the usage of adhesion promoter. Shown is the behavior comparison of Tcur1278-WT and Tcur1278-TA2 in high dilution of polyester-PUR nanoparticle suspension.

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3.3 Material and methods

Material and methods for this chapter were adapted from Islam, Apititus et al. 2019[144].

3.3.1 Chemicals

All chemicals used in this study were of analytical grade or higher quality and were purchased from AppliChem (Darmstadt, Germany), Sigma Aldrich Chemie (Taufkrichen, Germany) or VWR International (Darmstadt, Germany) unless specified. DNA polymerases were prepared in-house. All oligonucleotides were purchased from Eurofins MWG Operon (Ebersberg, Germany). Synthetic genes were ordered from Eurofins MWG Operons (Ebersberg, Germany). Polyester-PUR dispersion (Impranil® DLN-SD) was purchased from Covestro AG (Leverkusen, Germany). The expression strain Pichia pastoris (Komagataella phaffii) BSYBG11 and plasmid pBSYA1S1Z was purchased from bisy e.U. (Hofstätten/Raab, Austria). The host E. coli strains DH5α and BL21 Gold (DE3) were obtained from Agilent Technologies (Santa Clara, USA).

3.3.2 Use of eGFP-anchor fusion protein for binding tests

The reporter protein eGFP (enhanced green fluorescence protein) was used to detect anchor peptide binding to polyester-PUR and PET surfaces. eGFP-LCI and eGFP-TA2 proteins were obtained as previously reported (egfp was covalently fused to the N-terminus of the anchor peptides (LCI or TA2) using a stiff spacer helix (17 aa: AEAAAKEAAAKEAAAKA)[211] and a TEV cleavage site (7 aa: ENLYFQG)[212] as functional separators of both units[208b].

The binding of eGFP-anchor constructs to polyester-PUR layer and PET material was investigated with respect to the negative control eGFP (eGFP-17aa Helix-TEV) using cell- free extract (CFE) of eGFP fusion peptides (1.5 mg/mL lysozyme in 50 mM Tris-HCl buffer, pH 8.0, 1h, 37°C, and 900 rpm). The fluorescence of all CFEs was normalized to 100,000 RFU using the 96-well MTP reader FLUOstar Omega (BMG LABTECH GmbH, Ortenberg, Germany; excitation (Ex) 485 nm, emission (Em) 520 nm, gain 1000, 35 reads/well). Glass slides were coated with Impranil® DLN-SD (Covestro AG, Düsseldorf, Germany) and air dried to obtain polyester-PUR layer on a solid surface. eGFP-anchor and eGFP binding (50 µL) to polyester-PUR layer and PET were performed at room

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Engineering of a cutinase toward degradation of synthetic polymes temperature (10 min). All non-bound peptides and the negative control eGFP were washed off in three subsequent washing steps (1 mL 50 mM Tris-HCl buffer, pH 8.0). Unspecific binding of eGFP was minimized by washing the polyester-PUR layer containing glass slides and PET with sodium dodecylbenzene sulfonate (LAS) (1 mL 0.5 mM in 50 mM Tris-HCl buffer, pH 8.0) as previously described[208b]. Binding of eGFP-anchors to polyester- PUR and PET was confirmed by detection of the fusion partner eGFP (Leica TCS SP8 microscope, Ex: 485 nm, Em: 520 nm, gain 800; Leica Microsystems GmbH (Wetzlar, Germany)).

3.3.3 Construction of cutinase-anchor fusion protein

The wildtype (WT) tcur1278 (GenBank accession: CDN67545.1) derived from Thermomonospora curvata DSM43183[192] was introduced into a pBSYA1S1Z vector by phosphorothioate-based ligase-independent gene cloning (PLICing)[213]. The PLICing product was transformed into electrocompetent E. coli DH5α. The resulting plasmid, pBSYA1S1Z-Tcur1278 was isolated and transformed into freshly prepared electrocompetent Pichia pastoris BSYBG11 for gene expression. The plasmid, pBSYA1S1Z- Tcur1278 served as the vector backbone for the following fusion constructs: pBSYA1S1Z- Tcur1278-17x-TEV-LCI and pBSYA1S1Z-Tcur1278-17x-TEV-TA2. The PLICing primers listed in Table 12 were used to amplify the vector backbone and the gene sequence of anchor part including spacer, TEV restriction site and anchors LCI or TA2.

3.3.4 Production of Tcur1278-WT and Tcur1278-anchors in Pichia pastoris

Tcur1278-WT, Tcur1278-LCI, and Tcur1278-TA2 were produced in Pichia pastoris BSYBG11. The cultivation of pre-cultures was performed in 10 mL YPD medium (10 g L-1 yeast extract, 20 g L-1 peptone, 20 g L-1 D-glucose, 100 µg mL-1 zeocin; 100 mL Erlenmeyer flask) using fresh Pichia pastoris cells (30°C, 200 rpm, 70% humidity, 48 h; Multitron II, Infors GmbH, Einsbach, Germany). The main cultures (200 mL YPD medium supplemented with 100 µg/mL zeocin in 1 L Erlenmeyer flask) were inoculated using the pre-cultures as inoculum to an OD600nm of 0.5. After the cultivation (30°C, 200 rpm, 70% humidity, 72 h; Multitron II, Infors GmbH), the culture supernatant was separated from the cell broth by centrifuging (4°C, 11,279 x g, 15 min; Sorvall, ThermoFischer Scientific, Darmstadt, Germany). 200-400 mL supernatant was concentrated using an Amicon Ultra-15

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Centrifugal Filter Unit (Cut off 10 kDa) unit to 30-40 mL. Tcur1278-WT and Tcur1278-TA2 were purified via a cation exchange column (SOURCE™ 30S, GE Healthcare, Darmstadt, Germany) according to manufacturer’s protocol (running buffer: 50 mM HEPES buffer, pH 7.6, elution buffer: 2 M NaCl). Purified protein was dialyzed against reaction and storage buffer (100 mM Tris-HCl, pH 8.0).

3.3.5 Determination of cutinase activity via pNPB assay

Cutinase activity of Tcur1278-WT and Tcur1278-anchor fusion proteins was determined in 96-well MTP (PS-F-bottom, Greiner Bio-One, Frickenhausen, Germany). Clarified cell supernatant (10 µL) or purified protein (~0.05-1.0 µM) was transferred into MTP (PS-F- bottom, Greiner Bio-One) containing 85 µL reaction buffer (100 mM Tris-HCl, pH 8.0). The reaction mixture was incubated (1000 rpm, 5 min, RT) in an MTP shaking device (TiMix2, Edmund Bühler GmbH, Hechingen, Germany). Reactions were started by supplementing 5 µL of para-nitrophenyl butyrate (pNPB) to a final concentration of 0.5 mM. The release

-1 -1 of pNP was kinetically detected at 405 nm (εpNP=14.7 mM cm ) in a microtiter plate reader (cycles: 30, kinetic interval: 12 s, RT, Time: 5 min; TECAN Sunrise, Männerdorf, Switzerland). One unit (1 U) of cutinase activity is defined by the required amount of enzyme for the formation of 1 µmol pNP per minute.

3.3.6 Evaluation of polyester-PUR degradation

Continuous turbidimetric measurements were performed in 96-well MTP (PS-F-bottom, Greiner Bio-One). MTPs were prepared with 130 µL polyester-PUR dispersion diluted in buffer (dilutions between 1:100 and 1:1200 in 100 mM Tris-HCl, pH 8.0). Reactions were started by supplementing clarified cell supernatant (20 µL) or purified protein (0.6 nM). The degradation of polyester-PUR was kinetically detected at 600 nm in a microtiter plate reader (cycles: 700, kinetic interval: 120 s, RT, Time: 23 h; TECAN Sunrise).

3.3.7 Evaluation of PET-film degradation

Transparent PET-films (0.5 cm × 2.5 cm × 0.25 mm; ES301445, GoodFellow Cambridge Limited, Huntingdon, UK) were placed in a 2 mL Eppendorf tubes and incubated in a washing solution (0.1% w/v SDS, 50°C, 400 rpm, 30 min) using a thermomixer (Eppendorf thermomixer comfort, Hamburg, Germany). The films were washed in ethanol (50°C,

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700 rpm, 5 min) and subsequently in ddH2O (50°C, 700 rpm, 5 min). Afterwards, the films were dried in open Eppendorf tubes (50°C, 48 h) and individual weight was determined. Enzymatic reactions were initiated by incubating the films in culture supernatants containing the desired proteins/fusion proteins, which were diluted in reaction buffer (100 mM Tris-HCl, pH 8.0; 55°C, 650 rpm, 24-72 h). After the enzymatic treatment, the films were washed in three steps using washing solution (0.1 % w/v SDS, 0.5 % w/v Triton

X-100), ethanol, and ddH2O and dried as aforementioned. Finally, the dried films were weighed and the difference between the weight before and after enzymatic treatment was determined as weight loss[214].

3.3.8 Dynamic light scattering (DLS)

Dynamic light scattering (DLS) measurements were accomplished to determine the hydrodynamic radius (Rh) of polyester-PUR nanoparticles with an ALV/CGS-3 goniometer with an ALV/LSE 5004 tau digital correlator and a JDS Uniphase laser operating at 632.8 nm. All measurements were taken in triplicates at an angle of 90° and at 20°C after equilibrating the samples for at least 3 min.

3.3.9 Field emission scanning electron microscopy (FE-SEM)

The degradation of polyester-PUR by Tcur1278 and Tcur1278-TA2 was visualized with field emission scanning electron microscopy (FE-SEM) on a stainless-steel support coated with polyester-PUR. The stainless-steel support (1 cm2) was cleaned by 2-step sonication

(step 1: 1 M NaOH, 50 mL, 30 min and step 2: 500 mL ddH2O, 30 min; Bandelin Sonorex Digitec (Berlin, Germany)). Subsequently, the stainless steel was washed with absolute ethanol (50 mL) and air-dried. The polyester-PUR (10 µL) was coated on the steel support and air-dried as well. Negative control (Tris-HCl buffer, pH 8.0), Tcur1278 and Tcur1278- TA2 were added to the polyester-PUR layer (20 µM) and incubated for 24 h and 40 h, respectively. Incubation was performed at room temperature in sealed (Parafilm, IDL GmbH & Co. KG, Nidderau, Germany) Petri dishes (PS, 94/16 MM, Greiner Bio-One) to avoid evaporation.The samples were rinsed with ddH2O (3x 1 mL) and air-dried for FE- SEM analysis (S 4800 FE-SEM; accelerating voltage: 2 kV, working distance: 3.8-4.4 mm, magnification: 25080x, Hitachi, (Schaumburg, USA)).

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3.4 Results

The results are divided into 4 main parts as follows: i) selection of anchors for binding to target surfaces, ii) production of fusion proteins and determination of specific acivity, iii) polyester-PUR degradation studies and iv) evaluation of PET degradation.

3.4.1 Selection of anchors for binding to polyester-PUR and PET

Two previously known polymer binding anchor peptides, LCI and TA2[208b] were selected in this study for polyester-PUR and PET binding. eGFP as a reporter protein can be employed to test anchor peptide binding performance on a variety of target polymers (PE, PS, PP) easily by fluorescence[208b]. Therefore, eGFP-LCI and eGFP-TA2 binding was therefore visualized by the eGFP fluorescence. Glass slides with polyester-PUR layer and cloth made of 100% PET were coated with cell-free extract (normalized fluorescence to 100,000 RFU) of eGFP (negative control; Figure 39A and D), eGFP-LCI (Figure 39B and E), and eGFP-TA2 (Figure 39C and F). Samples were washed to remove unspecific protein binding, dried, and subsequently analyzed by detecting the fusion partner eGFP on the polyester-PUR surface or PET cloth via confocal microscopy.

Figure 39. Confocal microscopy of polyester-PUR and PET binding by eGFP-anchor. Upper left A) eGFP as negative control, B) eGFP-LCI and C) eGFP-TA2 for polyester-PUR binding. polyester- PUR (Impranil® DLN-SD) coated on a glass support was employed. Bottom left D) eGFP as negative control, E) eGFP-LCI and F) eGFP-TA2 for PET binding. A piece of 100% PET cloth (provided by Henkel AG & Co. KGaA) was used (Leica TCS SP8 microscope, Ex: 485 nm, Em: 520 nm, gain 750).

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As expected, eGFP without any anchor did not bind alone to the polyester-PUR surface. There is a high eGFP background visible for the employed PE cloth. Nevertheless, the fluorescence intensity with the eGFP-anchors on PET is significantly distinguishable compared to the eGFP negative control. TA2 was identified to be a very good anchor for polyester-PUR and PET due to the better binding performance of eGFP-TA2 compared to eGFP-LCI. LCI seems to be a good anchor for PET, but not for polyester-PUR. Accordingly, TA2 was selected as the anchor part to generate the fusion protein Tcur1278-TA2 for polyester-PUR and PET degradation. Tcur1278-LCI was generated as well to analyze PET degradation.

3.4.2 Cultivation of Tcur1278-WT and Tcur1278-anchors in Pichia pastoris

Tcur1278-WT, Tcur1278-LCI, and Tcur1278-TA2 were produced in Pichia pastoris BSYBG11 system (pBSYA1S1Z contains a secretion signal: prepro α-leader sequence), wherein the target proteins are secreted in the culture medium. In order to find out the optimum expression conditions for tcur1278-wt, three yeast culture media (YPD: yeast peptone dextrose; YPG: yeast peptone glycerin; and YNB: yeast nitrogen base) with different C-source were used at 20 and 30°C for cultivation time from 24 h to 96 h. Expression was analyzed by using the cutinase activity assay based on the hydrolysis of pNPB (Figure 40).

Figure 40. Principle of the pNPB-based screening system. para-nitrophenyl butyrate (pNPB) is hydrolyzed in the presence of an active hydrolase (here cutinase, Tcur0390/Tcur1278). The hydrolyzed products para-nitrophenol (pNP), which is colorimetrically detectable at 405 nm and butyric acid. YNB medium is clearly not suitable for Tcur1278-WT production (Figure 41A). YPD, as well as YPG medium, worked better at 30°C, wherein the Tcur1278-WT activity did not increase over the expression time while using YPG medium. Activity increase over the expression time was only observed in the case of YPD medium. The highest Tcur1278-WT activity was obtained in YPD medium at 30°C for a cultivation time of 96 h (Figure 41A). These

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Figure 41. Expression analysis of Tcur1278-WT and Tcur1278-anchors in Pichia pastoris BSYBG11 culture supernatants. A) Volumetric activity of Tcur1278-WT towards pNPB after cultivation in YPD, YPG, and YNB media for 24, 48, 72, and 96 h at 20 and 30°C. Best expression conditions: YPD medium, 30°C, 96 h. B) Expression of target constructs under the best conditions.

expression conditions were selected therefore for further cultivations. Finally, Tcur1278- WT and Tcur1278-anchors were cultivated under the best expression conditions and activity of the target proteins was determined via pNPB assay (Figure 41B). Tcur1278-LCI and Tcur1278-TA2 showed approximately 35% less pNPB activity than Tcur1278-WT. Due to the antimicrobial activity of selected anchors toward yeast, the expression level of the fusion proteins was less than that of Tcur1278-WT.

3.4.3 Determination of polyester-PUR degradation

Polyester-PUR and PET-film degradation were analyzed using Pichia pastoris culture supernatants, in which the pNPB activity was normalized to Tcur1278-LCI activity (lowest activity; see Figure 41B). The best fusion protein was selected by evaluating their polyester-PUR degradation performance in high dilution of polyester-PUR nanoparticles (1:400 (0.1% w/v) and 1:800 (0.05% w/v)). Since the dispersion of polyester-PUR nanoparticles is a turbid solution, a turbidity loss at 600 nm in case of degradation was expected. Culture supernatant containing no target protein (EV) showed in both polyester-PUR dilutions no degradation, as no turbidity loss was observed (Figure 42A and B). This experiment revealed that Tcur1278-WT is capable to degrade polyester-PUR under experimental conditions because the absorbance at 600 nm decreased continuously over time. Interestingly, Tcur1278-LCI showed very similar degradation

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Engineering of a cutinase toward degradation of synthetic polymes behavior compared to Tcur1278-WT indicating no anchor mediated stimulation of polyester-PUR degradation by LCI. On the other hand, the difference between Tcur1278- WT and Tcur1278-TA2 was clearly visible, wherein the degradation rate obtained by Tcur1278-TA2 (reciprocal slope of the turbidity measurement) was up to 2.8-fold higher than Tcur1278-WT (Table 9).

Figure 42. Polyester-PUR degradation by culture supernatants of Tcur1278-WT and Tcur1278- anchors. Activity was normalized to ~70 U/L and diluted 2 times (to 35 U/L). The final empolyed activity for polyester-PUR degradation was 4.67 U/L. A) polyester-PUR dispersion was diluted 400 times (0.1% w/v polyester-PUR nanoparticles) and B) 800 times (0.05% w/v polyester-PUR nanoparticles).

Table 9. Polyester-PUR degradation performance of Tcur1278-WT and Tcur1278-anchors.

Polyester-PUR 1:400 Polyester-PUR 1:800 Improvement Improvement Rate [1/h] Rate [1/h] [fold] [fold] Tcur1278-WT 0.0068 ± 3.2% 1.0 ± 4.5% 0.0070 ± 2.6% 1.0 ± 3.7% Tcur1278-LCI 0.0072 ± 2.9% 1.1 ± 3.9% 0.0076 ± 1.6% 1.1 ± 2.1% Tcur1278-TA2 0.0194 ± 1.5% 2.8 ± 0.7% 0.0189 ± 0.7% 2.7 ± 0.4%

3.4.4 Specific activity of Tcur1278-WT and Tcur1278-TA2

Tcur1278-WT and Tcur1278-TA2 were produced in Pichia pastoris BSYBG11 and purified via cation exchange chromatography due to their high isoelectric point (Tcur1278- WT: 9.53 and Tcur1278-TA2: 9.24). Finally, the specific activity of purified Tcur1278-WT and the fusion protein, Tcur1278-TA2 were obtained applying pNPB assay (Figure 43).

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Figure 43. Specific activity analysis of Tcur1278-WT and Tcur1278-TA2. Specific activity was determined by pNPB assay (0.5 mM pNPB was used as substrate).

Tcur1278-WT and Tcur1278-TA2 did achieve a specific activity of ~216 U/mg and ~204 U/mg, respectively confirming no negative effect on the cutinase activity by the fusion of anchor TA2.

3.4.5 Quantification of polyester-PUR degradation

Purified Tcur1278-WT and Tcur1278-TA2 for polyester-PUR degradation were characterized using three methods: 1) MTP-based turbidity assay, 2) Dynamic Light Scattering (DLS), and 3) Field Emission Scanning Electron Microscopy (FE-SEM).

3.4.5.1 MTP-based turbidity assay

The degradation of polyester-PUR nanoparticles is based on the hydrolytic cleavage of polyester bondage in polyester-PUR by Tcur1278. The degradation can continuously be monitored by the decrease of turbidity at 600 nm over time[215] A dilution series of polyester-PUR in a range between 1:100 (0.4% w/v) and 1:1200 (0.033% w/v) was conducted with equimolar protein concentration (0.6 nM) of Tcur1278-WT and Tcur1278- TA2. The continuous degradation of the particles at 1:200 (0.2% w/v), 1:300 (0.133% w/v), 1:600 (0.067% w/v), and 1:1200 (0.033% w/v) polyester-PUR dilution for approximately 24 h is shown as examples in Figure 44. In all measurements, no turbidity loss of the negative control (100 mM Tris-HCl, pH 8.0) was observed as expected proving no self- degradation of the nanoparticles. The decrease in absorbance was monitored for

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Tcur1278-WT and Tcur1278-TA2, wherein the loss of turbidity by Tcur1278-TA2 is significantly faster than that of Tcur1278-WT. Based on these results two performance indicators were defined: 1) degradation rate (r) and 2) half-life time of polyester-PUR

nanoparticles (t1/2). The degradation rate is defined as the reciprocal slope of the turbidity measurements, since the turbidity of the reaction decreases over time. All the absolute values of degradation rate at different polyester-PUR dilutions (0.033-0.4% w/v) are documented in Table 10. The main outcome of this set of experiment is the rapid degradation achieved by the adhesive fusion enzyme Tcur1278-TA2 compared to Tcur1278-WT at every dilution of polyester-PUR. The highest improvement (6.6-fold) by Tcur1278-TA2 was achieved at a dilution of 1:600 (0.067% w/v). Tcur1278-TA2 degraded polyester-PUR significantly better at all tested nanoparticle dilutions than Tcur1278-WT (p ≤ 0.01), which was confirmed by the determination of the statistical significance of the regression coefficients (Fischer’s test for analysis of variance; ANOVA; OriginPro 9.1).

Figure 44. Continuous turbidity measurement of polyester-PUR particle degradation. Enzymatic degradation of polyester-PUR dilutions of 1:200 (A), 1:300 (B), 1:600 (C), and 1:1200 (D) for about 24 h. Measurements of buffer control (grey), Tcur1278-WT (blue), and Tcur1278-TA2 (cyan) were performed in triplicates at 600 nm.

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The half-life time of the polyester-PUR particles was the second performance indicator representing the time point at which half of the initial particles is degraded. This indicator features a key value since the direct accelerated polyester-PUR degradation by the act of

anchor is highlighted by t1/2 (polyester-PUR). In Figure 45A, the t1/2 (polyester-PUR) of Tcur1278-WT and Tcur1278-TA2 in hours is shown for every polyester-PUR dilution. The

higher the polyester-PUR dilution, the lower the t1/2 (polyester-PUR), which is expected

due to the decreasing particle content with the increasing dilution. The t1/2 (polyester- PUR) value is lower for Tcur1278-TA2 compared to Tcur1278-WT for the complete polyester-PUR dilution series proving the anchor mediated faster degradation at any

polyester-PUR particle content. The improvement of t1/2 (polyester-PUR) by Tcur1278-TA2 showed a good correspondence to the improvement of the degradation rate as shown in Figure 45B. The best performance was accomplished by Tcur1278-TA2 at a dilution of

1:600 (0.067% w/v) showing a 6.7-times better t1/2 (6.2 h) compared to Tcur1278-WT (41.8 h).

Figure 45. Comparison of polyester-PUR degradation performance by Tcur1278 and Tcur1278- TA2. A) Half-life of polyester-PUR particles, at which the half of the initial particles are degraded by Tcur1278-WT (blue) and Tcur1278-TA2 (cyan). The improvement in reduction of t1/2 achieved by Tcur1278-TA2 is shown in green circles. B) Improvements achieved by Tcur1278-TA2 compared to Tcur1278-WT regarding degradation rate (cyan) and half-life (blue) is shown in bars. All values and errors were generated from triplicates.

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Table 10. Polyester-PUR degradation rates of Tcur1278-WT and Tcur1278-TA2 and improvements achieved by Tcur1278-TA2.

Polyester-PUR Tcur1278-WT [AU/h] Tcur1278-TA2 [AU/h] Improvement [fold] 1:100 (0.4%) 0.0033 ± 2.7% 0.0086 ± 5.9% 2.6 ± 6.5% 1:200 (0.2%) 0.0020 ± 5.0% 0.0065 ± 5.6% 3.2 ± 7.5% 1:300 (0.133%) 0.0013 ± 10.0% 0.0059 ± 7.5% 4.5 ± 12.5% 1:400 (0.1%) 0.0011 ± 10.3% 0.0062 ± 9.3% 5.5 ± 13.9% 1:500 (0.08%) 0.0011 ± 2.8% 0.0071 ± 4.7% 6.2 ± 5.5% 1:600 (0.067%) 0.0012 ± 0.9% 0.0078 ± 2.9% 6.6 ± 3.1% 1:700 (0.057%) 0.0012 ± 9.2% 0.0071 ± 10.1% 5.7 ± 13.7% 1:1200 (0.033%) 0.0012 ± 2.8% 0.0066 ± 7.3% 5.4 ± 7.9%

3.4.5.2 DLS of polyester-PUR particles

The hydrodynamic radii of degraded and non-degraded polyester-PUR particles were determined by DLS measurements after three days of enzymatic treatment. The used polyester-PUR suspension is a highly monodisperse suspension (PDI: 0.102 ± 0.013), wherein the hydrodynamic radius of nanoparticles is 95.3 nm (Figure 46B). A clear degradation of polyester-PUR particles is proven by the reduced the hydrodynamic radius to 66.3 - 67.7 nm of the particles due to the degradation activity of Tcur1278 (with and without anchor). Enzymatically degraded particles showed a comparable PDI (Tcur1278- WT: 0.093 ± 0.019; Tcur1278-TA2: 0.099 ± 0.012) to that of untreated particles indicating no change of monodispersity after degradation. As expected, smallest polyester-PUR

Figure 46. Hydrodynamic radii of untreated and treated polyester-PUR nanoparticles. The nanoparticle size distribution is represented in samples with no enzymatic treatment (dark grey), Tcur1278-WT (blue), and Tcur1278-TA2 (cyan). A) Degraded polyester-PUR particles between 0.01 and 10 nm and B) degraded polyester-PUR particles between 0.01 and 1000 nm.

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particles (up to 0.08 nm) were generated by Tcur1278-TA2 in comparison to Tcur1278- WT (up to 0.33 nm) showing once more the effectivity of the anchor fusion (Figure 46A).

3.4.5.3 FE-SEM analysis of polyester-PUR degradation

Stainless steel supports coated with polyester-PUR nanoparticles (Impranil® DLN-SD), which forms a dense film-like structure after drying (Figure 47). Duration of up to 40 h was considered to degrade the polyester-PUR film at ambient temperature by enzymatic treatment. As expected, no degradation of polyester-PUR films was observed in the reaction buffer (100 mM Tris-HCl, pH 8.0), when only buffer as a negative control was applied onto the film (Figure 47A and D). Degradation performance of Tcur1278-WT was not visible after 24 h (Figure 47B), while a very poor degradation was observed after 40 h (Figure 47E) of treatment. Interestingly, Tcur1278-TA2 was already able to significantly degrade the employed film within 24 h of incubation indicated by the big cracks on the film surface (Figure 47C; darker color). Almost a full degradation of the applied polymer film was recognized after 40 h by the visualization of the stainless-steel support underneath the film surface (Figure 47F).

Figure 47. FE-SEM analysis of polyester-PUR layers after enzymatic degradation. A Negative control Tris-HCL buffer (100 mM, pH 8.0), Tcur1278-WT, and Tcur1278-TA2 (10 µL and 20 µM each) were incubated on the polyester-PUR film (stainless steel support was coated with Impranil® DLN- SD) for 24 and 40 h. A) Buffer control (24 h), D) buffer control (40 h), B) Tcur1278-WT (24 h), E) Tcur1278-WT (40 h), C) Tcur1278-TA2 (24 h), and F) Tcur1278-TA2 (40 h).

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3.4.6 Broadening the targeted degradation by anchor peptides to PET

Evaluation of enzymatic PET degradation was performed by quantifying the weight loss of PET-film over time. As described for polyester-PUR degradation, the normalized activity of target proteins was employed for PET-film assay as well. PET-films (0.5 cm × 2.5 cm × 0.25 mm) were degraded by incubating them in the culture supernatant of EV, Tcur1278-WT, Tcur1278-LCI, and Tcur1278-TA2 at 55°C within 24 h to 72 h. In order to see the anchoring effect of LCI and TA2, two different dilutions of enzyme activity were considered. PET-film assay performed with 70 U/L enzyme activity showed increased weight loss over time for Tcur1278-WT and Tcur1278-TA2 (Figure 48A). A slightly increased PET-film degradation was observed for Tcur1278-TA2 after 24 h, but after 48 h and 72 h, the effect was not visible anymore. Surprisingly, Tcur1278-LCI did not

Figure 48. PET-film degradation by culture supernatants of Tcur1278-WT and Tcur1278-anchors. PET-films were treated with enzymes with A) 70 U/L and B) 14 U/L (1:5 dilution of 70 U/L) pNPB activity for 24, 48, and 72 h at 55°C. Weight loss was determined by subtracting the weight of the PET-film after enzyme incubation from the initial weight. show any improvement at any time point indicating no anchoring effect on PET degradation. The anchoring effect was clearer in diluted samples, wherein enzymes were employed only with 14 U/L (5 times diluted than 70 U/L). Already after 24 h, Tcur1278- TA2 was able to degrade > 0.5 mg PET-film while Tcur1278-WT did not show any significant degradation (Figure 48B) indicating a 16-fold accelerated PET-film degradation after 24 h. A higher degradation time (48 h-72 h) resulted in better degradation performance for both Tcur1278-WT and Tcur1278-TA2. However, the anchoring effect at 48 h and 72 h was decreased from 16-fold to 1.5 and 1.6-fold, respectively.

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Engineering of a cutinase toward degradation of synthetic polymes

3.5 Discussion

Microplastic (mostly PET, PP, and PUR foams) enters the aquatic environment through wastewater treatment plant (WWTP) effluents[216]. The analysis of microplastics in wastewater has revealed an average rate of 15.2 mg microplastic, which is released into the sewerage system per person per day[217]. A study reported up to 21 particles/m3 (<100 µM) in the rivers[217]. Often microplastic lingers in the aqueous environment in high dilutions, whereas targeted degradation systems of these microparticles are needed. Development of enzymes that can especially bind to the microplastic and subsequently depolymerize them represents a promising strategy for microplastic degradation in wastewater. Nature’s solution to degrade polymers such as cellulose relies on specialized hydrolases with a unique feature. Cellulases bind via an adhesion promotor (mostly known as cellulose binding domain; CBD) to cellulose, which allows the hydrolyzing domain (catalytic domain) to stick on the cellulose surface[26, 218]. Similar domains (e.g., carbohydrate binding modules (CBM) and polyhydroxyalkanoate binding modules (PBM)) are reported for enzymes depolymerizing natural carbohydrates like chitin[219] and polyesters like polyhydroxyalkanoate[30]. Studies reported increased depolymerization activity of hydrolases for instance chitinases[220], endoglucanases[221], glucoamylase[222] by introducing a CBM or CBM-like domains.

Synthetic polymer-degrading enzymes (e.g., esterases or cutinases) do not typically possess hydrophobic binding modules, which can act as an adhesion promoter for targeted polymer degradation. Mimicking nature’s CBD concept, a fusion of binding modules such as CBMs, PBMs, and hydrophobins to hydrolases (especially cutinases) have been reported for polymer degradation[200a, b]. An overview of enzymes and adhesion promoter is depicted in Table 11. CBMs were attached to different cutinases to enhance degradation activity toward substrates such as 3PET, PET-film, and PET fibers[200b, c]. Nature evolved CBMs for carbohydrate binding, which might not have a huge impact on synthetic polymer like PET. Therefore, the improvement of cutinase-CBM fusion compared to the bare cutinase is very moderate (1.4-1.5-fold)[200b, c]. On the other hand, PBMs are found in natural polyester degrading enzymes called polyhydroxyalkanoate depolymerases[30]. Accordingly, the fusion of PBMs to cutinases or polyamidases

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Engineering of a cutinase toward degradation of synthetic polymes stimulates a better hydrolysis of polymers counting PET and PUR compared to a fusion of CBMs (PET: 3.8-fold, PUR: 4-fold)[38, 200b]. In one study, PBM was utilized to degrade polyurethane-polyester co-polymers by polyamidase, wherein the hydrolysis was 4-fold higher compared to the native enzyme[38].

Table 11. Overview of fused enzyme-adhesion promotor in different studies.

Adhesion Protein T Improvement Enzyme Polymer Source promoter [mM] [°C] [fold] Cutinase 3PET PBM 25 50 3.8 [200b] (Thc_Cut1) PET-film Cutinase 3PET CBM 25 50 1.4 [200b] (Thc_Cut1) PET-film Cutinase Hydrophobin PET 5*10-3 50 16 [200a] (Thc_Cut1) Polyamidase PBM PUR pellet 2.5*10-3 50 4 [38] (PA) Cutinase Anchor Polyester- 6*10-6 RT 6.6 This study (Tcur1278) peptide PUR Cutinase Anchor PET-film 14 U/L1 55°C 16 This study (Tcur1278) peptide 1not purified; activity was normalized by pNPB assay

Hydrophobins, on the other hand, are not natural binding domains of a particular enzyme, but small surface-active proteins secreted by filamentous fungi (e.g., Trichoderma reesei)[223]. Characteristic of hydrophobins is to aggregate spontaneously to form amphipathic monolayers on interfaces[224]. This natural behavior of hydrophobins allows fungi to adhere, form surface layers, and decrease surface tension[201a]. Hydrophobins (HFB1, HFB2, HFB4, or HFB7) were fused to hydrolytic enzymes to utilize their nature to bind on hydrophobic surfaces like silanized glass, teflon, and PET[200a, 225]. PET-degrading cutinase (Thc_Cut1) fused to HFBs was able to degrade PET 16 times better than the cutinase without HFB[200a]. Antimicrobial peptides working as anchor peptides are versatile tools to adsorb to polymeric surfaces. Similar to the aforementioned enzyme- adhesion promotor studies, a cutinase (Tcur1278) and anchor peptides (LCI and TA2) were fused to each other to achieve an accelerated depolymerization of polyester-PUR nanoparticles and PET-films. In this thesis, PP-binding LCI and PS-binding TA2 were focused to quantify their ability to bind to polyester-PUR and PET surfaces. Using the reporter protein eGFP fused to LCI and TA2, it was possible to show that both LCI and TA2

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Engineering of a cutinase toward degradation of synthetic polymes bind to PET surface (here textile). A selectivity of anchoring was identified for TA2 while binding on a polyester-PUR film. The polyester-PUR as well as PET binding anchor peptide LCI and TA2 were fused to the cutinase Tcur1278 to achieve a targeted degradation of polyester-PUR nanoparticles and PET-films. In contrast to most reported studies, the degradation was performed at ambient temperature which is close to environmentally relevant conditions (e.g., wastewater).

Degradation kinetics of Tcur1278-TA2 was generally improved for all investigated polyester-PUR nanoparticle dilutions (1:100-1:1200) compared to Tcur1278-WT. In both cases, the higher the dilution (a smaller number of nanoparticles) the faster the degradation kinetics and the lower the half-life of nanoparticles. The degradation kinetics of Tcur1278-WT decreased from 0.0033 AU h-1 (first dilution 1:100) to an average of 0.0011 AU h-1 (final dilution 1:1200) retaining ~35% of initial degradation kinetics. In contrast, the degradation kinetics of Tcur1278-TA2 decreased from 0.0086 AU h-1 (first dilution 1:100) to an average of 0.0067 AU h-1 (final dilution 1:1200) retaining ~80% of initial degradation kinetics (Table 10). Thereby, the targeting property of Tcur1278-TA2 was demonstrated, whereas TA2 facilitates an accelerated degradation of nanoparticles in high dilutions by Tcur1278. Hence, the degradation kinetics of Tcur1278-TA2 was increased up to 6.6-fold (at 1:600 = 0.067% w/v nanoparticles) compared to the Tcur1278-

WT. The half-life of polyester-PUR nanoparticles (t1/2) was reduced from 41.8 to 6.2 h dilution of 1:700 (0.057% w/v) indicating a 6.7-fold improvement for Tcur1278-TA2 (Figure 44). Polyester-PUR nanoparticles were degraded in general by the act of the cutinase Tcur1278 to smaller nanoparticles (from ~95 to ~66 nm), which was confirmed by DLS measurements (Figure 46). Interestingly, 4-times smaller nanoparticles (Tcur1278- TA2: 0.08 nm vs. Tcur1278-WT: 0.33 nm) were identified for Tcur1278-TA2 in contrast to Tcur1278-WT after degradation proving an enhanced degradation activity of Tcur1278- TA2. Finally, FE-SEM enabled a deeper look at the degraded polyester-PUR films on a stainless-steel support. Tcur1278-TA2 degraded the films significantly faster compared to Tcur1278-WT. Under the experimental conditions (up to ~40 h, RT), Tcur1278-TA2 was able to degrade most of the polymer film while Tcur1278-WT showed a similar behavior to the buffer control (Figure 47).

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Engineering of a cutinase toward degradation of synthetic polymes

The principle of targeting microplastic via anchor peptide was expanded to PET-films to demonstrate the universality. Contrary to polyester-PUR nanoparticles, PET-films (0.5 cm × 2.5 cm × 0.25 mm) were used. Tcur1278-WT was already reported to degrade PET-films efficiently[192]. The principle is explained in Figure 49, wherein different dilution of protein/fusion protein amount play a crucial role. There is no difference between Tcur1278-WT and Tcur1278-TA2 in the highly concentrated protein/fusion protein sample since enough protein/fusion protein molecules are present for the degradation. The anchoring effect becomes visible in the diluted protein samples since distant Tcur1278- TA2 molecules can target PET-film via TA2 while Tcur1278 cannot effectively target without any adhesion promoter. The investigation in this study proved that LCI as adhesion promotor has no effect in targeting PET-films as the degradation performance (quantified by weight loss of the PET-films) of Tcur1278-LCI was worse than Tcur1278-WT (Figure 48). As expected, no significant difference between Tcur1278-WT and Tcur1278-

Figure 49. Principle of targeted PET-film degradation. Enough protein molecules are present for PET-film degradation in undiluted protein samples, so that the molecules do not need to “find” PET-film. Wild type cutinase (Tcur1278-WT) and fusion of cutinase and anchor peptide (Tcur1278- TA2) exhibit the same behavior. In the diluted protein samples, distant Tcur1278-WT molecules cannot reach PET-film, whereas Tcur1278-TA2 targets PET-film via the anchor peptide TA2 that binds specifically to PET. Thus, a targeted and thereby faster PET-film degradation is mediated by anchor peptides.

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Engineering of a cutinase toward degradation of synthetic polymes

TA2 was observed in high concentrated protein samples (70 U L-1) over 72 h. When the amount of applied protein was decreased 5-times (14 U L-1), a 16-fold higher degradation activity was documented for Tcur1278-TA2 after 24 h of treatment, whereas Tcur1278- WT was not able to degrade the PET-film (Figure 48). These experimental findings indicate that anchor peptide fusion allows effective PET-film degradation by cutinase with less protein amount and shorter time span, which is from economical point of view a smart way to biodegrade PET.

To date, this study represents the first systematic report on the degradation of polyester- PUR nanoparticles in highly diluted suspensions at ambient temperature and a very low protein concentration (0.6 nM). Furthermore, this principle was expanded to the synthetic polymer PET, wherein again low protein amount (corresponding to 14 U L-1) resulted in the highest targeted degradation of the used polymer. Low protein concentrations and applications at ambient temperature are very much appreciated for sustainable microplastic degradation processes. The binding performance can be further improved by peptide engineering for several synthetic polymers as already reported for the anchor peptides TA2 and LCI toward stronger PS and PP binding, respectively[208b, 226].

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Summary and conclusion

4. Summary and conclusion

Enzymatic functionalization of cellobiose and even cellulose might contribute to synthesize mimetics of the pharmaceutically valuable glycosaminoglycan (GAG). Since sulfation is one of the main functionalization of GAGs, building blocks of polysaccharides or GAGs can be targeted for selective sulfation by aryl sulfotransferases. Therefore, the engineering of a promising bacterial aryl sulfotransferase (ASTB) for selective saccharide sulfation was in the focus of the first part of this dissertation. The main challenge to re- engineer a novel enzyme is the availability of a robust and reliable directed evolution protocol. The well-known pNPS activity assay for sulfotransferases was advanced to a continuous screening system to operate efficiently. The step by step optimization of the pNPS screening system for effective sulfation of mono- and disaccharide acceptors led to a low coefficient of variation (14.3 and 10.5%, respectively). It is likely that the established pNPS screening system can be expanded to other saccharides (oligosaccharides) and substrate classes (antibiotics, steroids, flavonoids). The established protocol was validated in one round of directed ASTB evolution toward the glycosaminoglycan building block GlcNAc (monosaccharide), which allowed an identification of variants with an up to 3.4- fold improved specific activity. These results represented the first robust protocol for directed ASTB evolution toward a saccharide. Based on these findings, the engineering of ASTB was expanded to a KnowVolution campaign toward cellobiose (disaccharide). The KnowVolution strategy allowed a knowledge-driven directed evolution of ASTB. The specific activity of ASTB was increased 7.6-fold toward cellobiose after only one round of complete KnowVolution. Two crucial positions and thereby amino acid substitutions (L446P/V579K) were identified to exhibit a synergistic effect on ASTB activity toward disaccharides. MS analysis confirmed monosulfation in all cases. Computational studies (homology modelling) revealed ASTB’s homodimeric structure and suggested the roles of beneficial positions and substitutions. L446P increases the flexibility of the loop forming the entrance to the substrate-binding pocket and V579K contributes as a distal substitution. In order to demonstrate ASTB’s synthetic potentiality as a sulfation agent, synthesis of a sulfated product was performed in semi-preparative scale. GlcNAc-linker- tBoc was subjected to be sulfated by ASTB-WT, ASTB-V1 (best variant from directed evolution), and ASTB-M5 (best variant from KnowVolution). Conversion was improved

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Summary and conclusion from 27% (ASTB-WT) to 67.3% (ASTB-M5) and 87.1% (ASTB-V1), respectively. A structural elucidation of the monosulfated GlcNAc-linker-tBoc by NMR revealed the sulfation at rare positions (C-3/C-4). Thereby, ASTB opens an alternative green route to sulfate saccharides at C-3/C-4 positions that are challenging to achieve by direct chemical methods. As conclusion, the broad acceptor range of ASTB makes it to a highly attractive sulfotransferase for sulfation of mono-, di-saccharides, phenolic compounds, and likely sugar polymers such as cellulose. In future, sulfation of GlcNAc by ASTB might represent the first reaction step in the full-enzymatic cascade synthesis of glycosaminoglycan (dermatan sulfate and/or heparan sulfate) followed by chain elongation using glycosyltransferases.

Accumulation of microplastic in nature is a global challenge. More effective and fast degradation methods are needed to be implemented for microplastic degradation. The developed platform using anchor peptides to target microplastic for effective degradation might open up a new environmentally friendly way for microplastic management. The development of an accelerated and controlled synthetic polymer (PET and polyester-PUR) degradation by the means of anchor peptides was centralized in the second part of this dissertation. Anchor peptides were selected as adhesion promoters based on their binding ability to synthetic polymers. The anchor peptide TA2 served as adhesion promoter and was fused to the synthetic polymer degrading cutinase Tcur1278. The fusion protein Tcur1278-TA2 demonstrated a 6.6-fold increased degradation of polyester- PUR nanoparticles in a highly diluted suspension (0.067 % (w/v)) compared to the wild- type Tcur1278. The half-life of the nanoparticles was reduced from 41.8 h to 6.2 h (6.7-times faster). These results represent the first systematic study on the degradation of polyester-PUR nanoparticles in highly diluted suspensions at ambient temperature and low enzyme concentration. Furthermore, PET-films were degraded 16-times faster by Tcur1278-TA2 compared to Tcur1278-WT indicating the versatility of targeting polymers by anchor peptides. This proof of principle can be further developed by reengineering anchor peptides toward stronger polymer binding as already reported for PP and PS. In future, TA2 or other attractive anchor peptides can be evolved using PePevo (diversity generation protocol for short peptides) followed by the KnowVolution strategy to further enhance the binding to respective polymers and thereby the degradation by the fused

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Summary and conclusion hydrolase. Tailored anchor peptides will very likely enable an immobilization of polymer- degrading enzymes more efficiently to achieve an effective and controlled depolymerization. This might allow on the long run the development of sustainable biotechnological processes for not only degradation but also the conversion of plastics into value-added compounds.

Taken together, two scientific advancements in the field of protein engineering field were accomplished. On one hand, an aryl sulfotransferase was advanced to a synthetically attractive biocatalyst for selective sulfation of saccharides. On the other hand, a platform was developed to target synthetic polymers by anchor peptides as adhesion promoters for an effective depolymerization of synthetic polymers.

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Appendix

6. Appendix

6.1 List of abbreviation Abbreviation Full name aa Amino acid ASTA/ASTB Aryl sulfotransferase A/Aryl sulfotransferase B A(S)ST Aryl(sulfate) sulfotransferase bp Base pair CBD Cellulose binding domain CBM Carbohydrate-binding module cepPCR Casting error-prone PCR CS Chondroitin sulfate Da Dalton DLS Dynamic light scattering DMF Dimethylformamide DMSO Dimethylsulfoxide DNA Deoxyribonucleic acid DS Dermatan sulfate dsDNA double-stranded DNA epPCR error-prone PCR ESI-MS Electronspary ionization mass spectroscopy EV Empty vector FE-SEM Field emission scanning electron microscopy GAG Glycosaminoglycan GalNAc N-Acetylgalactosamine GlcA Glucuronic acid GlcNAc N-acetylglucosamine HA Hyaluronic acid/hyaluronan HPLC High-pressure liquid chromatography HS Heparan sulfate/heparosan IdoA Iduronic acid IPTG Isopropyl β-D-1-thiogalactopyranoside kDa Kilodalton KS Keratan sulfate LB Lysogeny Broth LCI Liquid chromatography peak I m/z Mass-to-charge ration MTP Microtiter plate NCBI National Center for Biotechnology Information NMR Nuclear magnetic resonance OD600 Optical density at 600 nm PAPS 3'-Phosphoadenosine-5'-phosphosulfate PCL Polycaprolactone PCR Polymerase chain reaction PDB Protein data bank PE Polyethylene PET Polyethylene terephthalate PHA Polyhydroxyalkanoate PHB Polyhydroxybutyrate PHV Polyhydroxyvalerate

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PLA Polylactic acid PLICing Phosphorothioate-based ligase-independent gene cloning pNPB para-Nitrophenyl butyrate pNPS para-Nitrophenyl sulfate PP Polypropylene PS Polystyrene PTO Phosphorothioate PUR Polyurethane RMSD Root mean square displacement RMSF Root mean square fluctuation rpm Rotations per minute SDM Site-directed mutagenesis SeSaM Sequence saturation mutagenesis ssDNA single-stranded DNA SSM Site-saturation mutagenesis SULT Sulfotransferase TA2 Tachystatin A2 TB Terrific Broth Tcur Thermomonospora curvata TdT deoxynucleotidyl transferase Tris tris(hydroxymethyl)aminomethane UDP Uridine diphosphate WT Wild-type

Amino acid Three letter code One letter code Alanine Ala A Arginine Arg R Asparagine Asn N Aspartic acid Asp D Cysteine Cys C Glutamine Gln Q Glutamic acid Glu E Glycine Gly G Histidine His H Isoleucine Ile I Leucine Leu L Lysine Lys K Methionine Met M Phenylalanine Phe F Proline Pro P Serine Ser S Threonine Thr T Tryptophan Trp W Tyrosine Tyr Y Valine Val V

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6.2 List of DNA and protein sequences

Aryl sulfotransferase B (ASTB) wild-type with C-terminal Strep II tag

1 atg cgt acc tac ctg aat acc gaa aaa cat ctg att acc ctg caa 45 1 Met Arg Thr Tyr Leu Asn Thr Glu Lys His Leu Ile Thr Leu Gln 15 Start 46 gct gaa tct gaa gaa cgt ttc ctg gcc gaa ctg cgt gct ggt aac 90 16 Ala Glu Ser Glu Glu Arg Phe Leu Ala Glu Leu Arg Ala Gly Asn 30

91 tac acc gcc gaa tca ccg ctg gtt gtg aaa aac ccg tat att atc 135 31 Tyr Thr Ala Glu Ser Pro Leu Val Val Lys Asn Pro Tyr Ile Ile 45

136 aat ccg ctg gcg gcc gtt att tgc ttt aat acg gat gaa gaa acc 180 46 Asn Pro Leu Ala Ala Val Ile Cys Phe Asn Thr Asp Glu Glu Thr 60

181 acg gcc gaa att acc gtc aaa ggc aaa gca atc gaa ggt gac ctg 225 61 Thr Ala Glu Ile Thr Val Lys Gly Lys Ala Ile Glu Gly Asp Leu 75

226 tct cat acc ttc gca gct gcg aaa gaa cac gtt ctg ccg gtc tat 270 76 Ser His Thr Phe Ala Ala Ala Lys Glu His Val Leu Pro Val Tyr 90

271 ggc ctg tac gat gac tat gtg aac acg gtc gtg atc aaa ctg agt 315 91 Gly Leu Tyr Asp Asp Tyr Val Asn Thr Val Val Ile Lys Leu Ser 105

316 aat ggt aaa acc agc gaa gtg aaa att gaa gtg gaa gaa ctg aac 360 106 Asn Gly Lys Thr Ser Glu Val Lys Ile Glu Val Glu Glu Leu Asn 120

361 gtt aat aaa gcc ctg tac tgc cgc acc acg ccg gaa tac ttc ggc 405 121 Val Asn Lys Ala Leu Tyr Cys Arg Thr Thr Pro Glu Tyr Phe Gly 135

406 aaa gat ttc atg ctg atc tca acc acg acc ccg ctg atc gaa tcg 450 136 Lys Asp Phe Met Leu Ile Ser Thr Thr Thr Pro Leu Ile Glu Ser 150

451 gct cgt acg gca ggc ttt gat tac gca ggt gac ctg cgt tgg tgt 495 151 Ala Arg Thr Ala Gly Phe Asp Tyr Ala Gly Asp Leu Arg Trp Cys 165

496 att acc aac ctg cag tca tgg gat atc aaa aaa ctg gaa aat ggt 540 166 Ile Thr Asn Leu Gln Ser Trp Asp Ile Lys Lys Leu Glu Asn Gly 180

541 cgc ctg ctg tat acg tcg cat cgt acc gtg cag aaa ccg tat tac 585 181 Arg Leu Leu Tyr Thr Ser His Arg Thr Val Gln Lys Pro Tyr Tyr 195

586 aac gtg ggc gtt atg gaa atg gat ttc tgt ggt aaa atc tac aaa 630 196 Asn Val Gly Val Met Glu Met Asp Phe Cys Gly Lys Ile Tyr Lys 210

631 gaa tac cgt ctg ccg ggc ggt tat cat cac gac gcg gtt gaa ctg 675 211 Glu Tyr Arg Leu Pro Gly Gly Tyr His His Asp Ala Val Glu Leu 225

676 gaa aac ggc aat att ctg gcc gca agt gat aac gac ttt aat gat 720 226 Glu Asn Gly Asn Ile Leu Ala Ala Ser Asp Asn Asp Phe Asn Asp 240

721 tcc gtg gaa gac ttc gtt gtc gaa att gaa cgc gcc acc ggc gca 765 241 Ser Val Glu Asp Phe Val Val Glu Ile Glu Arg Ala Thr Gly Ala 255

766 gtt atc aaa agt tgg gat ctg cag aaa att ctg ccg cgc ggc cag 810 256 Val Ile Lys Ser Trp Asp Leu Gln Lys Ile Leu Pro Arg Gly Gln 270

811 ggt aaa gct ggt gat tgg aac cat cac gac tgg ttt cat aac aat 855 271 Gly Lys Ala Gly Asp Trp Asn His His Asp Trp Phe His Asn Asn 285

856 gcg gtg tgg tac gat aaa ccg acg aat agc atc acc atg tct ggc 900 286 Ala Val Trp Tyr Asp Lys Pro Thr Asn Ser Ile Thr Met Ser Gly 300

901 cgc cac atg gac gct gtt att aac ttc gat tat gac agc ggt gcg 945 301 Arg His Met Asp Ala Val Ile Asn Phe Asp Tyr Asp Ser Gly Ala 315

946 ctg aat tgg atc ctg ggc gat ccg gaa ggt tgg tct gaa gaa tgg 990 316 Leu Asn Trp Ile Leu Gly Asp Pro Glu Gly Trp Ser Glu Glu Trp 330

991 cag aaa tac ttt ttc aaa aac gtg acc aaa ggc gat ttt gac tgg 1035 331 Gln Lys Tyr Phe Phe Lys Asn Val Thr Lys Gly Asp Phe Asp Trp 345

1036 cag tat gaa caa cat gct gcg cgt att ctg ccg aat ggc gat gtt 1080

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346 Gln Tyr Glu Gln His Ala Ala Arg Ile Leu Pro Asn Gly Asp Val 360

1081 ttt ctg ttc gac aac ggc acg tat cgc agt aaa aat gaa gct acc 1125 361 Phe Leu Phe Asp Asn Gly Thr Tyr Arg Ser Lys Asn Glu Ala Thr 375

1126 cgt gtg gat ccg gaa cag aat ttt tcc cgc ggt gtt att tac cgt 1170 376 Arg Val Asp Pro Glu Gln Asn Phe Ser Arg Gly Val Ile Tyr Arg 390

1171 atc gat acc gac aaa atg gaa atc gaa caa gtg tgg caa tat ggc 1215 391 Ile Asp Thr Asp Lys Met Glu Ile Glu Gln Val Trp Gln Tyr Gly 405

1216 aaa gaa cgc ggt gcc gaa ttc tac agc ccg tat atc tgc aac gtc 1260 406 Lys Glu Arg Gly Ala Glu Phe Tyr Ser Pro Tyr Ile Cys Asn Val 420

1261 gat tat tac ggc gaa ggt cat tac atg gtg cac tct ggc ggt att 1305 421 Asp Tyr Tyr Gly Glu Gly His Tyr Met Val His Ser Gly Gly Ile 435

1306 gcc acg tat cgt ggc aaa cac acc gat ggc ctg ggt gca atg ctg 1350 436 Ala Thr Tyr Arg Gly Lys His Thr Asp Gly Leu Gly Ala Met Leu 450

1351 ctg aac aaa tac aaa gac gaa cat atc cac ctg acg ctg gaa tca 1395 451 Leu Asn Lys Tyr Lys Asp Glu His Ile His Leu Thr Leu Glu Ser 465

1396 atc acc gtc gaa gtg cag aac gat caa ctg aaa tac gaa ctg aaa 1440 466 Ile Thr Val Glu Val Gln Asn Asp Gln Leu Lys Tyr Glu Leu Lys 480

1441 gtg cag ggc ggt aat tat tac cgc gca cgt cgc gtt tcg ccg tat 1485 481 Val Gln Gly Gly Asn Tyr Tyr Arg Ala Arg Arg Val Ser Pro Tyr 495

1486 gat gaa aaa acc aac ctg gtc ctg ggc aaa ggt gaa ctg ctg ggc 1530 496 Asp Glu Lys Thr Asn Leu Val Leu Gly Lys Gly Glu Leu Leu Gly 510

1531 ggt ttt ggt gtt acg ccg gaa ttt atg aaa gtc aat ttc aaa gat 1575 511 Gly Phe Gly Val Thr Pro Glu Phe Met Lys Val Asn Phe Lys Asp 525

1576 gcg gaa acc gaa ctg agc gaa aaa cat aac ctg aat gtc atc ctg 1620 526 Ala Glu Thr Glu Leu Ser Glu Lys His Asn Leu Asn Val Ile Leu 540

1621 gaa gaa gac cgt ctg gct att cgc gcg tca ttt cgt gaa ggc tcg 1665 541 Glu Glu Asp Arg Leu Ala Ile Arg Ala Ser Phe Arg Glu Gly Ser 555

1666 cag gtt ttc ctg gaa ctg aag ggt gcg gaa caa agt aaa ttt tat 1710 556 Gln Val Phe Leu Glu Leu Lys Gly Ala Glu Gln Ser Lys Phe Tyr 570

1711 aac att ccg acg gaa gtg cac gat gtt acc gcc gca tgt gtc tcc 1755 571 Asn Ile Pro Thr Glu Val His Asp Val Thr Ala Ala Cys Val Ser 585

1756 ttc gaa gaa cag aac gat aat gac ttt caa ttc tat gtg agc cgt 1800 586 Phe Glu Glu Gln Asn Asp Asn Asp Phe Gln Phe Tyr Val Ser Arg 600

1801 gaa ggc ctg tct ggt gaa ttc ggc atc tac ctg aac att gat agc 1845 601 Glu Gly Leu Ser Gly Glu Phe Gly Ile Tyr Leu Asn Ile Asp Ser 615

1846 aaa cgc tac gat acg cat ctg tct gtg aaa ctg gag ctc tct gca 1890 616 Lys Arg Tyr Asp Thr His Leu Ser Val Lys Leu Glu Leu Ser Ala 630

1891 tgg agc cat ccg cag ttc gaa aag tag 1917 631 Trp Ser His Pro Gln Phe Glu Lys A- StrepII tag Stop

Cutinase Tcur1278 wild type with N-terminal signal peptide

1 atg aga ttc cca tct att ttc acc gct gtc ttg ttc gct gcc tcc 45 1 Met Arg Phe Pro Ser Ile Phe Thr Ala Val Leu Phe Ala Ala Ser 15 Start 46 tct gca ttg gct gcc cct gtt aac act acc act gaa gac gag act 90 16 Ser Ala Leu Ala Ala Pro Val Asn Thr Thr Thr Glu Asp Glu Thr 30

91 gct caa att cca gct gaa gca gtt atc ggt tac tct gac ctt gag 135 31 Ala Gln Ile Pro Ala Glu Ala Val Ile Gly Tyr Ser Asp Leu Glu 45

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136 ggt gat ttc gac gtc gct gtt ttg cct ttc tct gct tcc att gct 180 46 Gly Asp Phe Asp Val Ala Val Leu Pro Phe Ser Ala Ser Ile Ala 60

181 gct aag gaa gag ggt gtc tct ctc gag aag aga gag gcc gaa gct 225 61 Ala Lys Glu Glu Gly Val Ser Leu Glu Lys Arg Glu Ala Glu Ala 75 Signal peptide (prepro α-leader sequence) 226 agt ctg cgt aaa agc ttt ggt ctg ctg agc gca acc gca gca ctg 270 76 Ser Leu Arg Lys Ser Phe Gly Leu Leu Ser Ala Thr Ala Ala Leu 90

271 gtt gca ggt ctg gtt gcc gca ccg cct gca cag gca gca gca aat 315 91 Val Ala Gly Leu Val Ala Ala Pro Pro Ala Gln Ala Ala Ala Asn 105

316 ccg tat cag cgt ggt ccg gat ccg acc gaa agc ctg ctg cgt gca 360 106 Pro Tyr Gln Arg Gly Pro Asp Pro Thr Glu Ser Leu Leu Arg Ala 120

361 gca cgt ggt ccg ttt gca gtt agc gaa cag agc gtt agc cgt ctg 405 121 Ala Arg Gly Pro Phe Ala Val Ser Glu Gln Ser Val Ser Arg Leu 135

406 agc gtg agc ggt ttt ggt ggt ggt cgt atc tat tat ccg acc aca 450 136 Ser Val Ser Gly Phe Gly Gly Gly Arg Ile Tyr Tyr Pro Thr Thr 150

451 acc agc cag ggc acc ttt ggt gca att gcc att agt ccg ggt ttt 495 151 Thr Ser Gln Gly Thr Phe Gly Ala Ile Ala Ile Ser Pro Gly Phe 165

496 acc gca agc tgg tca agc ctg gca tgg ctg ggt ccg cgt ctg gca 540 166 Thr Ala Ser Trp Ser Ser Leu Ala Trp Leu Gly Pro Arg Leu Ala 180

541 agc cat ggt ttt gtt gtt att ggt att gaa acc aac aca cgt ctg 585 181 Ser His Gly Phe Val Val Ile Gly Ile Glu Thr Asn Thr Arg Leu 195

586 gat cag ccg gat agc cgt ggt cgt cag ctg ctg gca gcc ctg gat 630 196 Asp Gln Pro Asp Ser Arg Gly Arg Gln Leu Leu Ala Ala Leu Asp 210

631 tat ctg acc cag cgt agc agc gtt cgt aat cgt gtt gat gca agc 675 211 Tyr Leu Thr Gln Arg Ser Ser Val Arg Asn Arg Val Asp Ala Ser 225

676 cgt ctg gca gtt gcc ggt cat agc atg ggt ggt ggt ggc acc ctg 720 226 Arg Leu Ala Val Ala Gly His Ser Met Gly Gly Gly Gly Thr Leu 240

721 gaa gca gca aaa agc cgt acc agc ctg aaa gca gcc att ccg att 765 241 Glu Ala Ala Lys Ser Arg Thr Ser Leu Lys Ala Ala Ile Pro Ile 255

766 gca ccg tgg aat ctg gat aaa acc tgg cct gaa gtt cgt acc ccg 810 256 Ala Pro Trp Asn Leu Asp Lys Thr Trp Pro Glu Val Arg Thr Pro 270

811 aca ctg att att ggt ggt gaa ctg gat agc att gca ccg gtt gcg 855 271 Thr Leu Ile Ile Gly Gly Glu Leu Asp Ser Ile Ala Pro Val Ala 285

856 acc cat agc att ccg ttt tat aac agc ctg acc aat gca cgt gaa 900 286 Thr His Ser Ile Pro Phe Tyr Asn Ser Leu Thr Asn Ala Arg Glu 300

901 aaa gca tat ctg gaa ctg aat aat gcc agc cat ttt ttt ccg cag 945 301 Lys Ala Tyr Leu Glu Leu Asn Asn Ala Ser His Phe Phe Pro Gln 315

946 ttt agc aat gat acc atg gcc aaa ttt atg atc agc tgg atg aaa 990 316 Phe Ser Asn Asp Thr Met Ala Lys Phe Met Ile Ser Trp Met Lys 330

991 cgc ttt atc gat gat gat acc cgc tat gat cag ttt ctg tgt ccg 1035 331 Arg Phe Ile Asp Asp Asp Thr Arg Tyr Asp Gln Phe Leu Cys Pro 345

1036 cct ccg cgt gca att ggt gat att agc gat tat cgt gat acc tgt 1080

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346 Pro Pro Arg Ala Ile Gly Asp Ile Ser Asp Tyr Arg Asp Thr Cys 360

1081 ccg cat acc agc gca tgg tca cat cct cag ttt gaa aaa tga taa 1125 361 Pro His Thr Ser Ala Trp Ser His Pro Gln Phe Glu Lys - - StrepII tag Stop

Tcur1278 wild-type with N-terminal signal peptide and C-terminal LCI

1 atg aga ttc cca tct att ttc acc gct gtc ttg ttc gct gcc tcc 45 1 Met Arg Phe Pro Ser Ile Phe Thr Ala Val Leu Phe Ala Ala Ser 15 Start 46 tct gca ttg gct gcc cct gtt aac act acc act gaa gac gag act 90 16 Ser Ala Leu Ala Ala Pro Val Asn Thr Thr Thr Glu Asp Glu Thr 30

91 gct caa att cca gct gaa gca gtt atc ggt tac tct gac ctt gag 135 31 Ala Gln Ile Pro Ala Glu Ala Val Ile Gly Tyr Ser Asp Leu Glu 45

136 ggt gat ttc gac gtc gct gtt ttg cct ttc tct gct tcc att gct 180 46 Gly Asp Phe Asp Val Ala Val Leu Pro Phe Ser Ala Ser Ile Ala 60

181 gct aag gaa gag ggt gtc tct ctc gag aag aga gag gcc gaa gct 225 61 Ala Lys Glu Glu Gly Val Ser Leu Glu Lys Arg Glu Ala Glu Ala 75 Signal peptide (prepro α-leader sequence) 226 agt ctg cgt aaa agc ttt ggt ctg ctg agc gca acc gca gca ctg 270 76 Ser Leu Arg Lys Ser Phe Gly Leu Leu Ser Ala Thr Ala Ala Leu 90

271 gtt gca ggt ctg gtt gcc gca ccg cct gca cag gca gca gca aat 315 91 Val Ala Gly Leu Val Ala Ala Pro Pro Ala Gln Ala Ala Ala Asn 105

316 ccg tat cag cgt ggt ccg gat ccg acc gaa agc ctg ctg cgt gca 360 106 Pro Tyr Gln Arg Gly Pro Asp Pro Thr Glu Ser Leu Leu Arg Ala 120

361 gca cgt ggt ccg ttt gca gtt agc gaa cag agc gtt agc cgt ctg 405 121 Ala Arg Gly Pro Phe Ala Val Ser Glu Gln Ser Val Ser Arg Leu 135

406 agc gtg agc ggt ttt ggt ggt ggt cgt atc tat tat ccg acc aca 450 136 Ser Val Ser Gly Phe Gly Gly Gly Arg Ile Tyr Tyr Pro Thr Thr 150

451 acc agc cag ggc acc ttt ggt gca att gcc att agt ccg ggt ttt 495 151 Thr Ser Gln Gly Thr Phe Gly Ala Ile Ala Ile Ser Pro Gly Phe 165

496 acc gca agc tgg tca agc ctg gca tgg ctg ggt ccg cgt ctg gca 540 166 Thr Ala Ser Trp Ser Ser Leu Ala Trp Leu Gly Pro Arg Leu Ala 180

541 agc cat ggt ttt gtt gtt att ggt att gaa acc aac aca cgt ctg 585 181 Ser His Gly Phe Val Val Ile Gly Ile Glu Thr Asn Thr Arg Leu 195

586 gat cag ccg gat agc cgt ggt cgt cag ctg ctg gca gcc ctg gat 630 196 Asp Gln Pro Asp Ser Arg Gly Arg Gln Leu Leu Ala Ala Leu Asp 210

631 tat ctg acc cag cgt agc agc gtt cgt aat cgt gtt gat gca agc 675 211 Tyr Leu Thr Gln Arg Ser Ser Val Arg Asn Arg Val Asp Ala Ser 225

676 cgt ctg gca gtt gcc ggt cat agc atg ggt ggt ggt ggc acc ctg 720 226 Arg Leu Ala Val Ala Gly His Ser Met Gly Gly Gly Gly Thr Leu 240

721 gaa gca gca aaa agc cgt acc agc ctg aaa gca gcc att ccg att 765 241 Glu Ala Ala Lys Ser Arg Thr Ser Leu Lys Ala Ala Ile Pro Ile 255

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766 gca ccg tgg aat ctg gat aaa acc tgg cct gaa gtt cgt acc ccg 810 256 Ala Pro Trp Asn Leu Asp Lys Thr Trp Pro Glu Val Arg Thr Pro 270

811 aca ctg att att ggt ggt gaa ctg gat agc att gca ccg gtt gcg 855 271 Thr Leu Ile Ile Gly Gly Glu Leu Asp Ser Ile Ala Pro Val Ala 285

856 acc cat agc att ccg ttt tat aac agc ctg acc aat gca cgt gaa 900 286 Thr His Ser Ile Pro Phe Tyr Asn Ser Leu Thr Asn Ala Arg Glu 300

901 aaa gca tat ctg gaa ctg aat aat gcc agc cat ttt ttt ccg cag 945 301 Lys Ala Tyr Leu Glu Leu Asn Asn Ala Ser His Phe Phe Pro Gln 315

946 ttt agc aat gat acc atg gcc aaa ttt atg atc agc tgg atg aaa 990 316 Phe Ser Asn Asp Thr Met Ala Lys Phe Met Ile Ser Trp Met Lys 330

991 cgc ttt atc gat gat gat acc cgc tat gat cag ttt ctg tgt ccg 1035 331 Arg Phe Ile Asp Asp Asp Thr Arg Tyr Asp Gln Phe Leu Cys Pro 345

1036 cct ccg cgt gca att ggt gat att agc gat tat cgt gat acc tgt 1080 346 Pro Pro Arg Ala Ile Gly Asp Ile Ser Asp Tyr Arg Asp Thr Cys 360

1081 ccg cat acc gca gaa gca gca gca aaa gaa gcc gct gcc aaa gaa 1125 361 Pro His Thr Ala Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu 375 17aa helix 1126 gcg gca gcg aaa gca gaa aat ctg tat ttt cag ggt gcc att aaa 1170 376 Ala Ala Ala Lys Ala Glu Asn Leu Tyr Phe Gln Gly Ala Ile Lys 390 TEV cleavage site 1171 ctg gtt cag agc ccg aat ggt aat ttt gca gca agc ttt gtt ctg 1215 391 Leu Val Gln Ser Pro Asn Gly Asn Phe Ala Ala Ser Phe Val Leu 405

1216 gat ggc acc aaa tgg atc ttc aaa agc aaa tac tat gac agc agc 1260 406 Asp Gly Thr Lys Trp Ile Phe Lys Ser Lys Tyr Tyr Asp Ser Ser 420

1261 aaa ggt tat tgg gtg ggt att tat gaa gtg tgg gat cgc aaa taa 1305 421 Lys Gly Tyr Trp Val Gly Ile Tyr Glu Val Trp Asp Arg Lys - 435 Liquid chromatography peak I (LCI) 1306 taa 1308 436 - Stop

Tcur1278 wild-type with C-terminal Tachystatin A2

1 atg aga ttc cca tct att ttc acc gct gtc ttg ttc gct gcc tcc 45 1 Met Arg Phe Pro Ser Ile Phe Thr Ala Val Leu Phe Ala Ala Ser 15 Start 46 tct gca ttg gct gcc cct gtt aac act acc act gaa gac gag act 90 16 Ser Ala Leu Ala Ala Pro Val Asn Thr Thr Thr Glu Asp Glu Thr 30

91 gct caa att cca gct gaa gca gtt atc ggt tac tct gac ctt gag 135 31 Ala Gln Ile Pro Ala Glu Ala Val Ile Gly Tyr Ser Asp Leu Glu 45

136 ggt gat ttc gac gtc gct gtt ttg cct ttc tct gct tcc att gct 180 46 Gly Asp Phe Asp Val Ala Val Leu Pro Phe Ser Ala Ser Ile Ala 60

181 gct aag gaa gag ggt gtc tct ctc gag aag aga gag gcc gaa gct 225 61 Ala Lys Glu Glu Gly Val Ser Leu Glu Lys Arg Glu Ala Glu Ala 75 Signal peptide (prepro α-leader sequence)

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Appendix

226 agt ctg cgt aaa agc ttt ggt ctg ctg agc gca acc gca gca ctg 270 76 Ser Leu Arg Lys Ser Phe Gly Leu Leu Ser Ala Thr Ala Ala Leu 90

271 gtt gca ggt ctg gtt gcc gca ccg cct gca cag gca gca gca aat 315 91 Val Ala Gly Leu Val Ala Ala Pro Pro Ala Gln Ala Ala Ala Asn 105

316 ccg tat cag cgt ggt ccg gat ccg acc gaa agc ctg ctg cgt gca 360 106 Pro Tyr Gln Arg Gly Pro Asp Pro Thr Glu Ser Leu Leu Arg Ala 120

361 gca cgt ggt ccg ttt gca gtt agc gaa cag agc gtt agc cgt ctg 405 121 Ala Arg Gly Pro Phe Ala Val Ser Glu Gln Ser Val Ser Arg Leu 135

406 agc gtg agc ggt ttt ggt ggt ggt cgt atc tat tat ccg acc aca 450 136 Ser Val Ser Gly Phe Gly Gly Gly Arg Ile Tyr Tyr Pro Thr Thr 150

451 acc agc cag ggc acc ttt ggt gca att gcc att agt ccg ggt ttt 495 151 Thr Ser Gln Gly Thr Phe Gly Ala Ile Ala Ile Ser Pro Gly Phe 165

496 acc gca agc tgg tca agc ctg gca tgg ctg ggt ccg cgt ctg gca 540 166 Thr Ala Ser Trp Ser Ser Leu Ala Trp Leu Gly Pro Arg Leu Ala 180

541 agc cat ggt ttt gtt gtt att ggt att gaa acc aac aca cgt ctg 585 181 Ser His Gly Phe Val Val Ile Gly Ile Glu Thr Asn Thr Arg Leu 195

586 gat cag ccg gat agc cgt ggt cgt cag ctg ctg gca gcc ctg gat 630 196 Asp Gln Pro Asp Ser Arg Gly Arg Gln Leu Leu Ala Ala Leu Asp 210

631 tat ctg acc cag cgt agc agc gtt cgt aat cgt gtt gat gca agc 675 211 Tyr Leu Thr Gln Arg Ser Ser Val Arg Asn Arg Val Asp Ala Ser 225

676 cgt ctg gca gtt gcc ggt cat agc atg ggt ggt ggt ggc acc ctg 720 226 Arg Leu Ala Val Ala Gly His Ser Met Gly Gly Gly Gly Thr Leu 240

721 gaa gca gca aaa agc cgt acc agc ctg aaa gca gcc att ccg att 765 241 Glu Ala Ala Lys Ser Arg Thr Ser Leu Lys Ala Ala Ile Pro Ile 255

766 gca ccg tgg aat ctg gat aaa acc tgg cct gaa gtt cgt acc ccg 810 256 Ala Pro Trp Asn Leu Asp Lys Thr Trp Pro Glu Val Arg Thr Pro 270

811 aca ctg att att ggt ggt gaa ctg gat agc att gca ccg gtt gcg 855 271 Thr Leu Ile Ile Gly Gly Glu Leu Asp Ser Ile Ala Pro Val Ala 285

856 acc cat agc att ccg ttt tat aac agc ctg acc aat gca cgt gaa 900 286 Thr His Ser Ile Pro Phe Tyr Asn Ser Leu Thr Asn Ala Arg Glu 300

901 aaa gca tat ctg gaa ctg aat aat gcc agc cat ttt ttt ccg cag 945 301 Lys Ala Tyr Leu Glu Leu Asn Asn Ala Ser His Phe Phe Pro Gln 315

946 ttt agc aat gat acc atg gcc aaa ttt atg atc agc tgg atg aaa 990 316 Phe Ser Asn Asp Thr Met Ala Lys Phe Met Ile Ser Trp Met Lys 330

991 cgc ttt atc gat gat gat acc cgc tat gat cag ttt ctg tgt ccg 1035 331 Arg Phe Ile Asp Asp Asp Thr Arg Tyr Asp Gln Phe Leu Cys Pro 345

1036 cct ccg cgt gca att ggt gat att agc gat tat cgt gat acc tgt 1080 346 Pro Pro Arg Ala Ile Gly Asp Ile Ser Asp Tyr Arg Asp Thr Cys 360

1081 ccg cat acc gca gaa gca gca gca aaa gag gca gcg gcg aaa gaa 1125 361 Pro His Thr Ala Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu 375 17aa helix

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Appendix

1126 gcc gca gcc aaa gcg gag aac ctg tac ttt cag ggc tat tcg cgc 1170 376 Ala Ala Ala Lys Ala Glu Asn Leu Tyr Phe Gln Gly Tyr Ser Arg 390 TEV cleavage site 1171 tgt caa ctg cag ggc ttt aac tgc gta gtc cgg tct tac ggc tta 1215 391 Cys Gln Leu Gln Gly Phe Asn Cys Val Val Arg Ser Tyr Gly Leu 405

1216 ccg acc att ccg tgt tgc cgt ggt ctg aca tgt cgc agc tac ttt 1260 406 Pro Thr Ile Pro Cys Cys Arg Gly Leu Thr Cys Arg Ser Tyr Phe 420

1261 ccc ggt agt acc tac ggt cgt tgc cag cgt tac tga taa 1299 421 Pro Gly Ser Thr Tyr Gly Arg Cys Gln Arg Tyr - - Tachystatin A2 (TA2) Stop

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Appendix

6.3 List of oligonucleotides

All oligonucleotides were purchased from Eurofins MWG Operon (Ebersberg, Germany). Nucleotide analogs, primers, and DNA polymerases along with their respective buffers for the generation of SeSaM library were obtained from SeSaM Biotech GmbH (Aachen, Germany). Table 12. Oligonucleotides used in this study. Letters in small case indicated the PTO-region and letters in bold show codons used for site-saturation and site-directed mutagenesis.

Primer name Sequence (5’-3’) Use

T7 forward TAATACGACTCACTATAGGG Sequencing primer

T7 reverse CTAGTTATTGCTCAGCGGT Sequencing primer

ASTB_M1 GGCAATATTCTGGCCGCAAGTG Sequencing primer

ASTB_M2 GGTTCCAATCACCAGCTTTACC Sequencing primer

ASTB_M3 CCACCTGACGCTGGAATCAATC Sequencing primer

ASTB_StrepII_F GTCTGTGAAACTGGAGCTCTCTGCATGGAGCCATC Strep-tag II insertion CGCAGTTCGAAAAGTAGCGTCGACAAGCTTGC

ASTB_StrepII_R GCAAGCTTGTCGACGCTACTTTTCGAACTGCGGAT Strep-tag II insertion GGCTCCATGCAGAGAGCTCCAGTTTCACAGAC

F1 CGACTCACTATAGGGGAATTGTGAGCGGA SeSaM library generation

R3 CGGGCTTTGTTAGCAGCCGGATCTCAG SeSaM library generation

SeSaM_F CACACTACCGCACTCCGTCG SeSaM library generation

SeSaM_R GTGTGATGGCGTGAGGCAGC SeSaM library generation

SeSaM_F1 CACACTACCGCACTCCGTCGCGACTCACTATAGGG SeSaM library generation GAATTGTGAGCGGA

SeSaM_R3 GTGTGATGGCGTGAGGCAGCCGGGCTTTGTTAGC SeSaM library generation AGCCGGATCTCAG

F1_up CGCCTGTCACCGACTCACTATAGGGGAATTGTGAG SeSaM library generation CGGA

R3_dn GCGGACAGTGCGGGCTTTGTTAGCAGCCGGATCT SeSaM library generation CAG

Bio_SeSaM_F [Biotin]CACACTACCGCACTCCGTCG SeSaM library generation

Bio_SeSaM_R [Biotin]GTGTGATGGCGTGAGGCAGC SeSaM library generation

V_F_PLIC catccgcagttcGAAAAGTAGCGTC Backbone amplification for PLICing of ASTB SeSaM library into pET22b(+)

V_R_PLIC ctatagtgagtcgTATTAATTTCGCGGGATCG Backbone amplification for PLICing of ASTB SeSaM library into pET22b(+)

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Appendix

ASTB_F_PLIC cgactcactatagGGGAATTGTGAGCGGATAAC Insert amplification for PLICing of ASTB SeSaM library into pET22b(+)

ASTB_R_PLIC gaactgcggatgGCTCCATGC Insert amplification for PLICing of ASTB SeSaM library into pET22b(+)

ASTB_SSM_111_F GTAAAACCAGCNNKGTGAAAATTGAAGTGG SSM at position 111

ASTB_SSM_111_R CCACTTCAATTTTCACMNNGCTGGTTTTA SSM at position 111

ASTB_SSM_144_F CTGATCTCAACCNNKACCCCGCTGATC SSM at position 144

ASTB_SSM_144_R GATCAGCGGGGTMNNGGTTGAGATCAG SSM at position 144

ASTB_SSM_446_F CGATGGCNNKGGTGCAATGCTGCTGAACAAATAC SSM at position 446

ASTB_SSM_446_R GTATTTGTTCAGCAGCATTGCACCMNNGCCATCG SSM at position 446

ASTB_SSM_579_F GCACGATNNKACCGCCGCATGTGTCTC SSM at position 579

ASTB_SSM_579_R GAGACACATGCGGCGGTMNNATCGTGC SSM at position 579

ASTB_SSM_608_F CTG TCT GGT GAA TTC NNK ATC TAC CTG SSM at position 608

ASTB_SSM_608_R CAGGTAGATMNNGAATTCACCAGACAG SSM at position 608

ASTB_SSM_615_F CTGAACATTGATNNKAAACGCTACGATACGCATCT SSM at position 615 GTC

ASTB_SSM_615_R GACAGATGCGTATCGTAGCGTTTMNNATCAATGT SSM at position 615 TCAG

ASTB_SSM_624_F ACGCATCTGTCTNNKAAACTGGAG SSM at position 624

ASTB_SSM_624_R CTCCAGTTTMNNAGACAGATGCGT SSM at position 624

ASTB_V579K_F GCACGATAAAACCGCCGCATGTGTCTC SDM VK at position 579

ASTB_V579K_R GAGACACATGCGGCGGTTTTATCGTGC SDM VK at position 579

ASTB_G608D_F CTG TCT GGT GAA TTC GAT ATC TAC CTG SDM GD at position 608

ASTB_G608D_R CAG GTA GAT ATC GAA TTC ACC AGA CAG SDM VK at position 608

ASTB_S615G_F CTGAACATTGATGGCAAACGCTACGATACGCATCT SDM SD at position 615 GTC

ASTB_S615G_R GACAGATGCGTATCGTAGCGTTTGCCATCAATGTT SDM SD at position 615 CAG

ASTB_H350A_F GATTTTGACTGGCAGTATGAACAAGCGGCTGCGC SDM HA at position 350 GTATTC

ASTB_H350A_R GAATACGCGCAGCCGCTTGTTCATACTGCCAGTCA SDM HA at position 350 AAATC

ASTB_G605stop_F CTGGTCCTGGGCAAATAAGAACTGCTG Stop codon at 506

ASTB_G605stop_R CAGCAGTTCTTATTTGCCCAGGACCAG Stop codon at 506

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Appendix

Tcur_PLIC_i_F gagagaggcggaagctAGTCTGCGTAAAAGCTTTG Insert (tcur1278) amplification

Tcur_PLIC_i_R cgaattcgctcttctTTATCATTTTTCAAACTGAG Insert (tcur1278) amplification

Tcur_PLIC_bb_F agaagagcgaattcgACGCGCCGGTAAG pBSYA1S1Z backbone amplification

Tcur_PLIC_bb_R agcttcggcctctctcTTCTCGAGGGAGAC pBSYA1S1Z backbone amplification

Tcur_LCI_PLIC_i_F cctgtccgcataccGCAGAAGCAGCAGCAAAAG Insert (17x-tev-lci and 17x- tev-ta2) amplification

Tcur_LCI_PLIC_i_R cgaattcgctcttctTTATTATTTGCGATCCCACAC Insert (17x-tev-lci) amplification

Tcur_TA2_PLIC_i_R cgaattcgctcttctTTATCAGTAACGCTGGCAACGAC Insert (17x-tev-ta2) amplification

Tcur_LCI_PLIC_bb_F agaagagcgaattcgACGCGCCGGTAAG pBSYA1S1Z (bb) amplification for PLICing of LCI and TA2

Tcur_LCI_PLIC_bb_R ggtatgcggacaggTATCACGATAATCG pBSYA1S1Z (bb) amplification for PLICing of LCI and TA2

Tcur_F GGGCGGACGCATGTCATGAGATTATTG Sequencing primer

Tcur_R CGACTTGTTGCATACATTCGATACC Sequencing primer

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Appendix

6.4 List of computational data

Figure 50. Sequence logo representation of multiple sequence alignment of ASSTs. Sequence logo was generated using WebLogo. Black arrows indicate conserved residues including catalytic histidine (H350) and binding sites (H220, H283, and R301). Red arrows indicate substituted residues (L446 and V579).

Figure 51. Comparison of ASTB-WT, ASTB-H350A, and ASTB-G506stop. The variant ASTB-H350A was generated to confirm the role of ctalytic histidine and G506stop to identify the improtance of C-terminal flexible loop. Activities were determined applying pNPS assay with cellobiose as acceptor and clarified crude cell lysate in 8 replicates. EV: empty vector as negative control.

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Appendix

Figure 52. Average RMSF and RMSD values of Cα atoms from MD simulations of ASTB. Mean residue-wise root mean square fluctuation (RMSF) obtained from three independent MD simulations of ASTB. (blue). Time evolution of the backbone root mean square displacement (RMSD) obtained from three independent MD simulations of ASTB (red).

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Appendix

6.5 List of analytical data

Figure 53. 1H NMR spectrum of 3-O-sulfo-GlcNAc-linker-tBoc and 4-O-sulfo-GlcNAc-linker-tBoc synthesized by ASTB-M5 as example.

Figure 54. 13C NMR spectrum of 3-O-sulfo-GlcNAc-linker-tBoc and 4-O-sulfo-GlcNAc-linker-tBoc synthesized by ASTB-M5 as example.

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Table 13. The 1H and 13C NMR data of 3-O-sulfo-GlcNAc-linker-tBoc. (600.23 MHz for 1H, 150.93 13 MHz for C, D2O, 30°C). The position of sulfation is marked in bold.

Atom C m. H nH m.

tBoc (CH3)3 27.96 Q 1.203 9 S C 81.38 S - 0 CO 158.48 S - 0 Linker 1‘ 44.48 T 3.44a 2 br s 2‘ 39.58 T 3.04a 2 br s CS 183.17 S - 0 GlcNAc 1 82.91 D 5.507, 1 br m 5.382 2 53.38 D 3.763 1 M 3 81.95 D 4.229 1 dd, ΣJ = 19.5

4 68.73 D 3.433 1 dd, ΣJ = 18.0 5 77.05 D 3.384 1 m 6 60.78 T 3.679 1 m 3.564 1 m CO 175.13 S - 0 2-Ac 22.31 Q 1.793x 3 s a … HSQC readout; x … might be interchanged

Table 14. The 1H and 13C NMR data of compound 4-O-sulfo-GlcNAc-linker-tBoc. (600.23 MHz for 1 13 H, 150.93 MHz for C, D2O, 30°C). The position of sulfation is marked in bold.

Atom C m. H nH m.

tBoc (CH3)3 27.96 Q 1.203 9 s C 81.38 S - 0 CO 158.48 S - 0 linker 1‘ 44.48 T 3.44a 2 br s 2‘ 39.58 T 3.04a 2 br s CS 183.17 S - 0 GlcNAc 1 82.91 D 5.390, 1 br m 2 54.54 D 5.2823.73a 1 m

3 72.80 D 3.652 1 m 4 76.99 D 4.005 1 dd, ΣJ = 19.0 .76.95 5 75.93 D 3.451 1 m 6 60.75 T 3.706 1 m 3.561 1 m CO 175.31 S - 0 2-Ac 22.49 Q 1.769x 3 s a … HSQC readout; x … might be interchanged

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Appendix

Figure 55. Expanded parts of 13C NMR spectra of products synthesized by ASTB-WT, ASTB-M5, and ASTB-V1. Synthesis ratios (3-O-sulfo-GlcNAc-linker-tBoc:4-O-sulfo-GlcNAc-linker-tBoc) for ASTB-WT, ASTB-M5, ASTB-V1 are 60:40, 55:45, and 57:43.

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Appendix

6.6 List of figures

Figure 1. Post-translation functionalization and modification...... 2 Figure 2. Microbial degradation of synthetic polymer...... 5 Figure 3. General scheme of a directed evolution campaign...... 7 Figure 4. Four phases of KnowVolution...... 10 Figure 5. General structure of glycosaminoglycans...... 14 Figure 6. General protein structure of cytosolic PAPS-dependent ST and catalyzed reaction...... 16 Figure 7. Protein structure of ASST from uropathogenic E. coli and its reaction mechanism...... 18 Figure 8. Sequence alignment between ASTA and ASTB from Desulfitobacterium hafniense...... 20 Figure 9. Engineering of aryl sulfotransferase B (ASTB) for higher sulfation activity toward saccharides. .. 21 Figure 10. Comparison of activity between ASTA and ASTB...... 32 Figure 11. Targeted reactions for establishment of pNPS-based screening system...... 33 Figure 12. Assay parameters of pNPS assay using GlcNAc and cellobiose as acceptors...... 34 Figure 13. Coefficient of variation of pNPS assay with GlcNAc and cellobiose as acceptors...... 35 Figure 14. Generation of astB SeSaM library...... 36 Figure 15. ASTB variants for GlcNAc sulfation...... 38 Figure 16. Determination of initial activities of ASTB-WT and ASTB-V1 with GlcNAc...... 39 Figure 17. Determination of the volumetric activity (U/L) of ASTB-WT and selected variants in the re- screening phase...... 40 Figure 18. ASTB structural model...... 42 Figure 19. Activity and SDS-PAGE analysis of the final single and recombination ASTB variants...... 43 Figure 20. SDS-PAGE and Native-PAGE analysis of ASTB and ASTB variants...... 44 Figure 21. Reaction kinetics of ASTB-WT and final variants with different acceptors...... 45 Figure 22. Electronspray ionization mass spectrometric analysis of enzymatically sulfated cellobiose...... 47 Figure 23. Targeted reaction for HPLC analysis of GlcNAc sulfation...... 48 Figure 24. Sulfation of GlcNAc-linker-tBoc by ASTB variants over time...... 49 Figure 25. Separation of reaction components by analytical HPLC and structures of novel sulfated compounds...... 50 Figure 26. HPLC and MS analysis of purified sulfated product...... 51 Figure 27. pH activity profile of ASTB-WT and variants within a pH range of 5.0-12.0...... 52 Figure 28. pH stability of ASTB-WT and variants after 6 h...... 53 Figure 29. Organic co-solvent resistance of ASTB-WT and variants...... 54 Figure 30. Temperature stability of ASTB-WT and variants between 4 and 67.1°C...... 55 Figure 31. Overview of KnowVolution campaign of ASTB toward cellobiose...... 58 Figure 32. Worldwide manufactured plastics from 1950 to 2016 and European plastic demand by the synthetic polymers in 2016...... 62 Figure 33. Chemical structure of polyethylene terephthalate (PET)...... 65 Figure 34. Chemical structure of polyurethane (PUR)...... 66

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Appendix

Figure 35. Structural model of the cutinase Tcur1278...... 68 Figure 36. Schematic presentation of cellulose degradation by cellulase...... 69 Figure 37. Different types of AMPs...... 71 Figure 38. Targeted microplastic degradation in high diulted suspension by the usage of adhesion promoter...... 72 Figure 39. Confocal microscopy of polyester-PUR and PET binding by eGFP-anchor...... 77 Figure 40. Principle of the pNPB-based screening system...... 78 Figure 41. Expression analysis of Tcur1278-WT and Tcur1278-anchors in Pichia pastoris BSYBG11 culture supernatants...... 79 Figure 42. Polyester-PUR degradation by culture supernatants of Tcur1278-WT and Tcur1278-anchors. ... 80 Figure 43. Specific activity analysis of Tcur1278-WT and Tcur1278-TA2...... 81 Figure 44. Continuous turbidity measurement of polyester-PUR particle degradation...... 82 Figure 45. Comparison of polyester-PUR degradation performance by Tcur1278 and Tcur1278-TA2...... 83 Figure 46. Hydrodynamic radii of untreated and treated polyester-PUR nanoparticles...... 84 Figure 47. FE-SEM analysis of polyester-PUR layers after enzymatic degradation...... 85 Figure 48. PET-film degradation by culture supernatants of Tcur1278-WT and Tcur1278-anchors...... 86 Figure 49. Principle of targeted PET-film degradation...... 90 Figure 50. Sequence logo representation of multiple sequence alignment of ASSTs...... 121 Figure 51. Comparison of ASTB-WT, ASTB-H350A, and ASTB-G506stop...... 121 Figure 52. Average RMSF and RMSD values of Cα atoms from MD simulations of ASTB...... 122 Figure 53. 1H NMR spectrum of 3-O-sulfo-GlcNAc-linker-tBoc and 4-O-sulfo-GlcNAc-linker-tBoc synthesized by ASTB-M5 as example...... 123 Figure 54. 13C NMR spectrum of 3-O-sulfo-GlcNAc-linker-tBoc and 4-O-sulfo-GlcNAc-linker-tBoc synthesized by ASTB-M5 as example...... 123 Figure 55. Expanded parts of 13C NMR spectra of products synthesized by ASTB-WT, ASTB-M5, and ASTB- V1...... 125

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Appendix

6.7 List of tables

Table 1. GAGs with their location and functions...... 15 Table 2. Two-step PCR program...... 22 Table 3. Sequencing analysis of mutants from astB SeSaM library...... 37 Table 4. Beneficial positions and substitutions with their effect on enzyme properties. Red letters indicate the best substitutions...... 41

Table 5. Kinetic constants (KM and Vmax) of ASTB-WT, ASTB-M1, ASTB-M2, and ASTB-M5 with resorcinol, GlcNAc, glucose, and cellobiose as acceptors...... 46 Table 6. Improvement of ASTB variant activity toward saccharides compared to ASTB-WT. V/WT indicates the ratio of volumetric activity between variant and WT...... 48 Table 7. Semi-preparative synthesis of sulfated GlcNAc-linker-tBoc by ASTB-WT, ASTB-M5, and ASTB-V1. 51 Table 8. Most widely used synthetic polymers and their applications...... 63 Table 9. Polyester-PUR degradation performance of Tcur1278-WT and Tcur1278-anchors...... 80 Table 10. Polyester-PUR degradation rates of Tcur1278-WT and Tcur1278-TA2 and improvements achieved by Tcur1278-TA2...... 84 Table 11. Overview of fused enzyme-adhesion promotor in different studies...... 88 Table 12. Oligonucleotides used in this study...... 118 Table 13. The 1H and 13C NMR data of 3-O-sulfo-GlcNAc-linker-tBoc...... 124 Table 14. The 1H and 13C NMR data of compound 4-O-sulfo-GlcNAc-linker-tBoc...... 124

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Declaration

7. Declaration

Hiermit versichere ich, dass ich die vorliegende Arbeit selbstständig verfasst und keine anderen als die angegebenen Quellen und Hilfsmittel benutzt habe, dass alle Stellen der Arbeit, die wörtlich oder sinngemäß aus anderen Quellen übernommen wurden, als solche kenntlich gemacht sind und dass die Arbeit in gleicher oder ähnlicher Form noch keiner Prüfungsbehörde vorgelegt wurde.

Aachen, den______Shohana Subrin Islam

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Author contribution

8. Author contribution

Islam S* & Apitius L*, Jakob F, Schwaneberg U (2018). Targeting microplastic in the void of diluted suspension. Environment international, 123, 428-435.

The manuscript has been published by Shohana Islam and Lina Apitius with a shared first authorship. The technology of enzymes (cutinases) fused to anchor peptides for enhanced microplastic degradation was jointly developed in a collaborative effort. Lina Apitius contributed to the binding tests of anchor peptides on surfaces. Shohana Islam contributed to the generation of enzyme and enzyme-anchor fusion constructs. Both contributed equally to the evaluation of the microplastic degradation performance, manuscript preparation, and writing. A detailed chapter is implemented in Shohana Islam´s dissertation and Lina Apitius included a short description in her thesis.

Aachen, den______Shohana Subrin Islam

______Lina Apitius

______Felix Jakob

______Ulrich Schwaneberg

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Acknowledgement

9. Acknowledgement

Eleven years ago, when I put my first step in Germany, I could not even imagine that this day will come true. My survival in this foreign country and in parallel higher education would have been impossible without all the kind support I have received here. I hope I can acknowledge all the people here.

I am very thankful to my doctor father Prof. Dr. Ulrich Schwaneberg for believing in me from the very beginning and giving me the opportunity to start my career in the field of biotechnology. I still remember the day he offered me a Ph.D. position and I could not hold my tears. Uli, thank you so much for always being very kind, for all the support, faith, guidance, and supervision.

I would like to thank Prof. Dr. Lothar Elling for being my second examiner in the Ph.D. defense committee and for an excellent collaboration within my Ph.D. thesis. It was wonderful to work with you and your group. Thanks to Prof. Dr. Andrij Pich for being my third examiner and Prof. Dr, Jan Schirwaski for being the chairperson of the committee.

I especially would like to thank Dr. Felix Jakob for your supervision throughout the whole Ph.D. time. I gratefully acknowledge your precious time, encouragement, and constructive criticism. Also, a very big thanks to you for always having an open ear for listening to the research as well as daily life problems. I am also very thankful to Dr. Diana Mate and Dr. Ronny Martínez for co-supervising me in the beginning and for your great support in my first publication. Especially, thank you, Diana, for being always a very good friend. A very special thank goes to Erik, who taught me all the important techniques for working in a biological lab appropriately during my bachelor thesis. It was a great pleasure for me to be a part of the biohybrid systems subgroup and thanks to all the people of the subgroup and DWI lab members for the nice working atmosphere. Special thanks to John, Catalina, Leticia, Lina, Kristin, Kati, Robin, and Patrizia for all the fun and discussions we had in and outside of the lab. Thank you, Catalina, for sharing the lunch for almost 3.5 years. Furthermore, I thank all the former and current members of the Schwaneberg group.

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Acknowledgement

I acknowledge the FuPol (Functionalization of polymers) alliance funded by German federal ministry of education and research (BMBF) for financial support. I would also like to thank Dr. Dominic Laaf for his great help regarding the sugar analytics, Dr. Helena Pelantová and Prof. Dr. Vladimír Křen for their efforts with the NMR measurements, Dr. Belén Infanzón and Dr. Mehdi Davari for the intensive computational analysis of a difficult protein, Thorsten Palmer for DLS, and Sabrina Mallmann for FE-SEM. I would like to especially thank Dr. Lina Apitius for a very nice collaboration and thank you Lina very much for teaching me anchor peptides and sarcasm.

I would like to also thank my “lab-twin” Dr. John Mandawe from the bottom of my heart for being such a true friend. Thank you for all the fun, support, and encouragement in good as well as difficult times. Your strength and curiosity, which I respect very much, will motivate me now and forever. My deepest thanks to my dearest friends Sara, Shishir, and Ketaki for their endless love and support and for making Aachen my home. A very special thank goes to Kornelia von Kaisenberg, without whom I would never able to get the DAAD award and scholarship from Bread for the World for my master studies. Thanks also to all the people who never believed in me, because you motivated me more and more to achieve my goals.

“Thanks” would be a very small word for you, Marco for your love, patience, and support. I am deeply thankful to have you in my life. No words can be appropriate for saying thanks to my mother and sister. Without their endless love and motivation, I would not have survived in Germany. Thank you very much for always believing in me, motivating me constantly, forgiving my mistakes, and standing by me no matter what. I am nothing without you and very proud to have you as my family. That’s why I dedicate this thesis to you, Ammu and Banhi!

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Curriculum vitae

10. Curriculum vitae

Personal Name Shohana Subrin Islam Date of birth 15.07.1988 Place of birth Dhaka, Bangladesh Nationality German

Working experience 2018 – Scientific coworker Institute of Biotechnology, RWTH Aachen University, Aachen, Germany 2014 – 2018 Ph.D. fellow in Biotechnology DWI – Leibniz Institute for Interactive Materials, Aachen, Germany Enzymatic functionalization and degradation of natural and synthetic polymers 2010 – 2014 Student assistant Institute of Biotechnology, RWTH Aachen University, Aachen, Germany Institute of Physics, RWTH Aachen University, Aachen, Germany

Education 2012 – 2014 Master of Science in Molecular and Applied Biotechnology RWTH Aachen University, Aachen, Germany Cloning, expression, and development of suitable MTP-based activity assays of industrially relevant sulfotransferases 2009 – 2012 Bachelor of Science in Biotechnology / Molecular Biotechnology RWTH Aachen University, Aachen, Germany

KM-value optimization of Glucose oxidase from Aspergillus niger for application in diabetes analytics

2007 – 2008 Studienkolleg for foreign students RWTH Aachen University, Aachen, Germany 2003 – 2005 Higher secondary school certificate Viqarunnisa Noon College, Dhaka, Bangladesh 1993 – 2003 Secondary school certificate Kadamtala Purba Bashabo High School, Dhaka, Bangladesh

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