A fusion protein composed of antibody fragments and β-lactamase was earlier created by Kojima et al. (2011), with antigen specificities against a bone disease marker and a pesticide. The enzyme was circularly permutated and fused to the variable heavy and light chain antibody fragments, thereby ensuring inactivity until binding of the target antigen triggered enzyme activation. Upon activation, the β-lactamase produced a colorimetric signal, which indicated antigen presence. In this work, a similar strategy was used to create two novel fusion proteins composed of circularly permuted β-lactamase and superfolder green fluorescent protein with anti-benzo[a]pyrene variable antibody fragments. The fusion proteins were designed and expressed in E. coli for the development of a single-step visual immunoassay. It was hypothesized that the cp reporter proteins would be activated once the binding of B[a]P to the variable antibody fragments occurred, and this interaction was expected to produce a detectable colorimetric or fluorescent signal. Although positive results were obtained in one instance, substantial supportive evidence in favour of the hypothesis could not be obtained. / SENTINEL Bioactive Paper Network, Natural Sciences and Engineering Research Council of Canada (NSERC), Canada Research Chairs Program.

A wide range of screening strategies have been employed to isolate antibodies and other proteins with specific attributes, including binding affinity, specificity, stability and improved expression. However, there remains no high-throughput system to screen for target-binding proteins in a mammalian, intracellular environment. Such a system would allow binding reagents to be isolated against intracellular clinical targets such as cell signalling proteins associated with tumour formation (p53, ras, cyclin E), proteins associated with neurodegenerative disorders (huntingtin, betaamyloid precursor protein), and various proteins crucial to viral replication (e.g. HIV-1 proteins such as Tat, Rev and Vif-1), which are difficult to screen by phage, ribosome or cell-surface display. This study used the â-lactamase protein complementation assay (PCA) as the display and selection component of a system for screening a protein library in the cytoplasm of HEK 293T cells. The colicin E7 (ColE7) and Immunity protein 7 (Imm7) Escherichia coli proteins were used as model interaction partners for developing the system. These proteins drove effective â-lactamase complementation, resulting in a signal-to-noise ratio (9:1 – 13:1) comparable to that of other â-lactamase PCAs described in the literature. The model Imm7-ColE7 interaction was then used to validate protocols for library screening. Single positive cells that harboured the Imm7 and ColE7 binding partners were identified and isolated using flow cytometric cell sorting in combination with the fluorescent â-lactamase substrate, CCF2/AM. A single-cell PCR was then used to amplify the Imm7 coding sequence directly from each sorted cell. With the screening system validated, it was then used to screen a protein library based the Imm7 scaffold against a proof-of-principle target. The wildtype Imm7 sequence, as well as mutants with wild-type residues in the ColE7- binding loop were enriched from the library after a single round of selection, which is consistent with other eukaryotic screening systems such as yeast and mammalian cell-surface display. In summary, this thesis describes a new technology for screening protein libraries in a mammalian, intracellular environment. This system has the potential to complement existing screening technologies by allowing access to intracellular proteins and expanding the range of targets available to the pharmaceutical industry.

Introduktion Antibiotikaresistens är ett globalt växande problem. Till gruppen β-laktamantibiotika hör piperacillin-tazobaktam och cefotaxim som båda verkar genom att försvaga cellväggen med kovalenta bindningar till peptidoglykanlagret som lyserar cellen. E. coli och K. pneumoniae tillhör gruppen Enterobacteriaceae, som är en del av den humana tarmfloran och ofta förekommande vid urinvägsinfektion och sepsis. Utvidgat Spektrum β-Laktamas (ESBL) är ett enzym som finns hos Enterobacteriaceae och som hydrolyserar β-laktamantibiotika. Matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) är en kvalitativ analysteknik för detektion av kemiska föreningar i avseende på massa och laddning. Kännedom om antibiotikametaboliters molekylvikt vid hydrolys möjliggör detektion. Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS) är en högsensitiv kvantifieringsmetod som separerar molekyler i avseende på polaritet för vidare detektion i avseende på massa och laddning. Syfte Syftet med denna studie var att vidareutveckla en snabb och effektiv metod för att påvisa nedbrytning av piperacillin-tazobaktam och cefotaxim i blodplasma med LC-MS/MS. Material och Metod Tiofaldigt sjunkande koncentrationer av piperacillin-tazobaktam från 2000 till 2 µg/ml, och cefotaxim med koncentrationerna 500 till 0,5 µg/ml analyserades med MALDI-TOF MS, dels intakt men även med bakterierna E. coli och K. pneumoniae med uttryck av olika resistensmekanismer. Vid optimerade koncentrationer spikades plasmaprover med nedbrutet antibiotika som sedan kvantifierades med LC-MS/MS. Resultat Lägsta detektionsgräns med MALDI-TOF MS för intakt och hydrolyserat piperacillin-tazobaktam var 20/2,5 µg/ml. För cefotaxim var lägsta gränsen 5 µg/ml. Med kliniskt relevanta blodkoncentrationer gick hydrolys inte att detektera för. Med tre bakteriekolonier/50 µl kunde dock hydrolys detekteras och kvantifieras med LC-MS/MS. Slutsats Detektion av β-laktamantibiotika är möjligt med både MALDI-TOF MS och LC-MS/MS. För att påvisa hydrolys krävdes större mängder bakterier än förväntat med LC-MS/MS. / Introduction Antibiotic resistance is a global growing problem. Piperacillin-tazobactam and cefotaxime are parts of the group β-lactam antibiotics. The common feature is to inhibit the cell wall synthesis by covalent bindings to the peptidoglycan layer and thereby causing lysis of the bacterial cell. E. coli and K. pneumoniae are members of the Enterobacteriaceae which is a part of the human normal flora but also are commonly associated with urinary tract infections which sometimes develops into to sepsis. Extended Spectrum β-Lactamases (ESBLs) are enzymes with hydrolytic abilities acting on β-lactam antibiotics, expressed by Enterobacteriaceae. The qualitative, Matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) can be used to detect chemical compounds in the ratio of mass to charge in accordance to their molecular weight. Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS) is a highly sensitive two-step method of quantification which first separate molecules by their polarity attraction force and then by the ratio of mass to charge. Aim The aim of this study was to develop a fast and efficient method to determine degradation of piperacillin-tazobactam and cefotaxime in blood plasma by LC-MS/MS. Method Tenfold dilution of piperacillin-tazobactam in concentrations of 2000 to 2 µg/ml, and cefotaxime in concentrations of 500 to 0,5 µg/ml where analyzed by MALDI-TOF MS, intact and also with the bacteria E. coli and K. pneumoniae with different expression of antibiotic resistance. Optimized concentrations where fixed in blood plasma and then quantified by LC-MS/MS. Result The detection limit by using MALDI TOF MS of hydrolyzed as well as non-hydrolyzed piperacillin-tazobactam was 20/2,5 µg/ml. The detection limit in cefotaxime was 5 µg/ml. Hydrolysis could not be detected in clinically fixed blood concentrations. Detection and quantification of hydrolysis by LC-MS/MS was possible in a concentration of three bacteria colonies/50 µl. Conclusion It is possible to detect hydrolysis in both MALDI TOF MS and LC-MS/MS. A larger amount of bacteria than expected was needed to demonstrate hydrolysis In LC-MS/MS.

Antibiotic resistance is a global and accelerating matter. Over time, the bacteria have evolved several defense mechanisms against the antibiotics. One of the defense mechanisms is that the bacteria can produce enzymes with the ability to hydrolyze the characteristic b-lactam ring of the antibiotics. These enzymes are called b-lactamases. There are three different generations of antibiotics clinically available, and b-lactamases have co-evolved with the antibiotics over the generations. The third generation of antibiotics are called the carbapenems and b-lactamases which hydrolyze carbapenems are called carbapenemases. Carbapenemases are promiscuous, which means that they hydrolyze a variety of antibiotics. The b-lactamase IMI-1 is an imipenem-hydrolyzing enzyme and imipenem is a carbapenem, hence IMI-1 is a carbapenemase. In this project, IMI-1 was investigated in complex with the carbapenems imipenem, meropenem and biapenem using computational methods. More specifically, a homology model of IMI-1 was generated and the carbapenems were docked into the model. The system was then used for MD simulations where the important molecular interactions were identified, and the binding free energies were calculated using the LIE method. The results indicate that IMI-1 has flexible loops that enables an open and a closed conformation of IMI- 1. All three carbapenems were docked and simulated in both conformations of IMI-1. The results indicate that open and closed conformations confirms the promiscuity of carbapenemases since the flexibility enables various initial binding mechanisms. in other words, the hydrolysis may occur so quickly that the binding does not have much bearing of the activity of the enzyme. Furthermore, the calculated binding free energies indicate that IMI-1 is optimized for the catalytic process rather than the binding affinity. In conclusion, IMI-1 and similar systems requires further research using computational methods to counteract antibiotic resistance based on knowledge.

Cette thèse comporte quatre fichiers vidéo. This thesis comes with four video files. / L'industrie chimique mondiale est en pleine mutation, cherchant des solutions pour rendre la synthèse organique classique plus durable. Une telle solution consiste à passer de la catalyse chimique classique à la biocatalyse. Bien que les avantages des enzymes incluent leur stéréo, régio et chimiosélectivité, cette sélectivité réduit souvent leur promiscuité. Les efforts requis pour adapter la fonction enzymatique aux réactions désirées se sont révélés d'une efficacité modérée, de sorte que des méthodes rapides et rentables sont nécessaires pour générer des biocatalyseurs qui rendront la production chimique plus efficace. Dans l’ère de la bioinformatique et des outils de calcul pour soutenir l'ingénierie des enzymes, le développement rapide de nouvelles fonctions enzymatiques devient une réalité. Cette thèse commence par un examen des développements récents sur les outils de calcul pour l’ingénierie des enzymes. Ceci est suivi par un exemple de l’ingénierie des enzymes purement expérimental ainsi que de l’évolution des protéines. Nous avons exploré l’espace mutationnel d'une enzyme primitive, la dihydrofolate réductase R67 (DHFR R67), en utilisant l’ingénierie semi-rationnelle des protéines. La conception rationnelle d’une librarie de mutants, ou «Smart library design», impliquait l’association covalente de monomères de l’homotétramère DHFR R67 en dimères afin d’augmenter la diversité de la librairie d’enzymes mutées. Le criblage par activité enzymatique a révélé un fort biais pour le maintien de la séquence native dans un des protomères tout en tolérant une variation de séquence élevée pour le deuxième. Il est plausible que les protomères natifs procurent l’activité observée, de sorte que nos efforts pour modifier le site actif de la DHFR R67 peuvent n’avoir été que modérément fructueux. Les limites des méthodes expérimentales sont ensuite abordées par le développement d’outils qui facilitent la prédiction des points chauds mutationnels, c’est-à-dire les sites privilégiés à muter afin de moduler la fonction. Le développement de ces techniques est intensif en termes de calcul, car les protéines sont de grandes molécules complexes dans un environnement à base d’eau, l’un des solvants les plus difficiles à modéliser. Nous présentons l’identification rapide des points chauds mutationnels spécifiques au substrat en utilisant l'exemple d’une enzyme cytochrome P450 industriellement pertinente, la CYP102A1. En appliquant la technique de simulation de la dynamique moléculaire par la force de polarisation adaptative, ou «ABF», nous confirmons les points chauds mutationnels connus pour l’hydroxylation des acides gras tout en identifiant de nouveaux points chauds mutationnels. Nous prédisons également la conformation du substrat naturel, l’acide palmitique, dans le site actif et nous appliquons ces connaissances pour effectuer un criblage virtuel d'autres substrats de cette enzyme. Nous effectuons ensuite des simulations de dynamique moléculaire pour traiter l’impact potentiel de la dynamique des protéines sur la catalyse enzymatique, qui est le sujet de discussions animées entre les experts du domaine. Avec la disponibilité accrue de structures cristallines dans la banque de données de protéines (PDB), il devient clair qu’une seule structure de protéine n’est pas suffisante pour élucider la fonction enzymatique. Nous le démontrons en analysant quatre structures cristallines que nous avons obtenues d’une enzyme β-lactamase, parmi lesquelles un réarrangement important des résidus clés du site actif est observable. Nous avons réalisé de longues simulations de dynamique moléculaire pour générer un ensemble de structures suggérant que les structures cristallines ne reflètent pas nécessairement la conformation de plus basse énergie. Enfin, nous étudions la nécessité de compléter de manière informatisée un hémisphère où l’expérimental n’est actuellement pas possible, à savoir la prédiction de la migration des gaz dans les enzymes. À titre d'exemple, la réactivité des enzymes cytochrome P450 dépend de la disponibilité des molécules d’oxygène envers l’hème du site actif. Par le biais de simulations de la dynamique moléculaire de type Simulation Implicite du Ligand (ILS), nous dérivons le paysage de l’énergie libre de petites molécules neutres de gaz pour cartographier les canaux potentiels empruntés par les gaz dans les cytochromes P450 : CYP102A1 et CYP102A5. La comparaison pour les gaz CO, N2 et O2 suggère que ces enzymes évoluent vers l’exclusion du CO inhibiteur. De plus, nous prédisons que les canaux empruntés par les gaz sont distincts des canaux empruntés par le substrat connu et que ces canaux peuvent donc être modifiés indépendamment les uns des autres. / The chemical industry worldwide is at a turning point, seeking solutions to make classical organic synthesis more sustainable. One such solution is to shift from classical catalysis to biocatalysis. Although the advantages of enzymes include their stereo-, regio-, and chemoselectivity, their selectivity often reduces versatility. Past efforts to tailor enzymatic function towards desired reactions have met with moderate effectiveness, such that fast and cost-effective methods are in demand to generate biocatalysts that will render the production of fine and bulk chemical production more benign. In the wake of bioinformatics and computational tools to support enzyme engineering, the fast development of new enzyme functions is becoming a reality. This thesis begins with a review of recent developments on computational tools for enzyme engineering. This is followed by an example of purely experimental enzyme engineering and protein evolution. We explored the mutational space of a primitive enzyme, the R67 dihydrofolate reductase (DHFR), using semi-rational protein engineering. ‘Smart library design’ involved fusing monomers of the homotetrameric R67 DHFR into dimers, to increase the diversity in the resulting mutated enzyme libraries. Activity-based screening revealed a strong bias for maintenance of the native sequence in one protomer with tolerance for high sequence variation in the second. It is plausible that the native protomers procure the observed activity, such that our efforts to modify the enzyme active site may have been only moderately fruitful. The limitations of experimental methods are then addressed by developing tools that facilitate computational mutational hotspot prediction. Developing these techniques is computationally intensive, as proteins are large molecular objects and work in aqueous media, one of the most complex solvents to model. We present the rapid, substrate-specific identification of mutational hotspots using the example of the industrially relevant P450 cytochrome CYP102A1. Applying the adaptive biasing force (ABF) molecular dynamics simulation technique, we confirm the known mutational hotspots for fatty acid hydroxylation and identify a new one. We also predict a catalytic binding pose for the natural substrate, palmitic acid, and apply that knowledge to perform virtual screening for further substrates for this enzyme. We then perform molecular dynamics simulations to address the potential impact of protein dynamics on enzyme catalysis, which is the topic of heated discussions among experts in the field. With the availability of more crystal structures in the Protein Data Bank, it is becoming clear that a single protein structure is not sufficient to elucidate enzyme function. We demonstrate this by analyzing four crystal structures we obtained of a β-lactamase enzyme, among which a striking rearrangement of key active site residues was observed. We performed long molecular dynamics simulations to generate a structural ensemble that suggests that crystal structures do not necessarily reflect the conformation of lowest energy. Finally, we address the need to computationally complement an area where experimentation is not currently possible, namely the prediction of gas migration into enzymes. As an example, the reactivity of P450 cytochrome enzymes depends on the availability of molecular oxygen at the active-site heme. Using the Implicit Ligand Sampling (ILS) molecular dynamics simulation technique, we derive the free energy landscape of small neutral gas molecules to map potential gas channels in cytochrome P450 CYP102A1 and CYP102A5. Comparison of CO, N2 and O2 suggests that those enzymes evolved towards exclusion of the inhibiting CO. In addition, we predict that gas channels are distinct from known substrate channels and therefore can be engineered independently from one another.

Tyrosine (4-hydroxyphenylalanine) is one of the naturally occurring 22 amino acids. The importance of tyrosine is due to the presence of its phenolic side chain. In biological systems, the tyrosyl residue in proteins is found to be sulfated, phosphorylated and nitrated. Upon oxidation with dioxygenases, Tyr residue forms dopaquinone which undergoes a series of reactions ultimately leading to the formation of melanin. Tyr is also a precursor to neurotransmitters (catechol amines namely dopamine, epinephrine and norepinephrine) and thyroid harmones T4 and T3. Tyr residue is also found to be cross linked with other amino acid residues in the active site of certain proteins. Tyr-Tyr cross link has also been associated with neurodegenerative diseases. Tyr residue in proteins has been targeted widely for site selective modifications. A series of chemical modifications like acylation, allylation, ene-type reaction, iodination with radiolabeled iodine, formation of Tyr-Tyr cross link with oxidants and aminoalkylation have been carried out on surface exposed Tyr residues in proteins. Apart from these chemical modifications of Tyr on protein surface, a couple of free Tyr-based scaffolds have also been developed for different applications. Similar to tyrosine-based scaffolds, several phenolate-based scaffolds have also been developed for various applications. Several phenolate-based binuclear metal complexes have been developed as mimics of the active site of metalloenzymes. Moreover, by varying the substituent in the phenolate scaffold, the redox properties of metal bound in these systems can be tuned. The thesis consists of five chapters. The first chapter gives general idea about tyrosine-and phenolate-based scaffolds. The first chapter also gives introduction to zinc(II)-containing enzymes metallo-β-lactamases (mβls) and phosphotriesterase (PTE) and their functional mimics. The importance of copper(II)-containing enzyme, catechol oxidase and its mimics has also been discussed. The significance and formation of o-dityrosine (Tyr-Tyr cross link) has also been briefly discussed. In chapters 2 and 3, a couple of phenolate-based ligands and their corresponding zinc(II)- and copper(II)- complexes have been synthesized and have been checked as mimics of zinc(II)-containing enzymes (mβl and PTE) and copper-containing enzyme catechol oxidase, respectively. In chapter 4, a series of tyrosine-based ligands have been designed and their in situ copper(II) complexes have been tested as mimics of catechol oxidase. In chapter 5, the effect of neighboring amino acid in the formation of Tyr-Tyr cross link has been studied. In chapter 2, a couple of zinc(II) complexes have been synthesized and studied as mimic of zinc(II)-containing enzymes mβl and PTE. Metallo-β-lactamases (mβls) are zinc(II)-containing enzymes which exist in both mono- and binuclear forms. Mβls are capable of hydrolyzing β-lactam ring in antibiotics and make them inactive (Scheme 1(A)). To date, an effective inhibitor for this enzyme is not known. Hence, in order to understand the nature of the enzyme a couple of synthetic mimics are known. However, in most of the synthetic mimics both the metal ions are in symmetrical environment. Therefore, we have attempted to design a few unsymmetrical phenolate- based ligands and their zinc(II) complexes. The unsymmetrical phenolate-based ligands HL1 and HL2 have been synthesized by sequential mannich reaction with formaldehyde and two different amines. Complexes 1 and 2 are obtained from ligands HL1 and HL2, respectively (Figure 1). For comparative purpose, the symmetrical ligands HL3 and HL4, and their zinc(II)-complexes 3 and 4 have been synthesized by reported procedures (Figure 1). The efficiency of the complexes 1-4 towards the hydrolysis of oxacillin has been studied. It has been observed that the binuclear zinc(II) complexes with metal-bound water molecule 1 and 4 are able to hydrolyze oxacillin at much faster rates compared to that of mononuclear complexes 2 and 3. However, between 1 and 4, there is no appreciable change in activity, indicating that the slight change in ligand environment has no significant role. PTE is a binuclear zinc(II)-containing enzyme, capable of hydrolyzing toxic organphosphotriesters to less toxic diesters (Scheme 1(B)). As the binuclear active site of mβl is comparable with that of phosphotriesterase (PTE), PTE activity of complexes 1-4 has been studied. Although the binuclear zinc(II)-complexes 1 and 4 are able to hydrolyze PNPDPP (p-nitrophenyl diphenyl phosphate) initially, these complexes are not able to effect complete hydrolysis. This is due to the inhibition of complexes 1 and 4 by hydrolyzed product, diester. However with mononuclear complexes 2 and 3 no such inhibitions is possible, and are capable of hydrolyzing PNPDPP at comparatively faster rates than 1 and 4. Scheme 1. Function of metallo-β-lactamase and phosphotriesterase. (A) Hydrolysis of β-lactam ring in antibiotics by metallo-β-lactamase. (B) Hydrolysis of organophosphotriesters to diesters by phosphotriesterase. Figure 1. Chemical structures of ligands HL1-HL4 and their corresponding zinc(II)complexes 1-4. In chapter 3, a couple of copper(II) complexes have been synthesized and their catechol oxidase activity has been studied. Catechol oxidase belongs to the class of oxidoreductase and it catalyzes the oxidation of a wide range of o-diphenols to o-quinones through the reduction of molecular oxygen to water (Scheme 2). A four new µ4-oxo-bridged tetranuclear copper(II) complexes (5-8) have been synthesized (Figure 2). The ability of these complexes to catalyze the oxidation of 3,5-DTBC (3,5-Di-tert-butylcatechol) to 3,5-DTBQ (3,5-Di-tert-butylquinone) has been studied. A detailed kinetic study has been carried out which reveals that the complexes with exogenous acetate ligands (5 and 6) are better catechol oxidase mimics compared to complexes with exogenous chloride ligands (7 and 8). This observation is due to the labile nature of acetate compared to chloride, as the displacement of exogenous ligand is essential for the binding of substrate to the catalyst. Based on mass spectral analysis a plausible mechanism has been proposed for the oxidation of 3,5-DTBC by these complexes. Scheme 2. Oxidation of catechol by catechol oxidase. Figure 2. Chemical structures of copper(II) complexes 5-8. In chapter 4, by following the analogy between phenol and tyrosine, a series of binucleating ligands of tyrosine or tyrosyl dipeptides (Figures 3 and 4) have been synthesized by Mannich reaction under mild conditions. The in situ complexation of these fifteen new binucleating ligands (HL5-HL19) with copper(II) chloride has been observed. In situ complexation was followed by UV-visible and mass spectral analysis. These in situ complexes were able to oxidize 3,5-DTBC at slower rate compared to that of the tetranuclear complexes reported in chapter 3. The catecholase activity has also been tested with the addition of base. A slight enhancement in activity of in situ complexes has been observed in the presence of base. Based on mass spectral evidences, a plausible mechanism for the oxidation of catechol by these in situ complexes has been proposed. Figure 3. Binucleating ligands (Mannich bases) of boc-protected tyrosine and tyrosyl dipeptides. Figure 4. Binucleating ligands (Mannich bases) of boc-deprotected tyrosyl dipeptides. In chapter 5 of the thesis, the effect of neighboring amino acid residue in the formation of o,o-dityrosine (Tyr-Tyr cross link) has been studied. o,o’-Dityrosine is a specific marker for oxidative/nitrosative stress. The increase in concentration of dityrosine is associated with several disease states. A detailed study has been carried out in order to find out the effect of neighboring amino acid residues in the rate of formation of dityrosine of several tyrosyl dipeptides. The formation of dityrosine has been carried out with horseradish peroxidase(HRP) and H2O2 (Scheme 3). Except Cys-Tyr, all other tyrosyl dipeptides, form corresponding dityrosine with HRP/ H2O2. With Cys-Tyr, the formation of corresponding disulfide is observed. The appreciably higher rate of dityrosine formation of Phe-Tyr is attributed to the presence of strong hydrophobic environment around the active site of HRP. Among the polar tyrosyl peptides, the positively charged peptides (Arg-Tyr, Lys-Tyr) undergo dityrosine formation at much faster rate compared to that of negatively charged dipepptides (Asp-Tyr, Glu-Tyr). This trend is in accordance with the pKa of neighboring amino acid residues. The positively charged neighboring residues with higher pKa stabilizes ionized tyrosine, hence the rate of dityrosine formation is higher for them. As positively charged neighboring residue enhances the rate of dityrosine formation, the effect of externally added L-Arg has been studied. A coupling of a few biologically relevant tyrosine derivatives has been studied. The derivatives in which one of the ortho-positions of tyrosine is blocked, does not undergo coupling under the experimental conditions employed. Scheme 3. Formation of dityrosine of Ile-Tyr from Ile-Tyr in the presence of H2O2 catalyzed by HRP. (For structural formula and figures pl refer the abstract pdf file)

MSc (Microbiology) / Department of Microbiology / Background: The occurrence of microorganisms and endocrine disrupting chemicals (EDCs) in water poses a serious concern due to their effects on humans, animals and environment. In recent years, EDCs have been increasingly reported in rivers that receive large amounts of wastewater effluents. Of all the EDCs, natural and synthetic hormones are among those that are recognized for their potential to mimic or interfere with normal hormonal functions of humans and animals. The present study aimed at assessing the occurrence of these hormones in relation to the molecular diversity of Aeromonas and evaluating the resistance of Aeromonas to antibiotics as well as to assess anti-bacterial activity of two selected traditional medicinal plants. Methods: Wastewater, water and fish samples were collected from various sources (rivers, wastewater treatment plants, taps, and dams) for the detection of hormones and isolation of Aeromonas species. The analysis of hormones from various organs of the fish and from water samples was conducted, after extraction using enzymelinked immunosorbent assays (ELISA). Different types of hormones including Estriol, Estradiol, Ethinylesradiol and Testosterone were detected, and their concentrations determined. Aeromonas spp were isolated rom the samples using microbiological methods and Conventional PCR was used for genotyping as well as for detection of the beta-lactamase genes. Kirby-bauer method was used to determine the susceptibility profiles of Aeromonas to different antibiotics. Microdilution assay was used to determine the Anti-bacterial activity of the plant (Annoniceae and Zornia milneana) extracts against Aeromonas species. Results: A total of 144 samples were collected from 23 different locations in two countries: South Africa and Zambia. These included wastewater and treated wastewater, River water, fish and tap water. 17α-ethinylestradiol (EE2) was detected in most of the samples (92.7%) with concentrations varying from 0.59 ng/ml to 65 ng/ml. The hormones were also detected from drinking water, with testosterone detected at high concentrations of up to 140 ng/ml in tap water. Most sewage treatment plants were not able to remove the EE2 from the wastewater as the concentration of this hormone in the final effluent was almost always higher than that in the influent. These homones were also detected in drinking water at high concentrations of up to 53.49 ng/ml in the tap water for EE2 and 1777 ng/ml for E2. The overall detection of Aeromonas species in the samples was 84.5%. A. caviae was the most prevalent species accounting for 73.6%, followed by A. veronii with 64.6%. The bacteria were completely resistant to cefuroxime accounting for 100% resistance. Aeromonas isolates also showed high resistance to trimethroprim (88.7% for A. hydrophila), cefazolin (highest 97.8% for A. cavie), and ceftazidime (83.9% for A. sobria). TEM was the most prevalent beta-lactamase gene with detection rate of 87%. All isolates lacked the presence of the CTX-M3 gene. Also, wastewater had the highest prevalence of A. veronni and A. caviae accounting for 87.5% and 82.5% respectively. Multiple antibiotic resistance was also observed with the Aeromonas isolates being resistant to up to 11 antibiotics. High prevalence of 77.1% of Aeromonas hydrophila was observed in the presence of ethinylestradiol (EE2). Aeromonas veronii and Aeromonas caviae were the most predominant species in the presence of total estriol, A. veronii had a prevalence of 57.1% and A. caviae had a prevalence of 52.8%. Aeromonas hydrophila and Aeromonas caviae had the lower prevalence in the presence of hormones with the percentages of 26.1% and 27.8% respectively. The methanol extracts of both Zornia milneana and Annona species showed good activity against the Aeromonas spp with the lowest MIC of 0.078 mg/ml. Ethyl acetate extracts were the least effective. Conclusion: This study has shown high occurrence of steroid hormones in all types of environmental samples tested. These included tap water, river water, wastewater and fish both in Zambia and South Africa. Therefore, steroid hormones constitute and important health problem in the Southern African Sub-Region. The incapacity of the wastewater treatment plants to remove EE2 is an important problem that needs to be tackled immediately. The prevalence of Aeromonas species is very high in our environmental water as well as in drinking water, with the highest prevalence observed in fish and wastewater. It was also revealed that there is relationship between steroid hormones and Aeromonas species, with the hormones supporting the growth of Aeromonas species. The presence of beta-lactamase genes which causes Aeromonas to be resistant to antibiotics was also noted. Methanol extracts of Zornia milneana and Annona spp were the most effective against Aeromonas spp and could serve as primary sources for the isolation of lead compounds. / NRF

L'ingénierie enzymatique est un outil très avantageux dans l'industrie biotechnologique. Elle permet d'adapter les enzymes à une activité ou à une condition de réaction spécifique. En outre, elle peut permettre de déchiffrer les éléments clés qui ont facilité leur modification. Bien que l'ingénierie enzymatique soit largement pratiquée, elle comporte encore plusieurs goulets d'étranglement. Certains de ces goulets d'étranglement sont techniques, comme le développement de méthodologies pour la création de banques de mutations ciblées ou la réalisation de criblages à haut débit, et d'autres sont conceptuels, comme le déchiffrage des caractéristiques clés pertinentes d'une protéine cible pour la réussite d'un projet d'ingénierie. Parmi ces défis, l'épistasie intra-génique, ou la non-additivité des effets phénotypiques des mutations, est une caractéristique qui entrave grandement la prévisibilité. L'amélioration de l'ingénierie enzymatique nécessite une approche multidisciplinaire qui inclut une meilleure compréhension des relations structure-fonction-évolution. Cette thèse vise à contribuer à l'avancement de l'ingénierie enzymatique en étudiant deux systèmes modèles. Premièrement, des variantes dynamiques de la ß-lactamase TEM-1 ont été choisies pour étudier le lien entre la dynamique des protéines et l'évolution. La ß-lactamase TEM-1 a été largement caractérisée dans la littérature, ce qui s'est traduit par des connaissances approfondies sur son mécanisme de réaction, ses caractéristiques structurelles et son évolution. Les variantes de la ß-lactamase TEM-1 utilisées comme système modèle dans cette thèse ont été largement caractérisées, montrant une dynamique accrue à l'échelle temporelle pertinente pour la catalyse (µs à ms) mais maintenant la reconnaissance du substrat. Dans cette thèse, l'évolution in vitro de ces variantes dynamiques a été réalisée par des cycles itératifs de mutagenèse et de sélection aléatoires pour permettre une exploration impartiale du paysage de ‘fitness’. Nous démontrons que la présence de ces mouvements particuliers au début de l'évolution a permis d'accéder à des voies de mutations connues. De plus, des interactions épistatiques connues ont été introduites dans les variantes dynamiques. Leur caractérisation in silico et cinétique a révélé que les mouvements supplémentaires sur l'échelle de temps de la catalyse ont permis d'accéder à des conformations conduisant à une fonction améliorée, comme dans le TEM-1 natif. Dans l'ensemble, nous démontrons que l'évolution de la b-lactamase TEM-1 vers une nouvelle fonction est compatible avec divers mouvements à l'échelle de temps µs à ms. Il reste à savoir si cela peut se traduire par d'autres enzymes ayant un potentiel biotechnologique. Deuxièmement, la lipase Cal-A, pertinente sur le plan industriel, a été choisie pour identifier les caractéristiques qui pourraient faciliter son ingénierie. La lipase Cal-A présente des caractéristiques telles que la polyvalence du substrat et une grande stabilité thermique et réactivité qui la rendent attrayante pour la modification des triglycérides ou la synthèse de molécules pertinentes dans les industries alimentaire et pharmaceutique. Contrairement à TEM-1, la plupart des études d'évolution in vitro de la lipase Cal-A ont été réalisées dans un but industriel, avec une exploration limitée de l'espace de mutation. Par conséquent, les caractéristiques qui définissent la fonction de la lipase Cal-A restent insaisissables. Dans cette thèse, nous faisons état de la mutagenèse ciblée de la lipase Cal-A, confirmant l'existence d'une région clé pour la reconnaissance du substrat. Cela a été fait en combinant une nouvelle méthodologie de création de bibliothèque basée sur l'assemblage Golden-gate avec une visualisation structurelle basée sur des scripts pour identifier et cartographier les mutations sélectionnées dans la structure 3D. La caractérisation et la déconvolution de deux des plus aptes ont révélé l'existence d'une épistasie dans l'évolution de la lipase Cal-A vers une nouvelle fonction. Dans l'ensemble, nous démontrons que l’identification d'une variété de propriétés suite à la mutagenèse ciblée peut grandement améliorer la connaissance d'une enzyme. Cette information peut être appliquée pour améliorer l'efficacité de l'ingénierie dirigée. / Enzyme engineering is a tool with great utility in the biotechnological industry. It allows to tailor enzymes to a specific activity or reaction condition. In addition, it can allow to decipher key elements that facilitated their modification. While enzyme engineering is extensively practised, it still entails several bottlenecks. Some of these bottlenecks are technical such as the development of methodologies for creating targeted mutational libraries or performing high-throughput screening and some are conceptual such as deciphering the key relevant features in a target protein for a successful engineering project. Among these challenges, intragenic epistasis, or the non-additivity of the phenotypic effects of mutations, is a feature that greatly hinders predictability. Improving enzyme engineering needs a multidisciplinary approach that includes gaining a better understanding of structure-function-evolution relations. This thesis seeks to contribute in the advancement of enzyme engineering by investigating two model systems. First, dynamic variants of TEM-1 ß-lactamase were chosen to investigate the link between protein dynamics and evolution. TEM-1 ß-lactamase has been extensively characterized in the literature, which has translated into extensive knowledge on its reaction mechanism, structural features and evolution. The variants of TEM-1 ß-lactamase used as model system in this thesis had been extensively characterized, showing increased dynamics at the timescale relevant to catalysis (µs to ms) but maintaining substrate recognition. In this thesis, in vitro evolution of these dynamic variants was done by iterative rounds of random mutagenesis and selection to allow an unbiased exploration of the fitness landscape. We demonstrate that the presence of these particular motions at the outset of evolution allowed access to known mutational pathways. In addition, known epistatic interactions were introduced in the dynamic variants. Their in silico and kinetic characterization revealed that the additional motions on the timescale of catalysis allowed access to conformations leading to enhanced function, as in native TEM-1. Overall, we demonstrate that the evolution of TEM-1 b-lactamase toward new function is compatible with diverse motions at the µs to ms timescale. Whether this can be translated to other enzymes with biotechnological potential remains to be explored. Secondly, the industrially relevant Cal-A lipase was chosen to identify features that could facilitate its engineering. Cal-A lipase presents characteristics such as substrate versatility and high thermal stability and reactivity that make it attractive for modification of triglycerides or synthesis of relevant molecules in the food and pharmaceutical industries. Contrary to TEM-1, most in vitro evolution studies of Cal-A lipase have been done towards an industrially-specified goal, with limited exploration of mutational space. As a result, features that define function in Cal-A lipase remain elusive. In this thesis, we report on focused mutagenesis of Cal-A lipase, confirming the existence of a key region for substrate recognition. This was done by combining a novel library creation methodology based on Golden-gate assembly with script-based structural visualization to identify and map the selected mutations into the 3D structure. The characterization and deconvolution of two of the fittest revealed the existence of epistasis in the evolution of Cal-A lipase towards new function. Overall, we demonstrate that mapping a variety of properties following mutagenesis targeted to specific regions can greatly improve knowledge of an enzyme that can be applied to improve the efficiency of directed engineering.

The open nature of Cell-Free Protein Synthesis (CFPS) systems has enabled flexible design, easy manipulation, and novel applications of protein engineering in therapeutic production, biocatalysis, and biosensors. This dissertation reports on three advances in the application of CFPS systems for 1) improving biocatalysis performance in industrial applications by site-specific covalent enzyme immobilization, 2) expressing and optimizing a difficult to express a mammalian protein in bacterial-based CFPS systems and its application for cost-effective, on-demand biosensors compatible with human body fluids, and 3) streamlining the procedure of an E. coli extract with built-in compatibility with human body fluid biosensors. Site-specific covalent immobilization stabilizes enzymes and facilitates recovery and reuse of enzymes which improves the net profit margin of industrial enzymes. Yet, the suitability of a given site on the enzyme for immobilization remains a trial-and-error procedure. This dissertation reports the reliability of several design heuristics and a coarse-grain molecular simulation in predicting the optimum sites for covalent immobilization of a target enzyme, TEM-1 ?-lactamase. This work demonstrates that the design heuristics can successfully identify a subset of favorable locations for experimental validation. This approach highlights the advantages of combining coarse-grain simulation and high-throughput experimentation using CFPS to efficiently identify optimal enzyme immobilization sites. Additionally, this dissertation reports high-yield soluble expression of a difficult-to-express protein (murine RNase Inhibitor or m-RI) in E. coli-lysate-based CFPS. Several factors including reaction temperature, reaction time, redox potential, and presence of folding chaperones in CFPS reactions were altered to find suitable conditions for m-RI expression. m-RI with the highest activity and stability was used to develop a lyophilized CFPS biosensor in human body fluids which reduced the cost of biosensor test by ~90%. Moreover, an E. coli extract with RNase inhibition activity was developed and tested which further streamlines the production of CFPS biosensors compatible with human body fluids.