Biocatalysis and enzyme technology represent significant research topics of contemporary biotechnology. The immobilisation of these catalysts on or in static supports serves the purpose of transforming the catalyst into a particle that can be handled through effortless mechanical operations, while the entrapment within a membrane or capsule leads to the restraint of the enzyme to a distinct space. This confinement leads to a catalyst with a superior stability, and cell durability under reaction conditions. Epoxide hydrolase is a widely available co-factor independent enzyme, which is known to have remarkable chemio-, regio- and stereoselectivity for a wide range of substrates. Recently it was found that certain yeasts, including Rhodosporidium toruloides, contain this enzyme and are able to enantioselectively catalyse certain hydrolysis reactions. The objective of this project was four-sided: a) to immobilise Rhodospridium toruloides in an optimised immobilisation matrix (calcium alginate beads), for the kinetic resolution of 1.2- epoxyoctane in order to obtain an optically pure epoxide and its corresponding vicinal diol, b) to determine the effect of immobilisation on activity as well as stability of the enzyme and gain better understanding of the parameters that influence enzyme activity in a support, c) to determine the effect of formulation parameters on some of the bead characteristics and, d) to gain some insight in the distribution of epoxide and diol in the water and bead phases and the formulation parameters that have an effect thereon. Rhodospridium toruloides was immobilised in calcium alginate beads consisting of different combinations of alginate and CaCl2 concentrations. Best results were obtained with a combination of 0,5 % (m/v) alginate and 0,2 M CaC12. The immobilised cells exhibited lower initial activity. but more than 40 times the residual activity of that of the free cells after a 12-hour storage period. Both the immobilised and free cells exhibited an increase in reaction rate (V) with an increase in substrate concentration. An increase in the alginate concentration lead to the formation of smaller beads, but a decrease in enzume activity, while an increase in the CaCl2 solution concentration had no effect on bead diameter or enzyme activity. Epoxide diffused preferentially into the beads (± 96 %), and the diol into the water phase, which leads to the natural separation of the epoxide and the diol. The CaCl2 concentration affected epoxide diffusion with no effect on diol diffusion, which opens up the possibility to regulate the diffusion of epoxide into the beads. Although only a very small fraction of the epoxide inside the beads could be extracted, the alginate proved to be chirally selective for the (R)-epoxide, improving the reaction efficiency by increasing the % ee, of the epoxide extracted from the beads between 26 % and 43 %. The possibility to develop a system where the product is formed, purified and concentrated in a one-step reaction by extracting the product from the bead phase was clearly demonstrated. / Thesis (M.Sc. (Pharm.) (Pharmaceutical Chemistry))--Potchefstroom University for Christian Higher Education, 2003.

Orientadores: Anete Pereira de Souza, Valéria Maia Merzel / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-24T03:05:12Z (GMT). No. of bitstreams: 1 Beloti_LilianLuzia_D.pdf: 11283696 bytes, checksum: dfedaea17b072c7d899caa1ccd4e9032 (MD5) Previous issue date: 2013 / Resumo: O resumo poderá ser visualizado no texto completo da tese digital / Abstract: The complete abstract is available with the full electronic document / Doutorado / Genetica de Microorganismos / Doutora em Genética e Biologia Molecular

Polyether compounds are a subgroup of natural products with regular occurrence of multiple C-O-C motifs. The biosynthetic origin of polycylic polethers has been studied and the majority of them are derived from polyketide or terpene pathways. Normally, the polycyclic polyethers can be divided into two groups based on their structural features: the first group features multiple rings that are interconnected by carbon-carbon single bond, which are produced by a biosynthetic cascade of exo epoxide-opening reactions; the other group has multiple fused cyclic ethers and are formed by an cascade of endo epoxide-opening reactions. Karenia brevis (K. brevis) is known as principle harmful bloom (HAB) organism of the Gulf of Mexico which can cause red tides. Brevetoxins (PbTx) are a suit of cyclic polyether ladder compounds produced by K. brevis. Brevetoxins are neurotoxins that can bind to voltage-gated sodium channels in nerve and muscle cells, resulting in disruption of normal neurological processes causing the human illness which is clinically described as neurotoxic shellfish poisoning (NSP). Inspired by Cane-Celmer-Wesley’s proposal regarding monensin biosynthesis, Nakanishi and Shimizu proposed a biosynthetic pathway for brevetoxin which suggests that PKS-mediated synthesis of the polyene is followed by epoxidation to afford a polyepoxide which then undergoes an epoxide-opening cascade, catalyzed by an epoxide hydrolase (EH). To find evidence to support the hypothesis that an epoxide hydrolase from polyether ladder producing dinoflagellates will catalyze the construction of the polyether ladder framework from polyepoxide substrates, and to study the role of epoxide hydrolase in the biosynthesis of polyether ladder compounds, it is necessary to identify and produce one or more epoxide hydrolase from dinoflagellates. The methods to detect epoxide hydrolase activity in K. brevis and different techniques to obtain epoxide hydrolases from K. brevis are discussed. A microsomal EH identified from a K. brevis EST library was cloned and expressed. The characterization of this EH, including substrate selectivity and enantioselectivity as well as its potential to catalyze the critical ento-tet cyclization epoxy alcohol, is discussed.

8 Abstract Epoxide hydrolases (EHs) demonstrating high degree of enantioselectivity or enantioconvergence are useful biocatalysts for the production of optically active epoxides and vicinal diols, which can serve as chiral building blocks for syntheses of biologically active drugs. EHs can play an important role also in degradations of xenobiotics. Genes encoding EHs Kau2 and Kau8 were expressed in E. coli host strains TOP10 and RE3. Enantioselectivities and regioselectivities of Kau2 and Kau8 in supernatants of desintegrated cells were determined for four substrates: tert-butylglycidyl ether, para-chlorostyrene oxide, para-nitrostyrene oxide, α-methylstyrene oxide. The highest values of enantioselectivity and regioselectivity were achieved with Kau2 and para-nitrostyrene oxide as a substrate. The Kau2 was chosen for further experiments on the basis of these results. Kau2 was overexpressed in the recombinant strain RE3(pSEKau2). We performed two batch cultures and one fed-batch culture in stirred bioreactor. The highest volumetric activity of 4500 U/l was obtained in the case of fed-batch culture. Two phase system consisting of polyethylenglycole 6000 and sodium citrate (pH 7.7) was used for Kau2 purification from the supernatant of desintegrated cells. Purification factor 2.6 +/- 0.3 was achieved and...

Epoxide hydrolase I from Solanum tuberosum (StEH1) and isolated variants thereof has been studied for mapping structure-function relationships with the ultimate goal of being able to in silico predict modifications needed for a certain activity or selectivity. To solve this, directed evoultion using CASTing and an ISM approach was applied to improve selectivity towards either of the enantiomeric product diols from (2,3-epoxypropyl)benzene (1). A set of variants showing a range of activites and selectivities was isolated and characterized to show that both enantio- and regioselectivity was changed thus the enrichment in product purity was not solely due to kinetic resolution but also enantioconvergence. Chosen library residues do also influence selectivity and activity for other structurally similar epoxides styrene oxide (2), trans-2-methyl styrene oxide (3) and trans-stilbene oxide (5), despite these not being selected for.    The isolated hits were used to study varying selectivity and activity with different epoxides. The complex kinetic behaviour observed was combined with X-ray crystallization and QM/MM studies, powerful tools in trying to explain structure-function relationships. Crystal structures were solved for all isolated variants adding accuracy to the EVB calculations and the theoretical models did successfully reproduce experimental data for activities and selectivities in most cases for 2 and 5.  Major findings from calculations were that regioselectivity is not always determined in the alkylation step and for smaller and more flexible epoxides additional binding modes are possible, complicating predictions and the reaction scheme further. Involved residues for the catalytic mechanism were confirmed and a highly conserved histidine was found to have major influence on activity thus suggesting an expansion of the catalytic triad to also include H104. Docking of 1 into the active site of the solved crystal structures was performed in an attempt to rationalize regioselectivity from binding. This was indeed successful and an additional binding mode was identified, involving F33 and F189, both residues targeted for engineering. For biocatalytic purpose the enzyme were was successfully immobilized on alumina oxide membranes to function in a two-step biocatalytic reaction with immobilized alcoholdehydrogenase A from Rhodococcus ruber, producing 2-hydroxyacetophenone from racemic 2.

Recent years have seen tremendous developments in methods for computational modeling of (bio-) molecular systems. Ever larger reactive systems are being studied with high accuracy approaches, and high-level QM/MM calculations are being routinely performed. However, applying high-accuracy methods to large biological systems is computationally expensive and becomes problematic when conformational sampling is needed. To address this challenge, classical force field based approaches such as free energy perturbation (FEP) and empirical valence bond calculations (EVB) have been employed in this work. Specifically: Force-field independent metal parameters have been developed for a range of alkaline earth and transition metal ions, which successfully reproduce experimental solvation free energies, metal-oxygen distances, and coordination numbers. These are valuable for the computational study of biological systems. Experimental studies have shown that the epoxide hydrolase from Solanum tuberosum (StEH1) is not only an enantioselective enzyme, but for smaller substrates, displays enantioconvergent behavior. For StEH1, two detailed studies, involving combined experimental and computational efforts have been performed: We first used trans-stilbene oxide to establish the basic reaction mechanism of this enzyme. Importantly, a highly conserved and earlier ignored histidine was identified to be important for catalysis. Following from this, EVB and experiment have been used to investigate the enantioconvergence of the StEH1-catalyzed hydrolysis of styrene oxide. This combined approach involved wildtype StEH1 and an engineered enzyme variant, and established a molecular understanding of enantioconvergent behavior of StEH1. A novel framework was developed for the Computer-Aided Directed Evolution of Enzymes (CADEE), in order to be able to quickly prepare, simulate, and analyze hundreds of enzyme variants. CADEE’s easy applicability is demonstrated in the form of an educational example. In conclusion, classical approaches are a computationally economical means to achieve extensive conformational sampling. Using the EVB approach has enabled me to obtain a molecular understanding of complex enzymatic systems. I have also increased the reach of the EVB approach, through the implementation of CADEE, which enables efficient and highly parallel in silico testing of hundreds-to-thousands of individual enzyme variants.

Epóxido hidrolases (EHs) são enzimas que catalisam a hidrólise de epóxidos a seus correspondentes dióis, apresentam potencial aplicação biotecnológica (separação de enantiômeros na produção de fármacos), estão envolvidas no metabolismo de ácidos graxos poliinsaturados e inibidores de EHs estão sendo estudados para possível utilização no tratamento de doenças. Uma enzima epóxido hidrolase (TrEH) do fungo filamentoso Trichoderma reesei QM9414 foi clonada, expressa, purificada e caracterizada funcionalmente e estruturalmente. A atividade de TrEH foi determinada com o substrato óxido de estireno (racêmico), demonstrando maior atividade nas temperaturas de 23 a 50 °C, no pH 7,2 a 37 °C, e as constantes cataliticas Km= 4,6 mM e kcat= 336 s-1. A enzima recombinante mostrou ser enantiosseletiva, pois hidrolisa preferencialmente (S)-(-)-óxido de estireno, (R)-(-)- epicloridrina e (S)-(-)-1,2-epoxibutano. Moléculas inibidoras da atividade de TrEH foram identificadas e algumas delas inibem até 60% o crescimento de T. reesei. A estrutura terciária de TrEH (1,7 Å) foi determinada por cristalografia, apresenta dobramento α/β-hidrolase e não tem alta homologia com nenhuma outra estrutura de EH. TrEH é uma nova enzima epóxido hidrolase solúvel cujas propriedades mostram seu potencial de utilização em aplicações biotecnológicas. / Epoxide hydrolases (EHs) are enzymes that catalyze the hydrolysis of epoxides to their corresponding diols, present a potential biotechnological application (separation of enantiomers for the production of drugs), they are involved in the metabolism of polyunsaturated fatty acids and EH inhibitors are being studied for possible use in the treatment of diseases. An epoxide hydrolase enzyme(TrEH) from the filamentous fungus Trichoderma reesei QM9414 was cloned, expressed, purified and functionally and structurally characterized. The activity of TrEH was determined with the substrate styrene oxide (racemic), showing higher activity at temperatures of 23 to 50 °C, at pH 7.2 at 37 °C, and the catalytic constants Km= 4.6 mM and kcat= 336 s-1. The recombinant enzyme has been shown to be enantioselective, because it preferably hydrolyzes (S)-(-)-styrene oxide, (R)-(-)-epichlorohydrin and (S)-(-)-1,2- epoxybutane. TrEH inhibitors have been identified and some of them inhibit up to 60% growth of T. reesei . The tertiary structure of TrEH (1.7 Å) was determined by crystallography, showing α/ β-hydrolase folding and low homology with any other EH structure. TrEH is a new soluble epoxide hydrolase enzyme whose properties show its potential for use in biotechnological applications.

The kinetic mechanism of an α/β hydrolase fold epoxide hydrolase from potato, StEH1, has been studied with the aims of explaining the underlying causes for enantio- and regioselectivity, both being important for product purity. Further effort has been laid upon understanding the causes of a hysteretic behavior discovered in the measurements leading to Paper I. The enantioselectivity was investigated with substrates differing only in substituent size at one carbon of the oxirane ring structure. In catalysis with trans-stilbene oxide and styrene oxide, enantioselectivity is the result of differences in alkylation rates. In pre-steady state measurement with trans-2-methylstyrene oxide (2-MeSO), a rate-limiting step involving slow transitions, referred to as hysteresis, was discovered. With this substrate enantioselectivity is proposed to be a consequence of the catalytic rate of (1R,2R)-enantiomer being more influenced by the hysteretic behavior than was the rate of the other enantiomer. In steady-state measurements with (1R,2R)-2-MeSO, at different temperatures and pH, hysteretic cooperativity was displayed. It can be concluded that this behavior is dependent on the relationship between kcat and the rate of transition between two Michaelis complexes. From the differences in pH dependence of kcat/KM in formation of the two diols resulting from low regioselectivity in catalysis of (1R,2R)-2-MeSO, it is suggested that hysteresis is a result of the substrates placed in different conformational modes within the active site cavity. Regioselectivity is proposed to be the result of specific interactions between the catalytically important Tyr and the substrate, with a link between KM-values and degree of regioselectivity. Furthermore, the hysteretic kinetic model proposed can explain hysteresis, cooperativity and regioselectivity resulting from StEH1 catalyzed hydrolysis of (1R,2R)-2-MeSO.

The work leading to this thesis has provided additional information and novel knowledge concerning structure-function relationship in the potato epoxide hydrolase. Epoxide hydrolases are enzymes catalyzing the hydrolysis of epoxides to yield the corresponding vicinal diols. The reaction mechanism proceeds via a nucleophilic attack resulting in a covalent alkylenzyme intermediate, which in turn is attacked by a base-activated water molecule, followed by product release. Epoxides and diols are precursors in the production of chiral compounds and the use of epoxide hydrolases as biocatalysts is growing. The promising biocatalyst StEH1, a plant epoxide hydrolase from potato, has been investigated in this thesis. In paper I the active site residue Glu35, was established to be important for the formation of the alkylenzyme intermediate, activating the nucleophile for attack by facilitated proton release through a hydrogen bond network. Glu35 is also important during the hydrolytic half reaction by optimally orienting the hydrolytic water molecule, aiding in the important dual function of the histidine base. Glu35 makes it possible for the histidine to work as both an acid and a base. In paper II a putative proton wire composed of five water molecules lining a protein tunnel was proposed to facilitate effective proton transfer from the exterior to the active site, aiding in protonation of the alkylenzyme intermediate. The protein tunnel is also proposed to stabilize plant epoxide hydrolases via hydrogen bonds between water molecules and protein. Enzyme variants with modified enantiospecificity for the substrate (2,3-epoxypropyl)benzene have been constructed by in vitro evolution using the CASTing approach. Residues lining the active site pocket were targeted for mutagenesis. From the second generation libraries a quadruple enzyme variant, W106L/L109Y/V141K/I155V, displayed a radical shift in enantioselectivity. The wild-type enzyme favored the S-enantiomer with a ratio of 2:1, whereas the quadruple variant showed a 15:1 preference for the R-enantiomer.

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