As biocélulas a combustível enzimáticas (BFCs) são dispositivos eletroquímicos que convertem energia química em energia elétrica, utilizando enzimas como biocatalisadores. Quando miniaturizada, uma BFC pode ser implantada em animais vertebrados e invertebrados, vislumbrando-se sua utilização na produção de energia elétrica para alimentar microdispositivos biomédicos e microssensores em pequenos insetos. No entanto, ainda é um desafio obter BFCs implantáveis e miniaturizadas, com uma potência suficiente (dezenas de microwatts) para alimentar microcircuitos eletrônicos de maneira estável e em longo prazo. Diante do exposto, esta tese de doutorado apresenta um estudo das propriedades eletroquímicas de eletrodos enzimáticos, visando a aplicação em BFCs de glicose/O2 miniaturizadas e implantáveis. Para isso, utilizaram-se fibras flexíveis de carbono (FCFs) modificadas com as enzimas bilirrubina oxidase (BOx) no cátodo e glicose desidrogenase (GDh) NAD-dependente no ânodo, a fim de se obter a redução de O2 e a oxidação de glicose, respectivamente. Os resultados obtidos mostram que FCFs previamente submetidas a um tratamento químico de oxidação com permanganato de potássio e com posterior eletrodepolimerização do mediador vermelho neutro produzem bioânodos estáveis e robustos. Estes eletrodos, combinados com biocátodos compostos por FCFs na ausência de mediadores redox, foram utilizados em BFCs miniaturizadas, que foram implantadas em formigas da espécie Atta sexdens rubrupilosa. A potência máxima da BFC operando in vivo foi 13,5 ± 3,8 µW cm-2 em 190 ± 58,9 mV, com corrente máxima de 143 ± 40,2 µA cm-2 e a voltagem de circuito aberto de 260 ± 99,6 mV. Acredita-se que estes valores ainda possam ser otimizados e este trabalho contribui para mostrar que a flexibilidade das FFC, a presença de um mediador de elétrons polimérico no ânodo, o uso do tratamento químico de oxidação com permanganato de potássio das fibras e a miniaturização dos eletrodos são elementos importantes, e que podem ser considerados no desenvolvimento de biocélulas a combustível implantáveis. / Enzymatic biofuel cells (BFCs) are electrochemical devices that convert chemical energy into electrical energy using enzymes as biocatalysts. When miniaturized, BFCs can be implanted in vertebrate and invertebrate animals and, their use to produce electrical energy to feed biomedical microdevices and micro-sensors in small insects can be observed. However, it is still challenging to obtain implantable and miniaturized BFCs, with sufficient power (tens of microwatts) to power electronic microcircuits in a stable and long-term manner. In view of the above, this PhD thesis presents a study of the electrochemical properties of enzymatic electrodes, aiming to use them in miniaturized and implantable glucose/O2 BFCs. In order to obtain a reduction in O2 and oxidation of glucose, flexible carbon fibers (FCFs) modified with bilirubin oxidase (BOx) enzymes in the cathode and glucose dehydrogenase (GDh) at the anode, respectively, were used. The results show that FCFs previously submitted to a chemical treatment of oxidation with potassium permanganate and, subsequently, electropolymerization of the neutral red mediator produce stable and robust bioanodes. These electrodes, combined with biocathodes consisting of FCFs in the absence of redox mediators, were used in miniaturized BFCs, which were implanted in Atta sexdens rubrupilosa ant species. The BFC maximum power source, operating in vivo, was 13.5 ± 3.8 μW cm-2 at 190 ± 58.9 mV, with a maximum current of 143 ± 40.2 μA cm-2 and the open circuit voltage was 260 ± 99.6 mV. Although these values can be optimized, this research shows that the flexibility of the FCF, the presence of a polymer electron mediator on the anode, using the chemical treatment of oxidation with potassium permanganate of the fibers and electrode miniaturization are important elements, which can be considered in the development of implantable biofuels.

<p>Polysaccharides are found in all forms of life and serve diverse purposes. They are enzymatically synthesised from activated monosaccharide precursors, nucleotide sugars. One such nucleotide sugar is UDP-glucuronic acid, which is formed from UDP-glucose by the UDP-glucose dehydrogenase (UGDH) enzyme. UGDH has been proposed to have a regulatory role in the biosynthesis of polysaccharides. The aim of the studies presented in this thesis was to investigate the role of UGDH in the polysaccharide biosynthesis in three different systems: human cell culture, bacterial cultures<i> </i>and growing<i> </i>plants<i>. </i>The effects of UGDH-overexpression on polysaccharide biosyntheses and, when achievable, on UDP-sugar levels, were investigated.</p><p>A mammalian UGDH was cloned from a kidney cDNA library. Transient expression of the cloned enzyme in mammalian cells led to an increased UGDH-activity. Northern blotting analyses revealed a single transcript of 2.6 kb in adult mouse tissues whereas human tissues expressed a predominant transcript of 3.2 kb and a minor transcript of 2.6 kb.</p><p>Overexpression of the bovine UGDH in mammalian cells induced increased synthesis of the glycosaminoglycans; heparan sulphate, chondroitin sulphate and hyaluronan, without changing their relative proportions. The effects on glycosaminoglycan synthesis caused by an increased demand of UDP-glucuronic acid were studied by overexpression of hyaluronan synthase (Has3), which requires UDP-glucuronic acid as substrate. Overexpression of Has3 and coexpression of Has3 and UGDH resulted in highly augmented production of hyaluronan without noticeably affecting heparan sulfate and chondroitin sulfate synthesis.</p><p>Expression of the bacterial UGDH in <i>E. coli</i> resulted in increased formation of UDP-glucuronic acid, but, unexpectedly, also to synthesis of fewer K5 polysaccharide chains. </p><p>Overexpression of UGD1, one of four <i>A. thaliana</i> UGDH genes, in <i>A. thaliana,</i> resulted in dwarfism. Analysis of the cell wall polysaccharides showed alteration in saccharide composition. Paradoxically, the UDP-sugars derived from UDP-glucuronic acid decreased in amount.</p>

<p>The glycosaminoglycan hyaluronan is an essential component of the extracellular matrix in all higher organisms, affecting cellular processes such as migration, proliferation and differentiation. Hyaluronan is synthesized by a plasma membrane bound hyaluronan synthase (HAS) which exists in three genetic isoforms. This thesis focuses on the understanding of the hyaluronan biosynthesis by studies on the expression and regulation of the HAS proteins.</p><p>In order to characterize the structural and functional properties of the HAS isoforms we developed a method to solubilize HAS protein(s) while retaining enzymatic activity. The partially purified HAS protein is, most likely, not asscociated covalently with other components. Cells transfected with cDNAs for HAS1, HAS2 and HAS3 were studied and all three HAS isozymes were able to synthesize high molecular weight hyaluronan chains in intact cells. The regulation of the hyaluronan chain length involves cell specific elements as well as external stimulatory factors. HAS3 transfected cells with high hyaluronan production exhibit reduced migration capacity and reduced amounts of a cell surface hyaluronan receptor molecule (CD44) compared to wild-type cells.</p><p>The three HAS isoforms were studied and shown to be differentially expressed and regulated in response to external stimuli. Platelet derived growth factor (PDGF-BB) and transforming growth factor (TGF-<i>β</i>1) are important regulators of HAS at both the transcriptional and translational level. The HAS2 isoform is the isoform most susceptible to external regulation.</p><p>The role of the UDP-glucose dehydrogenase in mammalian glycosaminoglycan biosynthesis was assessed. The enzyme is essential for hyaluronan, heparan sulfate and chondroitin sulfate biosynthesis, but does not exert a rate-limiting effect.</p>

The glycosaminoglycan hyaluronan is an essential component of the extracellular matrix in all higher organisms, affecting cellular processes such as migration, proliferation and differentiation. Hyaluronan is synthesized by a plasma membrane bound hyaluronan synthase (HAS) which exists in three genetic isoforms. This thesis focuses on the understanding of the hyaluronan biosynthesis by studies on the expression and regulation of the HAS proteins. In order to characterize the structural and functional properties of the HAS isoforms we developed a method to solubilize HAS protein(s) while retaining enzymatic activity. The partially purified HAS protein is, most likely, not asscociated covalently with other components. Cells transfected with cDNAs for HAS1, HAS2 and HAS3 were studied and all three HAS isozymes were able to synthesize high molecular weight hyaluronan chains in intact cells. The regulation of the hyaluronan chain length involves cell specific elements as well as external stimulatory factors. HAS3 transfected cells with high hyaluronan production exhibit reduced migration capacity and reduced amounts of a cell surface hyaluronan receptor molecule (CD44) compared to wild-type cells. The three HAS isoforms were studied and shown to be differentially expressed and regulated in response to external stimuli. Platelet derived growth factor (PDGF-BB) and transforming growth factor (TGF-β1) are important regulators of HAS at both the transcriptional and translational level. The HAS2 isoform is the isoform most susceptible to external regulation. The role of the UDP-glucose dehydrogenase in mammalian glycosaminoglycan biosynthesis was assessed. The enzyme is essential for hyaluronan, heparan sulfate and chondroitin sulfate biosynthesis, but does not exert a rate-limiting effect.

Polysaccharides are found in all forms of life and serve diverse purposes. They are enzymatically synthesised from activated monosaccharide precursors, nucleotide sugars. One such nucleotide sugar is UDP-glucuronic acid, which is formed from UDP-glucose by the UDP-glucose dehydrogenase (UGDH) enzyme. UGDH has been proposed to have a regulatory role in the biosynthesis of polysaccharides. The aim of the studies presented in this thesis was to investigate the role of UGDH in the polysaccharide biosynthesis in three different systems: human cell culture, bacterial cultures and growing plants. The effects of UGDH-overexpression on polysaccharide biosyntheses and, when achievable, on UDP-sugar levels, were investigated. A mammalian UGDH was cloned from a kidney cDNA library. Transient expression of the cloned enzyme in mammalian cells led to an increased UGDH-activity. Northern blotting analyses revealed a single transcript of 2.6 kb in adult mouse tissues whereas human tissues expressed a predominant transcript of 3.2 kb and a minor transcript of 2.6 kb. Overexpression of the bovine UGDH in mammalian cells induced increased synthesis of the glycosaminoglycans; heparan sulphate, chondroitin sulphate and hyaluronan, without changing their relative proportions. The effects on glycosaminoglycan synthesis caused by an increased demand of UDP-glucuronic acid were studied by overexpression of hyaluronan synthase (Has3), which requires UDP-glucuronic acid as substrate. Overexpression of Has3 and coexpression of Has3 and UGDH resulted in highly augmented production of hyaluronan without noticeably affecting heparan sulfate and chondroitin sulfate synthesis. Expression of the bacterial UGDH in E. coli resulted in increased formation of UDP-glucuronic acid, but, unexpectedly, also to synthesis of fewer K5 polysaccharide chains. Overexpression of UGD1, one of four A. thaliana UGDH genes, in A. thaliana, resulted in dwarfism. Analysis of the cell wall polysaccharides showed alteration in saccharide composition. Paradoxically, the UDP-sugars derived from UDP-glucuronic acid decreased in amount.

As biocélulas a combustível enzimáticas (BFCs) são dispositivos eletroquímicos que convertem energia química em energia elétrica, utilizando enzimas como biocatalisadores. Quando miniaturizada, uma BFC pode ser implantada em animais vertebrados e invertebrados, vislumbrando-se sua utilização na produção de energia elétrica para alimentar microdispositivos biomédicos e microssensores em pequenos insetos. No entanto, ainda é um desafio obter BFCs implantáveis e miniaturizadas, com uma potência suficiente (dezenas de microwatts) para alimentar microcircuitos eletrônicos de maneira estável e em longo prazo. Diante do exposto, esta tese de doutorado apresenta um estudo das propriedades eletroquímicas de eletrodos enzimáticos, visando a aplicação em BFCs de glicose/O2 miniaturizadas e implantáveis. Para isso, utilizaram-se fibras flexíveis de carbono (FCFs) modificadas com as enzimas bilirrubina oxidase (BOx) no cátodo e glicose desidrogenase (GDh) NAD-dependente no ânodo, a fim de se obter a redução de O2 e a oxidação de glicose, respectivamente. Os resultados obtidos mostram que FCFs previamente submetidas a um tratamento químico de oxidação com permanganato de potássio e com posterior eletrodepolimerização do mediador vermelho neutro produzem bioânodos estáveis e robustos. Estes eletrodos, combinados com biocátodos compostos por FCFs na ausência de mediadores redox, foram utilizados em BFCs miniaturizadas, que foram implantadas em formigas da espécie Atta sexdens rubrupilosa. A potência máxima da BFC operando in vivo foi 13,5 &plusmn; 3,8 &micro;W cm-2 em 190 &plusmn; 58,9 mV, com corrente máxima de 143 &plusmn; 40,2 &micro;A cm-2 e a voltagem de circuito aberto de 260 &plusmn; 99,6 mV. Acredita-se que estes valores ainda possam ser otimizados e este trabalho contribui para mostrar que a flexibilidade das FFC, a presença de um mediador de elétrons polimérico no ânodo, o uso do tratamento químico de oxidação com permanganato de potássio das fibras e a miniaturização dos eletrodos são elementos importantes, e que podem ser considerados no desenvolvimento de biocélulas a combustível implantáveis. / Enzymatic biofuel cells (BFCs) are electrochemical devices that convert chemical energy into electrical energy using enzymes as biocatalysts. When miniaturized, BFCs can be implanted in vertebrate and invertebrate animals and, their use to produce electrical energy to feed biomedical microdevices and micro-sensors in small insects can be observed. However, it is still challenging to obtain implantable and miniaturized BFCs, with sufficient power (tens of microwatts) to power electronic microcircuits in a stable and long-term manner. In view of the above, this PhD thesis presents a study of the electrochemical properties of enzymatic electrodes, aiming to use them in miniaturized and implantable glucose/O2 BFCs. In order to obtain a reduction in O2 and oxidation of glucose, flexible carbon fibers (FCFs) modified with bilirubin oxidase (BOx) enzymes in the cathode and glucose dehydrogenase (GDh) at the anode, respectively, were used. The results show that FCFs previously submitted to a chemical treatment of oxidation with potassium permanganate and, subsequently, electropolymerization of the neutral red mediator produce stable and robust bioanodes. These electrodes, combined with biocathodes consisting of FCFs in the absence of redox mediators, were used in miniaturized BFCs, which were implanted in Atta sexdens rubrupilosa ant species. The BFC maximum power source, operating in vivo, was 13.5 &plusmn; 3.8 &mu;W cm-2 at 190 ± 58.9 mV, with a maximum current of 143 &plusmn; 40.2 &mu;A cm-2 and the open circuit voltage was 260 &plusmn; 99.6 mV. Although these values can be optimized, this research shows that the flexibility of the FCF, the presence of a polymer electron mediator on the anode, using the chemical treatment of oxidation with potassium permanganate of the fibers and electrode miniaturization are important elements, which can be considered in the development of implantable biofuels.

Several bacteria isolated from the broccoli rhizosphere were assayed to compare their ability to solubilize phosphate and release pyrroloquinoline quinone (PQQ) into the surrounding media. Subsequently, their genomes were sequenced and analyzed for PQQ biosynthesis operon structure. PQQ biosynthesis genes pqqA-F were found in all isolates. The order of PQQ biosynthesis genes and predicted amino acid sequences were compared to each other and the host’s ability to solubilize phosphate and release PQQ. In all Pseudomonas species, two putative protease genes, pqqF, and pqqG, flanked the canonical pqqA-pqqE biosynthesis operon. No mechanistic studies have confirmed the function of pqqF and pqqG. Pseudomonas putida KT2440 is a versatile model organism, representing environmental, agronomical, and industrial interests. Like the broccoli isolates, P. putida KT2440 biosynthesizes and releases PQQ into its surroundings. To better understand their functions within PQQ synthesis in P. putida KT2440, ∆pqqF, ∆pqqG, and ∆pqqF/∆pqqG strains of P. putida KT2440 were generated and the resulting phenotypes were studied.

No description available.

Thesis (PhD (Genetics. Plant Biotechnology))--University of Stellenbosch, 2006. / The young internodes of sugarcane are ideal targets for altering metabolism, through genetic manipulation, to potentially control known fungal diseases such as Smut or to increase sucrose yields in these regions that are currently being discarded. At present, no regulatory sequences that specifically drive transgene expression in young developing sugarcane tissues are available. The objective of this study was therefore to isolate and evaluate such a sequence. The promoter targeted for isolation in this study regulates the expression of UDP-glucose dehydrogenase (EC 1.1.1.22), an enzyme which catalyses the oxidation of UDP-glucose to UDP-glucuronic acid, a precursor for structural polysaccharides which are incorporated into the developing cell wall. A strong correlation between the expression of UDP-glucose dehydrogenase and a demand for structural polysaccharides in developing tissues could therefore be expected. The first part of this study addressed the general practicality of promoter isolation from sugarcane, a complex polyploid. A gene encoding UDP-glucose dehydrogenase was isolated from a sugarcane genomic library. The gene contains an open reading frame (ORF) of 1443 bp, encoding 480 amino acids and one large intron (973 bp), located in the 5’-UTR. The derived amino acid sequence showed 88 – 98% identity with UDP-glucose dehydrogenase from other plant species, and contained highly conserved amino acid motifs required for cofactor binding and catalytic activity. Southern blot analysis indicates a low copy number for UDP-glucose dehydrogenase in sugarcane. The possible expression of multiple gene copies or alleles of this gene was investigated through comparison of sequences amplified from cDNA prepared from different tissues. Although five Single Nucleotide Polymorphisms (SNP) and one small-scale insertion/deletion (INDEL) were identified in the aligned sequences, hundred percent identity of the derived amino acid sequences suggested the expression of different alleles of the same gene rather than expression of multiple copies. The finding that multiple alleles are expressed to provide the required level of a specific enzyme, rather than the increased expression of one dominant allele, is encouraging for sugarcane gene and promoter isolation. In the second part of the study the suitability of UDP-glucose dehydrogenase as a target for the isolation of a developmentally regulated promoter was investigated. The contribution of UDP glucose dehydrogenase to pentan synthesis, as well as the expression pattern and subcellular localisation of the enzyme in mature sugarcane plants was studied at the tissue and cellular level. Radiolabelling with positionally labelled glucose was used to investigate the relative contributions of glycolysis, the oxidative pentose phosphate pathway and pentan synthesis to glucose catabolism. Significantly (P=0.05) more radiolabel was released as CO2 from [6-14C]- glucose than [1-14C]-glucose in younger internodes 3, 4 and 5, demonstrating a significant contribution of UDP-glucose dehydrogenase to glucose oxidation in the younger internodes. In addition, there was significantly (P=0.05) more radiolabel in the cell wall (fiber) component when the tissue was labelled with [1-14C]-glucose rather than [6-14C]-glucose. This also demonstrates a selective decarboxylation of glucose in position 6 prior to incorporation into the cell wall and is consistent with a major role for UDP-glucose dehydrogenase in cell wall synthesis in the younger internodes. Expression analysis showed high levels of expression of both the UDP-glucose dehydrogenase transcript and protein in the leafroll, roots and young internodes. In situ hybridisation showed that the UDP-glucose dehydrogenase transcript is present in virtually all cell types in the sugarcane internode, while immunolocalisation showed that the abundance of the protein declined in all cell types as maturity increased. Results obtained confirmed that this enzyme plays an important role in the provision of hemicellulose precursors in most developing tissues of the sugarcane plant, indicating that UDP-glucose dehydrogenase was indeed a suitable target for promoter isolation. Lastly, the promoter region and first intron, located in the 5’-untranslated region (UTR) of this gene, were isolated and subsequently fused to the GUS reporter gene for transient expression analysis and plant transformation. Transient expression analysis showed that the presence of the intron was essential for strong GUS expression. Analysis of stably transformed transgenic sugarcane plants, evaluated in a green house trial, showed that the isolated promoter is able to drive GUS expression in a tissue specific manner under these conditions.

The first goal of this work focused on the development of an amine dehydrogenase (AmDH) from a leucine dehydrogenase using site-directed mutagenesis. We aimed at reductively aminating a prochiral ketone to a chiral amine by using leucine dehydrogenase (LeuDH) as a starting template. This initial work was divided into two stages. The first focused mutagenesis to a specific residue (K68) that we know is key to developing the target functionality. Subsequently, mutagenesis focused on residues known to be in close proximity to a key region of the substrate (M65 and K68). This approach allowed for reduced library size while at the same time increased chances of generating alternate substrate specificity. An NAD+-dependent high throughput assay was optimized and will be discussed. The best variants showed specific activity in mU/mg range towards deaminating the target substrate. The second goal of this work was the development of a thermostable glucose dehydrogenase (GDH) starting with the wild-type gene from Bacillus subtilis. GDH is able to carry out the regeneration of both NADH and NADPH cofactors using glucose as a substrate. We applied the structure-guided consensus method to identify 24 mutations that were introduced using overlap extension. 11 of the tested variants had increased thermal stability, and when combined a GDH variant with a half-life ~3.5 days at 65℃ was generated--a ~10⁶increase in stability when compared to the wild-type. The final goal of this work was the characterization of GDH in homogeneous organic-aqueous solvent systems and salt solutions. Engineered GDH variants showed increased stability in all salts and organic solvents tested. Thermal stability had a positive correlation with organic solvent and salt stability. This allowed the demonstration that consensus-based methods can be used towards engineering enzyme stability in uncommon media. This is of significant value since protein deactivation in salts and organic solvents is not well understood, making a priori design of protein stability in these environments difficult.