Thesis supervisor: Mani Subramanian. Includes bibliographic references (p. 121-125).

Pyruvate is a valuable chemical intermediate in the production of fine chemicals used by agrochemical, pharmaceutical, and food industries. Current technology for production of pyruvic acid is based on conversion from tartaric acid and results in environmentally incompatible byproducts. An enzymatic approach to making pyruvate was developed by cloning the glycolate oxidase (GO) gene from spinach into Pichia pastoris (Payne, et al., (1995). High-level production of spinach glycolate oxidase in the methylotrophic yeast Pichia pastoris: Engineering a biocatalyst. Gene, 167(1-2), 215-219). GO is a flavoprotein (FMN dependent) which catalyzes the conversion of lactate to pyruvate with the equimolar production of hydrogen peroxide. Hydrogen peroxide can lower GO activity and make non-catalytic byproducts, so catalase was also cloned into P. pastoris to create a double transformant. Process development work was completed at the University of Iowa's Center for Biocatalysis and Bioprocessing. High-density P. pastoris fermentation (7.2 kg cells/L) was completed at the 100 L scale. Critical fermentation set-points were confirmed at 14 h glycerol feeding followed by methanol induction at 2 - 10 g/L for 30 h. After fermentation, these cells were permeabilized with benzalkonium chloride (BAC) to enable whole-cell biocatalysis and increase enzyme activity, yielding 100 U/g for GO. In 30 L enzyme reactions, permeabilized cells were recycled three times for over 92% conversion of 0.5 M lactate with an "enzyme to product" ratio of approximately 1:2 (Gough, et al., (2005). Production of pyruvate from lactate using recombinant Pichia pastoris cells as catalyst. Process Biochemistry, 40(8), 2597-2601). Though effective, the post-fermentation process for GO recovery involved several unit-operations, including multiple washing steps to remove residual BAC. The present work has focused on minimizing unit-operations by spray-drying the fermentation product to create a powdered biocatalyst. Optimal spray-drying conditions for the Buchi B-190 instrument were 150°C drying air, 15 mL/min liquid feed rate, and 600 mg cells/mL liquid feed. These conditions resulted in P. pastoris biocatalyst with activities of 80 - 100 U/g for GO and 180,000 - 220,000 U/g for catalase. The spray-dried cells retained nearly 100% of the enzyme activity compared to BAC treated cells as reported by Gough et al. Additionally, the spray-dried biocatalyst was stable at room temperature for 30 days, and no measurable enzyme leaching was observed. Then, P. pastoris was spray-dried under optimal conditions and tested for conversion of lactate to pyruvate for an improved "enzyme to product" ratio. Enzyme reaction optimization was done at the one-liter scale in DASGIP reactors. The DASGIP system contained four parallel reactors with control of temperature, pH, and dissolved oxygen. Other key variables included substrate loading, conducting the reaction in buffer or water, minimizing enzyme concentration, and maximizing the number of enzyme recycles. Optimal performance was achieved in water at pH 7.0 with an operating temperature of 25°C and 1.0 M substrate loading. Enzyme loading was at 12 g/L for the first two cycles, and subsequently, 2 - 3 g/L of fresh cells were added every alternate cycle to reach 15 cycles. Under these conditions, 75 - 95% conversion of lactate to pyruvate was accomplished for every 12 - 16 h reaction cycle. Based on these parameters, an "enzyme to product" ratio of 1:41 was achieved.

Thesis (M.Sc (Pharmaceutics))--North-West University, Potchefstroom Campus, 2009.

Much research was performed in order to find alternative energy sources. In the new concept presented in this thesis, methane was produced by a microbial consortium which is fed only by glycolate excreted by photosynthetic algae. It was unknown how the biogas production and the process stability are influenced by certain parameter shifts in glycolate feed, pH of the feed, oxygen input and temperature. Therefore, different parameter changes were applied to the reactor system. Gas formation and composition, pH, FosTac and organic acid content in the media were analyzed. Additionally, the community composition induced by the changes of the process parameters was analyzed. Therefore, the single cell level using flow cytometry and the genetic level using T-RFLP were observed. The R based tool flowCyBar was applied to follow community dynamics and to find key players in the process. Abiotic and biotic parameters were used for correlation analysis (Spearman´s Rho) to determine specific functions of sub-communities and the T-RFs found in the whole community. The cultivation at room temperature indicated that the biogas yield was 30 % lower in comparison to the cultivation at 37°C. The community seemed to be able to sustain pulsating oxygen input in case glycolate as substrate is available. Additionally, the system was able to recover after the oxygen application of one day. A linear correlation between the feed glycolate and the biogas production was observed (R^2 = 0.97). High biogas yields were obtained (up to 90 %) indicating that glycolate is a suitable substrate. The efficiency of the process was high with 41 ± 3 % methane in the biogas under defined conditions. It could be observed that biogas can be produced on glycolate as mono-substrate by a complex microbial community while it remained complex over the complete study (505 days cultivation) although glycolate was used as sole carbon source. Key sub-communities (e.g. cluster 1 and 3) and T-RFs (e.g. T-RF representing Planctomycetaceae or 67 bp) were defined and indicated the status of the bioreactor. The community was able to sustain certain parameter shifts, like changes in the pH of the feed (from 3 to 7), while others led to a complete crash of the system, like high glycolate feeds (3.6 g d^-1 L^-1). All in all, the stability of the process seemed to be susceptible on external influences.

Thesis (M.Sc (Pharmaceutics))--North-West University, Potchefstroom Campus, 2009.

Thesis (M.Sc (Pharmaceutics))--North-West University, Potchefstroom Campus, 2009.

Submitted by DANILO JOSÉ COURA GOMES null (danilogomes26@yahoo.com.br) on 2016-03-21T17:46:23Z No. of bitstreams: 1 Versão eletrônica.pdf: 37219010 bytes, checksum: 3db084a502fe18628a8958b5523753bd (MD5) / Rejected by Ana Paula Grisoto (grisotoana@reitoria.unesp.br), reason: Solicitamos que realize uma nova submissão seguindo as orientações abaixo: No campo “Versão a ser disponibilizada online imediatamente” foi informado que seria disponibilizado o texto completo porém no campo “Data para a disponibilização do texto completo” foi informado que o texto completo deverá ser disponibilizado apenas 24 meses após a defesa. Caso opte pela disponibilização do texto completo apenas 24 meses após a defesa selecione no campo “Versão a ser disponibilizada online imediatamente” a opção “Texto parcial”. Esta opção é utilizada caso você tenha planos de publicar seu trabalho em periódicos científicos ou em formato de livro, por exemplo e fará com que apenas as páginas pré-textuais, introdução, considerações e referências sejam disponibilizadas. Se optar por disponibilizar o texto completo de seu trabalho imediatamente selecione no campo “Data para a disponibilização do texto completo” a opção “Não se aplica (texto completo)”. Isso fará com que seu trabalho seja disponibilizado na íntegra no Repositório Institucional UNESP. Por favor, corrija esta informação realizando uma nova submissão. Agradecemos a compreensão. on 2016-03-22T16:56:26Z (GMT) / Submitted by DANILO JOSÉ COURA GOMES null (danilogomes26@yahoo.com.br) on 2016-03-22T17:53:54Z No. of bitstreams: 1 Versão eletrônica.pdf: 37215484 bytes, checksum: d91d471547bea0cdb42d7b423b2d9455 (MD5) / Approved for entry into archive by Juliano Benedito Ferreira (julianoferreira@reitoria.unesp.br) on 2016-03-23T14:24:19Z (GMT) No. of bitstreams: 1 gomes_djc_dr_araiq_par.pdf: 3387049 bytes, checksum: c79b9767bc573eb1f988cb8e70b185a6 (MD5) / Made available in DSpace on 2016-03-23T14:24:19Z (GMT). No. of bitstreams: 1 gomes_djc_dr_araiq_par.pdf: 3387049 bytes, checksum: c79b9767bc573eb1f988cb8e70b185a6 (MD5) Previous issue date: 2016-03-17 / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Foram sintetizados, no estado sólido, os compostos LnL3∙nH2O, sendo que Ln representa os lantanídeos trivalentes (La ao Lu) e o ítrio(III) e o L representa o glicolato (C2H4O2-). Os compostos foram sintetizados por adição de ligeiro excesso do ácido glicólico com carbonato básico de lantanídeos e ítrio, sob agitação e aquecimento. A caracterização dos compostos foi realizada utilizando técnicas como difratometria de raios X pelo método do pó, complexometria e as técnicas termoanalíticas como termogravimetria e calorimetria exploratória diferencial simultânea (TG-DSC) e termogravimetria acoplada à espectroscopia de absorção na região do infravermelho com transformada de Fourier (TG-FTIR). Também foram realizados cálculos teóricos para a elaboração de modelos estruturais mais prováveis e energeticamente favoráveis dos compostos sintetizados e a partir dessas estruturas foram gerados os espectros teóricos de infravermelho, para isso foram aplicados métodos da Teoria do Funcional de Densidade Eletrônica (DFT). Os resultados forneceram informações sobre o comportamento térmico, cristalinidade, estequiometria, sítios de coordenação e também pode identificar os principais produtos gasosos liberados durante o aquecimento dos compostos estudados. / Solid-state LnL3∙nH2O compounds, where Ln stands for trivalent lanthanides (La to Lu) or yttrium(III) and L is glycolate (C2H4O2 - ) have been synthesized. The compounds were synthesized by addition of slight excess of glycolic acid with basic carbonate of yttrium and lanthanides, under stirring and heating. The characterization of the compounds was performed using X-ray powder diffratometry, complexometry and thermoanalytical techniques such as simultaneous thermogravimetry and differential scanning calorimetry (TG-DSC) and thermogravimetry coupled to absorption spectroscopy in the region of infrared with Fourier transform (TG-FTIR). Theoretical calculations were also performed to elaborate more probable and energetically favorable structural models of the synthesized compounds and from these structures the theoretical infrared spectra were generated, with the application of methods of Functional Theory of Electron Density (DFT). The results provided information about thermal behaviour, crystallinity, stoichiometry, coordination sites and could also identify the main gaseous products released during heating of the compounds studied. / CNPq: 146916/2013-1

La bactérie Erwinia amylovora est responsable de la maladie du feu bactérien des Maleae (pommier, poirier…). Le pouvoir pathogène de cette bactérie dépend d'une seringue moléculaire appelé système de sécrétion de type 3 (SSTT). Ce SSTT permet à la bactérie d’injecter des effecteurs dans les cellules de la plante. Parmi les effecteurs injectés, DspA/E est l'effecteur indispensable au pouvoir pathogène d’E. amylovora. Cet effecteur est à lui seul capable de provoquer la mort des cellules chez le pommier et le tabac et permet à la bactérie de se multiplier de manière transitoire chez A. thaliana. L’objectif de ce travail de thèse était de comprendre la fonction de DspA/E dans la cellule végétale et d’identifier des facteurs végétaux impliqués dans la toxicité de DspA/E. Afin de répondre à cette question, des plantes transgéniques exprimant DspA/E sous contrôle d’un promoteur inductible à l’estradiol ont été construites.Dans un premier temps, la caractérisation phénotypique des lignées exprimant DspA/E a été effectuée. Les résultats obtenus montrent que DspA/E est toxique lorsqu'il est exprimé in planta (il provoque la mort des cellules, inhibe la germination, la croissance racinaire et la traduction) et permet la multiplication in planta d’un mutant dspA/E. Un crible de mutants suppresseurs de la toxicité de l'effecteur DspA/E a été effectué sur une lignée transgénique exprimant DspA/E dans le but d'identifier un ou plusieurs gènes impliqués dans la toxicité de DspA/E. Ce crible suppresseur a permis d'identifier un candidat potentiel impliqué dans la photo-respiration, la glycolate oxydase 2 (GOX2). L’analyse fonctionnelle réalisée sur le mutant gox2-2 a permis de montrer que le gène GOX2 est un régulateur positif des réponses de défense d’A. thaliana en réponse à l’infection par E. amylovora.Enfin, la caractérisation du stress oxydant a permis de montrer que plusieurs formes actives de l’oxygène (H2O2 et O2.-) s’accumulent au cours de l’interaction entre A. thaliana et E. amylovora. Ceci a permis également de comprendre le rôle de DspA/E sur ce stress oxydant. Nos résultats suggèrent que la glycolate oxydase 2 participerait à l’induction du stress oxydant en perturbant le métabolisme des sucres. / The bacterium E. amylovora is responsible for the fire blight disease of Maleae (apple, pear…). The pathogenicity of this bacterium relies on a molecular syringe, the type three secretion system (TTSS). This TTSS allows the bacterium to inject effector proteins into the plant cell. Among these effectors, DspA/E is essential for the pathogenicity of E. amylovora. This effector can provoke cell death on apple and tobacco and allows the bacterium to multiply transiently in A. thaliana.The purpose of the thesis was to understand the function of DspA/E in the plant cell and to identify plant factors involved in the toxicity of DspA/E. To answer this question, transgenic plants which express DspA/E under an estradiol-inducible promoter were built.At first, phenotypical characterization of DspA/E transgenic lines was performed. Our results showed that DspA/E is toxic when expressed in planta (it provokes cell death, inhibits germination, root growth and translation) and allows in planta multiplication of dspA/E bacterial mutant. A screening for suppressor mutants of DspA/E toxicity was performed on a DspA/E transgenic line in order to identify one or several genes involved in the toxicity of DspA/E. This screening allowed us to identify a potential candidate involved in photorespiration, the glycolate oxidase 2 (GOX2). Functional analysis performed on the gox2-2 mutant allowed us to show that the GOX2 gene is a positive regulator of A. thaliana responses against E. amylovora.Finally, characterization of oxidative stress allowed us to show that several reactive oxygen species (H2O2 et O2.-) accumulate during A. thaliana and E. amylovora interaction. This allowed us to understand the role of DspA/E in the oxidative stress. Our results suggest that the glycolate oxidase 2 could be involved in the induction of the oxidative stress by disrupting sugar metabolism.

Much research was performed in order to find alternative energy sources. In the new concept presented in this thesis, methane was produced by a microbial consortium which is fed only by glycolate excreted by photosynthetic algae. It was unknown how the biogas production and the process stability are influenced by certain parameter shifts in glycolate feed, pH of the feed, oxygen input and temperature. Therefore, different parameter changes were applied to the reactor system. Gas formation and composition, pH, FosTac and organic acid content in the media were analyzed. Additionally, the community composition induced by the changes of the process parameters was analyzed. Therefore, the single cell level using flow cytometry and the genetic level using T-RFLP were observed. The R based tool flowCyBar was applied to follow community dynamics and to find key players in the process. Abiotic and biotic parameters were used for correlation analysis (Spearman´s Rho) to determine specific functions of sub-communities and the T-RFs found in the whole community. The cultivation at room temperature indicated that the biogas yield was 30 % lower in comparison to the cultivation at 37°C. The community seemed to be able to sustain pulsating oxygen input in case glycolate as substrate is available. Additionally, the system was able to recover after the oxygen application of one day. A linear correlation between the feed glycolate and the biogas production was observed (R^2 = 0.97). High biogas yields were obtained (up to 90 %) indicating that glycolate is a suitable substrate. The efficiency of the process was high with 41 ± 3 % methane in the biogas under defined conditions. It could be observed that biogas can be produced on glycolate as mono-substrate by a complex microbial community while it remained complex over the complete study (505 days cultivation) although glycolate was used as sole carbon source. Key sub-communities (e.g. cluster 1 and 3) and T-RFs (e.g. T-RF representing Planctomycetaceae or 67 bp) were defined and indicated the status of the bioreactor. The community was able to sustain certain parameter shifts, like changes in the pH of the feed (from 3 to 7), while others led to a complete crash of the system, like high glycolate feeds (3.6 g d^-1 L^-1). All in all, the stability of the process seemed to be susceptible on external influences.

No description available.