by Leung Sau-mei. / With: Two articles bound together subsequent to publication. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1990. / Bibliography: leaves 101-108. / Abstract --- p.i / Acknowledgements --- p.iv / List of Abbreviations --- p.v / Table of content --- p.vi / Chapter Section 1 --- Introduction / Chapter 1.1 --- The phenomenon of host controlled restriction and modification in bacteria --- p.2 / Chapter 1.2 --- Classification of restriction and modification systems --- p.3 / Chapter 1.3 --- The nomenclature system for restriction and modification systems --- p.8 / Chapter 1.4 --- Variety of type II restriction and modification systems --- p.9 / Chapter 1.5 --- Properties of type II restriction endonucleases --- p.11 / Chapter 1.5.1 --- Biological function --- p.11 / Chapter 1.5.2 --- Protein structure --- p.12 / Chapter 1.5.3 --- Genetics --- p.16 / Chapter 1.5.4 --- Cleavage mechanism --- p.18 / Chapter 1.5.5 --- Factors affecting optimal activity --- p.21 / Chapter 1.6 --- Aim of study --- p.27 / Chapter Section 2 --- Materials and methods / Chapter 2.1 --- Screening for type II restriction endonucleases --- p.28 / Chapter 2.1.1 --- Sources --- p.28 / Chapter 2.1.2 --- Preparation of crude enzyme extract --- p.29 / Chapter 2.1.3 --- Assay of restriction enzyme activity --- p.30 / Chapter 2.1.4 --- Characterization of strains --- p.31 / Chapter 2.2 --- Purification of restriction endonucleases --- p.31 / Chapter 2.2.1 --- Preparation of column materials --- p.32 / Chapter 2.2.2 --- Purification of BcoI --- p.33 / Chapter 2.2.3 --- "Purification of Bcol0278I, BspI, Bsu8646I and PvuHKU" --- p.33 / Chapter 2.2.4 --- Purification of Bsu8565I and Pei9403I --- p.33 / Chapter 2.2.5 --- Purification of Sol3335I --- p.34 / Chapter 2.3 --- Characterization of discovered restriction endonucleases --- p.34 / Chapter 2.3.1 --- Determination of recognition specificity --- p.34 / Chapter 2.3.2 --- Determination of cleavage specificity of BcoI --- p.35 / Chapter 2.3.3 --- Unit definition --- p.36 / Chapter 2.3.4 --- Assays for ionic requirement and optimal temperature --- p.37 / Chapter 2.3.5 --- Heat inactivation --- p.37 / Chapter 2.4 --- Construction of Bacillus coagulans SM1 genomic library --- p.38 / Chapter 2.4.1 --- Preparation of chromosomal DNA --- p.38 / Chapter 2.4.1.1 --- Restriction digestion of chromosomal DNA --- p.40 / Chapter 2.4.2 --- Large scale preparation of vector pBR322 DNA --- p.43 / Chapter 2.4.2.1 --- Restriction digestion of vector DNA --- p.44 / Chapter 2.4.2.2 --- Preparation of lambda insert DNA for control tests --- p.45 / Chapter 2.4.3 --- Ligation of insert and vector DNA --- p.46 / Chapter 2.4.4 --- Transformation --- p.46 / Chapter 2.4.4.1 --- Preparation of electro-competent cells --- p.46 / Chapter 2.4.4.2 --- Electro-transformation --- p.47 / Chapter 2.4.5 --- Rapid screening for the presence of plasmid --- p.49 / Chapter Section 3 --- Results / Chapter 3.1 --- Presence of type II restriction endonucleases --- p.50 / Chapter 3.2 --- Strain identification --- p.50 / Chapter 3.3 --- Purification and properties of the discovered restriction endonucleases --- p.52 / Chapter 3.3.1 --- BcoI --- p.55 / Chapter 3.3.2 --- "Bco10278I, BspI, Bsu8646I and PvuHKUI" --- p.59 / Chapter 3.3.3 --- Bsu8565I and Pei9403I --- p.65 / Chapter 3.3.4 --- Sol3335I --- p.70 / Chapter 3.4 --- Construction of Bacillus coagulans SM1 genomic library --- p.73 / Chapter 3.4.1 --- Preparation of chromosomal DNA --- p.73 / Chapter 3.4.1.1 --- Generation of 4-10 kb insert fragments --- p.73 / Chapter 3.4.2 --- Preparation of plasmid DNA --- p.75 / Chapter 3.4.3 --- Ligation of insert and vector DNA --- p.76 / Chapter 3.4.4 --- Rapid screening for the presence of plasmid --- p.77 / Chapter Section 4 --- Discussion / Chapter 4.1 --- Screening of type II restriction endonucleases --- p.79 / Chapter 4.1.1 --- Methods for screening of type II restriction endonucleases --- p.79 / Chapter 4.1.2 --- Factors affecting the detection of restriction endonucleases --- p.83 / Chapter 4.2 --- Purification of the discovered restriction endonucleases --- p.87 / Chapter 4.3 --- Characterization of discovered restriction endonucleases --- p.91 / Chapter 4.3.1 --- Determination of recognition site --- p.91 / Chapter 4.3.2 --- Determination of cleavage specificity --- p.93 / Chapter 4.4 --- Characteristics of the discovered restriction endonucleases --- p.95 / Chapter 4.5 --- Molecular cloning of BcoI --- p.96 / Chapter 4.6 --- Future prospects --- p.99 / References --- p.101 / Appendix I --- p.109

這篇論文研究了幾種基於聚二甲基硅氧烷（PDMS）的微流控芯片，及其對於酶反應和蛋白質結晶的應用。 / 本論文分為兩部份。首先，論文第一部份介紹了兩種微流控芯片。利用到PDMS透氣的性質，第一種微流控芯片是一種基於脫氣PDMS的納升液體進液系統。在第一種微流控芯片的基礎上，我們引進一種可由普通注射器控制的氣閥控制系統，可對反應液體的分配和混合進行更方便和精確的操作。兩種芯片均成功應用於酶反應動力學的測定。在一次實驗中，只需要3~5微升的反應物就可以得到鹼性磷酸酶（alkaline phosphatase）的Michaelis-Menten動力學。 / 論文第二部份研究了“體積效“應對於蛋白質結晶的影響。首先，基於微孔的微流控芯片的蛋白質結晶篩選實驗揭示了在小體積（納升）下，蛋白質的結晶條件比在大體積（微升）下更多。在液滴里的蛋白質結晶實驗結果說明大體積的蛋白質液滴結晶速度更快。最後，蛋白質結晶實驗成功在雙乳液（double emulsion）的內核中進行。 / This thesis describes the design and development of micro dispensing systems for enzyme assay and for protein crystallization. The micro dispensing systems were fabricated by the soft-lithography method with the widely used material poly(dimethylsiloxane) (PDMS), which is gas and water permeable elastomer. / This thesis contains two major parts. In the first part, enzyme assay was performed in two micro dispensing systems, one based on microwells and the other based on pneumatic valves. The complete Michaelis-Menten kinetics measurement of the alkaline phosphatase (AP) with different fluorescein diphosphate (FDP) was achieved in one chip for each system using the fluorescence detection. These micro dispensing systems’ fabrication and operation were simple, and the total sample consumption was about 3~5 μL. / The second part reports the study of the volume effect on protein crystallization. Three micro dispensing systems, the microwell-based, droplet-based and double emulsion-based systems, were developed to perform the protein crystallization. Lysozyme and thaumatin were chosen as the model proteins. First, the protein crystallization screening experiments showed that protein crystallized in more precipitant conditions in the microchip than in conventional microbatch system. Second, the protein crystallization results carried out in droplets showed that protein crystallized faster in larger droplets. Finally, the protein crystallization in double emulsions was demonstrated. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Zhou, Xiaohu. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 87-91). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Abstract --- p.i / 摘要 --- p.iii / Acknowledgements --- p.iv / Table of Contents --- p.vi / List of figures --- p.viii / Chapter Chapter 1 --- Introduction to microfluidics --- p.1 / Chapter 1.1 --- Fabrication of microfluidic devices --- p.2 / Chapter 1.1.1 --- PDMS-based microfluidic device --- p.2 / Chapter 1.1.2 --- PMMA microfluidic device --- p.6 / Chapter 1.1.3 --- Assembly of the microfluidic device --- p.8 / Chapter 1.2 --- Degassed PDMS pumping method --- p.9 / Chapter 1.3 --- Droplet-based microfluidics --- p.13 / Chapter 1.4 --- Thesis organization --- p.16 / Chapter Chapter 2 --- Micro dispensing systems for enzyme assay --- p.17 / Chapter 2.1 --- Introduction to enzyme assay --- p.17 / Chapter 2.1.1 --- Introduction to micro platforms for enzyme assay --- p.17 / Chapter 2.1.2 --- Introduction to enzyme kinetics and Michaelis-Menten kinetics --- p.21 / Chapter 2.2 --- Experimental --- p.26 / Chapter 2.2.1 --- Design of the micro dispensing systems --- p.26 / Chapter 2.2.2 --- Fabrication of the microfluidic devices --- p.30 / Chapter 2.2.3 --- Reagents and operation --- p.32 / Chapter 2.3 --- Results and discussion --- p.32 / Chapter 2.3.1 --- Microwell-based dispensing system --- p.32 / Chapter 2.3.2 --- Micro dispensing system based on pneumatic valves --- p.40 / Chapter 2.4 --- Conclusions --- p.45 / Chapter Chapter 3 --- Micro dispensing systems for protein crystallization --- p.47 / Chapter 3.1 --- Introduction to protein crystallization --- p.47 / Chapter 3.1.1 --- Principle of protein crystallization --- p.49 / Chapter 3.1.2 --- From macrofluidics to microfluidics --- p.51 / Chapter 3.2 --- Experimental --- p.56 / Chapter 3.2.1 --- Design of the micro dispensing systems --- p.56 / Chapter 3.2.2 --- Fabrication of the microfluidic devices --- p.58 / Chapter 3.2.3 --- Reagents and operation --- p.60 / Chapter 3.2.4 --- Layer-by-layer modification --- p.61 / Chapter 3.3 --- Results and discussion --- p.65 / Chapter 3.3.1 --- Demonstration of the micro dispensing systems --- p.65 / Chapter 3.3.2 --- Protein crystallization screening results --- p.69 / Chapter 3.3.3 --- Protein crystallization in droplets --- p.73 / Chapter 3.3.4 --- Protein crystallization in w/o/w double emulsions --- p.77 / Chapter 3.4 --- Conclusions --- p.80 / Appendix --- p.82 / References --- p.87

Chez Arabidopsis thaliana, deux gènes codent des enzymes de branchement (BE) BE2.2 et BE2.1 responsables de la formation des liaisons α-1,6 de l’amidon transitoire synthétisé dans la feuille de la plante. Ces enzymes, appartenant à la famille GH13_8 de la classification CAZy, agissent en clivant une liaison α-1,4 d’un glucane puis en transférant la chaîne clivée en position α-1,6 selon un mécanisme d’action qui peut être intra ou intermoléculaire. Dans ce travail, une caractérisation enzymatique et structurale des BEs d’A. thaliana (classées de type II d’après leur séquence en acides aminés) a été réalisée et les résultats comparés à ceux de la BE d’E. coli (GlgB, enzyme de type I).L'état oligomérique, la forme en solution et l’organisation structurale des BEs ont été évalués par une approche SAXS. Par des analyses spectrophotométriques, le pH, la température, mais aussi le KM pour l’amylose et l’amylopectine ont été déterminés. Une analyse sur gel de polyacrylamide, en conditions natives, a permis d’évaluer le comportement électrophorétique des BEs en présence ou en absence de ces substrats et d’en déterminer leur constante d’affinité (Ks). Notre étude révèle que les BEs d’ A. thaliana ont plus d'affinité pour l’amylopectine que pour l’amylose contrairement à GlgB. En présence d’un substrat branché, des changements d’oligomérie et/ou de la conformation des BEs d’A. thaliana ont été observés. Finalement, des analyses en chromatographie échangeuse d'anions ont permis de déterminer la taille minimale du substrat nécessaire à l’activité des BEs et la taille des chaînes transférées. Les résultats obtenus pointent vers un mécanisme d’action intramoléculaire de BE2.2. / BE2.2 and BE2.1 are the two genetically independent branching enzymes (BE) isoforms involved in transitory starch synthesis in A. thaliana and belong to family GH13_8 (according to CAZy database). Both are classified as type II BE due to their amino acid sequence. In Arabidopsis leaves, they are the only enzymes that catalyze the formation of α-1,6 branch points by cleaving α-1,4 linkages and transferring the newly formed reducing end in α-1,6 position through an intra or intermolecular mechanism. In this work, we report in vitro enzymatic characterization and structural analysis of A. thaliana BEs, these results were compared to E. coli BE enzymatic analysis (GlgB, type I enzyme).Structural analysis using SAXS approach was used to evaluate A. thaliana BEs oligomeric state, shape in solution and to determine BE organization. In vitro enzymatic analyses were performed using spectrophotometry assays to establish their catalytic parameters such as pH, temperature and also KM for amylose and amylopectin. Native PAGE analyses were also used to assess BEs behaviour in the presence or absence of substrates and to determine their affinity constant (Ks) for amylopectin and amylose. Enzymatic characterization reveals that both A. thaliana BEs have more affinity for amylopectin than for amylose, contrary to GlgB. Moreover, interaction of A. thaliana BEs with branched substrates induces protein oligomerization and/or conformational changes. Finally, determination of the minimal length of their substrate and characterization of reaction products were performed using anions exchange chromatography analyses.Taken together, our data point to an intramolecular mechanism of action of BE2.2.

Typescript (photocopy). / Digitized by Kansas Correctional Industries

Enzymes are desirable catalysts as they may exhibit high activity, high selectivity, and may be easily engineered. Additionally, enzymes can be mass-produced recombinantly making them a potentially less expensive option than their organic or inorganic counterparts. As a result, they are being used more in industrial applications making their relevance ubiquitous. In this work, various engineering approaches were developed to control the activity and selectivity of enzymes for multi-step catalysis. Unlike nature, many industrial processes require multiple steps to produce the desired product, which is both timely and expensive. Through the use of enzymes, biosynthesis can be used to develop efficient multi-step catalytic cascades. The majority of this work focused on engineering a hyperthermophilic enzyme from the aldo-keto reductase (AKR) superfamily, alcohol dehydrogenase D (AdhD) from Pyrococcus furiosus, to develop approaches to control activity and selectivity. As the AKR superfamily contains many unifying characteristics, such as a conserved catalytic tetrad, (α/β)8-barrel quaternary fold, conserved cofactor binding pocket, and varying substrate loops, the approaches developed here can be applied to many enzymes. AKR members participate in a broad range of redox reactions, such as those involving aldehydes, hydrocarbons, xenobiotics, and many more, and are necessary in physiological processes in all living systems, making these enzymes industrially relevant. AdhD in particular can oxidize alcohols or reduce aldehydes/ketones in the presence of NAD(P)(H). Furthermore, the tools utilized here are modular and can be used to develop pathways with enzymes from different superfamilies’ to expand their current capabilities. In our initial engineering efforts, AdhD cofactor selectivity was broadened or reversed through site directed mutations or insertions in substrate loop B, on the back side of the cofactor binding pocket. To further examine how substrate loops affect cofactor selectivity, allosteric control was added to AdhD through the insertion of a calcium-dependent repeat-in-toxin domain from Bordetella pertussis. Through the chimeric protein, β-AdhD, we demonstrated that the addition of calcium shifts cofactor selectivity in real-time, reminiscent of a protein dimmer. Our next focus shifted towards unnatural amino acid incorporation to add an extra level of selectivity to AdhD. This was done by merging the properties of AdhD and an organic catalyst, TEMPO, for selective alcohol oxidation. We also demonstrated the ability to impart enzymatic selectivity onto an organic catalyst. This was done both in solution and in AdhD hydrogels for added functionality. The next study focused on increasing catalytic efficiency while retaining AdhD structure by engineering the microenvironment of AdhD with supercharged superfolder GFP (sfGFP). The complex interplay between salt, pH, and protein charge was studied and it was determined that catalysis is a function of protein charge, which can affect apparent local ionic strength. The final study focused on utilizing the previous tools examined to engineer substrate channeling in a multi-step cascade with hexokinase II (HK2), sfGFP, and glucose-6-phosphate dehydrogenase (G6PD). In conclusion, we have utilized a myriad of tools to develop engineering approaches to regulate AdhD activity and selectivity. These tools were then extended to engineer substrate channeling in a three-enzyme system. These approaches are modular and provide a foundation for the development of multi-step catalytic cascades.

Orientador: Rogério Lopes Vieites / Banca: Flavia Aparecida de Carvalho Mariano Nassser / Banca: Juliana Audi Giannoni / Resumo: A atemoia é um fruto climatérico altamente perecível, e para sua comercialização é necessário que se faça uso de métodos de conservação pós colheita. O presente trabalho teve como objetivo a conservação da Atemoia com a utilização de 1-metilciclopropeno (1-MCP). Os frutos após a colheita foram selecionados e submetidos a diferentes concentrações de 1-MCP (200 µL.L-1, 300 µL.L-1 e 400 µL.L-1) e após o procedimento foram armazenados sob refrigeração em câmara fria a 15ºC±1 e 90±5% de UR. Foram avaliados pH, acidez titulável (AT), sólidos solúveis (SS), índice de maturação, perda de massa fresca, atividade respiratória, açúcares redutores, amido, coloração, ácido ascórbico, compostos fenólicos totais, capacidade antioxidante, atividade das enzimas polifenoloxidase (PFO), peroxidase (POD), poligalacturonase (PG) e pectinametilesterase (PME), análise visual e sensorial. As análises foram realizadas nos frutos em triplicata a cada 3 dias, durante 18 dias. O delineamento utilizado foi inteiramente casualizado em esquema fatorial (4x7), com 3 repetições por tratamento. O uso de 1-metilciclopropeno na maior dose, de 400 µL.L-1 juntamente com o armazenamento refrigerado é recomendado para conservação e aumento da vida de prateleira. / Abstract: The atemoya is a highly perishable climacteric fruit, and marketing is necessary to make use of post harvest conservation methods. This work aimed at the conservation of atemóia with the use of 1-methylcyclopropene (1-MCP). The fruits after harvest were selected and subjected to different concentrations of 1-MCP (200 ppm, 300 ppm and 400 ppm) and after the procedure were stored under refrigeration in cold storage at 15 ° C ± 1 and 90 ± 5% RH. pH were evaluated, titratable acidity (TA), soluble solids (SS), maturation index, loss of weight, respiratory activity, reducing sugars, starch, coloring, ascorbic acid, total phenolic compounds, antioxidant capacity determined by DPPH, activity enzyme polyphenol oxidase (PPO), peroxidase (POD), polygalacturonase (PG) and pectin methyl esterase (PME), visual and sensory analysis. The analyzes were performed in triplicate in fruits every 3 days for 18 days. The design was completely randomized with three replicates per treatment. The use of 1-methylcyclopropene at all doses along with the refrigerated refrigerator is recommended for shelf life and shelf life. / Mestre

All organisms must maintain an adequate level of thymidylate, which gets phosphorylated twice and then utilized by DNA polymerases for DNA replication that must precede cell division. Most organisms rely on classical thymidylate synthase (TSase) for this function. However, a subset of microorganisms – including a number of notable, widespread human pathogens – relies on an enzyme with a distinct structure and catalytic strategy. This enzyme is termed flavin-dependent thymidylate synthase (FDTS), as the flavin is required for thymidylate production. Because of this considerable orthogonality between FDTS and classical TSase, FDTS serves as a promising target for new therapeutics – one that could have only mild adverse effects on the host organism. FDTS catalyzes the reductive methylation of uridylate (2′-deoxyuridine-5′-monophosphate; dUMP) to yield thymidylate (2′-deoxythymidine-5′-monophosphate; dTMP). The methylene originally resides on CH2H4folate and is eventually transferred to the nucleotide. This methylene’s route to dUMP is unique in enzymology, and our experiments described herein strive to gain an understanding of the molecular details of its transfer. Compounds that mimic intermediates and transition states along this path are likely to bind FDTS tightly and could be leads for drugs, and our new insights could facilitate this. After methylene transfer is complete, a hydride transfer from flavin to the nucleotide occurs. We utilized rapid quench flow techniques in heavy water to follow the hydrogen transfers in FDTS; solvent isotope effects were measured and analyzed, furnishing evidence that the hydride transfer contributes to rate limitation. Reconstitution of the enzyme with unnatural flavins both reinforced these conclusions and suggested new hypotheses and experiments.

Título original: Hidrólise enzimática de hemicelulose com endoxilanase I de Aspergillus versicolor para produção de xilooligossacarídeos e avaliação do seu efeito prebiótico / Orientador: Eleonora Cano Carmona / Coorientador: Michel Brienzo / Banca: Fernando Masarin / Banca: Sandra Regina Caccato Antonini / Resumo: Hemiceluloses são polissacarídeos com ligações do tipo β-1,4 sendo o principal tipo encontrado em gramíneas as xilanas, estruturas com cadeia principal de xilose. O pseudocaule de bananeira é um resíduo agrícola da colheita de banana que apresenta em média 19,1 % de xilana em sua composição. Xilanas apresentam grande potencial econômico e uma importante aplicação dessa macromolécula é a produção de xilo-oligossacarídeos (XOS). XOS são oligômeros de xilose, reconhecidos por seu efeito prebiótico, além de possuírem diversas propriedades biológicas que promovem a saúde. A produção de XOS pode ser realizada por hidrólise enzimática através da ação de endoxilanase na cadeia da xilana. No entanto, é importante que a hemicelulose seja extraída da biomassa lignocelulósica e que o extrato enzimático seja livre de β-xilosidase, enzima que hidrolisa XOS a xilose. Tendo em vista o aumento da demanda de compostos que previnem doenças e promovem a saúde, o objetivo desse trabalho foi a produção de xilo-oligossacarídeos, a partir da hidrólise enzimática de hemiceluloses extraídas de pseudocaule de bananeira, usando a endoxilanase I de Aspergillus versicolor e avaliar a ação prebiótica dos compostos produzidos. A xilanase I de Aspergillus versicolor foi produzida pelo crescimento do microrganismo em meio contendo xilana proveniente de farelo de trigo. A enzima foi purificada por cromatografia de troca iônica (DEAE-Sephadex A-50) e cromatografia de exclusão molecular (Sephadex G-75). Previame... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Hemicelluloses are polysaccharides with β-1,4 bonds and the main type found in grasses is xylan, xylose main chain structures. The banana pseudostem is an agricultural residue of the banana crop that presents on average 19.1% of xylan in its composition. Xylans present great economic potential and an important application of this macromolecule is the production of xylooligosaccharides (XOS). XOS are sugar oligomers composed of xylose units recognized for their prebiotic effect, besides having diverse biological properties that promote the health. The production of XOS can be carried out by enzymatic hydrolysis through the action of endoxylanase on the xylan chain. However, it is important that the hemicellulose is extracted from the lignocellulosic biomass and that the enzyme extract is free from β-xylosidase, an enzyme that hydrolyzes XOS to xylose. Because of the increase demand for compounds that prevent disease and promote health, the objective of this study was xylo-oligosaccharides production from the enzymatic hydrolysis of hemicelluloses extracted from banana pseudostem using Aspergillus versicolor endoxylanase I and evaluate the prebiotic action of the compounds produced. A xylanase from Aspergillus versicolor was produced by the growth of the microorganism in medium containing xylan (wheat bran). The enzyme was purified through ion exchange chromatography (DEAE-Sephadex A-50) and molecular exclusion chromatography (Sephadex G-75). Pior to the hemicellulose extractio... (Complete abstract click electronic access below) / Mestre

The enzymatic production of R-phenylacetylcarbinol (R-PAC), with either whole cells or partially purified pyruvate decarboxylase (PDC) as the biocatalyst, requires high PDC activity and an inexpensive source of pyruvate for an economical feasible biotransformation process. Microbial pyruvate produced by a vitamin auxotrophic strain of Candida glabrata was selected as a potential substrate for biotransformation. With an optimal thiamine concentration of 60 ??g/l, a pyruvic acid concentration of 43 g/l and yield of 0.42 g/g glucose consumed were obtained. Using microbially-produced unpurified pyruvate resulted in similar PAC concentrations to those with commercial pure substrate confirming its potential for enzymatic PAC production. To obtain high activity yeast PDC, Candida utilis was cultivated in a controlled bioreactor. Optimal conditions for PDC production were identified as: fermentative cell growth at initial pH at 6.0 followed by pH downshift to 3.0. Average specific PDC carboligase activity of 392 ?? 20 U/g DCW was achieved representing a 2.7-fold increase when compared to a constant pH process. A mechanism was proposed in which the cells adapted to the pH decrease by increasing PDC activity to convert the accumulated internal pyruvic acid via acetaldehyde to ethanol thereby reducing intracellular acidification. The effect of pH shift on specific PDC activity of Saccharomyces cerevisiae achieved a comparable increase of specific PDC carboligase activity to 335 U/g DCW. The effect of pyruvic acid at pH 3.0 on induction of PDC activity was confirmed by cultivation at pH 3 with added pyruvic acid. Using microarray techniques, genome-wide transcriptional analyses of the effect of pH shift on S. cerevisiae revealed a transient increased expression of PDC1 after pH shift, which corresponded to the increase in specific PDC activity (although the latter was sustained for a longer period). The results showed significant gene responses to the pH shift with approximately 39 % of the yeast genome involved. The induced transcriptional responses to the pH shift were distinctive and showed only limited resemblance to gene responses reported for other environmental stress conditions, namely increased temperature, oxidative conditions, reduced pH (succinic acid), alkaline pH and increased osmolarity.

Thesis (Ph. D.)--Chemistry and Biochemistry, Georgia Institute of Technology, 2006. / Barefield, E. Kent, Committee Member ; Beckham, Haskell W., Committee Member ; Doyle, Donald F., Committee Member ; Weck, Marcus, Committee Member ; Seley, Katherine L., Committee Chair.