Submitted by Gabriel Mamoru Marques Shinohara (shinohara@fct.unesp.br) on 2016-09-01T07:03:59Z No. of bitstreams: 1 Tese_Doutorado_Gabriel_Mamoru_Marques_Shinohara.pdf: 7611898 bytes, checksum: 7047b413755e309ae225c6adc00005a2 (MD5) / Approved for entry into archive by Ana Paula Grisoto (grisotoana@reitoria.unesp.br) on 2016-09-02T13:56:07Z (GMT) No. of bitstreams: 1 shinohara_gmm_dr_araiq_par.pdf: 5169602 bytes, checksum: e9ff84ff42bb20906dae94562a15b2a6 (MD5) / Made available in DSpace on 2016-09-02T13:56:07Z (GMT). No. of bitstreams: 1 shinohara_gmm_dr_araiq_par.pdf: 5169602 bytes, checksum: e9ff84ff42bb20906dae94562a15b2a6 (MD5) Previous issue date: 2016-08-18 / Neste trabalho investigou-se a influência da concentração dos agentes complexantes etilenoglicol (EG) e sorbitol (SB) nas propriedades estruturais e fotoluminescentes do luminóforo Y2O3:Eu3+ (2 mol%) obtido pelo método Pechini modificado. A quimiometria, em especial, planejamento de experimentos (Design of Experiments – DOE), foi aplicada para planejar a proporção dos precursores dos luminóforos. Todos os dados estruturais, morfológicos e espectroscópicos relacionados aos luminóforos preparados pela variação dos parâmetros de síntese foram sistematicamente interpretados pelo uso de ferramentas de quimiometria de forma a gerar uma análise comparativa consistente entre todos os óxidos. Padrões de difração de todas as amostras são característicos da estrutura forma-C de óxido de ítrio, e a equação de Scherrer aplicada aos dados de difração de raios X indicaram que ambos agentes complexantes resultaram em partículas de Y2O3:Eu3+ com tamanho de cristalito similares no intervalo de 19 a 28 nm. Por imagens de microscopia eletrônica de varredura e de transmissão foi possível confirmar que independente do precursor utilizado, são formados aglomerados de partículas esferoidais com diâmetro de 10 a 30 nm no caso de óxidos obtidos via etilenoglicol e diâmetros maiores de 10 a 50 nm para partículas preparadas utilizando sorbitol. Espectros de fotoluminescência de todos os luminóforos exibiram as emissões no vermelho no intervalo de 550–750 nm esperadas atribuídas a todas as transições 4f–4f do Eu3+. Apesar do fato de nenhum diferença detectável ter sido observada no perfil dos espectros, o que indica que o ambiente ocupado pelo Eu3+ deve ser idêntico em todas as amostras independente do tipo de agente complexante utilizado, luminóforos preparados a partir de sorbitol apresentaram maior intensidade relativa de emissão. A análise dos dados ópticos também permitiu estabelecer as melhores proporções dos precursores do luminóforo que resultaram nos emissores de maior intensidade relativa. Em adição, as amostras com maior intensidade de luminescência preparadas com etilenoglicol foram aquelas com menor valor de energia liberada no principal pico exotérmico da curva DSC e com menor tamanho de cristalito. E as amostras preparadas com sorbitol com maior intensidade de luminescência, foram aquelas produzidas com menor valor de energia liberada no principal pico exotérmico da curva DSC e com maior tamanho de cristalito. Além disso, é possível concluir que o luminóforo vermelho Y2O3:Eu3+ preparado com sorbitol possui intensidade relativa de emissão maior do que as amostras preparadas com etilenoglicol, além disso, o sorbitol é um reagente de menor custo, não tóxico como o EG e o seu uso tornou a síntese bem mais rápida, tais vantagens reduzem consideravelmente os custos da síntese. / The investigation of ethylene glycol (EG) and sorbitol (SB) complexing agents concentration on structural and photoluminescence (PL) properties of Y2O3:Eu3+ (2 mol%) oxides obtained by Pechini method is reported. The chemometrics, in special, Design of Experiments (DOE), was applied to plan phosphor precursor’s ratio. All structural, morphological and spectroscopic data related to the phosphors produced by synthesis parameters variation were systematically interpreted by using chemometrics tools in order to provide a consistent comparative analysis of all oxides. Diffraction patterns for all samples are characteristic of yttrium oxide C-form structure, and Scherrer's equation applied to X-ray diffraction data indicated that both complex agents yielded Y2O3:Eu3+ particles with similar crystallite size in the range of 19 to 28 nm. By SEM and TEM images it was possible to confirm that particles, independently on the precursor used, form agglomerates of spheroidal particles with a diameter in the range of 10 to 30 nm in the case of the oxides obtained via ethylene glycol and a larger diameter range of 10 to 50 nm for the particles produced by using sorbitol. Photoluminescence data for all phosphors exhibit the expected red emission in the range of 550–750 nm ascribed to all Eu3+ 4f–4f set of transitions. Despite the fact that no detectable difference is observed in the spectra profile indicating that Eu3+ environment should be identical for all samples independent on the agent complex used, phosphors prepared using sorbitol showed the higher relative intensity emission. Samples with higher intensity of luminescence prepared with ethylene glycol were those with the lowest amount of energy released in the main exothermic peak of the DSC curve and less crystallite size and the samples prepared with sorbitol with higher intensity of luminescence, were those produced with lower amount of energy released in the main exothermic peak of the DSC curve and larger crystallite size . Furthermore, it is possible to conclude that the red phosphor Y2O3:Eu3+ prepared with sorbitol has higher relative emission intensity than the samples prepared with ethylene glycol, in addition, sorbitol is a less costly reagent, non-toxic and its use has a much faster synthesis, such advantages significantly reduce the costs of synthesis.

Made available in DSpace on 2014-12-17T14:13:50Z (GMT). No. of bitstreams: 1 MaizaRA_TESE.pdf: 1687383 bytes, checksum: 6a89b25caa7db5fc536b4051629db0e3 (MD5) Previous issue date: 2013-12-06 / Candid?ase ? um problema de import?ncia crescente, devido o aumento do n?mero de indiv?duos imunocomprometidos e o surgimento de cepas resistentes aos antif?ngicos convencionais. ? de fundamental import?ncia a busca por novos agentes antif?ngicos mais eficazes, menos t?xicos, sendo os ?leos essenciais (OEs) excelentes alternativas para esse prop?sito. Esse estudo investigou a atividade biol?gica do OE de Mentha spicata L. sobre Candida guilliermondii de origem anal e vaginal. Para tanto foram determinadas a Concentra??o Inibit?ria M?nima (CIM), Concentra??o Fungicida M?nima (CFM), cin?tica do crescimento das leveduras (Time-Kill), altera??es micromorfol?gicas (t?cnica do microcultivo em c?mara ?mida) e investiga??o do mecanismo de a??o antif?ngico, utilizando o bioensaio do sorbitol. O OE de M. spicata foi obtido pelo processo de extra??o por destila??o a vapor. Na an?lise fitoqu?mica desse ?leo foi observada a presen?a de carvona com 84,32%, seguida pelo limoneno (13,70%) e tra?os de iso-dihidrocarvona (0,82%). Os resultados da an?lise da CIM variou entre 32 e 128 μg/mL. A CFM variou entre 64 e 1024 μg/mL. Na avalia??o da a??o de OE e da nistatina 100UI/mL, o antif?ngico padr?o apresentou o efeito fungicida a partir de 4 horas e para OE de M. spicata foi observado efeito fungist?tico na CIM, CIMX2 e CIMX4 frente ?s cepas avaliadas. O OE de M. spicata apresentou forte atividade antif?ngica contra as cepas de C. guilliermondii, promovendo altera??es micromorfol?gicas vis?veis por microscopia ?ptica, nas concentra??es testadas (CIM, CIMx2), resultado semelhante ao que se observou com a nistatina (100UI/mL). Na investiga??o do mecanismo de a??o antif?ngico foi constatado que houve altera??o da CIM na presen?a de sorbitol, com eleva??o dos valores quatro vezes maior que a concentra??o inicial, o que indica que os componentes desse OE apresentam a??o direta sobre a parede celular das leveduras. Conclui-se que o OE de Mentha spicata ? um potencial agente terap?utico no tratamento de candid?ase

Resumo: Recentemente foi demonstrado que o boro exibe rápida e significante mobilidade no floema de algumas espécies produtoras de polióis. Os polióis como os açúcares alcoóis sorbitol, dulcitol e manitol possuem a capacidade de complexar o B conferindo a esse elemento alguma mobilidade dentro da planta. As espécies selecionadas foram o cajueiro (não produtora de sorbitol) e o pessegueiro (produtora de sorbitol). O trabalho teve por objetivo verificar a mobilidade de B aplicado via foliar em mudas de cajueiro e pessegueiro. As espécies foram conduzidas separadamente em dois experimentos em casa de vegetação pertencente a UNESP/FEIS no município de Ilha Solteira-SP. O delineamento experimental foi em blocos ao acaso, com três repetições, em parcelas subdivididas, em duas épocas (30 e 60 dias). Foram avaliados nas parcelas cinco tratamentos e nas subparcelas dois tipos de folha (folhas velhas e folhas novas). Os tratamentos estudados constituiram-se da adubação via solo e/ou via foliar com boro marcado ou não com 10B, da seguinte forma: sem adubação via solo e foliar com B (Sol -B Fol -B); sem adubação via solo com B e adubação foliar com 10B (Sol -B Fol 10 B); adubação via solo com B e sem adubação foliar (Sol B Fol -B); adubação via solo com B e foliar com 10B (Sol B Fol10B) e adubação via solo com 10B e foliar com B (Sol10B Fol B). Na adubação via solo o mesmo foi acrescido de B na dose de 0,5 mg kg-1 de solo e na adubação via foliar aplicação de solução com B na doses de 255 mg L-1, utilizando-se como fonte de B o ácido bórico e ácido bórico enriquecido com átomos de 10 B (99%). Foram avaliados quinzenalmente a altura e o número de folhas e o teor total de B e a porcentagem de B na folha velha e folha nova proveniente do fertilizante aos 30 e 60 dias após a aplicação dos tratamentos / Abstract: Recently it was demonstrated the boron mobility in the phloem of some species producing polyols. The polyols or such alcohols sugar as sorbitol, mannitol and dulcitol are capable of complexing B giving to this element some mobility within the plant. This work had the objective to study the effect of fertilization with boron in the soil and leaves of cashew and peach seedling. They were conducted two experiments for 120 days in the greenhouse at UNESP, Ilha Solteira/SP. The experimental design was randomized blocks with three replications in a split plot, in two times for collection of leaves (30 and 60 days after application of treatments). We studied five treatments in plots and two ages of leaves in the subplots (old leaves and new leaves). The treatments studied were: no fertilizer with B in the soil and leaves (Sol -B Fol -B); without fertilizer with B in the soil and leaves with 10B (Sol - B Fol10B); fertilizing in the soil with B and leaves without B (Sol B Fol -B); fertilizing in the soil with B and leaves with 10B (Sol B fol10B) and fertilizing in the soil with 10 B and leaves with B (Sol10B Fol B). Were evaluated the height and the number of leaves biweekly and B total content and B percentage at the leaf from the fertilizer (% Bfopf) at 30 and 60 days. There was no fertilization effect with boron in the cashew seedling growing . The foliar fertilization with boron showed higher increase in the leaves number on peach seedlings at 60 days after fertilization. Boron foliar application is restricted or intermediate mobility in mobile cashew and peach evaluated at 30 and 60 days after fertilization / Orientador: Salatiér Buzetti / Coorientador: Regina Célia Faria Simão Canesin / Banca: Luiz de Souza Corrêa / Banca: Cassio Hamilton Abreu Junior / Mestre

Este trabalho teve como objetivo estudar o potencial do uso de farelo obtido do resíduo da extração do pigmento de cúrcuma (Cúrcuma longa L.) na elaboração de filmes biodegradáveis. Primeiramente, foi conduzido um estudo da extração de corante de cúrcuma por método Soxhlet para simular o processo industrial, dessa forma, utilizaram-se dois tipos de solventes, etanol: isopropanol (1:1) e metanol: acetona (1:1). A partir das tortas foi feito um processo de moagem e peneiragem na tentativa de isolar o amido presente, apesar de não ter obtido sucesso, este processo permitiu obter diferentes frações de farelo de cúrcuma. As frações foram divididas em 80 (F1), 200 (F2), 270 (F3), 400 (F4) mesh e fração centrifugada (F5). A caracterização das tortas de cúrcuma e das frações consistiu em análise da composição centesimal, MEV, difração de raios X e FTIR. A partir destas frações foram elaborados filmes pela metodologia casting. Os filmes foram caracterizados por MEV, difração de raios X, FTIR, análise antioxidante, propriedades mecânicas, solubilidade em água, umidade e permeabilidade ao vapor de água. Os filmes feitos a partir das frações (metanol:acetona) apresentaram melhores propriedades mecânicas e de barreira do que os feitos a partir da frações (etanol:isopropanol), porém industrialmente o solvente utilizado para a extração do pigmento é o etanol:isopropanol. Assim, o filme elaborado a partir da fração FE2 (200 mesh) (etanol:isopropanol) foi o escolhido para realizar o estudo de otimização do processo de elaboração destes filmes por ter as melhores propriedades mecânicas e de barreira do que os filmes feitos a partir das frações FE3 e FE5 . Para o estudo da otimização foi desenvolvido um planejamento experimental 22 com quatro pontos axiais e três pontos centrais para estudar o efeito da temperatura de aquecimento (Ta) e do pH da solução filmogênica sobre as propriedades mecânicas, umidade, solubilidade e permeabilidade ao vapor de água dos filmes plastificados com glicerol (22 g de glicerol/100 g da fração) e sorbitol (30 g de sorbitol/100 g da fração). Os resultados foram avaliados utilizando a metodologia de superfície de resposta e análise de multi-resposta. As condições ótimas obtidas para obter filmes com melhores propriedades mecânicas e funcionais foram: T=84,7ºC e pH 8,3, para filmes com glicerol e T=87,6ºC e pH 8,5 para filmes com sorbitol. Os filmes com glicerol quando comparados com o sorbitol, apresentaram menor índice de cristalinidade, menor teor de curcuminóides pela análise por HPLC e DPPH. Os filmes de farelo de cúrcuma com sorbitol foram mais resistentes, pouco elongáveis e menos permeáveis ao vapor de água do que filmes elaborados com glicerol. / This work aimed to study the potential of the bran residue obtained from the extraction of the pigment from turmeric (Curcuma longa L.) in the preparation of biodegradable films. The extraction of the turmeric dye was conducted by Soxhlet procedure in orer to simulate the industrial process. Two types of solvents were employed ethanol isopropanol (1:1) and methanol acetone (1:1). The pies were crushed and sieved in an attempt to isolate the starch, but no success was achieved. However, this process yielded different fractions, which ensured better characterization of the material. The fractions were divided into 80 (F1), 200 (F2), 270 (F3) and 400 (F4) mesh subfractions and centrifuged (F5). The analysis of pies and turmeric fraction were characterized by chemical composition, SEM, XRD and spectroscopy. Films were prepared from these fractions by film casting methodology, and were characterized by SEM, X-ray diffraction and FTIR analysis; they were also analyzed in terms of their antioxidant, mechanical, water solubility, moisture permeability, and water vapor properties. The films prepared from fractions (methanol: acetone) showed better mechanical and barrier properties than those prepared from the fractions (ethanol: isopropanol), but industrially the solvent used for the extraction of pigment is ethanol: isopropanol. Thus, the film prepared from fractional FE2 (200 mesh) (ethanol: isopropanol) which have the best mechanical and barrier properties than the films prepared from fractions from FE3 and FE5, was chosen for the optimization of the preparation of these films. To this end, we developed an experimental design 22 with four axial points and three focal points, for evaluation of the effect of the heating temperature (Ta) and the filmogenic solution pH on the mechanical properties, moisture, solubility, and permeability to water vapor of the films plasticized with glycerol (22 g fraction glycerol/100 g) and sorbitol (30 g sorbitol/100 g fraction). Results were examined by using response surface methodology and analysis multi-response analysis. The optimum conditions for attainment of films with better mechanical properties and functional characteristics were: T = 84.7 º C and pH 8.3 for films with glycerol, and T = 87.6 ° C and pH 8.5 for films with sorbitol. Compared to films plasticized with sorbitol, the films containing glycerol, had lower crystallinity index, lower content of curcuminoids as verified by HPLC analysis and DPPH. The turmeric bran films plasticized with sorbitol were more resistant, less elongated, and slightly permeable to water vapor compared to films prepared with glycerol.

Une nouvelle voie de synthèse de carburants à partir de biomasse a été récemment proposée : la transformation directe du sorbitol (sucre hydrogéné d'origine lignocellulosique) en alcanes légers (six atomes de carbone au maximum) en phase aqueuse par catalyse hétérogène bifonctionnelle métal/acide, suivant la réaction : C6O6H14 + 6 H2 → C6H14 + 6 H2O (déshydratation/hydrogénation du sorbitol en hexane). L'enjeu de la thèse est d'identifier des systèmes catalytiques bifonctionnels stables, actifs et sélectifs pour les hydrocarbures à 5 ou 6 atomes de carbone, valorisables dans le pool essence.Les premiers systèmes étudiés sont des catalyseurs à base de platine et de ruthénium supportés sur silice-alumine. Ces catalyseurs ne sont pas stables en milieu hydrothermal et catalysent des réactions indésirables de rupture C-C : la décarbonylation dans le cas du platine (conduisant au CO2) et l'hydrogénolyse ou la méthanation dans le cas du ruthénium. C'est pourquoi de nouveaux systèmes catalytiques ont été préparés par des mélanges mécaniques de catalyseurs métalliques (Pt, Pd, Ir) déposés sur zircone et d'oxydes tungstés (ZrO2-WOx, Al2O3-WOx, TiO2-WOx). Ces systèmes catalytiques présentent une stabilité en phase aqueuse meilleure que pour les catalyseurs à base de silice-alumine. Les sélectivités varient en fonction de la nature de l'oxyde et de la nature du métal utilisés. En particulier, Un nouveau système catalytique, Pt/ZrO2 + TiO2-WOx, (brevet 12/01.546) s'est révélé actif et très sélectif pour les composés en C6. Enfin, les mécanismes réactionnels mis en jeu pour la réaction de transformation du sorbitol ont été discutés en s'appuyant sur des tests de réactivité modèle. / A new way of biofuels synthesis was recently proposed: the direct transformation of sorbitol (ex-lignocellulose hydrogenated sugar) into light hydrocarbons (maximum six carbon atoms) in aqueous phase by metal/acid bifunctional heterogeneous catalysis, following the reaction : C6O6H14 + 6 H2 → C6H14 + 6 H2O (dehydration/hydrogenation of sorbitol into hexane). The aim of the PhD work is to identify stable and active bifunctional catalytic systems with selectivity to 5-6 carbon atoms hydrocarbons, which can be further upgraded into gasoline. The first studied systems are platinum and ruthenium catalysts supported on silica-alumina. These catalysts are not stable in hydrothermal medium and catalyse undesired C-C cleavage reactions such as decarbonylation for platinum (leading to CO2) and hydrogenolysis or methanation for ruthenium. That is why new catalytic systems have been prepared by mechanical mixing of metallic catalysts (Pt, Pd, Ir) supported on zirconia and tungstated oxides (ZrO2-WOx, Al2O3-WOx, TiO2-WOx). These catalytic systems present an improved hydrothermal stability compared to the silica-alumina based catalysts. Various selectivities are obtained depending on the oxide and metal natures. Particularly, A new catalytic system, Pt/ZrO2 + TiO2-WOx, (patent 12/01.546), was found to be active and very selective to C6 compounds. Finally, the reaction mechanisms involved in the sorbitol transformation reaction have been discussed, referring to model reactivity tests.

La presente Tesis Doctoral se ha centrado en el uso de materiales lignocelulósicos como materia prima renovable para la generación de biocombustibles y productos químicos de alto valor añadido. Para ello, se ha investigado la conversión catalítica de biomasa en alcoholes C6 mediante hidrogenación hidrolítica empleando catalizadores novedosos, basados en materiales carbonosos y rutenio. Los objetivos propuestos en este estudio, incluyendo profundizar en la comprensión del proceso, analizar su rendimiento catalítico y estudiar algunos parámetros que influyen en la selectividad y la conversión con el fin de mejorarlos.

Pressurized hot water and DTPA-Sorbitol are two relatively new soil boron (B) extraction methods with potential to replace the cumbersome hot water extraction. The objective of this research is to produce data in support of acceptance or rejection of these two alternative B extractions. The three soil tests were used to extract B from samples of calcareous sand and silt loam and limed, loamy fine sand treated with 10 levels of B and incubated for 7 and 28 d. As B application increased so did extractable B with each extraction method. High correlations (r of 0.977 to 0.999) were observed between extractable B and rate of B application with all three methods. Hot water generally extracted the least and pressurized hot water the most B regardless of soil type, rate of application or duration of incubation. Greenhouse and field experiments were conducted on one limed acid and two alkaline soils naturally low in B to test alfalfa response to B fertilizer. Values from the three soil extraction methods were correlated to yield, B tissue concentration and total B removal of alfalfa. In greenhouse studies with varying levels of soil applied B, highly significant relationships exist between extractable soil B and both tissue B concentration and total B removal. Correlations between yield and extractable soil B were impossible to obtain because of a lack of alfalfa yield responses to applied boron. All three methods accurately predict plant B tissue concentrations and total B removal. The field experiment produced a significant positive relationship between total alfalfa yield and extractable B using hot water and pressurized hot water extractions, but not using DTPA-Sorbitol. The results observed in this research support pressurized hot water extraction as the better of the two alternatives to replace hot water extraction in a broad range of soil types.

Subsurface microbes are extremophiles adapted to thrive in deep, resource-limited environments, performing crucial roles in a myriad of biogeochemical processes. The extremozymes they produce might play a pivotal role in catalyzing these processes. Identifying and characterizing those enzymes could contribute to the advancements in industrially important biocatalytic reactions. Among various enzymes, sorbitol dehydrogenases are enzymes that catalyze the reversible conversion of sorbitol into fructose in the presence of NAD+. In this study, we focus on the exploration of a sorbitol dehydrogenase (SDHSIUC1) derived from the novel strictly anaerobic, thermophilic, subsurface bacterium, Caldiatribacterium inferamans SIUC1, which is one of the first cultured members from the candidate phylum Atribacteria OP9. As SDHSIUC1 originated from a subsurface microbe, we hypothesized that the enzyme has industrially beneficial characteristics such as higher thermostability and can be used for bioindustry applications such as synthesis of rare sugars and chiral alcohols. We successfully cloned, expressed, and purified the functional SDHSIUC1 enzyme aerobically using E. coli BL21(DE3) and did biochemical assays to characterize its properties. Additionally, in combination with the findings of biochemical characterization, we applied in silico approaches such as molecular modeling and molecular docking to describe the functional mechanism of the enzyme. Initial phylogenetic tree analysis using a pool of 24 amino acid sequences showed that the closest relative for SDHSIUC1 is a Candidatus Caldiatribacterium californiense, which is an uncultured member of the Atribacteria phylum. Size exclusion chromatography and Native-PAGE suggested that SDHSIUC1 is a hexamer with a size of 225 kDa. Kinetic characterization of the SDHSIUC1 showed that the enzyme has a higher affinity for sorbitol and fructose in the presence of NAD+ and NADH, respectively. Furthermore, SDHSIUC1 enzyme is promiscuous as it could utilize other polyols (i.e., glycerol, xylitol, inositol), diols (i.e., butanediol), aldehydes (i.e., glycolaldehyde), and ketoses (i.e., sorbose) in the presence of NAD+/NADH cofactors. We observed a significant increase in enzyme activity in the presence of Zn2+, where other metal ions such as Mn2+ and Mg2+ also resulted in rate improvements. The enzyme is an alkaline dehydrogenase that prefers a higher pH above 8. The effect of temperature on SDHSIUC1 activity showed that it’s a thermophilic enzyme with activity at 85 ℃. The thermal denaturation points of the enzyme at 85 ℃ was increased when the enzyme was preincubated at 85 ℃ in the presence of Zn2+. Notably, the enzyme preincubated 25 min at 85 ℃ in the presence of Zn2+ prefers fructose conversion and ceased the sorbitol conversion. We identified the presence of a structural Zn2+ binding site in SDHSIUC1 in addition to a catalytic Zn2+ binding site. We speculated that the structural Zn2+ involves thermal stability of the enzyme. Hence, we mutated the cysteine with serine of potential structural Zn2+ binding site (Cys96, Cys99, Cys102, and Cys110). Indeed, the Inductively coupled plasma mass spectrometry (ICP-MS) analysis revealed the mutated enzyme contains a lower amount of Zn2+ relative to the native enzyme. The data revealed that the mutated enzyme has low melting temperature (78 ℃) relative to the native enzyme (92 ℃), suggesting that structural Zn2+ is key to enhance the thermal stability of the SDHSIUC1. Surprisingly, we observed that the mutant enzyme completely lost its activity. The data suggests the role of structural Zn2+ binding site on both the structural and functional stability of SDHSIUC1. In consistence with the in-vitro data, the preliminary computational modeling data suggest that the losing structural Zn2+ unstable the enzyme and we are conducting in depth in-silico study to unveil the mechanism(s). We envisioned that the mechanisms behind the thermal stability of SDHSIUC1 could be used as basic model to enhance thermostable protein for the industrial application (e.g., design thermostable plastic hydrolyzing enzymes). To further demonstrate the potential applications of the SDHSIUC1, we genome-integrated it into the industrially important microorganism Pseudomonas putida KT2440. The resulting strain exhibited significantly increased growth in the presence of sorbitol compared to the wild-type P. putida KT2440, highlighting the potential of this enzyme for industrial applications such as enabling sorbitol catabolism or establishing xylose reductase pathway in P. putida KT2440 (i.e., leverage xylitol dehydrogenase activity of SDHSIUC1). In summary, this study has uncovered a novel thermostable sorbitol dehydrogenase from a subsurface microbe, which could have potential applications in the bioindustry where thermostable sorbitol dehydrogenases are required for the application in food and beverage industry, pharmaceutical industry, biofuel production etc. as it would be advantageous for the industrial processes.

No description available.

The GADD45 family of proteins consists of three small nuclear proteins, GADD45A, GADD45B, and GADD45G, which are implicated in modulating the cellular response to various types of genotoxic/physiological stress. This family of proteins has been shown to interact with and modulate the function of cell-cycle control proteins, such as p21 and cdc2/cyclin B1, the DNA repair protein, PCNA, key stress response MAP kinases, including MEKK4 (an upstream regulator of JNK kinase), and p38 kinase. Despite similarities in amino acid sequence, structure and function, each gadd45 gene is induced differentially, depending on the type of stress stimuli. For example, the alkylating agent, methylmethane sulfonate (MMS), rapidly induces all three genes, whereas hydrogen peroxide and sorbitol preferentially induce gadd45a and gadd45b, respectively. Studies of the mechanisms of the stress-mediated induction of the gadd45 genes have predominantly focused on gadd45a, with knowledge of gadd45b and gadd45g regulation lacking. Thus, in order to generate a more complete understanding of the collective regulation of the gadd45 genes, a comprehensive analysis of the stress-mediated induction of gadd45b has been carried out. Towards this end, a gadd45b promoter-reporter construct was generated, consisting of 3897bp sequence upstream of the transcription start site of gadd45b, fused to a luciferase reporter. In a human colorectal carcinoma cell line (RKO), in which gadd45b mRNA levels profoundly increase by various stress stimuli, we observe similar, high levels of induction of the gadd45b-luciferase construct with MMS or UVC treatments, but surprisingly not with sorbitol or anisomycin. Linker-scanning mutagenesis of the gadd45b promoter reveals several important MMS and UVC cis-acting responsive elements contained within the proximal promoter, including a GC-rich region and the CCAAT box. Furthermore, we have identified three constitutively bound transcription factors, Sp1, MZF1, and NFY, and one inducible factor, Egr1, which bind to these regions and which contribute to MMS-responsiveness. In contrast, a post-transcriptional mechanism appears to regulate gadd45b induction upon sorbitol treatment, as this treatment increases the gadd45b mRNA half-life, compared to MMS treatment. Interestingly, with the exception of a common cis-element, the stress-mediated induction of gadd45b appears to be mechanistically distinct from gadd45a. In conclusion, this study provides novel evidence that gadd45b induction by distinct stress agents, MMS and sorbitol, is regulated differentially at the level of mRNA transcription or mRNA stability, respectively. / Molecular Biology and Genetics