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Made available in DSpace on 2014-06-11T19:27:22Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-03-29Bitstream added on 2014-06-13T19:35:22Z : No. of bitstreams: 1 sato_vs_me_jabo.pdf: 522350 bytes, checksum: 34c42a9a6f174fa4f2002e3d48b07c50 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / A capacidade de bactérias endofíticas em solubilizar fosfato inorgânico é alvo de grande interesse por parte dos microbiologistas, uma vez que as fosfatases são responsáveis por hidrolisar compostos orgânicos produzindo fósforo solúvel. Dessa forma, a fosfatase ácida ligada à membrana (MBAP) foi obtida a partir de Enterobacter sp. isolada de raízes de Cattleya walkeriana (Orchidaceae) e identificada pelo seqüenciamento do gene 16S rRNA. A expressão da enzima mostrou-se estritamente regulada pelo fósforo (expressão ideal em 7 mm). O pH ótimo aparente (3,5) não foi afetado pela concentração de p-nitrofenilfosfato. Em pH 3,5, a enzima é uma fosfomonidrolase inespecífica capaz de hidrolisar os substratos PNPP (61,2 U/mg), ATP (19,7 U/mg), e o pirofosfato (29,7 U/mg), com K0.5 de 0,06 mM, 0,11 mM e 0,08 mM, respectivamente. A enzima exibi cinética Michaelina para o pNPP (n=1,2). Para o ATP e o pirofosfato interações sítio-sítio foram observadas com n=1,6 e 2,3, respectivamente. Os íons de magnésio foram potentes estimuladores (K0.5=2,2 mM), enquanto o arsenato e o fosfato foram potentes inibidores competitivos. A atividade PNPPase foi inibida pelo EDTA, mas não pelo cálcio, levamisol, zinco, cobalto e phidroximercuribenzoato. A entalpia de inativação térmica foi da ordem de 77,5 kcal.mol- 1. Os resultados sugerem que a produção da fosfatase ácida ligada à membrana representa um mecanismo de solubilização do fosfato mineral aumentando a disponibilidade de nutrientes para as plantas / The ability of endophytic bacteria in solubilizing inorganic phosphate is of great interest by microbiologists since phosphatases are responsible for catalyzing the hydrolysis of organic compounds producing soluble phosphorus. Thus, the membranebound acid phosphatase (MBAP) was obtained from Enterobacter sp. isolated from Cattleya walkeriana (Orchidaceae) roots and identified by the 16S rRNA gene sequencing analysis. The enzyme expression was demonstrated to be strictly regulated by phosphorus (optimal expression at 7 mM). The enzyme was obtained by centrifugation at 100.000g for 1 h at 4ºC. The apparent optimal pH (3.5) was not affect by p-Nitrophenyl phosphate concentration. At pH 3.5, the enzyme showed a broad substrate specificity hydrolyzing different substrates such as PNPP (61.2 U/mg), ATP (19.7 U/mg), and pyrophosphate (29.7 U/mg), with K0.5 values of 0.06 mM, 0.11 mM and 0.08 mM, respectively. The hydrolysis of PNPP by the enzyme exhibited Michaelian kinetics with n= 1.2. For ATP and pyrophosphate site-site interactions were observed with n= 1.6 and 2.3, respectively. Although magnesium ions were stimulatory (K0.5= 2.2 mM), arsenate and phosphate were a powerful competitive inhibitor. The PNPPase activity was inhibited EDTA but not by calcium, levamisole, zinc, cobalt and phydroxymercurybenzoate. The ΔH for thermal inactivation was 77.5 kcal.mol-1. Our results suggest that the production of a membrane-bound acid phosphatase might be one mechanism of mineral phosphate solubilization turn it´s nutrients availability to plants

Vakuoläre PPasen (V-PPase) in Landpflanzen dienen dem Transport von Protonen in die Vakuole und dem Aufbau eines elektrochemischen Gradienten, während sie gleichzeitig durch Hydrolyse eine Anreicherung des toxischen PPi im Cytosol verhindern. Zahlreiche Publikationen bewiesen bereits positive Effekte der stabilen V-PPase-Überexpression in Pflanzen. Unter anderem zeigte die Ackerschmalwand, Tabak, Reis und Tomate eine erhöhte Biomasse und gesteigerte Stresstoleranz auf Grund einer erhöhten stabilen V-PPase Ex-pression. Um die zugrundeliegenden Prozesse ohne potenzielle pleiotropische Effekte während der Pflanzenentwicklung zu analysieren, wurden in der vorliegenden Dissertation die physiologischen Auswirkungen einer transienten V-PPase-Überexpression in Nicotiona benthamiana Blättern und die Einflussnahme von NaCl quantitativ erfasst. Zu diesem Zweck wurden zwei endogene V-PPasen (NbVHP1 und NbVHP2) aus N. bentha-miana zunächst bioinformatisch und dann auf Transkriptionsebene mittels quantitativer Real-Time-PCR identifiziert. Die endogenen V-PPasen wurden mittels der Agrobakterien-Infiltrationstechnik transient in N. benthamiana Blättern und ihre vakuoläre Lokalisation mit Hilfe von Fluoreszenzmarkern bestätigt. Die Protonenpump-Funktion der überexprimierten NbVHPs konnte mit der Patch-Clamp-Technik anhand des vier-fach erhöhten Protonenpump-stroms in den isolierten Mesophyllvakuolen verifiziert werden. Im Zuge der elektro-physiologischen Charakterisierung der endogenen N. benthamiana V-PPasen konnte die für V-PPasen typische Sensitivität gegenüber cytosolischem Calcium bestätigt werden, welche sich bei einem erhöhten Calcium-Spiegel in einer Hemmung der Pumpströme äußerte. Ferner wurde ihre gleichartige Substrataffinität (Km von 65 µM PPi) unabhängig des vakuolären pHs zwischen 5,5 und 7,5 festgestellt. Der Vergleich dieser Ergebnisse mit analog durchgeführten Messungen an der bereits publizierten AtVHP1 von A. thaliana bestätigte die große Homo-logie der V-PPasen von Landpflanzen. Im Gegensatz zu den erwünschten Auswirkungen der stabilen V-PPase Überexpression resultierte diese starke transiente Überexpression nach drei Tagen im Absterben makroskopischer Blattbereiche. Das Ausmaß dieser Nekrosen wurde anhand des vorhandenen PhotosystemII in den transformierten Blättern mit der Puls-Amplituden-Modulations-Technik quantifiziert. Die analoge transiente Überexpression einer löslichen PPase (IPP1) führte allerdings zu keinerlei negativen Effekten für die Pflanze, wodurch die erhöhte Protonentransportaktivität im Gegensatz zur Hydrolyseaktivität der V-PPasen als Ursache des Zellsterbens verifiziert werden konnte. Aufgrund dieser unerwarteten negativen Auswirkungen der transienten V-PPase-Überex-pression auf die Blattvitalität wurde zusätzlich die Salzstresstoleranz der Blätter untersucht. Unter Berücksichtigung des kurzen Transformations- und damit Beobachtungszeitfensters wurde ein Salzapplikationsverfahren etabliert, bei dem simultan mit der Agrobakterien-infiltration 200 mM NaCl direkt in den Blattapoplasten eingeführt wurde. Anhand einer Zu-nahme in sowohl der Transskriptmenge der V-PPase als auch des PPi-induzierten Protonen-pumptransportes über den Tonoplasten wurde gezeigt, dass die NaCl-Anwesenheit im Blatt eine erhöhte Aktivität der endogenen V-PPasen des N. benthaminan Pflanzen bewirkte. Der gleichzeitige tendenzielle Rückgang der V-ATPase-Pumpaktivität in salzbehandelten Mesophyllvakuolen lässt vermuten, dass die V-PPasen eine größere Rolle bei der Bewahrung des vakuolären pH-Wertes und der protonenmotorische Kraft (PMF) unter Salzstress ein-nimmt. Interessanterweise führte die Salzapplikation bei einer V-PPase-Überexpression zu keinen additiven negativen Effekten, sondern verhinderte sogar das Auftreten der Nekrosen. Um dieses Phänomen zu ergründen, wurde zunächst mit Hilfe von Apoplastenwaschungen und Natrium-Konzentrationsmessungen bestätigt, dass das injizierte NaCl im Blatt verblieb und von den Blattzellen aufgenommen wurde. Für weitere Studien der Ursachen der Nekrosen wurden in-vivo-pH-, Membranpotenzial- und Metabolitmessungen durchgeführt. Während in V-PPase-überexprimierenden Zellen der vakuoläre pH-Wert zu Kontrollvakuolen signifikant sank, blieb er mit zusätzlicher Salzbehandlung auf Kontrollniveau. Des Weiteren schwächte die Salzapplikation die starke Depolarisation der Plasmamembran nach V-PPase-Über-expression um mehr als die Hälfte ab. Hingegen konnten keine nennenswerten Ver-änderungen im Metabolit- und Ionengehalt des Blattgewebes bei V-PPase-Überexpression festgestellt werden. Lediglich der Natrium- und Chlorid-Spiegel waren bei salz-behandelten Blättern erwartungsgemäß erhöht. Diese Ergebnisse bekräftigten, dass der stark erhöhte V-PPase-vermittelte Protonenpumpstrom und weniger metabolische Veränderungen für die Nekrosen von V-PPase-überexprimierte Pflanzen verantwortlich ist. Diese negativen Auswirkungen werden offensichtlich durch die Salzbehandlung stark vermindert, da die Aufnahme der Salz-ionen über Protonen-Na+/K+-Antiporter wie NHX antagonistisch auf die V-PPase verursachte Protonenanreicherung und die daraus folgende Veränderung des Membran-potentials und der PMF entgegenwirkt. In diese Arbeit wurde in einem neuen Blickwinkel deutlich, dass die natürliche Expressions-kontrolle der V-PPase in ausdifferenzierten Pflanzenzellen sich den Umweltbedingungen anpasst, um das Gleich-gewicht zwischen den positiven und negativen Auswirkungen der Pumpaktivität zu halten. / The plant vacuolar PPases (V-PPase) transport protons across the tonoplast from the cytosol into the vacuole. This establishes a trans-tonoplast electrochemical gradient and prevents the accumulation of toxic PPi in the cytosol. Many publications have already shown positive effects of stable V-PPase overexpression in plants. Arabidopsis, tobacco, rice and tomato for example, all showed increased biomass and greater stress tolerance with stable V-PPase overexpression. To understand the underlying processes without potential pleiotropic effects during plant development, we characterised V-PPase in Nicotiana benthamiana leaves using an electrophysiological approach, and investigated its physiology in overexpressing plants, concentrating on its regulation by NaCl. Two endogenous V-PPases (NbVHP1 and NbVHP2) from N. benthamiana were identified using bioinformatics and quantified with real-time PCR. Endogenous V-PPases were transiently overexpressed using Agrobacteria in N. benthamiana leaves, with their vacuolar localisation confirmed by fluorescence labelling. The proton pump activity of the NbVHPs was verified by a fourfold increase in the patch clamp-recorded proton current response of isolated mesophyll vacuoles. For the electrophysiological characterisation of the endogenous V-PPases, the V-PPase-typical cytosolic calcium sensitivity was confirmed; the proton pump became inhibited with increased calcium levels. Furthermore, their similar substrate affinity, a Km of 65 μM PPi, was found to be independent of the vacuolar pH between 5.5 and 7.5. Both attributes were comparable to the already published AtVHP1 of Arabidopsis thaliana, showing a high homology of plant V-PPases. The physiological effects of V-PPase overexpression was obvious from the time-delayed death of transformed cells. In contrast to the desired effects of stable V-PPase over-expression, this strong transient overexpression resulted in macroscopic dead leaf areas after three days. The extent of these necroses was quantified by counting the existing photosystems in the transformed leaves using the pulse-amplitude modulation technique (PAM). In contrast, no necrosis was found in plants where a soluble PPase (IPP1) was over-expressed. This supports the hypothesis that the increased proton transport activity of V-PPases, not the hydrolysis activity, is the cause of cell death. Because of these unexpected negative effects of transient V-PPase over-expression on leaf vitality, the salt stress tolerance of the leaves was also examined. The short transformation and observation time window meant we needed to establish a new way of salt application; we injected 200 mM NaCl directly into the leaf apoplast simultaneously with the agrobacterial material. Absorption of NaCl by leaf cells was confirmed by apoplast washing and sodium concentration measurements. The NaCl treatment increased the amount of transcripts of endogenous V-PPases in tobacco and increased the PPi-induced proton transport across the vacuolar membrane. The concomitant decline in V-ATPase pumping activity in saline-treated mesophyll vacuoles indicates that V-PPases have a significant role in maintaining the vacuolar pH and proton motive force (PMF) under saline conditions. Interestingly, simultaneous salt administration with V-PPase overexpression did not result in an additive negative effect but did prevent the occurrence of necrotic leaf areas. For further analysis of the necrosis, in vivo pH, membrane potential and metabolite measurements were performed. Under V-PPasen over-expression and salt treatment, the vacuolar pH remained constant and did not decrease as it did in non-salt-treated V-PPase over-expressing cells. Furthermore, the salt application attenuated the strong depolarisation of the plasma membrane by more than a half in V-PPase overexpressing plants, but no noteworthy changes in cell metabolism could be detected by metabolite and ion concentration measurements. Only the Na+ and Cl- levels were, as expected, increased in salt-treated leaves. This decrease in vacuolar pH alongside a membrane depolarisation confirms that the strong proton pump current of the V-PPase overexpressing vacuoles is the cause of the observed necrosis. These negative effects are obviously greatly reduced by salt treatment, since the uptake of the salt ions via proton:Na+/K+ antiporters such as NHX acts as an antagonist to V-PPase-induced proton accumulation in the vacuole and the consequent change in membrane potential and PMF. This work reveals in a new perspective, that the natural expression control of V-PPase in differentiated plant cells adapts to environmental conditions to balance the positive and negative effects of pumping activity.

The enzyme inosine triphosphate pyrophosphatase (ITPase) is responsible for controlling the levels of the by-products guanosine monophosphate (GMP) and adenosine monophosphate (AMP) through their precursor inosine monophosphate (IMP). ). Human ITPase consists of a 194-amino acid homodimer which relies upon either an Mg2+ ion or a Mn2+ ion for catalytic activity, and orthologs of this protein have been found in many different organisms. The purpose of this project was to try out methods learned throughout the education and to use this knowledge to gather new data about the human protein inosine triphosphate pyrophosphatase (ITPase). The protein was expressed in BL21/DE3 cells from a pre-made vector. Experiments performed during this project include secondary- and tertiary stability measurements, tryptophan fluorescence spectra, binding curve and thermic stability to ITPase with ANS and methotrexate. The Tm-value of human ITPase was examined with Trp-Fluorescence, ANS-fluorescence and Near-UV and Far-UV circular dichroism (CD). The stability of ITPase monitored by Near-UV as well as Far-UV coincides, indicating that secondary- and tertiary-unfolding occur simultaneously without any intermediates. The results of Trp-fluorescence showed that the tryptophans were already exposed and thus it did not yield a reliable result. The binding properties of ANS and MTX to ITPase were also examined.

The human patogen Streptococcus pneumoniae encodes a single copy of eukaryotic-like Ser/Thr protein kinase StkP. StkP regulates virulence, competence, stress resistence, gene expression and plays a role in the regulation of cell division cycle. Analysis of phosphoproteome maps of the wild type and stkP mutant strain of S. pneumoniae showed that in vivo StkP phosphorylates several putative substrates including Mn-dependent inorganic pyrophosphatase PpaC. Mass spectrometry analysis identified two phosphorylation sites in an active site of the protein. Pyrophosphatases are essential enzymes that catalyze hydrolysis of inorganic pyrophosphate produced during various biosynthetic reactions that utilize ATP. Changes in pyrophosphatase activity have been described to have global effects on cell metabolism, growth and division of bacteria. The aim of this thesis was to investigate the phosphorylation of inorganic pyrophosphatase PpaC in S. pneumoniae. Gene ppaC was cloned, expressed in E. coli and protein was purified via affinity chromatography. Phosphorylation of PpaC by StkP was examined in a kinase assay but we did not confirm that PpaC is a direct substrate of StkP in vitro. Further we prepared a set of mutants in ppaC gene. We replaced two presumable phosphoaminoacids identified by mass-spectrometry with...

Resumo: A capacidade de bactérias endofíticas em solubilizar fosfato inorgânico é alvo de grande interesse por parte dos microbiologistas, uma vez que as fosfatases são responsáveis por hidrolisar compostos orgânicos produzindo fósforo solúvel. Dessa forma, a fosfatase ácida ligada à membrana (MBAP) foi obtida a partir de Enterobacter sp. isolada de raízes de Cattleya walkeriana (Orchidaceae) e identificada pelo seqüenciamento do gene 16S rRNA. A expressão da enzima mostrou-se estritamente regulada pelo fósforo (expressão ideal em 7 mm). O pH ótimo aparente (3,5) não foi afetado pela concentração de p-nitrofenilfosfato. Em pH 3,5, a enzima é uma fosfomonidrolase inespecífica capaz de hidrolisar os substratos PNPP (61,2 U/mg), ATP (19,7 U/mg), e o pirofosfato (29,7 U/mg), com K0.5 de 0,06 mM, 0,11 mM e 0,08 mM, respectivamente. A enzima exibi cinética "Michaelina" para o pNPP (n=1,2). Para o ATP e o pirofosfato interações sítio-sítio foram observadas com n=1,6 e 2,3, respectivamente. Os íons de magnésio foram potentes estimuladores (K0.5=2,2 mM), enquanto o arsenato e o fosfato foram potentes inibidores competitivos. A atividade PNPPase foi inibida pelo EDTA, mas não pelo cálcio, levamisol, zinco, cobalto e phidroximercuribenzoato. A entalpia de inativação térmica foi da ordem de 77,5 kcal.mol- 1. Os resultados sugerem que a produção da fosfatase ácida ligada à membrana representa um mecanismo de solubilização do fosfato mineral aumentando a disponibilidade de nutrientes para as plantas / Abstract: The ability of endophytic bacteria in solubilizing inorganic phosphate is of great interest by microbiologists since phosphatases are responsible for catalyzing the hydrolysis of organic compounds producing soluble phosphorus. Thus, the membranebound acid phosphatase (MBAP) was obtained from Enterobacter sp. isolated from Cattleya walkeriana (Orchidaceae) roots and identified by the 16S rRNA gene sequencing analysis. The enzyme expression was demonstrated to be strictly regulated by phosphorus (optimal expression at 7 mM). The enzyme was obtained by centrifugation at 100.000g for 1 h at 4ºC. The apparent optimal pH (3.5) was not affect by p-Nitrophenyl phosphate concentration. At pH 3.5, the enzyme showed a broad substrate specificity hydrolyzing different substrates such as PNPP (61.2 U/mg), ATP (19.7 U/mg), and pyrophosphate (29.7 U/mg), with K0.5 values of 0.06 mM, 0.11 mM and 0.08 mM, respectively. The hydrolysis of PNPP by the enzyme exhibited "Michaelian" kinetics with n= 1.2. For ATP and pyrophosphate site-site interactions were observed with n= 1.6 and 2.3, respectively. Although magnesium ions were stimulatory (K0.5= 2.2 mM), arsenate and phosphate were a powerful competitive inhibitor. The PNPPase activity was inhibited EDTA but not by calcium, levamisole, zinc, cobalt and phydroxymercurybenzoate. The ΔH for thermal inactivation was 77.5 kcal.mol-1. Our results suggest that the production of a membrane-bound acid phosphatase might be one mechanism of mineral phosphate solubilization turn it's nutrients availability to plants / Orientador: João Martins Pizauro Junior / Coorientador: Cecília Maria Costa do Amaral / Banca: Mariana Carina Frigieri / Banca: Jesus Aparecido Ferro / Mestre

abstract: The remarkable conservation of molecular and intra-/inter-cellular pathways underpinning the fundamental aspects of sugar partitioning in two evolutionarily divergent organisms – a non-vascular moss Physcomitrella patens and a vascular cereal crop Oryza sativa (rice) – forms the basis of this manuscript. Much of our current knowledge pertaining to sugar partitioning in plants mainly comes from studies in thale cress, Arabidopsis thaliana, but how photosynthetic sugar is loaded into the phloem in a crop as important as rice is still debated. Even less is known about the mechanistic aspects of sugar movement in mosses. In plants, sugar either moves passively via intercellular channels called plasmodesmata, or through the cell wall spaces in an energy-consuming process. As such, I first investigated the structure of plasmodesmata in rice leaf minor vein using electron tomography to create as of yet unreported 3D models of these channels in both simple and branched conformations. Contrary to generally held belief, I report two different 3D morphotypes of simple plasmodesmata in rice. Furthermore, the complementary body of evidence in arabidopsis implicates plasma membrane localized Proton Pyrophosphatase (H+-PPase) in the energy-dependent movement of sugar. Within this wider purview, I studied the in situ ultrastructural localization patterns of H+-PPase orthologs in high-pressure frozen tissues of rice and physcomitrella. Were H+-PPases neo-functionalized in the vascular tissues of higher plants? Or are there evolutionarily conserved roles of this protein that transcend the phylogenetic diversity of land plants? I show that H+-PPases are distinctly expressed in the actively growing regions of both rice and physcomitrella. As expected, H+-PPases were also localized in the vascular tissues of rice. But surprisingly, H+-PPase orthologs were also prominently expressed at the gametophyte-sporophyte junction of physcomitrella. Upon immunogold labeling, H+-PPases were found to be predominantly localized at the plasma membrane of the phloem complexes of rice source leaves, and both the vacuoles and plasma membrane of the transfer cells in the physcomitrella haustorium, linking H+-PPases in active sucrose loading in both plants. As such, these findings suggest that the localization and presumably the function of H+-PPases are conserved throughout the evolutionary history of land plants. / Dissertation/Thesis / 3D MODEL OF SIMPLE PLASMODESMATA / 3D MODEL OF COMPLEX PLASMODESMATA / MODELING SIMPLE PLASMODESMATA IN IMOD / MODELING COMPLEX PLASMODESMATA IN IMOD / Doctoral Dissertation Biology 2016

Made available in DSpace on 2014-06-11T19:27:22Z (GMT). No. of bitstreams: 0 Previous issue date: 2008-08-11Bitstream added on 2014-06-13T20:56:20Z : No. of bitstreams: 1 rombola_th_me_jabo.pdf: 290913 bytes, checksum: 08f244b9cd35a229582ac7563c4b4ff9 (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Espécies de Burkholderia filogenéticamente distantes do complexo B. cepacia, são versáteis solubilizadores de minerais insolúveis, através da produção de ácidos, viabilizando nutrientes para as plantas. Descrevemos a purificação e caracterização da fosfatase ácida ligada à membrana, produzida pela bactéria do gênero Burkholderia, isolada de solo agrícola em Ponta grossa-PR-BRASIL, e identificada através da análise do 16s rDNA. A expressão da enzima se mostrou estritamente dependente ao fósforo (expressão ótima a 5 mM). A enzima ligada à membrana foi purificada por ultracentrifugação a 100.000g por 1 hora a 4°C e testada com atividade enzimática para p-nitrofenilfosfato (PNFF) e Pirofosfato. O pH ótimo da enzima foi 6 e não foi afetado pela concentração de PNFF. Em pH 6 a hidrolise do PNFF seguiu os seguintes parâmetros cinéticos com n=1.5, Vm= 103.8 U.mg-1 e K0,5=0,06 mM em uma faixa de 0,003 e 10 mM de PNFF. Estudos do pH sobre os parâmetros cinéticos demonstraram 26 pontos de variação na faixa de 3,5 a 6 e em seguida diminui até pH 8 enquanto K0,5 não mostrou variação nesta faixa. O H para hidrólise do PNFF foi de 5,74 kcal.mol-1. A atividade PNFFase foi inibida pelo arsenato e Cálcio, mas não por levamisol, EDTA, zinco e cobalto. A hidrolise do pirofosfato na presença de 2 mM de MgCl2 seguiu os parâmetros cinéticos de Michaelis-Menten” com Km= 0.142 mM e Vm= 237 U.mg-1.Os resultados sugerem que a produção da fosfatase ácida ligada à membrana pode ser um mecanismo de solubilização de fosfato mineral por essa bactéria. / Burkholderia species phylogenetically greatly distant from the B. cepacia complex species are versatile organisms that solubilize insoluble minerals through the production of acids, increasing the availability of nutrients to plants. Here we describe the purification and characterization a membrane-bound acid phosphatase produced by bulkholderia isolated from an agricultural soil in Ponta grossa-PR-BRASIL and identified through analysis of the 16S rDNA gene. The expression of this enzyme was demonstrated to be strictly regulated by phosphorus (optimal expression at 5 mM). The membrane bound enzyme was purified by centrifugation at 100.000g for 1 h at 4ºC and the enzyme was assayed for p-Nitrophenyl phosphatase (PNPPase) and pyrophosphatase activities. The optimal pH (6.0) was not affect by p-Nitrophenyl phosphate concentration. At pH 6.0 the hydrolysis of PNPP following a cooperative kinetics with n= 1.5, Vm= 103.8 U.mg-1 and K0.5= 0.60 mM in the range between 0.003 and 10 mM. Studies of pH effects on kinetics parameters revealed a 26-folds variation in the range of pH 3.5 to 6 following decreases until pH 8.0, wile K0,5 and cooperativity did not varied considerably in this range. The H for hydrolysis of PNPP was 5,74 kcal.mol-1. PNPPase activity was inhibited by arsenate, phosphate and calcium, but not for levamisole, EDTA, zinc, magnesium and cobalt. The hydrolysis of pyrophosphate in presence of 2 mM MgCl2 follows Michaelis-Menten” kinetics with Km= 0.142 mM and Vm= 237 U.mg-1. Our results suggesting that the production of membranebound acid phosphatase might be one mechanism of mineral phosphate solubilization by this genus of Bacterium.

Orientador: João Martins Pizauro Junior / Banca: Maria Inês Tiraboschi Ferro / Banca: Pietro Ciancaglini / Resumo: Espécies de Burkholderia filogenéticamente distantes do complexo B. cepacia, são versáteis solubilizadores de minerais insolúveis, através da produção de ácidos, viabilizando nutrientes para as plantas. Descrevemos a purificação e caracterização da fosfatase ácida ligada à membrana, produzida pela bactéria do gênero Burkholderia, isolada de solo agrícola em Ponta grossa-PR-BRASIL, e identificada através da análise do 16s rDNA. A expressão da enzima se mostrou estritamente dependente ao fósforo (expressão ótima a 5 mM). A enzima ligada à membrana foi purificada por ultracentrifugação a 100.000g por 1 hora a 4°C e testada com atividade enzimática para p-nitrofenilfosfato (PNFF) e Pirofosfato. O pH ótimo da enzima foi 6 e não foi afetado pela concentração de PNFF. Em pH 6 a hidrolise do PNFF seguiu os seguintes parâmetros cinéticos com n=1.5, Vm= 103.8 U.mg-1 e K0,5=0,06 mM em uma faixa de 0,003 e 10 mM de PNFF. Estudos do pH sobre os parâmetros cinéticos demonstraram 26 pontos de variação na faixa de 3,5 a 6 e em seguida diminui até pH 8 enquanto K0,5 não mostrou variação nesta faixa. O H para hidrólise do PNFF foi de 5,74 kcal.mol-1. A atividade PNFFase foi inibida pelo arsenato e Cálcio, mas não por levamisol, EDTA, zinco e cobalto. A hidrolise do pirofosfato na presença de 2 mM de MgCl2 seguiu os parâmetros cinéticos de "Michaelis-Menten" com Km= 0.142 mM e Vm= 237 U.mg-1.Os resultados sugerem que a produção da fosfatase ácida ligada à membrana pode ser um mecanismo de solubilização de fosfato mineral por essa bactéria. / Abstract: Burkholderia species phylogenetically greatly distant from the B. cepacia complex species are versatile organisms that solubilize insoluble minerals through the production of acids, increasing the availability of nutrients to plants. Here we describe the purification and characterization a membrane-bound acid phosphatase produced by bulkholderia isolated from an agricultural soil in Ponta grossa-PR-BRASIL and identified through analysis of the 16S rDNA gene. The expression of this enzyme was demonstrated to be strictly regulated by phosphorus (optimal expression at 5 mM). The membrane bound enzyme was purified by centrifugation at 100.000g for 1 h at 4ºC and the enzyme was assayed for p-Nitrophenyl phosphatase (PNPPase) and pyrophosphatase activities. The optimal pH (6.0) was not affect by p-Nitrophenyl phosphate concentration. At pH 6.0 the hydrolysis of PNPP following a cooperative kinetics with n= 1.5, Vm= 103.8 U.mg-1 and K0.5= 0.60 mM in the range between 0.003 and 10 mM. Studies of pH effects on kinetics parameters revealed a 26-folds variation in the range of pH 3.5 to 6 following decreases until pH 8.0, wile K0,5 and cooperativity did not varied considerably in this range. The H for hydrolysis of PNPP was 5,74 kcal.mol-1. PNPPase activity was inhibited by arsenate, phosphate and calcium, but not for levamisole, EDTA, zinc, magnesium and cobalt. The hydrolysis of pyrophosphate in presence of 2 mM MgCl2 follows "Michaelis-Menten" kinetics with Km= 0.142 mM and Vm= 237 U.mg-1. Our results suggesting that the production of membranebound acid phosphatase might be one mechanism of mineral phosphate solubilization by this genus of Bacterium. / Mestre