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11	Electrophoretic Evidence of Esterase Inhibition in Larval Caddisflies Exposed to Inorganic Mercury Benton, Michael J., Guttman, Sheldon I. 01 January 1997 (has links) In an previous 72-hr mercury bioassay with the larval caddisfly Nectopsyche albida, electrophoretically detectable esterase activity was absent in exposed individuals that succumbed to mercury toxicity, while nine other enzymes remained active hours after death. Esterase activity also persisted in unexposed individuals (Benton and Guttman, 1992a.b). To test the effects of mercury exposure duration on esterase activity, additional larval N. albida were exposed under conditions identical to those in the earlier bioassay, and esterase activity in live individuals was tested electrophoretically every 12 hr. To test the effects of mercury concentration on esterase activity, unexposed N. albida larvae were electrophoresed, and the esterase-specific stain was spiked with various concentrations of mercury. Electrophoretic banding patterns were then densitometrically quantified to identity changes in esterase activity with exposure duration and mercury concentration. Results suggest that: inorganic mercury inhibited esterase activity in N. albida, inhibition increased with exposure duration, and inhibition increased with mercury concentration. electrophoresis esterase inhibition mercury trichoptera
12	Functional characterisation of a novel ferulic acid esterase from Malawian hot spring metagenome Ngobeni, Rhulani January 2011 (has links) <p><!--[if gte mso 9]><xml> <o:OfficeDocumentSettings> <o:AllowPNG /> </o:OfficeDocumentSettings> </xml><![endif]--><!--[if gte mso 9]><xml> <w:WordDocument> <w:View>Normal</w:View> <w:Zoom>0</w:Zoom> <w:TrackMoves /> <w:TrackFormatting /> <w:PunctuationKerning /> <w:ValidateAgainstSchemas /> <w:SaveIfXMLInvalid>false</w:SaveIfXMLInvalid> <w:IgnoreMixedContent>false</w:IgnoreMixedContent> <w:AlwaysShowPlaceholderText>false</w:AlwaysShowPlaceholderText> <w:DoNotPromoteQF /> <w:LidThemeOther>EN-ZA</w:LidThemeOther> <w:LidThemeAsian>X-NONE</w:LidThemeAsian> <w:LidThemeComplexScript>X-NONE</w:LidThemeComplexScript> <w:Compatibility> <w:BreakWrappedTables /> <w:SnapToGridInCell /> <w:WrapTextWithPunct /> <w:UseAsianBreakRules /> <w:DontGrowAutofit /> <w:SplitPgBreakAndParaMark /> <w:EnableOpenTypeKerning /> <w:DontFlipMirrorIndents /> 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,&quot / sans-serif&quot / ">There has been a decline in the global fossil fuel reserves, due to an increasing demand for petroleum. Biofuels can be used as an alternative source of energy whereby biomass is converted to liquid fuels such as bioethanol. There is considerable interest in lipolytic enzymes because of their broad substrate range and for this purpose these enzymes have potential in a variety of biotechnological application. Lipolytic enzymes include esterases (E.C 3.1.1.1) and lipases (E.C3.1.1.3). Esterases preferentially hydrolyse short chain (&lt / C10) ester-containing molecules that are partly soluble in water, while lipases hydrolyse a broad range of substrates preferably water-insoluble fatty acyl molecules (&gt / C10). The aim of this study was to express, purify and characterise the lipolytic enzyme present on afosmid, Try 11, previously isolated from a metagenomic library of a Malawian hotspring, which conferred activity on tributyrin and ethyl ferulate. Bioinformatic analysisof the fosmid insert sequence predicted an open reading frame consisting of 951 bp,designated RHgene34, encoding a 317 amino acid protein with 41 % similarity to the</span><span style="font-size: 10.0pt / font-family:&quot / Arial&quot / ,&quot / sans-serif&quot / mso-fareast-font-family:ArialOOEnc">&alpha / /&beta / hydrolase fold-3 domain protein of </span><i><span style="font-size:10.0pt / font-family:&quot / Arial&quot / ,&quot / sans-serif&quot / ">Burkholderia </span></i><span style="font-size:10.0pt / font-family:&quot / Arial&quot / ,&quot / sans-serif&quot / ">sp. The RHgene34 protein </span><span style="font-size:10.0pt / font-family:&quot / Arial&quot / ,&quot / sans-serif&quot / ">contains conserved motifs of esterases/lipases, such as HGGG (residues 95-98),GxSxG (residues 167 - 171) and the putative catalytic triad composed of Ser157,Asp255 and His285. The gene was cloned and expressed in pET21a(+), and transformed into <i>Escherichia coli </i>Rosetta. <i>p</i>-Nitrophenol (<i>p</i>-Np) fatty acyl esters of different carbon chain lengths were used for kinetic characterisation of RHgene34. </span><span style="font-size:10.0pt / font-family:&quot / Arial&quot / ,&quot / sans-serif&quot / ">Kinetic analysis revealed that RHgene34 had a broad range activity on the <i>p</i>-Npesters, from C2 - C14. RHgene34 operates optimally at 45 &ordm / C, pH 9.0 and has a half life of 30 mins at 45 &ordm / C. This study demonstrates that functional screening combined with the sequence analysis is a useful approach for isolating novel enzymes from ametagenome.</span></p> <p style="margin-bottom:0cm / margin-bottom:.0001pt / text-align: justify / line-height:normal / mso-layout-grid-align:none / text-autospace:none" class="MsoNormal"><span style="font-size:12.0pt / font-family:&quot / Arial&quot / ,&quot / sans-serif&quot / "><br type="_moz" /> </span></p> <br /> <p style="margin-bottom:0cm / margin-bottom:.0001pt / text-align: justify / line-height:normal / mso-layout-grid-align:none / text-autospace:none" class="MsoNormal"><span style="font-size:12.0pt / font-family:&quot / Arial&quot / ,&quot / sans-serif&quot / "><br type="_moz" /> </span></p> <br />
13	Functional characterisation of a novel ferulic acid esterase from Malawian hot spring metagenome Ngobeni, Rhulani January 2011 (has links) <p><!--[if gte mso 9]><xml> <o:OfficeDocumentSettings> <o:AllowPNG /> </o:OfficeDocumentSettings> </xml><![endif]--><!--[if gte mso 9]><xml> <w:WordDocument> <w:View>Normal</w:View> <w:Zoom>0</w:Zoom> <w:TrackMoves /> <w:TrackFormatting /> <w:PunctuationKerning /> <w:ValidateAgainstSchemas /> <w:SaveIfXMLInvalid>false</w:SaveIfXMLInvalid> <w:IgnoreMixedContent>false</w:IgnoreMixedContent> <w:AlwaysShowPlaceholderText>false</w:AlwaysShowPlaceholderText> <w:DoNotPromoteQF /> <w:LidThemeOther>EN-ZA</w:LidThemeOther> <w:LidThemeAsian>X-NONE</w:LidThemeAsian> <w:LidThemeComplexScript>X-NONE</w:LidThemeComplexScript> <w:Compatibility> <w:BreakWrappedTables /> <w:SnapToGridInCell /> <w:WrapTextWithPunct /> <w:UseAsianBreakRules /> <w:DontGrowAutofit /> <w:SplitPgBreakAndParaMark /> <w:EnableOpenTypeKerning /> <w:DontFlipMirrorIndents /> 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,&quot / sans-serif&quot / ">There has been a decline in the global fossil fuel reserves, due to an increasing demand for petroleum. Biofuels can be used as an alternative source of energy whereby biomass is converted to liquid fuels such as bioethanol. There is considerable interest in lipolytic enzymes because of their broad substrate range and for this purpose these enzymes have potential in a variety of biotechnological application. Lipolytic enzymes include esterases (E.C 3.1.1.1) and lipases (E.C3.1.1.3). Esterases preferentially hydrolyse short chain (&lt / C10) ester-containing molecules that are partly soluble in water, while lipases hydrolyse a broad range of substrates preferably water-insoluble fatty acyl molecules (&gt / C10). The aim of this study was to express, purify and characterise the lipolytic enzyme present on afosmid, Try 11, previously isolated from a metagenomic library of a Malawian hotspring, which conferred activity on tributyrin and ethyl ferulate. Bioinformatic analysisof the fosmid insert sequence predicted an open reading frame consisting of 951 bp,designated RHgene34, encoding a 317 amino acid protein with 41 % similarity to the</span><span style="font-size: 10.0pt / font-family:&quot / Arial&quot / ,&quot / sans-serif&quot / mso-fareast-font-family:ArialOOEnc">&alpha / /&beta / hydrolase fold-3 domain protein of </span><i><span style="font-size:10.0pt / font-family:&quot / Arial&quot / ,&quot / sans-serif&quot / ">Burkholderia </span></i><span style="font-size:10.0pt / font-family:&quot / Arial&quot / ,&quot / sans-serif&quot / ">sp. The RHgene34 protein </span><span style="font-size:10.0pt / font-family:&quot / Arial&quot / ,&quot / sans-serif&quot / ">contains conserved motifs of esterases/lipases, such as HGGG (residues 95-98),GxSxG (residues 167 - 171) and the putative catalytic triad composed of Ser157,Asp255 and His285. The gene was cloned and expressed in pET21a(+), and transformed into <i>Escherichia coli </i>Rosetta. <i>p</i>-Nitrophenol (<i>p</i>-Np) fatty acyl esters of different carbon chain lengths were used for kinetic characterisation of RHgene34. </span><span style="font-size:10.0pt / font-family:&quot / Arial&quot / ,&quot / sans-serif&quot / ">Kinetic analysis revealed that RHgene34 had a broad range activity on the <i>p</i>-Npesters, from C2 - C14. RHgene34 operates optimally at 45 &ordm / C, pH 9.0 and has a half life of 30 mins at 45 &ordm / C. This study demonstrates that functional screening combined with the sequence analysis is a useful approach for isolating novel enzymes from ametagenome.</span></p> <p style="margin-bottom:0cm / margin-bottom:.0001pt / text-align: justify / line-height:normal / mso-layout-grid-align:none / text-autospace:none" class="MsoNormal"><span style="font-size:12.0pt / font-family:&quot / Arial&quot / ,&quot / sans-serif&quot / "><br type="_moz" /> </span></p> <br /> <p style="margin-bottom:0cm / margin-bottom:.0001pt / text-align: justify / line-height:normal / mso-layout-grid-align:none / text-autospace:none" class="MsoNormal"><span style="font-size:12.0pt / font-family:&quot / Arial&quot / ,&quot / sans-serif&quot / "><br type="_moz" /> </span></p> <br />
14	Esterases in malting barley Humberstone, Fiona Jane January 2000 (has links) No description available. 664
15	Screening, isolating and characterizing acetyl xylan esterase enzymes from a novel ecological niche Marais, Charl 03 1900 (has links) Thesis (MSc)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: Hemicellulose, a plant cell wall polysaccharide composed amongst other 6-carbon sugars of xylose, a 5-carbon fermentable sugar, has various side-chains which hinder breakdown by the hydrolytic enzyme xylanase. We executed a screen of a metagenomic library established from heat treated saw dust to isolate novel genes for plant cell wall esterases and isolated two clones that potentially could be utilised as acetyl xylan esterases: HEL12 and HEL37 from Citrobacter farmeri and Bacillus vallismortis respectively are confirmed short-chain acetyl xylan esterases (C2-C4) with an optimal temperature of 30°C and 35°C and pH 8.0. HEL12 (33kDa) and HEL37 (25kDa) are small, dextrous acetyl xylan esterases with HEL37: Km of 1.621mM for p-nitrophenyl acetate and Km of 3.571mM for the substrate p-nitrophenyl palmitate with a Vmax of 2.462 mMol/min/mg protein and Vmax of 0.4363 mMol/min/mg protein respectively. In addition HEL12 was active on other substrates with a Km of 1.321mM protein for p-nitrophenyl acetate and Km of 1.692mM for the substrate p-nitrophenyl butyrate with a Vmax of 3.812 and Vmax of 1.523 mMol/min/mg protein respectively. Both enzymes were assayed on various acetylated polymers including acetylated xylan to indicate their ability to hydrolyse plant lignocellulosic polymers. / AFRIKAANSE OPSOMMING: Met die herwinde interisering in die energie krisis, is “herwinbare energie” vinnig besig om die nuwe modewoord te raak tussen verskeie industriële bedrywighede. Hemicellulose, ‘n plant sel wand polisakkaride opgemaak uit xilose, ‘n 5-koolstof fermenteerbare suiker, het verskillende sykettings wat ‘n hindernis veroorsaak in die hidrolase van die komponent deur xilanase ensieme. HEL12 en HEL37 van bakterieë Citrobacter farmeri en Bacillus vallismortis afsonderlik, is deel van die hidrolitiese groep van ensieme wat die hidrolisasie van ester sy-bindings op die xilaan suikerstring kataliseer. Die ensieme was primêr geassesseer vir aktiwiteit deur middel van sintetiese substraat 4-nitrofeniel asetaat. HEL12 en HEL37 was gevind om kort-ketting asetiel xilaan esterase (C2-C4) te wees, met ‘n optimale temperatuur van 30°C en 35°C afsonderlik, beide met ‘n optimale pH van 8.0. Met ‘n geskatte gewig van omtrent +- 30kDa is beide die ensieme van die kleiner meer behendige asetiel xilaan esterase, met HEL37: Km = 1.621 op 4-nitrofeniel asetaat en Km = 3.571 op substraat 4-nitrofeniel palmetaat, en Vmax = 2.462 met ‘n Vmax = 0.4363 afsonderlik, en HEL12: Km = 1.321 op 4-nitrofeniel asetaat en Km = 1.692 op sustraat 4-nitrofeniel buteraat met ‘n Vmax = 3.812 en Vmax = 1.523 afsonderlik. Biofuel production UCTD
16	Efeito citotóxico do sistema HRP/Indóis em células McCoy in vitro / Pereira, Débora Helena. January 2008 (has links) Resumo: A terapia pró-droga/enzima direcionada por anticorpo (ADEPT) consiste em uma primeira etapa , no direcionamento de uma enzima veiculada por anticorpo à uma célula tumoral. Numa segunda etapa uma pró-droga inócua é administrada, e, na presença da enzima, produz compostos citotóxicos restritos à localização do tumor. O par enzima/pró-droga horseradish peroxidase (HRP)/ ácido 3- indol acético (IAA) tem sido aplicada nas estratégias ADEPT. Nesta combinação, o hormônio de planta não tóxico IAA é ativado para espécies citotóxicas pela ação catalítica da HRP. A elucidação das etapas e produtos da reação IAA/HRP levou uma série de moléculas produto a serem apontadas como responsáveis pelos efeitos citotóxicos sem que, até o presente momento, o mecanismo de citotoxicidade tenha sido elucidado. Nesse trabalho, utilizando-se células McCoy como alvo, foi constatado um efeito citotóxico dose dependente do sistema IAA/HRP, por necrose. Esse efeito é quase completamente abolido com a utilização de substâncias antioxidantes ou em anaerobiose. Também foi estudado o uso de um Ester derivado do IAA, o Etil Ester do IAA, como uma nova combinação citotóxica pró-droga/ enzima. Foi constatado que a HRP isolada não consegue catalizar a oxidação do Etil Ester do IAA na ausência de uma enzima adicional (esterase). Dessa forma, pode-se controlar a citotoxicidade do IAA pelo uso de duas enzimas, HRP e esterase. Finalmente, foram apresentadas evidências da aplicação potencial da tríade: Etil Ester IAA/ esterase/ HRP como uma estratégia potencial para a metodologia ADEPT e correlata. / Abstract: The antibody-directed enzyme pro-drug therapy (ADEPT) in a first stage, it's directed to an enzyme carried to an antibody to a tumor cell. In a second stage a pro-drug harmless is administered, and in the presence of the enzyme, produces cytotoxic compounds restricted the location of the tumor. The pair enzyme / pro-drug horseradish peroxidase (HRP)/ 3 - indole acetic acid (IAA) has been applied in ADEPT strategies. In this combination, the nontoxic plant hormone nontoxic IAA is activated for cytotoxic species by the action of catalytic HRP. The elucidation of the steps and products of the reaction IAA/ HRP led to a series of product molecules identified as being responsible for cytotoxic effects, without, so far, the mechanism of cytotoxicity has been elucidated. In this work, using cells McCoy as a target, we have seen a cytotoxic effect dosedependent system IAA/ HRP, for necrosis. This effect is almost completely abolished with the use of antioxidant substances or oxygen depletion. We also studied the use of an Ester derived from the IAA, the Ethyl Ester of the IAA, as a new combination cytotoxic pro-drug/ enzyme. We have seen that the HRP alone can not catalyze the oxidation of Ethyl Ester of the IAA in the absence of an additional enzyme (esterase). Thus, we can control the cytotoxicity of the IAA for the use of two enzymes, HRP and esterase. Finally, we showed evidence of the potential application of the triad: Ethyl Ester IAA/esterase/ HRP as a potential strategy for the methodology ADEPT and correlates. / Orientador: Luiz Marcos da Fonseca / Coorientador: Valdecir Farias Ximenes / Banca: Maria das Graças Carvalho / Banca: Eliana Aparecida Varanda / Mestre Esterase-6. McCoy cells - Cytotoxic effect. eng
17	Functional characterisation of a novel ferulic acid esterase from malawian hot spring metagenome Ngobeni, Rhulani January 2011 (has links) >Magister Scientiae - MSc / There has been a decline in the global fossil fuel reserves, due to an increasing demand for petroleum. Biofuels can be used as an alternative source of energy whereby biomass is converted to liquid fuels such as bioethanol. There is considerable interest in lipolytic enzymes because of their broad substrate range and for this purpose these enzymes have potential in a variety of biotechnological application. Lipolytic enzymes include esterases (E.C 3.1.1.1) and lipases (E.C3.1.1.3). Esterases preferentially hydrolyse short chain (<C10) ester-containing molecules that are partly soluble in water, while lipases hydrolyse a broad range of substrates preferably water-insoluble fatty acyl molecules (>C10). The aim of this study was to express, purify and characterise the lipolytic enzyme present on afosmid, Try 11, previously isolated from a metagenomic library of a Malawian hotspring, which conferred activity on tributyrin and ethyl ferulate. Bioinformatic analysisof the fosmid insert sequence predicted an open reading frame consisting of 951 bp,designated RHgene34, encoding a 317 amino acid protein with 41 % similarity to theα/β hydrolase fold-3 domain protein of Burkholderia sp. The RHgene34 protein contains conserved motifs of esterases/lipases, such as HGGG (residues 95-98),GxSxG (residues 167 - 171) and the putative catalytic triad composed of Ser157,Asp255 and His285. The gene was cloned and expressed in pET21a(+), and transformed into Escherichia coli Rosetta. p-Nitrophenol (p-Np) fatty acyl esters of different carbon chain lengths were used for kinetic characterisation of RHgene34. Kinetic analysis revealed that RHgene34 had a broad range activity on the p-Npesters, from C2 - C14. RHgene34 operates optimally at 45 ºC, pH 9.0 and has a half life of 30 mins at 45 ºC. This study demonstrates that functional screening combined with the sequence analysis is a useful approach for isolating novel enzymes from ametagenome. Biofuels Lipolytic enzyme Esterase Lipase Esters
18	Evaluation of Novel Carbamate Insecticides for Neurotoxicity to Non-Target Species Jiang, Ying 03 March 2011 (has links) Malaria (vector: Anopheles gambiae) is a major infectious disease that kills about 1 million people each year. For the improvement of its treatment and vector control during the past decades, several issues such as high medicine cost, insecticide resistance, and lack of an effective vaccine have prevented adequate control of malaria. Additionally, the low selectivity of malaria vector insecticides also presents a public health problem. The purpose of developing novel carbamate insecticides in our laboratory is to offer effective and selective insecticide options to achieve the ultimate goal of malaria control. First, 50% inhibition concentration (IC50) data was collected from three mammalian AChEs with eight commercial carbamate insecticides by using the Ellman assay. The IC50 values varied from 57 nM to 7358 nM. The AChE sensitivity pattern and level were shown to be similar between the recombinant mouse and ICR male mouse brain cortex homogenate (slope = 0.99, R2 = 0.96). Then eight novel carbamate insecticides that are possible malaria vector control agents were selected for further neurotoxicity testing in non-target organisms. For commercial carbamate insecticides, the IC50 varied from 9.1 nM to 2,094 nM. For the novel carbamate insecticides, it varied from 58 nM to 388,800 nM. Based on IC50 data from previous work on A. gambiae, the selectivity index (IC50 of non-target species / IC50 A. gambiae) ranged from 0.17 to 5.64 and from 0.47 to 19,587 for commercial and novel carbamate insecticides, respectively. Subsequently, the AChE protein sequence alignment comparison and cladogram were used to compare the genetic and evolutionary relationship among five different organisms. The alignment score ranged from 88 for mouse vs. human to 54 for hen vs. T. californica. The evolutionary relationships among species was obtained from the cladogram. Recombinant mouse vs. recombinant human was shown to have the most similar inhibitor potency profiles (alignment score = 88, closest taxa position on cladogram, similar AChE sensitivity pattern [R2 = 0.81] and level [P > 0.05] to the novel carbamates). Neurotoxic esterase (NTE) assay showed that the novel carbamates did not significantly inhibit NTE, inhibition of which underlies a significant hazard for anticholinesterases, especially organophosphates, in several nontarget vertebrate organisms. The NTE activity in the presence of novel carbamate insecticides ranged from 93% to 116% of the control, while in the commercial group, bendiocarb significantly inhibited NTE, leaving only 76.5% of the initial reactivity at 1 mM inhibitor concentration. Further in vivo bioassay using Daphnia magna was conducted to compare the aquatic toxicity of commercial and novel carbamates. The data showed that except for PRC331 (3-tert-butylphenylmethylcarbamate), all novel carbamates were of similar potency as bendiocarb (LC50 = 611 nM) for aquatic toxicity, and their LC50 values ranged from 172 nM (PRC331) to 1109 nM. In conclusion, the novel carbamate insecticides would appear to be an improvement over commercial carbamate insecticides because of greater selectivity, negligible NTE inhibition capacity, but in some cases with potent in vivo toxicity to Daphnia magna. However, since the envisioned usage of these compounds is in bednets or as indoor residual sprays (IRS), any environmental exposures to nontarget aquatic organisms are expected to be minimal. / Master of Science neurotoxic esterase acetylcholinesterase carbamate insecticides neurotoxicity
19	Estrutura do Gene da Esterase do Hormônio Juvenil de Apis Mellifera e seu Papel Durante o Desenvolvimento Pós-Embrionário e a Diferenciação de Castas. / Honey bee (Apis mellifera) Juvenile Hormone Esterase Gene Structure and its Rule During Post-Embryonic Development and Caste Differentiation. Santos, Aline Mackert dos 16 July 2004 (has links) Os hormônios juvenis (HJ) são uma classe de sesquiterpenóides que executam um papel crucial no desenvolvimento dos insetos. O HJ modula a ação de ecdisona, prevenindo a metamorfose nos estágios larvais. Os títulos deste honnônio são determinados pela sua síntese nos Corpora Allata e pela atividade hidrolítica de uma esterase específica (EHJ -Esterase do Hormônio Juvenil), um membro da família das carboxiesterases (3.1.1.1), que transforma o HJ em um metabólito considerado inativo (HJ-ácido). O HJ está intimamente envolvido no desenvolvimento e diferenciação de castas em A. mellifera; os títulos de hormônio diferem consideravelmente durante o desenvolvimento das castas. A metodologia ORESTES (Open-Reading-Frame-Expressed-Sequence- Tags) foi usada para a obtenção da seqüência do gene da EHJ. Vinte e seis clones que mostraram homologia com a seqüência da EHJ de outros insetos foram usados para a construção de primers para a análise da expressão do gene em experimentos de RT-PCR. O fragmento obtido pela amplificação utilizando estes primers mostrou alta identidade com as EHJ de Drosophila melanogaster e Tenebrio molitor em nível de aminoácidos. A primeira fita de cDNA foi sintetizada usando RNA total e usada como molde para PCR. A normalização foi feita utilizando-se a expressão do gene da actina de A. mellifera. O gene da EHJ é mais expresso em corpo gorduroso e epitélío do intestino. O pico de expressão do gene em operárias foi observado nos estágios que antecedem a metamorfose (L5F e L5S), após este período ocorre diminuição de expressão do gene em pré-pupas e pupas jovens e um aumento de expressão no final do período pupal e adultos de até 15 dias. A atividade do gene da EHJ está relacionada aos títulos de HJ durante o desenvolvimento, o que sugere a importância da EHJ para que a metamorfose ocorra normalmente. Os níveis de mRNA da EHJ foram quantificados nas castas e sexos. Operárias mostraram a maior expressão do gene durante os estágios de L3, L4, L5F1 e L5S1. Em rainhas, a expressão aumenta em pré-pupa, ao contrário do que ocorre em operárias. Os menores níveis de expressão ocorrem em zangões. A expressão do gene da EHJ é menor quando o HJ é essencial para o desenvolvimento das características de rainhas, o que ocorre nos estágios larvais mais jovens, podendo ser estabelecida relação direta entre o HJ e os níveis de mRNA da EHJ durante o desenvolvimento e manutenção de características de cada casta. O gene mostrou menor expressão em ovários de rainhas nos estágios larvais, isto pode ter importância na manutenção dos níveis de HJ para que este órgão seja protegido de degeneração, garantindo seu desenvolvimento normal. Já que os níveis de HJ são diferentes nas castas e sexos, a atividade diferencial do gene da EHJ aparentemente é um elemento chave na manutenção dos tipos morfológicos nesta complexa sociedade. O gene foi iníbido pela aplicação de 20E em pupas, assim, sugerimos que o gene é induzido pela presença de HJ, como ocorre nas fases larvais jovens e após a emergência, e inibido na presença de ecdisteróides, já que os resultados obtidos neste trabalho mostram que o gene da EHJ está reprimido quando os títulos de ecdisteróides estão elevados nas fases pupais. / The juvenile hormones (JH) are a class of sesquiterpenoids that play a crucial role in insect development. JH modulate the activity of ecdysone, preparing for metamorphosis at the end of the larval phase. The titers of this hormone are mainly determined by synthesis in the corpora allata and by the hydrolytic activity of a specific esterase (JHE - Juvenile Hormone Esterase), a carboxylesterase family member (3.1.1.1), which transforms JH into a metabolite considered inactive (JHacid). JH is intimately involved in Apis mellifera development and caste differentiation; the hormone titers differ considerably in developing queens and workers. The ORESTES (Open-Reading-Frame-Expressed-Sequence-Tags) methodology was used to obtain the JHE gene sequence. Twenty six clone sequences that showed homology with JHEs of other insects were used to construct specific primers to perform RT-PCR, in order to analyze JHE gene expression. The fragment amplified using these primers showed high identity with the JHE of Drosophila melanogaster and Tenebrio molitor at amino acid level. First strand cDNA was synthesized using total RNA and used as template for PCR. A. mellifera actin gene expression levels were used for normalization. The JHE gene is highly expressed in fat body and gut epithelium. The highest peak of JHE gene expression in workers was observed in the stages before metamorphosis, i.e. L5F and L5S, after which there is a decrease in the gene expression of pre-pupae and young pupae, with a increase at the end of pupal stages, and in the adult stages (until 15 days). The JHE gene activity is extremely related with the JH titers during the development, what suggests the importance of JHE enzyme activity to the normal metamorphosis. We quantified JHE mRNA levels in the castes and sexes of A. mellifera. Workers have the highest JHE gene expression levels during L3, L4, L5F1 and L5S1. In queens, there is an increase of JHE gene expression in pre-pupae, otherwise in works this stage shows a decrease in JHE expression. The lowest expression levels occur in drones. JHE expression is lower when JH is essential for the development of queen characteristics, what occurs during the early phases. Therefore it is possible to establish a direct relationship between JH and JHE mRNA levels during development and maintenance of the characteristics in each caste. The gene shows low expression levels in queens ovaries during larval stages where it may be important to the maintenance of JH levels, in order to protect this organ from degeneration, and to warrant a normal development. Since the levels of JH are different in the castes and sexes, the differential activity of the JHE gene apparently plays a key role in the maintenance of the morphotypes of this complex insect society. The gene was inhibited by 20E application in pupae, so we can suggest that the gene is induced by JH presence like we detected during larval stages and after emergence, and inhibited by ecdysteroids, since the data obtained in this work suggest that the JHE gene is repressed when the ecdysteroids titers are elevated. Apis mellifera Apis mellifera Desenvolvimento. Development. Esterase do hormônio juvenil. Juvenile hormone esterase.
20	Caracterização funcional e estrutural de uma enzima lipolítica encontrada na biblioteca metagenômica de solo de Terra Preta de Índio. / Functinal and structural characterization of lipolytic enzyme present in soil metagenomic library of Terra Preta de Índio. Carvalho, Cecília Fonseca 07 August 2015 (has links) A construção de bibliotecas metagenômicas tornou possível o acesso ao potencial biotecnológico de micro-organismos não cultiváveis. O solo é um habitat pouco explorado, com elevada diversidade bacteriana que possuem genes codificadores de enzimas de interesse industrial, chamadas de biocatalisadores naturais. Dentre estas, destacam-se as enzimas lipolíticas, lipases e esterases, por promoverem a hidrólise de matérias-primas de alto valor agregado e com muitas aplicações biotecnológica. Devido a necessidade, existe uma constante busca para isolar novos genes com diferentes especificações. Neste trabalho, o gene lip4, isolado de biblioteca metagenômica de solo, foi superexpresso, purificado e identificado como membro da família V das lipases bacterianas. Tem atividade ótima hidrolisando triacilglicerol de cadeia média em pH alcalino sem presença de íons. A estrutura cristalográfica obtida identificou o dobramento conservado das α/β hidrolases e a tríade catalítica, Ser94-Asp217-His245, além da presença do domínio CAP, comum nas esterases. / Metagenomic libraries construction make possible the access to the biotechnological potential of not cultivated microorganisms. Soil environment has a big bacterial diversity with genes encoding industrial interest enzymes, named natural biocatalysts. Among these, lipolytic enzymes, that includes lipases and estarases, are able to catalyse different biochemistry reaction and promoting raw material hydrolysis with added values. In this overview, a search for new genes with different specifications, allowed isolation by functional screening in tributyrin agar, a gene lip4 from a soil metagenomic library. These gene was overexpressed, purified and identified as a member to family V of bacterial lipases. Presents better activity in alkaline pH with medium chain triacyglycerol without ions. Three dimensional structure of Lip4 identified a conserved α/β hydrolase backbone and the catalytic triad Ser94-Asp217-His245, besides the presence of CAP domain, common structure in esterase. Cristalografia de raio-X Esterase Esterase Metagenomic Metagenômica Tributirina Tributyrin X-ray Crystallography

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