Dissertations / Theses on the topic 'Pectinase enzyme'
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Göğüş, Nihan Tarı Canan. "Effect of The Morphology of Aspergillus Sojae on Pectinase Enzyme and The Optimization of Fermentation Conditions/." [s.l.]: [s.n.], 2006. http://library.iyte.edu.tr/tezlerengelli/master/gidamuh/T000578.pdf.
Full textDalagnol, Luíza Merlini Garcia. "Avaliação do uso do ultrassom na extração do mosto da uva cabernet sauvignon e na atividade enzimática." reponame:Biblioteca Digital de Teses e Dissertações da UFRGS, 2017. http://hdl.handle.net/10183/158743.
Full textGrape processing for juice and wine production encompasses different technological processes, such as enzymatic treatment, that usually uses commercial enzyme preparations to improve clarification and extraction processes. However, this is a treatment that demands high processing time, being relevant the search for new technologies in order to improve the whole process. Ultrasound (US) can be used as an alternative to reduce the processing time and improve the quality of the product, moreover, it can provide benefits in the combined use with enzymes, favoring the enzymatic reactions. In this work, the combined use of enzymatic treatment (ET), ultrasound (US), and mechanical agitation (MS) was studied in order to understand the effects of the techniques on Cabernet Sauvignon must extraction. Initially, nine commercial enzyme preparations were characterized according to their enzymatic activities and applied to must extraction, evaluating temperature (40, 50 and 60 °C), time (15 and 30 min), and enzyme concentration (0.01 to 2.0 Ug-1). The best results were obtained for the preparation Zimopec PX5® under conditions of 1.0 U.g-1 of pectinase at 50 °C for 30 min. A significant increase on quality parameters (color, °Brix, yield, antioxidant capacity, total anthocyanins) was obtained to extraction combining the three methods (US, MS and ET), mainly when US were applied. Based on these previous results, a new study was conducted to investigate the influence of sonication on the catalytic efficiency of the enzymes pectinase (PE), xylanase (XLN) and cellulase (CE). The enzymatic activities were evaluated at different pH and in samples of enzyme and substrates submitted to a previous treatment of sonication by determined times (0 - 30 min). The kinetic and thermodynamic parameters were estimated, showing a positive effect of the ultrasonic treatment on the enzymatic activity. Previous sonication of the substrate contributed significantly to the increase in XLN activity, whereas the previous sonication of the enzyme improved the activity of CE and PE. This research showed the potential benefits of ultrasound treatment on reaction rate and catalytic efficiency (Vmax/Km) improvement of enzymes, attesting that US can be used to increase the catalytic efficiency of pectinase, xylanase and cellulase enzymes. According to the obtained results, it can be affirmed that ultrasound improved the extraction process, and can be applied as an alternative technique, as well as provided beneficial effects on the enzymatic activity.
Gacura, Matthew David. "Drivers of Fungal Community Composition and Function In Temperate Forests." Kent State University / OhioLINK, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=kent1543579763552776.
Full textByarugaba-Bazirake, George William. "The effect of enzymatic processing on banana juice and wine." Thesis, Link to the online version, 2008. http://hdl.handle.net/10019/1633.
Full textPedrolli, Danielle Biscaro [UNESP]. "Caracterização físico-química de pectinases extracelulares purificadas de Aspergillus giganteus." Universidade Estadual Paulista (UNESP), 2008. http://hdl.handle.net/11449/95014.
Full textFundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
O fungo Aspergillus giganteus produz uma única poligalacturonase (PG) quando cultivado em meio líquido com pectina de citrus como única fonte de carbono, e pelo menos três pectina liases (PL) quando cultivado em meio líquido com resíduo de laranja como fonte de carbono. A PG de A. giganteus foi purificada em duas etapas: precipitação de proteínas com 70% de saturação com sulfato de amônio e troca aniônica. A PG purificada apresentou massa molar de 69,7±0,07 kDa. A máxima atividade da enzima foi observada em pH 6,0 a 55-60ºC, sendo essa estável em meio neutro e alcalino. A PG apresentou meias-vidas de 115, 18 e 6 min quando incubada a 40, 50 e 55 ºC, respectivamente. A enzima mostrou-se ativa sobre substratos de qualquer grau de metilação, com maior especificidade para substratos de baixa ou nenhuma metilação, apresentando Vmax 669,6 e 602,8 μmol/mg.min, Km 3,25 e 1,16 mg/mL, kcat 770 e 690 s-1 sobre pectina de citrus 34 % esterificada e ácido poligalacturônico, respectivamente. A PG apresentou atividade exo liberando apenas ácido galacturônico como produto de hidrólise. 2-Mercaptoetanol, DTT, Co2+, Mn2+, Mg2+, NH4 + e Na+ agiram como estimulantes da atividade PG. Já Hg2+, EDTA, citrato de sódio, ácido iodoacético, SDS, Ba2+, Cu2+, Pb2+ e Zn2+ inibiram essa atividade enzimática. A principal pectina liase de A. giganteus, a PL I, foi purificada em três etapas: troca aniônica, troca catiônica e filtração em gel. A massa molar da PL I foi estimada em 55,7±1,4 kDa. A máxima atividade da enzima foi obtida em pH 8,5 a 50ºC. A PL I apresentou meias-vidas de 19, 9 e 6 min a 40, 45 e 50ºC, respectivamente. As maiores atividades da PL I foram observadas em pectinas de citrus com 34 e 72% de metilação, nas quais apresentou Vmax 1.488,1 e 1.129,8 U/mg.min, respectivamente, e Km 4,8 mg/mL para ambos os substratos. O padrão de degradação da pectina...
Aspergillus giganteus produces one polygalacturonase (PG) on liquid medium with citrus pectin as the only carbon source, and at least three pectin lyases (PL) with orange waste. Homogenous PG was obtained after two steps: protein precipitation with 70 % ammonium sulphate saturation and anionexchange chromatography. The purified PG showed molecular weight of 69.7±0,07 kDa. The enzyme exhibited maximal activity at pH 6.0 and 55-60 °C, and was stable in neutral and alkaline medium. The half-live for PG at 40, 50 and 55 °C was 115, 18 and 6 min, respectively. The enzyme was active on substrates with any methyl-esterification degree, and hydrolysed better low esterified and not esterified substrates, showing Vmax 669.6 and 602.8 μmol. mg-1.min-1, Km 3.25 and 1.16 mg/mL, kcat 770 and 690 s-1 on 34 % esterified citrus pectin and polygalacturonic acid, respectively. The unique soluble product released in the reaction with pectin and polygalacturonic acid was monogalacturonic acid, and according to this results the enzyme can be classified as exoPG. PG activity enhanced in presence of 2-mercaptoethanol, DTT, Co2+, Mn2+, Mg2+, NH4 + and Na+, and was completely inhibited in presence of Hg2+. EDTA, sodium citrate, iodoacetic acid, SDS, Ba2+, Cu2+, Pb2+ and Zn2+ inhibited enzyme activity. The main PL from A. giganteus was called PL I and was purified after three steps: anion-exchange and cation-exchange chromatographies, and gel filtration. The purified PL I showed molecular weight of 55.7±1.4 kDa. The enzyme exhibited maximal activity at pH 8.5 and 50 °C, and was stable in neutral and acid medium. The half-live for PL I at 40, 45 and 50 °C was 19, 9 and 6 min, respectively. The enzyme was more active on citrus pectins with 34 and 72% of esterification, showing Vmax 1,488.1 and 1,129.8 U. mg-1.min-1, repectively, and Km 4.8 mg/mL on both substrates. The degradation pathern on 34% esterified...(Complete abstract click electronic access below)
Hassan, Sozan. "Caractérisation de nouvelles enzymes impliquées dans la dégradation de polysaccharides végétaux à partir de la bactérie Dickeya dadantii 3937." Thesis, Lyon, INSA, 2011. http://www.theses.fr/2011ISAL0115.
Full textThe phytopathogenic bacterium Dickeya dadantii is responsible for soft rot diseases of various plants. It secretes in the external medium a large array of enzymes which are able to degrade the constituents of the plant-cell wall. The first part of my work was related to the féruloyl esterases FaeD and FaeT. Feruloyl esterases are responsible for the hydrolysis of ester linkages between ferulic acid and the xylan or pectin chains. By cleaving these linkages, they improve the complete degradation of the plant-cell wall. The importance of these enzymes led us to search whether D. dadantii produces such esterases. A gene bank screening using a specific detection test for the feruloyl esterase activity allowed us to identify two genes which were characterized. While faeT is weakly transcribed in all the conditions, the faeD transcription is strongly induced in the presence of ferulic acid and it is controlled by the regulator FaeR. Whereas FaeT is a cytoplasmic protein, FaeD is secreted by the Out system responsible for the secretion of several pectinases. The enzymes FaeD and FaeT were overproduced in E. coli and their main biochemical properties were determined. The determination of the complete sequence of the D. dadantii genome makes it possible to develop functional genomic studies. This sequence confirms the presence of genes encoding the previously characterized pectinases and it reveals that this genome encodes new potential pectinases. The second part of my work was related to the gene pelN identified by genome analysis. Pectate lyases cleave the glycosidic bounds in the polygalacturonate chain by a β-elimination reaction, generating unsaturated products. This reaction mechanism requires cations as cofactor, generally Ca2+. After cloning of the gene pelN, the protein PelN was overproduced in E. coli. Its pectate lyase activity was demonstrated by its capacity to produce unsaturated derivatives from polygalacturonate or pectins. This study showed that PelN is the first pectate lyase that uses Fe2+ ions as the preferential cofactor. In D. dadantii, the pelN expression depends on various regulators controlling the pectinase synthesis, such as PecS or GacA. PelN is an extracellular protein secreted by the Out system. These studies contribute to increase the knowledge on the respective role of the different enzymes involved in the degradation of the plant-cell wall and the cooperative interactions in this pluri-enzymatic system
Zhang, Jing. "Biochemical Study and Technical Applications of Fungal Pectinase." Doctoral thesis, Uppsala : Acta Universitatis Upsaliensis : Universitetsbiblioteket [distributör], 2006. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-6295.
Full textAltan, Asena Yenidünya Ali Fazıl. "Isolation And Molecular Characterization of Extracellular Lipase And Pectinase Producing Bacteria From Olive Oil Mills/." [s.l.]: [s.n.], 2004. http://library.iyte.edu.tr/tezler/master/biyoteknoloji/T000497.pdf.
Full textSykes, Melissa. "Do zoospores of Phytophthora cinnamomi produce enzymes such as cutinases, cellulases and pectinases?" Thesis, Sykes, Melissa (1995) Do zoospores of Phytophthora cinnamomi produce enzymes such as cutinases, cellulases and pectinases? Honours thesis, Murdoch University, 1995. https://researchrepository.murdoch.edu.au/id/eprint/32817/.
Full textSandri, Ivana Greice. "Enzimas pectinolíticas : seleção de linhagens fúngicas produtoras, caracterização e aplicação em processos da indústria de alimentos." reponame:Repositório Institucional da UCS, 2010. https://repositorio.ucs.br/handle/11338/516.
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In this work, fungus samples isolated from decomposing vegetables were tested with respect to their capacity of producing pectinases to be applied to the enzymatic treatment of apple and bilberry juices. Taking in account the secretion of endo-polygalacturonase (endo-PG), two isolates which were identified and named Aspergillus niger LB23 e Aspergillus fumigatus LB39J were selected for solid-state and submerged cultivations, respectively. The enzymatic extracts from these strains did not present total aflatoxin (B1, B2, G1, G2) levels up to detection limit of 2 μg/Kg. The enzyme preparation obtained from solid-state cultivation of A. niger LB23 showed the largest activities of both endo-PG and exo-polygalacturonase (exo-PG) at pH 4.0, whereas the reaction with submerged A. fumigatus L39J enzymatic extracts was favoured at pH between 4.0 and 5.0 for endo-PG and 5.0 for exo-PG. With respect to the reaction temperature for A. niger LB23 pectinolytic preparation, endo and exo-PG showed the best performances at the ranges 40-50ºC and 50-60ºC, respectively. For A. fumigatus L39J polygalacturonases, a range from 40 to 60ºC, for endo-PG, and 60ºC, for exo-PG, were defined as the best temperatures. When the thermal stability of polygalacturonases present in both preparations was evaluated, preservation of about 70% of activities was observed after exposing the enzymes to temperatures up to 40ºC for 150 minutes. The influence of different glucose and pectin concentrations on the results of the solid-state process with A. niger LB23 was assessed and an increment in endo and exo-PG activities were observed in medium containing 6% (w/w) of pectin and absence of glucose, with activities of 65 units per gram of dry medium (U/gdm) and 198 U/gdm being achieved. In submerged cultivation of A. fumigatus LB39, highest enzyme activities were obtained in medium with 20 g/L of pectin and without glucose: 42 U/mL for endo-PG and 32 U/mL for exo-PG. In the conditions defined for the submerged process, different medium initial pH values were tested. Maximum activities were observed at initial pH of 3.0 for endo-PG (44 U/mL) and 4.0 for exo-PG (37 U/mL). Furthermore, in bench bioreactor, it was observed that an initial pH of 4.0, with uncontrolled minimum value and, afterwards, maximum value controlled at 3.5 led to the largest enzyme production with 81 U/mL of endo-PG and 49 U/mL of exo-PG. By comparing the enzymatic preparations produced by the selected strains, it was noticed that the pectinase produced by A. niger LB23 were more efficient for the hydrolysis of pectic substances present in both apple and bilberry juices, since it improved reduction of turbidity (90 and 40%) and viscosity (40 and 60%) and increasing clarification (90 and 55%), respectively. These results indicate the potential of A. niger LB23 pectinases for the enzymatic treatment of fruit juices, being remarkable that both the level of phenolic compounds and antioxidant capacity of juices were mostly preserved after treatment.
Silva, Dênis [UNESP]. "Purificação e caracterização bioquímica de poligalacturonases produzidas pelo fungo Penicillium viridicatum RFC3 em fermentação em estado sólido e submersa." Universidade Estadual Paulista (UNESP), 2006. http://hdl.handle.net/11449/103949.
Full textFundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
A poligalacturonase (PG) é uma pectinase que degrada a molécula de pectina atuando internamente , ao acaso, liberando oligossacarídeos (Endo-PG) ou por ataque a partir da extremidade não redutora da cadeia, liberando moléculas de ácido galacturônico (Exo-PG). As pectinases, como várias outras enzimas, têm sido obtidas a partir de microrganismos, em processos fermentativos em estado sólido (FES) e fermentação submersa (FSM). A purificação e caracterização destas enzimas podem possibilitar o estudo das diferenças de suas propriedades funcionais, bem como, permitem avaliar suas possibilidades de aplicação, em diferentes condições, em processos industriais. Este trabalho teve por objetivos estudar a produção, purificação e caracterização da PG produzida por Penicillium viridicatum RFC3 através de FES e FSM. Para FSM foram testados os resíduos agroindustriais (farelo de trigo, bagaço de laranja, uma mistura de ambos) a 3% (p/v) além da água amarela (resíduo do processamento da indústria cítrica) no seu estado bruto e com diluições (2, 4 e 10x). Os pHs iniciais foram corrigidos para 4,5, 5 e 5,5 e a fermentação ocorreu por 24 - 96 h. A ANOVA mostrou que o extrato do bagaço de laranja (pH 5,5 e 96 h) foi o melhor meio para a produção da PG em FSM. A purificação da PG produzida em FSM iniciou-se pela ultrafiltração do extrato bruto (membranas de 50 e 10 kDa, repectivamente) seguida pela aplicação da amostra em uma coluna de filtração em gel Sephadex G75 e posterior aplicação em uma Q Sepharose. A fração isolada e desorvida revelou, após 2 eletroforese (PAGE-SDS) a homogeneidade da banda cuja massa molar foi estimada em 92,24 kDa e pI de 5,4. A enzima mostrou ser uma exopoligalacturonase (Exo-PG) com pH e temperatura ótimos em 5,0 e 50-55ºC.
The polygalacturonase (PG) is a pectinase that degrade pectin acting internally, releasing olygossacharide (Endo-PG) or by atack to a non reducing chain extremity, releasing galacturonic acid (Exo-PG). The pectinases, like many others enzymes, have been obtained from microorganisms by solid state fermentation (SSF) and submerged fermentation (SMF). The purification and characterization of these enzymes besides the study of differences of its functional properties permit its application on industrial process, on different conditions. This work aimed the study of production, purification and characterization of PG from Penicillium viridicatum RFC3 by SSF and SMF. In SMF they were assayed agroindustrial wastes (wheat bran, orange bagasse and a mixture of both) at 3% (w/v 1:1) and citric industry liquid waste (yellow water) in crude state and with dilutions (2, 4 and 10x). It was also evaluated the variation of the initial pH of the medium (4.5, 5.0 nd 5.5) and the time of fermentation (24 96 h). The ANOVA test revealed that the culture medium composed by orange bagasse extract, at pH 5.5 and 96 h, was the best condition for production of PG in SMF. The purification process of PG from SMF was carried out by the ultrafiltration of the crude extract (50 and kDa membranes, respectively) and by gel filtration in Sephadex G75 and Q Sepharose columns. The fraction with PG activity show by eletrophoresis (SDSPAGE), an homogeinity of a band with estimated molar mass of 92.24 KDa and pI 5.4. The enzyme revealed to be an Exo-PG with optimal pH and temperature of 5.0 and 50-55 °C. It also revealed to be Ca2+ dependent, which increased its 4 activity and stability. The Km of the enzyme was 1.30 and 1.16 mg/ml on Ca2+ presence. The Vmax was 1.76 and 2.07 ?mol/min/mg when Ca2+ was added. The better fermentation conditions in SSF was wheat bran mixed with orange bagasse (1:1 w/w) at 80% moisture as an culture medium and 336h of fermentation.
Poletto, Patrícia. "Produção, recuperação e avaliação de pectinases de Aspergillus niger LB-02-SF obtidas em biorreator de tambor rotativo." reponame:Repositório Institucional da UCS, 2015. https://repositorio.ucs.br/handle/11338/951.
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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior.
In the present work, the production of pectinases by Aspergillus niger LB-02-SF in solid-state cultivation was studied using a rotating drum bench bioreactor. Furthermore, extraction and concentration of enzymes by ultrafiltration were evaluated. Formulations with the concentrated extract, KCl and glycerol were assayed with respect to the maintenance of activity level and the efficiency in the treatment of fruit juices. With respect to the production of enzymes in bioreactor, the operational parameters evaluated were agitation, cultivation temperature, occupation volume (30, 50 and 70%) and specific air flow (0.18, 0.36, 0.54, 0.72 e 0.90 LkgM – liter of air per kilogram of medium per minute). Tests without and with agitation of the bioreactor (1 rpm per 5 minutes each 120 minutes) indicated that, with occupation volume of 30%, agitation leads to increasing fungal growth and decreasing enzyme production, whereas with 50% of occupation an opposite behavior was noticed. With 70% of occupation volume, no significant difference was observed among the pectinolytic activities achieved with and without agitation, the production being hindered in both cases by the insufficient aeration of medium. The interval between agitation events was evaluated for an occupation volume of 70% and no significant difference in enzyme production was observed for the intervals of 90 and 120 minutes (standard condition). For the interval of 60 minutes, the activity was reduced in approximately 58%. The cultivations with temperature control at 34oC and without control indicated that enzyme production was favored with the natural increase of medium temperature, an aspect that could be related to a stress condition that would lead to a higher enzyme production. Specific air flows between 0.18 and 0.90 LkgM were evaluated for 30 and 50% of bioreactor occupation volume. Larger fungal growth was observed when the bioreactor was loaded with the smallest occupation volume. With the intermediate flow of 0.54 LkgM, the highest enzyme activities were achieved for both occupation volumes; however, the occupation volume of 50% resulted the higher activity of 178.2 U/g, with an activity of 133.4 U/g being measured for 30% of occupation. The results achivied indicated that for a higher enzyme production, the bioreactor should be conducted with 50% of occupation volume, specific airflow of 0.54 LkgM and interval between the agitations of 120 minutes. In the study on the extraction of pectinases from the solid medium with water pH 4, under agitation, it was observed that extraction times from 15 to 120 minutes presented similar results. The extraction temperatures of 20, 30 and 40oC did not influence protein extraction, but pectinase activity was reduced in 40% at 40oC, possibly due to the low enzyme thermostability. Among the extraction ratios of solid/liquid assessed, the value 1/10 resulted in the highest enzyme activity solubilized, with recovery of 81% of the pectinolytic activity that was present in the medium. In the concentration tests, ultrafiltration of concentrated extract with no previous treatment resulted in 59% of enzyme activity recovery and in a final permeate flux of about 11 L/m²/h. On the other hand, the operation protocol that included the use of activated charcoal, microfiltration and ultrafiltration presented enzyme recovery of 74% and final permeate flux of 24 L/m2/h, results that demonstrate the importance of pre-treatments in this process. Previously to the preparation of enzyme formulations, a volume of 30 L of enzyme extract was 103-fold concentrated by ultrafiltration, with 69% of enzyme recovery, and a final 663-U/mL extract was obtained. This concentrated extract was formulated with 20, 30, 40 and 50% (w/w) of glycerol and 2% (w/w) of KCl and evaluated for 59 weeks with respect to the enzyme activity conservation at 5ºC. At the end of the test period, the blank sample (with no additives) presented an activity decrease of 25% and also microbial contamination, whereas the formulations preserved 100% of the initial activity along the experiment. The 50%-glycerol formulation was used for Bordô grape maceration and for depectinization of apple, Rosé grape and blackberry juices, showing statistically similar results to those obtained with the commercial preparations Novozym 33095 and Pectinex Ultra SP-L used as reference. The results attained in this work pointed out to the possibility of scaling-up this process, from the enzyme production in solid-state cultivation to the achievement of an enzyme formulation that has shown potential to be used in the industry of fruit juice.
Ali, Ahmed. "Use of pectinases to improve the nutritive value of lupins for poultry." University of Western Australia. School of Animal Biology, 2009. http://theses.library.uwa.edu.au/adt-WU2010.0094.
Full textSoriano, Lasheras Margarita. "Análisis de sistemas pectinolíticos bacterianos. Aislamiento y caracterización de las pectinasas PelA de "Paenibacillus" sp. BP-23 E YvpA de "Bacillus subtilis"." Doctoral thesis, Universitat de Barcelona, 2004. http://hdl.handle.net/10803/2391.
Full textEn los últimos años ha aumentado de forma importante la aplicación de enzimas microbianos en la industria. La mayoría de estos enzimas son degradadores de polisacáridos de la pared celular vegetal, como celulosas, hemicelulosas y pectinas.
La pectina es un polímero formado principalmente por cadenas de ácido galacturónico, parcialmente esterificado. Las pectinasas son las enzimas que degradan la pectina.
El principal objetivo de la tesis fue caracterizar pectinasas con potencial aplicación industrial. Para ello, primero se realizó el análisis de los sistemas degradadores de pectina de las cepas bacterianas Paenibacillus sp. BP-23 y Bacillus sp. BP-7, aisladas previamente por el grupo de investigación a partir de suelo de arrozal del Delta del Ebro.
El sistema pectinolítico de ambas cepas mostró una multiplicidad de bandas, motivo por el cual, se procedió a la clonación, purificación y caracterización de pectinasas a partir de las cepas analizadas y de Bacillus subtilis 168. Los estudios realizados indican que PelA de Paenibacillus sp. BP-23 e YvpA de Bacillus subtilis son enzimas nuevos, con características diferentes a las pectato liasas conocidas, que permiten identificar un subgrupo de enzimas dentro de las pectato liasas de la familia PL3. La inusual elevada actividad sobre pectina de los dos enzimas estudiados en este trabajo los hace buenos candidatos para aplicaciones biotecnológicas relacionadas con la degradación de pectina de sustratos naturales.
En el contexto de la utilización creciente de enzimas en la industria se planteó la producción del más activo de ellos, la pectato liasa PelA, en cantidades elevadas para poder realizar ensayos de aplicación industrial, utilizando cepas huésped de Bacillus subtilis. Con este fin se construyeron vectores lanzadera que replican tanto en Bacillus subtilis como en Escherichia coli.
Los resultados obtenidos en este trabajo suponen una aproximación a la tarea de sobreproducir pectato liasas, lo que ha permitido la identificación de problemas específicos de la expresión de los enzimas en estudio y posibilitarán la construcción futura de nuevas cepas recombinantes de productividad y rendimiento aumentado.
Pectins are linear polymers of alpha-D-galacturonic acid. They are found in the middle lamella and primary cell wall of plant tissues and they are degraded by pectinases.
The aim of the thesis was the caracterization of pectinases with a potencial applications in the industry.
Therefore, we analysed the petinolytic system of the bacterial strains Paenibacillus BP-23 and Bacillus BP-7, previously isolated from a rice field.
The pectinolytic system of both strains a multiple glycanase system, some enzymes of which have been cloned and characterized.
Paenibacillus sp BP-23 PelA and Bacillus subtilis YvpA, belong to pectate lyases PL3 family, and show and unusual features among pectin and pectate lyases. For this reason, we constructed several shuttles which were used to overexpres PelA in Bacillus subtilis.
Freitas, Paula Mendes de [UNESP]. "Produção de poligalacturonase termoestável pelo fungo Rhizomucor pusillus A 13.36 em fermentação em estado sólido, purificação e caracterização da enzima." Universidade Estadual Paulista (UNESP), 2009. http://hdl.handle.net/11449/103941.
Full textConselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)
O fungo Rhizomucor pusillus A 13.36, quando cultivado por fermentação em estado sólido utilizando farelo de algodão como fonte de carbono por 72 horas, produziu altos níveis de poligalacturonase (18,6U/g). Essa enzima apresentou atividade máxima em pH 5,5 e em 55oC. A poligalacturonase bruta apresentou estabilidade na faixa de pH entre 4,5 e 6,5, com uma atividade residual de 91,3% no pH 6,5, e também até 70oC por 1h, com atividade residual de 80% nas temperaturas de 55 a 60oC. Esta enzima foi purificada por concentração e uma combinação de procedimentos de gel filtração e cromatografia de troca-iônica. A massa molecular da enzima foi 53,7kDa. A focalização isoelétrica mostrou uma única banda cujo ponto isoelétrico (pI) foi 3,8. A atividade máxima da enzima purificada também ocorreu em pH 5,5 e a 55oC. A enzima purificada foi estável na faixa de pH 4,5 a 6,5, com atividade residual na faixa de 80 a 100%, e também até 70oC por 1h, com atividade residual de 100% nas temperaturas de 55 a 60oC. A enzima purificada foi totalmente inibida por Ca2+, Cu2+, Hg2+ e 80% inibida por Fe2+ e Ag+. Mg2+ estimulou a atividade da poligalacturonase em 100%. Entre os substratos avaliados, a pectina de citrus (D.E. 92%) foi o que mais estimulou a ação da enzima. A afinidade por pectinas de alta esterificação e a ausência de atividade quando o ácido poligalacturônico foi usado como substrato, indica que a enzima purificada é uma polimetilgalacturonase. Os resultados obtidos pela cromatografia de papel demonstraram que a polimetilgalacturonase purificada possui o modo de clivagem endo/exo misto. O Km com pectina de citrus foi 10,78mg mL-1 e a Vmax foi 2379,04μmolmin-1mg-1. As possíveis aplicações desta enzima incluem composição de detergentes, processamento têxtil e de fibras de celulose, degradação ou modificação de material vegetal, aditivo...
Rhizomucor pusillus A 13.36 produced high levels of polygalacturonase (18.6U/g) in solid state fermentation in medium composed by cotton bran for 72 hours. This enzyme showed maximal activity at pH 5.5 and 55oC. Crude polygalacturonase was stable from pH 4.5 to 6.5, with remaining activity of 91,3% at pH 6.5, and stable until 70oC for 1h, with remaining activity of 80% at 55-60oC. This enzyme was purified by concentration and a combination of gel filtration and ion exchange chromatographic procedures. The molecular mass of the enzyme was 53.7kDa. Isoelectric focusing showed a single band with an isoelectric point (pI) of 3.8. The purified enzyme also showed maximum activity at pH 5.5 and 55oC. The purified enzyme was stable from pH 4.5 to 6.5, with remaining activity range of 80 to 100%, and stable until 70oC for 1h, with remaining activity of 100% at 55-60oC. The purified enzyme was totally inhibited by Ca2+, Cu2+, Hg2+ and 80% inhibited by Fe2+ and Ag+. Mg2+ increased poligalacturonase activity in 100%. Citrus pectin (D.E. 92%) was found to be the preferred substrate among the different substrates tested in the enzyme assay. The affinity for high pectin esterification and the lack of activity when polygalacturonic acid was used as substrate, indicates that the enzyme is a polymethylgalacturonase. The results obtained by paper chromatography showed that the purified polymethylgalacturonase has the mixed endo/exo mode of cleavage. The Km with citrus pectin was 10.78mg mL-1 and the Vmax was 2379.04μmolmin-1mg- 1. The possible applications of this enzyme include the composition of detergents, textile and cellulosic fiber processing, degradation or modification of plant material, animal feed additive and wine and juice processing.
Freitas, Paula Mendes de. "Produção de poligalacturonase termoestável pelo fungo Rhizomucor pusillus A 13.36 em fermentação em estado sólido, purificação e caracterização da enzima /." Rio Claro : [s.n.], 2009. http://hdl.handle.net/11449/103941.
Full textBanca: Eleonora Cano Carmona
Banca: Hamilton Cabral
Banca: Daniela Alonso Bocchini Martins
Banca: Maria de Lourdes T. de M. Polizeli
Resumo: O fungo Rhizomucor pusillus A 13.36, quando cultivado por fermentação em estado sólido utilizando farelo de algodão como fonte de carbono por 72 horas, produziu altos níveis de poligalacturonase (18,6U/g). Essa enzima apresentou atividade máxima em pH 5,5 e em 55oC. A poligalacturonase bruta apresentou estabilidade na faixa de pH entre 4,5 e 6,5, com uma atividade residual de 91,3% no pH 6,5, e também até 70oC por 1h, com atividade residual de 80% nas temperaturas de 55 a 60oC. Esta enzima foi purificada por concentração e uma combinação de procedimentos de gel filtração e cromatografia de troca-iônica. A massa molecular da enzima foi 53,7kDa. A focalização isoelétrica mostrou uma única banda cujo ponto isoelétrico (pI) foi 3,8. A atividade máxima da enzima purificada também ocorreu em pH 5,5 e a 55oC. A enzima purificada foi estável na faixa de pH 4,5 a 6,5, com atividade residual na faixa de 80 a 100%, e também até 70oC por 1h, com atividade residual de 100% nas temperaturas de 55 a 60oC. A enzima purificada foi totalmente inibida por Ca2+, Cu2+, Hg2+ e 80% inibida por Fe2+ e Ag+. Mg2+ estimulou a atividade da poligalacturonase em 100%. Entre os substratos avaliados, a pectina de citrus (D.E. 92%) foi o que mais estimulou a ação da enzima. A afinidade por pectinas de alta esterificação e a ausência de atividade quando o ácido poligalacturônico foi usado como substrato, indica que a enzima purificada é uma polimetilgalacturonase. Os resultados obtidos pela cromatografia de papel demonstraram que a polimetilgalacturonase purificada possui o modo de clivagem endo/exo misto. O Km com pectina de citrus foi 10,78mg mL-1 e a Vmax foi 2379,04μmolmin-1mg-1. As possíveis aplicações desta enzima incluem composição de detergentes, processamento têxtil e de fibras de celulose, degradação ou modificação de material vegetal, aditivo... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: Rhizomucor pusillus A 13.36 produced high levels of polygalacturonase (18.6U/g) in solid state fermentation in medium composed by cotton bran for 72 hours. This enzyme showed maximal activity at pH 5.5 and 55oC. Crude polygalacturonase was stable from pH 4.5 to 6.5, with remaining activity of 91,3% at pH 6.5, and stable until 70oC for 1h, with remaining activity of 80% at 55-60oC. This enzyme was purified by concentration and a combination of gel filtration and ion exchange chromatographic procedures. The molecular mass of the enzyme was 53.7kDa. Isoelectric focusing showed a single band with an isoelectric point (pI) of 3.8. The purified enzyme also showed maximum activity at pH 5.5 and 55oC. The purified enzyme was stable from pH 4.5 to 6.5, with remaining activity range of 80 to 100%, and stable until 70oC for 1h, with remaining activity of 100% at 55-60oC. The purified enzyme was totally inhibited by Ca2+, Cu2+, Hg2+ and 80% inhibited by Fe2+ and Ag+. Mg2+ increased poligalacturonase activity in 100%. Citrus pectin (D.E. 92%) was found to be the preferred substrate among the different substrates tested in the enzyme assay. The affinity for high pectin esterification and the lack of activity when polygalacturonic acid was used as substrate, indicates that the enzyme is a polymethylgalacturonase. The results obtained by paper chromatography showed that the purified polymethylgalacturonase has the mixed endo/exo mode of cleavage. The Km with citrus pectin was 10.78mg mL-1 and the Vmax was 2379.04μmolmin-1mg- 1. The possible applications of this enzyme include the composition of detergents, textile and cellulosic fiber processing, degradation or modification of plant material, animal feed additive and wine and juice processing.
Doutor
Gaffé, Joël. "Contribution à l'étude des pectine-méthyl-estérases des parois végétales du lin." Rouen, 1992. http://www.theses.fr/1992ROUES039.
Full textSilva, Dênis. "Purificação e caracterização bioquímica de poligalacturonases produzidas pelo fungo Penicillium viridicatum RFC3 em fermentação em estado sólido e submersa /." Rio Claro : [s.n.], 2006. http://hdl.handle.net/11449/103949.
Full textBanca: Eleonora Cano Carmona
Banca: Rubens Monti
Banca: Iracema de Oliveira Moraes
Banca: Márcia Luzia Rizzatto
Resumo: A poligalacturonase (PG) é uma pectinase que degrada a molécula de pectina atuando internamente, ao acaso, liberando oligossacarídeos (Endo-PG) ou por ataque a partir da extremidade não redutora da cadeia, liberando moléculas de ácido galacturônico (Exo-PG). As pectinases, como várias outras enzimas, têm sido obtidas a partir de microrganismos, em processos fermentativos em estado sólido (FES) e fermentação submersa (FSM). A purificação e caracterização destas enzimas podem possibilitar o estudo das diferenças de suas propriedades funcionais, bem como, permitem avaliar suas possibilidades de aplicação, em diferentes condições, em processos industriais. Este trabalho teve por objetivos estudar a produção, purificação e caracterização da PG produzida por Penicillium viridicatum RFC3 através de FES e FSM. Para FSM foram testados os resíduos agroindustriais (farelo de trigo, bagaço de laranja, uma mistura de ambos) a 3% (p/v) além da água amarela (resíduo do processamento da indústria cítrica) no seu estado bruto e com diluições (2, 4 e 10x). Os pHs iniciais foram corrigidos para 4,5, 5 e 5,5 e a fermentação ocorreu por 24 - 96 h. A ANOVA mostrou que o extrato do bagaço de laranja (pH 5,5 e 96 h) foi o melhor meio para a produção da PG em FSM. A purificação da PG produzida em FSM iniciou-se pela ultrafiltração do extrato bruto (membranas de 50 e 10 kDa, repectivamente) seguida pela aplicação da amostra em uma coluna de filtração em gel Sephadex G75 e posterior aplicação em uma Q Sepharose. A fração isolada e desorvida revelou, após 2 eletroforese (PAGE-SDS) a homogeneidade da banda cuja massa molar foi estimada em 92,24 kDa e pI de 5,4. A enzima mostrou ser uma exopoligalacturonase (Exo-PG) com pH e temperatura ótimos em 5,0 e 50-55ºC
Abstract: The polygalacturonase (PG) is a pectinase that degrade pectin acting internally, releasing olygossacharide (Endo-PG) or by atack to a non reducing chain extremity, releasing galacturonic acid (Exo-PG). The pectinases, like many others enzymes, have been obtained from microorganisms by solid state fermentation (SSF) and submerged fermentation (SMF). The purification and characterization of these enzymes besides the study of differences of its functional properties permit its application on industrial process, on different conditions. This work aimed the study of production, purification and characterization of PG from Penicillium viridicatum RFC3 by SSF and SMF. In SMF they were assayed agroindustrial wastes (wheat bran, orange bagasse and a mixture of both) at 3% (w/v 1:1) and citric industry liquid waste (yellow water) in crude state and with dilutions (2, 4 and 10x). It was also evaluated the variation of the initial pH of the medium (4.5, 5.0 nd 5.5) and the time of fermentation (24 96 h). The ANOVA test revealed that the culture medium composed by orange bagasse extract, at pH 5.5 and 96 h, was the best condition for production of PG in SMF. The purification process of PG from SMF was carried out by the ultrafiltration of the crude extract (50 and kDa membranes, respectively) and by gel filtration in Sephadex G75 and Q Sepharose columns. The fraction with PG activity show by eletrophoresis (SDSPAGE), an homogeinity of a band with estimated molar mass of 92.24 KDa and pI 5.4. The enzyme revealed to be an Exo-PG with optimal pH and temperature of 5.0 and 50-55 °C. It also revealed to be Ca2+ dependent, which increased its 4 activity and stability. The Km of the enzyme was 1.30 and 1.16 mg/ml on Ca2+ presence. The Vmax was 1.76 and 2.07 ?mol/min/mg when Ca2+ was added. The better fermentation conditions in SSF was wheat bran mixed with orange bagasse (1:1 w/w) at 80% moisture as an culture medium and 336h of fermentation
Doutor
Romanová, Kristýna. "Proteomická identifikace enzymů degradující rostlinnou biomasu." Master's thesis, Vysoké učení technické v Brně. Fakulta chemická, 2011. http://www.nusl.cz/ntk/nusl-216797.
Full textTRINDADE, L. C. "Simulação computacional do efeito da pressão sobre a enzima pectina metilesterase do tomate." Universidade Federal do Espírito Santo, 2018. http://repositorio.ufes.br/handle/10/7374.
Full textEste trabalho descreve o desenvolvimento de uma simulação computacional, desenvolvida através do programa GROMACS. Mais especificamente, estudamos os resultados da simulação computacional referente a evolução do raio de giro da proteína Pectina Metilesterase (PME) do tomate em função da pressão aplicada, mantendo a temperatura fixa. Foi realizada uma descrição sobre os modelos físicos utilizados pelo programa. Também foi descrito, de forma suscinta, características sobre sistemas e estruturas dos aminoácidos e proteínas. As simulações computacionais consideraram uma temperatura de 26, 85oC e pressões aplicadas de 1 bar, 1 kbar, 3 kbar, 5 kbar, 7 kbar, 9 kbar e 10 kbar. As simulações trabalharam com um tempo de ação da pressão de até 100 pico segundos. Os resultados da simulação computacional indicaram uma redução não linear do raio de giro da enzima Pectina Metilesterase (PME) do tomate de acordo com o aumento da pressão aplicada, mantendo temperatura fixa.
Monteiro, Alexandre Costa [UNESP]. "Produção de poligalacturonases por fungos termofílicos e mesofílicos em fermentação em estado sólido e comparação das isoformas da enzima produzidas por cada grupo." Universidade Estadual Paulista (UNESP), 2008. http://hdl.handle.net/11449/94968.
Full textConselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)
As pectinases são enzimas com importantes aplicações econômicas, especialmente na indústria alimentícia. Pectinases termoestáveis, produzidas por microrganismos termofílicos, apresentam uma série de características que favorecem sua aplicação em processos industriais, como, por exemplo, estabilidade em ampla faixa de pH e temperatura e maior tolerância a agentes químicos desnaturantes de proteínas. Contudo, os principais microrganismos empregados na produção de poligalacturonases (PGs) são fungos mesofílicos. O presente trabalho teve por objetivo realizar um estudo comparativo de produção de PGs em fermentação em estado sólido (FES) por fungos mesofílicos e termofílicos. Foram utilizados os fungos mesofílicos Aspergillus niger NRRL 3112 e Penicillium itallicum IZ 1584, reconhecidos como bons produtores de PGs, os termofílicos Thermomyces lanuginosus ROB e Rhizomucor pusillus A13.36 e o termodúrico Aspergillus fumigatus N31. Os microrganismos foram cultivados em FES em farelo de trigo, os mesofílicos a 27ºC e os demais a 45ºC, por períodos de 10 a 15 dias. Os maiores produtores de PG foram P. itallicum IZ 1584 (51U/g) e A. fumigatus N31 (59,40 U/g). As PGs dos fungos termofílicos apresentaram maiores valores de temperatura ótima e maior termoestabilidade que as dos mesofílicos. T. lanuginosus produziu PG com maior valor de temperatura ótima (70ºC). A. fumigatus produziu a PG mais termoestável, que conservou 100% da atividade original após 1 h de incubação a 50ºC em ausência de substrato. Cromatografias em filtração em gel e zimogramas revelaram a expressão de 7 isoformas de PG em A. niger NRRL 3112, 3 isoformas em P. itallicum IZ 1584, T. lanuginosus ROB e R. pusillus, e 2 isoformas em A. fumigatus N31.
Pectinases are enzymes with important economic applications, especially in food industry. Termostable pectinases, produced by termophilic microorganisms, display an array of characteristics that support their application in industrial processes, such as stability through a wide pH and temperature range, as well as higher tolerance to protein denaturating chemical agents. However, the major microoganisms employed in polygalacturonases (PGs) prodution are mesophilic fungi. The present work aimed to make a comparative study of PG production by mesophilic and thermophilc fungi in Solid-State Fermentation (SSF). The fungi employed were the mesophilic Aspergillus niger NRRL 3112 and Penicillium itallicum IZ 1584, both known as good PGs producers, the thermophilic Thermomyces lanuginosus ROB and Rhizomucor pusillus A13.36 and the termoduric Aspergillus fumigatus N31. The fungi were bred in wheat bran in SSF, the mesophilic at 27ºC and the others at 45ºC, during periods of 10 to 15 days. The higher PGs producers where P. itallicum IZ 1584 (51U/g) and A. fumigatus N31 (59,40 U/g). PGs of thermophilc fungi have shown higher values of optimum temperature and termostability than mesophilic PGs. T. lanuginosus produced PG with the higher value of optimum temperature for activity (70ºC). A. fumigatus have shown the most thermostable PG, which mantained 100% of its original activity after 1 h of incubation at 50ºC in absence of substrate. Gel filtration chromatography and zymograms revealed the expression of 7 PG isoforms for A. niger, 3 isoforms for P. itallicum IZ 1584, T. lanuginosus ROB and R. Pusillus, and 2 isoforms for A. fumigatus N31.
Paixão, Airles Regina da Costa. "Ação da pectina metil esterase e cloreto de cálcio no armazenamento e controle da podridão-mole em pimentão." Universidade Federal de Sergipe, 2016. https://ri.ufs.br/handle/riufs/3011.
Full textThe pepper (Capsicum annuum L.) has commercial importance, by having sources of vitamins, minerals and fiber. However, a post-harvest problem, excessive softening which reduces the life and favors action pathogens such as Pectobacterium caratovorum subsp. caratovorum- Pcc, causal agent of soft rot, a major disease of post-harvest chili that is favored by the reduction of firmness. One technique that has recently been used for firmly maintaining the application is pectin methyl esterase (PME) with the addition of a calcium solution prologando thus reducing its lifetime and pathogen attack. The objective of this study was to use methyl pectin esterase (PME) exogenous associated with calcium chloride in maintaining firmness and control of Pectobacterium caratovorum subsp. caratovorum about pepper. The first experiment was conducted in a completely randomized design in a 4x5 factorial scheme with three replications for 12 days, evaluated every three days. In the second experiment, the test was conducted in a completely randomized design in a factorial 5x5 with three replications. In the first study the fruits of pepper were subjected to vacuum infusion method with pressure of 200 mmHg for 5 minutes, and following evaluated the loss of weight (PMF), fruit firmness (FF), skin color ( CC), soluble solids (SS), pH, total acidity (TA) and activity of SMEs. In the second test the fruits of pepper were subjected to vacuum infusion method with pressure of 200 mmHg for five minutes, then the fruits were inoculated with the PCC then held the fruit firmness analysis (FF), SME activity and the severity of the disease in chili (SD). The fruits obtained in general a reduction of over time firmly in all treatments but was found significant effect in maintaining the fruit firmness when treated in vacuum infusion with calcium chloride, without altering the physicochemical characteristics, as soluble solids content, total acidity and activity of SMEs, slowing the fruit ripening process. The fruits when treated with vacuum infusion with calcium chloride associated with pectin methyl esterase was not favorable because it altered the physicochemical properties chili, highlighting the decline of firmness, thus deteriorating the quality of the fruit. Regarding inoculation Pcc in the fruit was observed inhibition of growth of this pathogen, prolongation of fruit and better firmness treated fruits infusion over calcium inoculation Pcc chloride.
O pimentão (Capsicum annuum L.) tem grande importância comercial, por possuir fontes de vitaminas, minerais e fibras. Contudo, possui problema pós-colheita, o amolecimento excessivo que reduz a vida útil e favorece ação de patógenos como a Pectobacterium caratovorum subsp. caratovorum- Pcc, agente causal da podridão-mole, uma das principais doenças da pós-colheita do pimentão que é favorecida pela redução da firmeza. Uma técnica que vem sendo utilizada recentemente para a manutenção da firmeza é aplicação da pectina metil esterase (PME) com a adição de solução de cálcio prologando, assim, a sua vida útil e diminuindo o ataque do patógeno. Assim, o objetivo do trabalho foi utilizar a pectina metil esterase (PME) exógena associada ao cloreto de cálcio na manutenção da firmeza e no controle da Pectobacterium caratovorum subsp. caratovorum sobre o pimentão. O primeiro experimento foi realizado em delineamento inteiramente casualizado em esquema fatorial 4x5 com três repetições, durante 12 dias, avaliados a cada 3 dias. No segundo experimento, o ensaio foi realizado em delineamento inteiramente casualizado no esquema fatorial 5x5 com três repetições. No primeiro trabalho os frutos de pimentão foram submetidos ao método de infusão a vácuo com pressão de 200 mmHg por 5 minutos, e por seguinte avaliou-se a perda de massa fresca (PMF), firmeza do fruto (FF), cor da casca (CC), teor de sólidos solúveis (SS), pH, acidez total, (AT) e atividade de PME. No segundo ensaio os frutos de pimentão foram submetidos ao método de infusão a vácuo com pressão de 200 mmHg por cinco minutos, posteriormente os frutos foram inoculados com a Pcc em seguida realizou-se as análises de firmeza do fruto (FF), atividade de PME e a severidade da doença no pimentão (SD). Os frutos obtiveram de forma geral uma redução da firmeza ao longo do tempo em todos os tratamentos, porém foi verificado o efeito significativo na manutenção da firmeza dos frutos quando tratados em infusão a vácuo com cloreto de cálcio, não alterando as características físico-químicas, como o teor de sólidos solúveis, acidez total e atividade de PME, retardando o processo de amadurecimento do fruto. Os frutos quando tratados com infusão a vácuo com cloreto de cálcio associado à pectina metil esterase não foi favorável, pois alterou as propriedades físico-químicas do pimentão, com destaque para o declínio da firmeza, deteriorando assim a qualidade do fruto. Em relação à inoculação da Pcc no fruto, observou-se uma inibição do crescimento desse patógeno, prolongamento do fruto e uma melhor firmeza nos frutos tratados com infusão de cloreto de cálcio mais a inoculação da Pcc.
Leschevin, Maïté. "Implication de la paroi végétale et plus particulièrement des enzymes de modification des pectines dans la tolérance au stress salin chez Arabidopsis." Thesis, Amiens, 2021. http://www.theses.fr/2021AMIE0027.
Full textSoil salinization is a alarming situation encountered in several regions of the world where the pressure on water is becoming increasingly strong, especially due to climate change and the need to increase crop yields to face a global growing population. Excess of salt in soil affects plant physiological mechanism thus reducing plant production. A better knowledge of plant defense mechanism in response to salt stress is crucial to provide efficient strategies in crop yield. The plant cell wall is the first physical barrier between the plant cell compartment and the environment and plays an essential role in cell growth and development but also in response to various stresses, including salt stress. The cell wall is a highly complex and dynamic structure, mainly composed of polysaccharides (cellulose, hemicelluloses and pectins). Pectins can be methylesterified and acetylated, and their degree of methylesterification (DM) and acetylation (DA) can be modulated in muro by specific enzymes, pectin methylesterases (PMEs, EC 3.1.1.11) and acetylesterases (PAEs, EC 3.1. 1.6). Some parcelar data from the literature showed the role of pectins and their degree of methylesterification in tolerance to salt stress. The aim of this work was to provide new insights on the role of the cell wall in response to salt stress in the glycophyte Arabidopsis thaliana. Three distinct strategies were developed. Firstly, the natural variation between two common accessions of Arabidopsis thaliana (Wassilewskija, Ws and Columbia, Col-0) in response to salt stress has been characterized using an integrative approach establishing a correlation between physiological, biochemical, metabolomics and proteomics analyses. The results showed a better tolerance to salt stress associated with the genetic background Ws with an older developmental stage, a more efficient detoxification of reactive oxygen species and a higher content of xylan, mannan and lignin within the wall. Secondly, a reverse genetics approach has been developed to determine the contribution of two pectin remodeling enzymes, AtPME3 and AtPAE7 in salt tolerance. The results showed changes in the cell wall sugar composition as a reduction in homogalacturonan and an increase in arabinan in both atpme3 and atpae7 mutants after a long exposure to salt. Additionaly, salt stress induces a modulation of the PRE activities with an alteration of the pectin methylesterification pattern indicating a role of PME and PAE in cell wall integrity under salinity. Finally, a more informative approach combining cell wall metabolism, pectin remodeling enzymes, sodium ion detoxification pathway, and impact of calcium ions on cell wall integrity was carried out to characterize the role of the cell wall in the sodium hypersensitive mutant Atsos1. The SOS1 gene encodes a Na+/H+ antiporter which is involved in Na + exclusion. Preliminary results revealed that PME and PAE activities remained unchanged in atsos1 unlike the wild-type where the activites increased. That was associated with a reduction in pectin and mannan in atsos1, which was recovered by Ca2+ supply. All these data suggest the key role of atsos1 to maintain cell wall integrity under salt stress
Monteiro, Alexandre Costa. "Produção de poligalacturonases por fungos termofílicos e mesofílicos em fermentação em estado sólido e comparação das isoformas da enzima produzidas por cada grupo /." Rio Claro : [s.n.], 2008. http://hdl.handle.net/11449/94968.
Full textBanca: Eleonora Cano Carmona
Banca: Hamilton Cabral
Resumo: As pectinases são enzimas com importantes aplicações econômicas, especialmente na indústria alimentícia. Pectinases termoestáveis, produzidas por microrganismos termofílicos, apresentam uma série de características que favorecem sua aplicação em processos industriais, como, por exemplo, estabilidade em ampla faixa de pH e temperatura e maior tolerância a agentes químicos desnaturantes de proteínas. Contudo, os principais microrganismos empregados na produção de poligalacturonases (PGs) são fungos mesofílicos. O presente trabalho teve por objetivo realizar um estudo comparativo de produção de PGs em fermentação em estado sólido (FES) por fungos mesofílicos e termofílicos. Foram utilizados os fungos mesofílicos Aspergillus niger NRRL 3112 e Penicillium itallicum IZ 1584, reconhecidos como bons produtores de PGs, os termofílicos Thermomyces lanuginosus ROB e Rhizomucor pusillus A13.36 e o termodúrico Aspergillus fumigatus N31. Os microrganismos foram cultivados em FES em farelo de trigo, os mesofílicos a 27ºC e os demais a 45ºC, por períodos de 10 a 15 dias. Os maiores produtores de PG foram P. itallicum IZ 1584 (51U/g) e A. fumigatus N31 (59,40 U/g). As PGs dos fungos termofílicos apresentaram maiores valores de temperatura ótima e maior termoestabilidade que as dos mesofílicos. T. lanuginosus produziu PG com maior valor de temperatura ótima (70ºC). A. fumigatus produziu a PG mais termoestável, que conservou 100% da atividade original após 1 h de incubação a 50ºC em ausência de substrato. Cromatografias em filtração em gel e zimogramas revelaram a expressão de 7 isoformas de PG em A. niger NRRL 3112, 3 isoformas em P. itallicum IZ 1584, T. lanuginosus ROB e R. pusillus, e 2 isoformas em A. fumigatus N31.
Abstract: Pectinases are enzymes with important economic applications, especially in food industry. Termostable pectinases, produced by termophilic microorganisms, display an array of characteristics that support their application in industrial processes, such as stability through a wide pH and temperature range, as well as higher tolerance to protein denaturating chemical agents. However, the major microoganisms employed in polygalacturonases (PGs) prodution are mesophilic fungi. The present work aimed to make a comparative study of PG production by mesophilic and thermophilc fungi in Solid-State Fermentation (SSF). The fungi employed were the mesophilic Aspergillus niger NRRL 3112 and Penicillium itallicum IZ 1584, both known as good PGs producers, the thermophilic Thermomyces lanuginosus ROB and Rhizomucor pusillus A13.36 and the termoduric Aspergillus fumigatus N31. The fungi were bred in wheat bran in SSF, the mesophilic at 27ºC and the others at 45ºC, during periods of 10 to 15 days. The higher PGs producers where P. itallicum IZ 1584 (51U/g) and A. fumigatus N31 (59,40 U/g). PGs of thermophilc fungi have shown higher values of optimum temperature and termostability than mesophilic PGs. T. lanuginosus produced PG with the higher value of optimum temperature for activity (70ºC). A. fumigatus have shown the most thermostable PG, which mantained 100% of its original activity after 1 h of incubation at 50ºC in absence of substrate. Gel filtration chromatography and zymograms revealed the expression of 7 PG isoforms for A. niger, 3 isoforms for P. itallicum IZ 1584, T. lanuginosus ROB and R. Pusillus, and 2 isoforms for A. fumigatus N31.
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Haddaoui, Asmaa. "Dégradation des pigments anthocyaniques des jus de fruits rouges par les activités [Bêta]-glycosidasiques présentés dans les préparations pectinolytiques industrielles d'origine fongique." Vandoeuvre-les-Nancy, INPL, 1995. http://www.theses.fr/1995INPL151N.
Full textDelmas, Frédéric. "L'acide 3-Désoxy-D-Manno-2octulosonique 8-Phosphate (KDO-8-P) synthase chez les plantes : caractérisation fonctionnelle d'une enzyme impliquée dans la biosynthèse d'un composé pectinique des parois végétales." Bordeaux 2, 2004. http://www.theses.fr/2004BOR21104.
Full textThe KDSA gene codes for the 3-deoxy-D-manno-octulosonic acid 8-phosphate (Kdo-8-P) synthase which synthesizes the phosphorylated precursor of Kdo, an essential and indissociable component of the outer membrane in Gram-negative bacteria. In plants, the role of Kdo is largely misunderstood ; however it is a constituent of a complex pectic polysaccharide : rhamnogalacturonan II, essential for the structuration of the pectin bnetwork in primary cell wall. The study of the KDSA enzyme has been performed in tomato (Lycopersicon esculentum Mill. ) and in Arabidopsis thaliana. The expression of KDSA was shown to be preferentially associated with dividing cells. Its function is thought to be of great importance since no null mutant plants, lacking the Kdo-8P synthase activity, could be isolated. A preliminary study of the Kdo transferase has been performed. This enzyme is the last one of the biosynthetic pathway of Kdo and the first results will be discussed
Evangelista, Danilo Elton. "Produção recombinante, caracterização enzimática e estudos sobre a ocorrência de pectinases no bicudo da cana-de-açúcar (Sphenophorus levis, Curculionidae)." Universidade Federal de São Carlos, 2012. https://repositorio.ufscar.br/handle/ufscar/5498.
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Plant cell wall confers to the cell plant, structural support as well as protection against pathogens and phytophagous. Among the cell wall polysaccharides includes pectic substances, which are composed of partially methyl-esterified galacturonic acid residues linked by α-1,4 glycosidic bonds. These enzymes are often synthesized by phytophatogenic micro-organisms for invasion of host plant or by own plant for modeling plant cell wall. The pectic substances are the major component of middle lamella and are natural degraded by pectinases action. Pectin methylesterase (PME) catalysis removes methyl-ester groups, and the Endo-polygalacturonase (Endo-PG) promoves the randomly hydrolysis reaction of α-1,4 bonds. One of the most importante agricultural pest species of the family Curculionidae (Coleoptera: Curculionidae) is Sphenophorus levis, the sugarcane weevil. The larvae of this insect penetrate into the rhizome and build galleries in the stem, decreasing productivity and causing the death of the plant. Large damages to the crop like that are significant in the costs of products derived from sugarcane. Considering the impact of this pest in the sugarcane crop and the absence of efficient method for control, new strategies for controlling are still necessary. The analysis of the cDNA library of S. levis larvaes shows the presence of one PME and one Endo-PG genes that we called Sl-PME and Sl-EndoPG respectively. Considering the importance of studies of insect pests and the extensively use of theses pectinases in different industry fields, we performed the characterization of genomic sequences coding for S. levis pectinases (Sl-Pectinases). It was also carried out the production and characterization of a Sl-PME and a Sl-EndoPG recombinant, expressed in heterologous system. We also accomplished analysis of gene expression by qRTPCR in different stages of development as well as different tissues, and phylogenetic studies between Sl-Pectinases and other pectinases from different kingdoms. The Sl- Pectinases sequences identified, show more similar to homologous insect genes deposited in the GenBank, especially with Sitophilus oryzae. The phylogenetic analysis indicates that the insect group is more correlated with bacteria group and fungi group respectively to PMEs and EndoPGs sequences. Pectinases genomic sequences revealed two introns for Sl-EndoPG gene with 53 and 166 bp, but no one for Sl-PME gene. Both of Sl-Pectinases Recombinant showed catalytic activity. The recombinant Sl-EndoPG shows optimal activity at pH 5,06 ± 0,27 and 49,74 ± 2,49 oC, but extremely low thermostability. For the polygalacturonic acid no-methylated as substract, the enzyme revealed Km = 3,88 mg.mL-1, Vmax = 21.96 μM.s-1 e Kcat = 3.137 s-1; for the citrus pectin partially methylated as substract, the enzyme presented Km = 4,98 mg.mL-1, Vmax = 17,19 μM.s-1 e Kcat = 2.456 s-1. Results in expression analysis suggest that S. levis pectinases have a digestive enzymes role, actting on the midgut. The present work represents the first pectinases of insect produced in Pichia pastoris heterologous system and characterized as optimal conditions of activity, thermostability and kinetic parameters.
A parede celular vegetal confere à célula vegetal suporte estrutural, proteção contra patógenos e fitófagos. Dentre os polissacarídeos da parede celular vegetal são inclusas as substâncias pécticas, as quais são compostas por resíduos de ácido galacturônico, parcialmente esterificados, ligados em série via ligações glicosídicas α- 1,4. As substâncias pécticas são o maior componente da lamela média e são naturalmente degradadas pela ação enzimática das pectinases. A Pectina metilesterase (PME) catalisa a remoção dos grupos metil-ester, e a Endo- Poligalacturonase (Endo-PG) promove a reação de hidrólise aleatória das ligações α- 1,4. Essas enzimas são comumente sintetizadas por micro-organismos fitopatógenos para invasão ao hospedeiro ou pelas próprias plantas para modelamento da parede celular vegetal. Na agricultura, uma das mais importantes espécies pragas da família Curculionidae (Coleoptera: Curculionidae) é o Sphenophorus levis, o bicudo da canade- açúcar. As larvas deste inseto penetram no rizoma e constroem galerias ao longo do colmo, causando queda na produtividade ou até mesmo a morte da planta. Grandes danos na cultura como esses são significativos nos custos de produtos derivados da cana-de-açúcar. Considerando o impacto dessa praga na cultura e a ausência de eficientes métodos de controle, novas estratégias ainda são necessárias no combate à praga. A análise de uma biblioteca de cDNA de larvas do inseto S. levis mostrou a presença de genes codificantes para uma PME e uma Endo-PG, os quais nomeamos de Sl-PME e Sl-EndoPG respectivamente. Devido a importância nos estudos de insetos pragas e a extensa aplicação das pectinases em diversos campos industriais, foi promovida a caracterização das sequências genômicas codificantes para as pectinases de S. levis (Sl-Pectinases). Também foi realizada a produção e caracterização de uma Sl-PME e uma Sl-EndoPG recombinantes, expressas em sistema heterólogo. Além disso, foram conduzidas análises de expressão gênica por qRT-PCR em diferentes estágios de desenvolvimento e diferentes tecidos; e estudos filogenéticos entre as Sl-Pectinases e outras pectinases de diferentes reinos. As sequências das Sl-Pectinases identificadas apresentaram maior similaridade com genes homólogos de insetos depositados no GenBank, principalmente como o Sitophilus oryzae. A análise filogenética indicou que o grupo dos insetos é mais correlacionado com o grupo das bactérias e com o grupo de fungos, respectivamente para as sequências PMEs e Endo-PGs. As sequências genômicas das pectinases revelaram dois introns para Sl-EndoPG com 53 e 166 pb, mas nenhum para o gene Sl- PME. Ambas as Sl-Pectinases Recombinantes apresentaram atividade catalítica. A Sl- EndoPG recombinante mostrou maior atividade em pH 5,06 ± 0,27 e 49,74 ± 2,49 oC, mas baixa termoestabilidade. Para o substrato ácido poligalacturônico não metilado, a enzima revelou Km = 3,88 mg.mL-1, Vmax = 21.96 μM.s-1 e Kcat = 3.137 s-1, para o substrato pectina de citrus parcialmente metilada, a enzima apresentou Km = 4,98 mg.mL-1, Vmax = 17,19 μM.s-1 e Kcat = 2.456 s-1. Os resultados da análise de expressão sugerem que as pectinases de S. levis são enzimas digestivas atuantes no intestino médio. Este trabalho representa as primeiras pectinases de inseto produzidas em sistema heterólogo de Pichia pastoris e caracterizadas quanto a condições ótimas de atividade, termoestabilidade e parâmetros cinéticos.
Carpentier, Laurence. "Étude des interactions entre protéines : application à la séparation d’enzymes pectinolytiques." Compiègne, 1986. http://www.theses.fr/1986COMPD024.
Full textDufour, Dominique. "Contribution à l'étude de la physiologie des champignons pectolytiques, cultivés en milieu solide, en relation avec la respiration et la synthèse de pectinases." Compiègne, 1990. http://www.theses.fr/1990COMPD307.
Full textBarnavon, Laurent. "Etude d'enzymes et de l'expression des gènes correspondants impliqués ans l'évolution des polysaccharides pariétaux au cours du développement de la baie de raisin." Montpellier 2, 1999. http://www.theses.fr/1999MON20173.
Full textMassiot, Patrice. "Caracterisation structurale et degradation enzymatique des polysaccharides de parois cellulaires de la racine de carotte (daucaus carota l. )." Rennes 1, 1988. http://www.theses.fr/1988REN10089.
Full textLegentil, Anne. "Étude de la composition chimique des pectines de la paroi cellulaire de la fraise et de leur solubilisation par des préparations enzymatiques industrielles : application à la liquéfaction de la fraise." Vandoeuvre-les-Nancy, INPL, 1996. http://www.theses.fr/1996INPL018N.
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