{"id":71311,"date":"2018-03-09T23:15:55","date_gmt":"2018-03-09T23:15:55","guid":{"rendered":"https:\/\/www.deberes.net\/tesis\/sin-categoria\/nad-glutamato-deshidrogenasa-de-haloferax-mediterranei-clonaje-secuenciacion-y-expresion-purificacion-y-propiedades-de-la-enzima-nativa-y-recombinante\/"},"modified":"2018-03-09T23:15:55","modified_gmt":"2018-03-09T23:15:55","slug":"nad-glutamato-deshidrogenasa-de-haloferax-mediterranei-clonaje-secuenciacion-y-expresion-purificacion-y-propiedades-de-la-enzima-nativa-y-recombinante","status":"publish","type":"post","link":"https:\/\/www.deberes.net\/tesis\/quimica\/nad-glutamato-deshidrogenasa-de-haloferax-mediterranei-clonaje-secuenciacion-y-expresion-purificacion-y-propiedades-de-la-enzima-nativa-y-recombinante\/","title":{"rendered":"Nad-glutamato deshidrogenasa de haloferax mediterranei: clonaje, secuenciaci\u00f3n y expresi\u00f3n. purificaci\u00f3n y propiedades de la enzima nativa y recombinante."},"content":{"rendered":"<h2>Tesis doctoral de <strong> Susana D\u00edaz Gonz\u00e1lez <\/strong><\/h2>\n<p>Haloferax mediterranei es un organismo hal\u00f3filo extremo perteneciente al dominio archaea y que presenta diferentes actividades glutamato deshidrogenasa, enzima que cataliza la interconversi\u00f3n entre catabolitos tan importantes como el 2-oxoglutarato y el l-glutamato. Una de ellas, la nadp-dependiente, presenta  un m\u00e1ximo de actividad a 2 m de nacl y 1 m de kcl, siendo ligeramente mayor en presencia de kcl que en nacl. Tambi\u00e9n se ha determinado actividad glutamato deshidrogenasa nad-dependiente, con la que se han realizado estudios de purificaci\u00f3n y caracterizaci\u00f3n, as\u00ed como el clonaje, secuenciaci\u00f3n y expresi\u00f3n de su gen.       Con la finalidad de obtener enzima en concentraciones elevadas que permitiesen realizar estudios de cristalizaci\u00f3n, el gen de la nad-glutamato deshidrogenasa de hfx. Mediterranei, se expres\u00f3 en el hu\u00e9sped mes\u00f3filo e. Coli, obteni\u00e9ndose la prote\u00edna en forma de agregados insolubles. La renaturalizaci\u00f3n de la nad-glutamato deshidrogenasa recombinante se llev\u00f3 a cabo mediante diluci\u00f3n r\u00e1pida empleando tampones con una elevada concentraci\u00f3n de sal, siendo \u00e9sta \u00f3ptima a 4 m de nacl.    Se purific\u00f3 y caracteriz\u00f3 la nad-glutamato deshidrogenasa nativa observando que las propiedades moleculares y cin\u00e9ticas determinadas para esta enzima son pr\u00e1cticamente id\u00e9nticas a las obtenidas para la enzima recombinante. La nad-glutamato deshidrogenasa de hfx. Mediterranei, es un homohex\u00e1mero con un tama\u00f1o de subunidad de aproximadamente 48 kda y una masa molecular de 290 kda. La enzima presenta un comportamiento halof\u00edlico y tambi\u00e9n cierto car\u00e1cter termof\u00edlico, con un m\u00e1ximo de actividad a      4 m de nacl y una temperatura \u00f3ptima de 60 \u00c2\u00bac. Adem\u00e1s el an\u00e1lisis pept\u00eddico realizado mediante la t\u00e9cnica de nanoelectrospray lc\/ms, permite la confirmaci\u00f3n de forma inequ\u00edvoca de la identidad de la enzima nativa con la enzima obtenida mediante clonaje, secuenciaci\u00f3n y expresi\u00f3n.El an\u00e1lisis comparativo<\/p>\n<p>&nbsp;<\/p>\n<h3>Datos acad\u00e9micos de la tesis doctoral \u00ab<strong>Nad-glutamato deshidrogenasa de haloferax mediterranei: clonaje, secuenciaci\u00f3n y expresi\u00f3n. purificaci\u00f3n y propiedades de la enzima nativa y recombinante.<\/strong>\u00ab<\/h3>\n<ul>\n<li><strong>T\u00edtulo de la tesis:<\/strong>\u00a0 Nad-glutamato deshidrogenasa de haloferax mediterranei: clonaje, secuenciaci\u00f3n y expresi\u00f3n. purificaci\u00f3n y propiedades de la enzima nativa y recombinante. <\/li>\n<li><strong>Autor:<\/strong>\u00a0 Susana D\u00edaz Gonz\u00e1lez <\/li>\n<li><strong>Universidad:<\/strong>\u00a0 Alicante<\/li>\n<li><strong>Fecha de lectura de la tesis:<\/strong>\u00a0 19\/11\/2004<\/li>\n<\/ul>\n<p>&nbsp;<\/p>\n<h3>Direcci\u00f3n y tribunal<\/h3>\n<ul>\n<li><strong>Director de la tesis<\/strong>\n<ul>\n<li>Mar\u00eda Jos\u00e9 Bonete  P\u00e9rez<\/li>\n<\/ul>\n<\/li>\n<li><strong>Tribunal<\/strong>\n<ul>\n<li>Presidente del tribunal: Juan  Luis Serra ferrer <\/li>\n<li>arturo Manjon rubio (vocal)<\/li>\n<li>Jos\u00e9 Antonio Ferragut rodriguez (vocal)<\/li>\n<li>M\u00aa dolores Arriaga giner (vocal)<\/li>\n<\/ul>\n<\/li>\n<\/ul>\n<p>&nbsp;<\/p>\n","protected":false},"excerpt":{"rendered":"<p>Tesis doctoral de Susana D\u00edaz Gonz\u00e1lez Haloferax mediterranei es un organismo hal\u00f3filo extremo perteneciente al dominio archaea y que presenta [&hellip;]<\/p>\n","protected":false},"author":1,"featured_media":0,"comment_status":"open","ping_status":"open","sticky":false,"template":"","format":"standard","meta":{"site-sidebar-layout":"default","site-content-layout":"","ast-site-content-layout":"","site-content-style":"default","site-sidebar-style":"default","ast-global-header-display":"","ast-banner-title-visibility":"","ast-main-header-display":"","ast-hfb-above-header-display":"","ast-hfb-below-header-display":"","ast-hfb-mobile-header-display":"","site-post-title":"","ast-breadcrumbs-content":"","ast-featured-img":"","footer-sml-layout":"","theme-transparent-header-meta":"","adv-header-id-meta":"","stick-header-meta":"","header-above-stick-meta":"","header-main-stick-meta":"","header-below-stick-meta":"","astra-migrate-meta-layouts":"default","ast-page-background-enabled":"default","ast-page-background-meta":{"desktop":{"background-color":"var(--ast-global-color-4)","background-image":"","background-repeat":"repeat","background-position":"center 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