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Deslignificación De Pasta Kraft De Pinus Radiata Con Una Levadura Genéticamente Modificada Para Producir Lacasa A

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Deslignificación De Pasta Kraft De Pinus Radiata Con Una Levadura Genéticamente Modificada Para Producir Lacasa A Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA) Forest Systems 2010 19(2), 234-240 Available online at www.inia.es/forestsystems ISSN: 2171-5068 eISSN: 2171-9845 Deslignificación de pasta kraft de Pinus radiata con una levadura genéticamente modificada para producir lacasa A. Arana-Cuenca1*, A. Téllez-Jurado1, S. Yagüe2, E. Fermiñán3, J. M. Carbajo4, A. Domínguez5, T. González4, J. C. Villar5 y A. E. González2 1 Universidad Politécnica de Pachuca. Ctra. Pachuca-Cd. Sahagún, km 20. Rancho Luna. Ex Hacienda de Sta. Bárbara. Municipio de Zempoala. 43830 Hidalgo. Mexico 2 CBMSO-CSIC. C/ Nicolás Cabrera, 1. Cantoblanco-UAM. 28033 Madrid. España 3 Departamento de Microbiología y Genética. Universidad de Salamanca. 37007 Salamanca. España 4 CIFOR-INIA. Laboratorios de Celulosa y Papel. Ctra. A Coruña, km 7,5. 28040 Madrid. España 5 Instituto Cubano de Investigaciones de los Derivados de la Caña de Azúcar (ICIDCA). Cuba Resumen Una posible aplicación biotecnológica de las lacasas fúngicas es el blanqueo de pasta de papel mediante la degra- dación de la lignina en la pasta cruda. Para ello es necesaria su producción a bajo coste y a concentraciones superio- res a las secretadas por los hongos de forma natural. La expresión heteróloga en levaduras es una alternativa para su producción a mayor escala. En el presente trabajo se ha realizado la expresión heteróloga del gen cglcc1, responsable de la producción de lacasa en el hongo basidiomiceto Coriolopsis gallica, en la levadura Kluyveromyces lactis. Pos- teriormente y para verificar si la capacidad deslignificante se mantiene en la levadura, se ha tratado una pasta kraft de Pinus radiata con la cepa recombinante. Tras el tratamiento, el número kappa disminuyó en un 13%, mientras que el contenido en lignina lo hizo en un 22%, corroborando así el efecto deslignificador. Se concluye que el empleo de microorganismos carentes de actividades celulolíticas y genéticamente modificados para producir altos niveles de ac- tividad lacasa constituye una opción para deslignificar aplicable a procesos de blanqueo de pasta de celulosa. Palabras clave: lignina, bioblanqueo, pastas kraft, lacasa heteróloga, Kluyveromyces lactis, Coriolopsis gallica Abstract Delignification of Pinus radiata kraft pulp by treatment with a yeast genetically modified to produce laccases Cellulose pulp bleaching is one of the main biotechnological applications of fungal laccases due to their capacity to degrade lignin from unbleached pulp. This application requires low cost enzyme production and higher enzyme concentrations than those obtained from the natural fungal producers. Heterologous expression of laccase in yeasts is an option for producing these enzymes on an industrial scale. In this work, we have demostrated the heterologous expression of the cglcc1 gene, responsible for laccase production in the basidiomicetous fungus Coriolopsis gallica, in the yeast Kluyveromyces lactis. In order to know if the transformed yeast has delignificant capability, a Pinus radiata kraft pulp has been incubated with it. After the treatment, a significant decrease in kappa number (13%) and in lignin content (22%) was observed. These results showed the delignificant capability of this transformed yeast. It can be concluded that the use of genetically modified microorganisms that do not demonstrate cellulolitic activity can produce high laccase levels and delignify cellulose pulps with a potential applications in cellulose pulp bleaching. Key words: lignin, biobleaching, kraft pulp, heterologous laccase, Kluyveromyces lactis, Coriolopsis gallica. Introducción tructura, tienen baja especificidad por sustrato, reducen el oxígeno molecular a agua y pueden oxidar diferentes Las lacasas (EC 1.10.3.2) son enzimas del grupo oxi- compuestos fenólicos y no fenólicos (González et al., dasas azules que contienen átomos de cobre en su es- 2005). Las lacasas secretadas por los hongos participan en varios procesos celulares, incluyendo deslignifica- * Corresponding author: [email protected] ción, esporulación, producción de pigmentos, forma- Received: 15-01-10; Accepted: 24-06-10. ción del cuerpo fructífero y en mecanismos de patoge- Bio-deslignificación de pasta kraft de P. radiata 235 nicidad (Arana et al., 2002). Debido a su baja especifi- codifica para la lacasa de Coprinellus congregatus au- cidad de sustrato se han propuesto varias aplicaciones menta la supervivencia de S. cereviseae frente al estrés industriales como el mantenimiento de la calidad en vi- oxidativo (Kim et al., 2006). Trichoderma reesei, trans- nos, el aumento de la digestibilidad de piensos obteni- formado con el gen lacasa de Phlebia radiata, puede dos a partir de residuos agrícolas y la conversión de in- modificar la fracción soluble de una pasta kraft (Salo- termedios químicos (Rodríguez Couto y Toca Herrera, heimo y Niku-Paavola, 1991). 2006). No obstante, la función más estudiada de estas Coriolopsis gallica es un hongo basidiomiceto de po- enzimas es su participación en el proceso de desligni- dredumbre blanca con capacidad ligninolítica (Calvo et ficación natural, por lo que se ha propuesto emplearla al., 1998). Se ha probado que puede decolorar y dismi- en la industria de pasta de papel en procesos de biopul- nuir la DQO de efluentes de la industria cervecera con peo y de bioblanqueo (Bajpai et al., 2007) donde susti- alto contenido fenólico (Yagüe et al., 2000) y decolo- tuiría total o parcialmente el uso de reactivos químicos rar colorantes textiles (Robinson et al., 2001). Se ha clo- con la consecuente mejora medioambiental. Kirk y Yang nado y caracterizado el gen cglcc1 que codifica para (1979) reportaron que Phanerochaete chrysosporium una lacasa y su ADNc correspondiente (GenBank, No. puede deslignificar parcialmente pulpa no blanqueada de acceso AF297874, http://www.ncbi.nlm.nih.gov) y y se han realizado numerosos estudios usando hongos se ha estudiado la expresión de dicho gen usando ácido ligninolíticos o las enzimas que secretan para reducir la tánico como inductor (Carbajo et al., 2002). Por otra cantidad de lignina. parte, Kluyveromyces lactis es una levadura muy estu- El número kappa disminuyó 11 puntos tras 8 días de diada en la industria alimentaria, específicamente en la incubación con Phlebia sp. MG-60 (Li et al., 2002); producción de β-galactosidasa (lactasa). Su habilidad además Tavares et al. (2004) reportan 50% de reducción para crecer en sustratos baratos como lactosa, aumenta usando un tratamiento multietapa con aumento a 110°C, su potencial como huésped para producir proteínas he- seguido de un tratamiento con la lacasa de una pasta de terólogas. Esta levadura ya ha sido usada con éxito pa- eucalipto. Según Balakshin et al. (2001) se reduce de ra producir lacasa de modo heterólogo (Piscitelli et al., 13,7 a 8,5 el número kappa de una pasta kraft de euca- 2005; Faraco et al., 2008). lipto usando la lacasa, aumentando la temperatura a El objetivo del presente trabajo ha sido producir ac- 65°C y con una presión parcial de oxígeno de 5 bar. tividad lacasa en la levadura Kluyveromyces lactis, ca- Para que estos procesos puedan ser rentables es ne- rente de actividades celulolíticas. El propósito ha sido cesaria una fuente de proteínas de bajo costo. Dado obtener un microorganismo eficaz para deslignificar que en la mayoría de las ocasiones la producción de pasta de celulosa, donde el empleo directo de hongos esta enzima por las cepas silvestres es baja, es difícil de pudrición blanca como Coriolopsis gallica está des- alcanzar los niveles mínimos necesarios para que su aconsejado pues provoca a su vez la degradación de la producción sea rentable y se investigan posibles alter- celulosa. Para ello, se pretende introducir el ADNc del nativas, siendo una de las más factibles su expresión, gen cglcc1, responsable de la producción de la proteí- de forma regulada, como proteína heteróloga en orga- na lacasa del hongo C. gallica, en K. lactis, una leva- nismos modelo. dura de interés industrial. Una vez comprobada la pro- La expresión heteróloga de la lacasa se ha estudiado ducción activa de la proteína heteróloga, el siguiente desde 1990 (Kojima et al., 1990) y las lacasas de dife- paso será comprobar la capacidad deslignificante de la rentes microorganismos se han expresado en levaduras levadura transformada, para lo que ésta se aplicará a una y hongos. La empresa Novo Nordish Co. comercializa pasta kraft de Pinus radiata y se medirá el efecto pro- el producto DeniLite II consistente en la lacasa de Myce- ducido y sus repercusiones sobre un blanqueo posterior. liophtora thermophila expresada heterológamente en Aspergillus oryzae usado en la industria textil para el destintado del azul índigo. También se han realizado es- Material y métodos tudios de producción de lacasa recombinante en Sa- charomyces cerevisiae que, transformada con el gen que Obtención y caracterización de la pasta codifica para la lacasa de Trametes versicolor, puede fermentar azúcares transformándolos en etanol sin in- La madera de P. radiata se recibió en forma de tro- hibir su crecimiento por la presencia de fenoles (Lars- zas, fue descortezada manualmente y astillada en una son et al., 2001). La expresión heteróloga del gen que astilladora semi-industrial de volante de dos cuchillas. 236 A. Arana-Cuenca et al. / Forest Systems (2010) 19(2), 234-240 Las astillas fueron posteriormente tamizadas para re- Construcción del plásmido y transformación coger la fracción entre 8 mm y 30 mm. de K. lactis La cocción kraft de las astillas se realizó en un di- gestor de 25 L de capacidad con recirculación forza- En primer lugar se construyó el «cassete» promotor- da de las lejías y calentamiento indirecto por vapor. La gen-terminador. Para ello se uso el plásmido pBlues- temperatura se ha controlado desde un ordenador pro- cript II SK (+) donde se insertó el promotor del gen visto de una interfase y una aplicación informática di- klpho5, el ADNc del gen cglcc1 y finalmente el termi- señada a tal efecto. Previamente a la cocción, las asti- nador del gen klpho5. Para las ligaciones se usaron 50 llas se sometieron a un tratamiento con vapor a 100°C ng de plásmido con una relación molar 1:3 (plásmi- durante 10 min.
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