Caracterización bioquímica y molecular de una esterasa fúngica. Aplicación en la industria papelera

Leída en Universidad Complutense de Madrid. Facultad de Ciencias Biológicas el 06-26-2006; 217 págs.En este trabajo se describe una nueva esterasa fúngica producida en cultivos líquidos del hongo Ophiostoma piceae. Los preparados enzimáticos comercializados hasta la fecha, aunque son eficaces en el control biológi-co del pitch en coníferas, no rinden buenos resultados en las maderas de frondosas. Este hecho motivó la búsqueda de una enzima capaz de de-gradar los compuestos involucrados en la formación de depósitos en es-tas especies vegetales. Estudios preliminares pusieron de manifiesto la actividad enzimática del hongo O. piceae sobre ésteres de p-nitrofenol y oleato de colesterilo. Éste último fue escogido como modelo de los éste-res de esteroles presentes en los extraíbles de la madera de Eucaliptus globulus, especie empleada mayoritariamente en España para la produc-ción de pasta de papel. Inicialmente se estudiaron varias actividades enzimáticas, utilizando un medio basal que contenía glucosa, como fuente de carbono y aceite de oliva, como inductor. En estas condiciones, se purificó y caracterizó u-na única enzima. Ésta mostraba una elevada afinidad sobre triglicéridos y ésteres de esteroles. La degradación de mezclas complejas de estos compuestos, presentes en los extraíbles y pastas de papel de diferentes especies vegetales, evidenciaron la posible aplicación de la esterasa en el control biológico del pitch en madera de frondosas y coníferas. La determinación del extremo N-terminal de la proteína y péptidos in-ternos, resultado de la hidrólisis de la misma, permitieron obtener una sonda de DNA específica. De esta forma, se pudo llevar a cabo la se-cuenciación del gen de la esterasa de O. piceae. La comparación con los genes de otras esterasas mostró una homología de aproximadamente un 40% con los que codifican las lipasas de Candida rugosa y Geotrichum candidum. A partir de estos estudios, se pudieron identificar los residuos implicados en la catálisis de la enzima. El modelo estructural de la este-rasa, construido a partir de las estructuras cristalográficas de las lipasas CRL1 y CRL3 de C. rugosa, permitió englobar a la enzima en la familia de las α/β hidrolasas, así como identificar algunos de los aminoácidos responsables de su especificidad de sustrato.Beca Técnicos especializados Comunidad de Madrid y contrato adscrito proyecto europeo QLRT-1999-01357Peer reviewe

Characterization and application of a sterol esterase immobilized on polyacrylate epoxy-activated carriers (DilbeadsTM)

The sterol esterase from the ascomycete Ophiostoma piceae was immobilized on novel polyacrylate-based epoxy-activated carriers (DilbeadsTM). Six supports with particle sizes between 120-165 micrometers were prepared varying the composition of monomers, crosslinkers and porogens. Their surface areas and porosities were determined by N2 adsorption and mercury intrusion porosimetry. The pore volumes ranged from 0.63 to 1.32 cm3/g, but only DilbeadsTM RS and NK had narrow pore size distributions (with maxima at 33.5 and 67.0 nm, respectively). The distribution of the enzyme in the support was studied by fluorescence confocal microscopy. The immobilized esterase on DilbeadsTM TA showed a significant pH and thermal stability and was assayed in the continuous hydrolysis of cholesteryl esters -present in the pulp industry process waters-.We thank Mª Teresa Seisdedos (Centro de Investigaciones Biologicas, CSIC) for help with the confocal microscopy. This research was supported by the Spanish Ministry of Education and Science (Projects BIO2002-00337 and BIO2003-00621) and Comunidad de Madrid (Project S-0505/AMB0100). We thank CSIC for a research fellowshipPeer reviewe

Gestión del conocimiento. Perspectiva multidisciplinaria. Volumen 10

El libro “Gestión del Conocimiento. Perspectiva Multidisciplinaria”, Volumen 10, de la Colección Unión Global, es resultado de investigaciones. Los capítulos del libro, son resultados de investigaciones desarrolladas por sus autores. El libro es una publicación internacional, seriada, continua, arbitrada de acceso abierto a todas las áreas del conocimiento, que cuenta con el esfuerzo de investigadores de varios países del mundo, orientada a contribuir con procesos de gestión del conocimiento científico, tecnológico y humanístico que consoliden la transformación del conocimiento en diferentes escenarios, tanto organizacionales como universitarios, para el desarrollo de habilidades cognitivas del quehacer diario. La gestión del conocimiento es un camino para consolidar una plataforma en las empresas públicas o privadas, entidades educativas, organizaciones no gubernamentales, ya sea generando políticas para todas las jerarquías o un modelo de gestión para la administración, donde es fundamental articular el conocimiento, los trabajadores, directivos, el espacio de trabajo, hacia la creación de ambientes propicios para el desarrollo integral de las instituciones

Caracterización bioquímica y molecular de una esterasa fúngica : aplicación en la industria papelera

Depto. de Microbiología y ParasitologíaFac. de Ciencias BiológicasTRUEunpu

Apolipoprotein E genotyping method by real time PCR, a fast and cost-effective alternative to the TaqMan and FRET assays

The apolipoprotein E gene (APOE) polymorphism genotyping has an allegedly important predictive value for coronary heart disorders and Alzheimer’s disease. We developed a simple, fast, cost-effective and suited for high-throughput protocol for determining APOE genotypes by Real Time PCR monitored by SYBR® Green. The method is based on differential amplification by allele-specific primers. These primers have variations in their 3 -end nucleotides such that are specific for one of the two variants in each polymorphic position. By this protocol,we obtained a 100% concordance with the APOE genotypes determined by sequencing analysis. The main advantages of this method are its relative simplicity and the reduced cost compared to other methodologies, such as the TaqMan® and FRET assays.Fundingwas provided by grants FIS 05/0912 from the Ministerio de Sanidad, CIEN Foundation and the Spanish CIBERNED network.Peer reviewe

Additional mechanisms conferring genetic susceptibility to Alzheimer’s disease

Familial Alzheimer’s disease (AD), mostly associated with early onset, is caused by mutations in three genes (APP, PSEN1, and PSEN2) involved in the production of the amyloid βpeptide. In contrast, the molecular mechanisms that trigger the most common late onset sporadic AD remain largely unknown. With the implementation of an increasing number of case-control studies and the upcoming of large-scale genomewide association studies there is a mounting list of genetic risk factors associated with common genetic variants that have been associated with sporadic AD. Besides apolipoprotein E, that presents a strong association with the disease (OR~4), the rest of these genes have moderate or low degrees of association, with OR ranging from 0.88 to 1.23. Taking together, these genes may account only for a fraction of the attributable AD risk and therefore, rare variants and epistastic gene interactions should be taken into account in order to get the full picture of the genetic risks associated with AD. Here, we review recent whole-exome studies looking for rare variants, somatic brain mutations with a strong association to the disease, and several studies dealing with epistasis as additional mechanisms conferring genetic susceptibility to AD. Altogether, recent evidence underlines the importance of defining molecular and genetic pathways, and networks rather than the contribution of specific genes.This work was supported by CIBERNED, CIEN and Reina Sofia Foundations and the Carlos III Health Institute (PI12/00045).Peer Reviewe

Genetic variability of the gene cluster CALHM1-3 in sporadic Creutzfeldt-Jakob disease

Perturbations of calcium homeostasis have been associated with several neurodegenerative disorders. A common polymorphism (rs2986017) in the CALHM1 gene, coding for a regulator of calcium homeostasis, is a genetic risk factor for the development of Alzheimer disease (AD). Although some authors failed to confirm these results, a meta-analysis has shown that this polymorphism modulates the age at disease onset. Furthermore, a recent association study has explored the genetic variability of CALHM1 gene and two adjacent paralog genes (CALHM3 and CALHM2) in an Asian population. Since several lines of evidence suggest that AD and prion diseases share pathophysiologic mechanisms, we investigated for the first time the genetic variability of the gene cluster formed by CALHM1 and its paralogs in a series of 235 sporadic Creutzfeldt-Jakob disease (sCJD) patients, and compared the genotypic and allelic frequencies with those presented in 329 controls from the same ancestry. As such, this work also represents the first association analysis of CALHM genes in sCJD. Sequencing analysis of the complete coding regions of the genes demonstrated the presence of 10 single nucleotide polymorphisms (SNP) within the CALHM genes. We observed that rs4918016-rs2986017-rs2986018 and rs41287502-rs41287500 polymorphic sites at CALHM1 were in linkage disequilibrium. We found marginal associations for sCJD risk at CALHM1 polymorphic sites rs41287502 and rs41287500 [coding for two linked missense mutations p.(Met323Ile); (Gly282Cys)], and rs2986017 [p.(Leu86Pro)]. Interestingly, a TGG haplotype defined by the rs4918016- rs2986017-rs2986018 block was associated with sCJD. These findings underscore the need of future multinational collaborative initiatives in order to corroborate these seminal data. © 2012 Landes Bioscience.Ministerio de Ciencia e Innovación; Ministerio de Sanidad, Política Social e Igualdad; CIBERNEDPeer Reviewe

Spruce pulp treatment with an esterase from Ophiostoma piceae significantly decreases the content of both triglycerides and sterol esters responsible for pitch deposits

An esterase has been isolated from the ascomycete Ophiostoma piceae showing high affinity (Km around 1 mM) and activity on both glycerol and sterol esters of long chain fatty acids (kcat around 290 s-1 for triolein and 138 s-1 for cholesteryl oleate). This enzyme could have high potential for the enzymatic control of pitch in paper pulp manufacturing from different raw materials. Northern spruce (Picea abies) pulp containing triglycerides and sterol esters as the main lipophilic compounds, was treated with the O. piceae esterase (a control with boiled enzyme was included). The treated pulps were lyophilized, Soxhlet extracted with acetone, and the chloroform soluble compounds analyzed by gas chromatography-mass spectrometry. After 3-h treatment, 87% of triglycerides and 46% of sterols esters in the spruce pulp were degraded by the O. piceae esterase. These results suggest that O. piceae esterase could be used for pitch biocontrol in both hardwood and softwood paper pulp manufacturingThis work has been funded by the EU projects FAIR CT95-560 and QLK5-99-1357 and the Spanish project 1FD97-0742. O. Calero thanks the financial support received from the Spanish Comunidad de Madrid.Peer reviewe

Hydrolysis of sterol esters by an esterase from Ophiostoma piceae: Application for pitch control in pulping of Eucalyptus globulus wood

1Centro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu 9, E-28040 Madrid, Spain; 2Instituto de Recursos Naturales y Agrobiología, CSIC, Reina Mercedes 10, PO Box 1052, E-41080, Seville, Spain; 3Instituto de Catálisis, CSIC, Cantoblanco, E-28049 Madrid, Spain.A sterol esterase purified from cultures of the sapstain fungus Ophiostoma piceae was able to hydrolyse sterol esters and glycerides. The kinetics of sterol esters and triglyceride hydrolysis by this new esterase, estimated using a pH-stat, showed a Km app and a kcat app in the range of 0.9–1.1 mM and 70–300 s–1, respectively. Its ability to hydrolyse both pure sterol esters and natural mixtures of saponifiable lipids from eucalypt wood was compared with those of commercial sterol esterases from other microbial sources. Its specific activity on sterol esters was higher than that found with all the commercial esterases assayed, and the highest hydrolysis of eucalypt sterol esters was also attained using the O. piceae esterase. This sterol esterase could be of biotechnological interest for the hydrolysis of sterol esters that form pitch deposits in paper pulp manufacturing.This study has been supported by ENCE, the EU-Contract QLK5-99-1357, and the Spanish projects BIO2003-00621 and AGL2002-00393. AG acknowledges a ‘Ramón y Cajal’ contract of the Spanish MCYT.Peer reviewe