Thermophiles and Thermozymes

Interest in the study of life in hot environments, both with respect to the inhabiting microorganisms and the enzymes they produce, is currently very high. The biological mechanisms responsible for the resistance to high temperatures are not yet fully understood, whereas thermostability is a highly required feature for industrial applications. In this e-book, the invited authors provide diverse evidence contributing to the understanding of such mechanisms and the unlocking of the biotechnological potential of thermophiles and thermozymes

Utilización de screencasts para un aprendizaje activo

[Resumen] La presente innovación consistió en la elaboración por los estudiantes de screencasts, es decir, vídeos cortos en los que se graba parte o la totalidad de la pantalla del ordenador, acompañados por audios con las explicaciones correspondientes, acerca de temas propuestos por los profesores de la materia. Se recomendó el formato PechaKucha: 20 diapositivas de 20 segundos cada una. Al finalizar la experiencia, los estudiantes respondieron un cuestionario en la plataforma de teleenseñanza Moodle. De las respuestas obtenidas destacamos: la mayoría necesitó menos de 2 horas para la elaboración del screencast; sólo el 5% había utilizado antes este recurso; a todos los estudiantes la elaboración del screencast les ayudó a sintetizar los contenidos del tema; al 95% les ayudó a preparar la presentación oral; sólo el 10% optó por tener a su disposición los screencasts elaborados por sus compañeros para el estudio, prefiriendo resúmenes de los temas; el 80% opinó que 20 segundos por diapositiva era poco tiempo para desarrollar los contenidos; al 58% le gustaría disponer de los temas impartidos por los profesores en formato vídeo; el 90% asistiría igualmente a las clases presenciales aun disponiendo de los vídeos, si bien la mitad con menor frecuencia[Abstract] The present innovation consisted of the preparation by the students of screencasts, i.e. short videos recording the computer screen (full or partial) with the corresponding explanations in audio, about themes proposed by the professors. The PechaKucha format was suggested: 20 slides of 20 seconds each one. At the end of the experience, the students answered a questionnaire in Moodle. From the responses obtained we outline: the majority needed less than 2 hours to record the screencast; only 5% had used this resource previously; all the students considered the screencast usefull to synthesize the contents of the theme; 95% considered it usefull to prepare the oral presentation; only 10% chose the screencasts made by their colleagues to study the themes, being summaries the preferred option; 80% thought that 20 seconds per slide was insufficient time to develop the contents; 58% would appreciate to have available the lectures taught by the professors in video format; 90% would continue assisting to presential master classes, although half less often

Diseño de una materia de máster para la docencia a distancia

[Resumen] Se han elaborado una serie de contenidos para adaptar una materia de máster a la modalidad de docencia a distancia utilizando como plataforma de teleenseñanza Moodle. La materia se estructuró en unidades didácticas y en cada una de estas unidades se pusieron a disposición del estudiante diversos materiales como, entre otros, presentaciones del tema, lecturas y vídeos breves realizados con la herramienta gratuita Screencast-o-matic. Para compartir los diferentes contenidos con los estudiantes de una manera visual y atractiva se utilizó la herramienta Padlet. Así mismo, se habilitó un foro de dudas por cada una de las unidades. El alumno dispuso también de tutorías online a demanda. Como actividad obligatoria, el estudiante tuvo que responder a unos cuestionarios por cada unidad. Para la parte práctica de la materia se utilizó la metodología de aprendizaje basado en métodos de casos. Finalmente, el estudiante tuvo que completar un examen a través de Moodle. Para la calificación final se tuvo en cuenta además del examen, las respuestas a los cuestionarios y la memoria con la resolución del caso práctico planteado. Los resultados fueron muy satisfactorios ya que no hubo diferencias significativas con respecto a los resultados obtenidos mediante docencia presencial.[Abstract] A series of contents have been developed to adapt a master's subject to the modality of distance teaching using Moodle as online platform. The subject was structured in didactic units and in each one of these units various materials were made available to the student, such as, among others, subject presentations, readings and short videos made with the free tool Screencast-o-matic. To share the different contents with the students in a visual and attractive way, the Padlet tool was used. Moreover, a doubts forum was established for each one of the units. The student also had online tutorials on demand. As a compulsory activity, the student had to answer some questionnaires for each unit. For the practical part of the subject, the case study method was used. Finally, the student had to complete an exam through Moodle. For the final grade, the answers to the questionnaires and the report with the resolution of the case study were taken into account in addition to the exam. The results were very satisfactory since there were no significant differences with respect to the results obtained through faceto- face teaching

Microbial diversity analysis and screening for novel xylanase enzymes from the sediment of the Lobios Hot Spring in Spain

[Abstract] Here, we describe the metagenome composition of a microbial community in a hot spring sediment as well as a sequence-based and function-based screening of the metagenome for identification of novel xylanases. The sediment was collected from the Lobios Hot Spring located in the province of Ourense (Spain). Environmental DNA was extracted and sequenced using Illumina technology, and a total of 3.6 Gbp of clean paired reads was produced. A taxonomic classification that was obtained by comparison to the NCBI protein nr database revealed a dominance of Bacteria (93%), followed by Archaea (6%). The most abundant bacterial phylum was Acidobacteria (25%), while Thaumarchaeota (5%) was the main archaeal phylum. Reads were assembled into contigs. Open reading frames (ORFs) predicted on these contigs were searched by BLAST against the CAZy database to retrieve xylanase encoding ORFs. A metagenomic fosmid library of approximately 150,000 clones was constructed to identify functional genes encoding thermostable xylanase enzymes. Function-based screening revealed a novel xylanase-encoding gene (XynA3), which was successfully expressed in E. coli BL21. The resulting protein (41 kDa), a member of glycoside hydrolase family 11 was purified and biochemically characterized. The highest activity was measured at 80 °C and pH 6.5. The protein was extremely thermostable and showed 94% remaining activity after incubation at 60 °C for 24 h and over 70% remaining activity after incubation at 70 °C for 24 h. Xylanolytic activity of the XynA3 enzyme was stimulated in the presence of β-mercaptoethanol, dithiothreitol and Fe3+ ions. HPLC analysis showed that XynA3 hydrolyzes xylan forming xylobiose with lower proportion of xylotriose and xylose. Specific activity of the enzyme was 9080 U/mg for oat arabinoxylan and 5080 U/mg for beechwood xylan, respectively, without cellulase activity

Thermus thermophilus as a Source of Thermostable Lipolytic Enzymes

Lipolytic enzymes, esterases (EC 3.1.1.1) and lipases (EC 3.1.1.3), catalyze the hydrolysis of ester bonds between alcohols and carboxylic acids, and its formation in organic media. At present, they represent about 20% of commercialized enzymes for industrial use. Lipolytic enzymes from thermophilic microorganisms are preferred for industrial use to their mesophilic counterparts, mainly due to higher thermostability and resistance to several denaturing agents. However, the production at an industrial scale from the native organisms is technically complicated and expensive. The thermophilic bacterium Thermus thermophilus (T. thermophilus) has high levels of lipolytic activity, and its whole genome has been sequenced. One esterase from the T. thermophilus strain HB27 has been widely characterized, both in its native form and in recombinant forms, being expressed in mesophilic microorganisms. Other putative lipases/esterases annotated in the T. thermophilus genome have been explored and will also be reviewed in this paper.Xunta de Galicia; PGIDIT06REM38202PRXunta de Galicia; 09MDS037383P

Analítica de aprendizaje de una materia optativa del grado de Biología

[Resumen] El uso de plataformas virtuales de docencia por los estudiantes genera una gran cantidad de huellas y el reto consiste en analizar estos datos para sacar conclusiones del proceso de enseñanza-aprendizaje. En el presente trabajo, nos hemos propuesto analizar los registros que dejan los estudiantes en los espacios virtuales de una materia optativa de cuarto curso del grado de Biología con la finalidad de conocer las actividades que han presentado un mayor número de interacciones y que han despertado por tanto un mayor interés por parte del alumnado.[Abstract] The use of virtual teaching platforms by students generates a large number of footprints and the challenge is to analyze these data to obtain conclusions about the teaching-learning process. In the present work, we have proposed to analyze the records that students leave in the virtual spaces of an elective subject in the fourth year of the Biology degree in order to know the activities that have presented a greater number of interactions and that have attracted more interest from the students.http://hdl.handle.net/2183/2879

Optimization of Saccharomyces cerevisiae α-galactosidase production and application in the degradation of raffinose family oligosaccharides

[Abstract] Background: α-Galactosidases are enzymes that act on galactosides present in many vegetables, mainly legumes and cereals, have growing importance with respect to our diet. For this reason, the use of their catalytic activity is of great interest in numerous biotechnological applications, especially those in the food industry directed to the degradation of oligosaccharides derived from raffinose. The aim of this work has been to optimize the recombinant production and further characterization of α-galactosidase of Saccharomyces cerevisiae. Results: The MEL1 gene coding for the α-galactosidase of S. cerevisiae (ScAGal) was cloned and expressed in the S. cerevisiae strain BJ3505. Different constructions were designed to obtain the degree of purification necessary for enzymatic characterization and to improve the productive process of the enzyme. ScAGal has greater specificity for the synthetic substrate p-nitrophenyl-α-D-galactopyranoside than for natural substrates, followed by the natural glycosides, melibiose, raffinose and stachyose; it only acts on locust bean gum after prior treatment with β-mannosidase. Furthermore, this enzyme strongly resists proteases, and shows remarkable activation in their presence. Hydrolysis of galactose bonds linked to terminal non-reducing mannose residues of synthetic galactomannan-oligosaccharides confirms that ScAGal belongs to the first group of α-galactosidases, according to substrate specificity. Optimization of culture conditions by the statistical model of Response Surface helped to improve the productivity by up to tenfold when the concentration of the carbon source and the aeration of the culture medium was increased, and up to 20 times to extend the cultivation time to 216 h. Conclusions: ScAGal characteristics and improvement in productivity that have been achieved contribute in making ScAGal a good candidate for application in the elimination of raffinose family oligosaccharides found in many products of the food industry.Xunta de Galicia; ED431C 2016–01

Test for the production and assay of the proteolytic activities of halophilic bacteria and archaea isolated from algerian hypersaline environments

[Abstract] The present work was carried out on 133 halophilic strains isolated on MGM (Modified Growth Medium) medium with 12 and 23% (w/v) of salt. A screening of the extracellular proteolytic activities, carried out on the same medium supplemented with casein or gelatin at 1% (w/v), allowed us to select 24 bacterial and 21 archaeal strains presenting a precipitate around the colonies for casein and/or a translucent halo (after addition of Frazier’s reagent) for gelatin. The enzymatic test was performed on liquid medium in microculture in a 2 mL Eppendorf tube. The assay of the proteolytic activity, using Azocasein as substrate, followed two protocols—the first with PBS and the second with Tris HCl, with positive and negative controls—and demonstrated interesting results for 10 strains among the 45 tested including five bacteria and five archaea. These underwent morphological, physiological and molecular characterizations based on amplification and sequencing of the 16S ribosomal RNA gene

Exploring the Taxonomical and Functional Profile of As Burgas Hot Spring Focusing On Thermostable β-galactosidases

[Abstract] In the present study we investigate the microbial community inhabiting As Burgas geothermal spring, located in Ourense (Galicia, Spain). The approximately 23 Gbp of Illumina sequences generated for each replicate revealed a complex microbial community dominated by Bacteria in which Proteobacteria and Aquificae were the two prevalent phyla. An association between the two most prevalent genera, Thermus and Hydrogenobacter, was suggested by the relationship of their metabolism. The high relative abundance of sequences involved in the Calvin–Benson cycle and the reductive TCA cycle unveils the dominance of an autotrophic population. Important pathways from the nitrogen and sulfur cycle are potentially taking place in As Burgas hot spring. In the assembled reads, two complete ORFs matching GH2 beta-galactosidases were found. To assess their functional characterization, the two ORFs were cloned and overexpressed in E. coli. The pTsbg enzyme had activity towards o-Nitrophenyl-β-D-galactopyranoside (ONPG) and p-Nitrophenyl-β-D-fucopyranoside, with high thermal stability and showing maximal activity at 85 °C and pH 6, nevertheless the enzyme failed to hydrolyze lactose. The other enzyme, Tsbg, was unable to hydrolyze even ONPG or lactose. This finding highlights the challenge of finding novel active enzymes based only on their sequence.Xunta de Galicia; ED431C 2020/08Ministerio de Ciencia, Innovación y Universidades; RTI2018-099249-B-I00Ministerio de Educación Cultura y Deporte; FPU12/0505