Cloning, Expression, Characterization and Immobilization of a Recombinant Carboxylesterase from the Halophilic Archaeon, Halobacterium salinarum NCR-1.

Only a few halophilic archaea producing carboxylesterases have been reported. The limited research on biocatalytic characteristics of archaeal esterases is primarily due to their very low production in native organisms. A gene encoding carboxylesterase from Halobacterium salinarum NRC-1 was cloned and successfully expressed in Haloferax volcanii. The recombinant carboxylesterase (rHsEst) was purified by affinity chromatography with a yield of 81%, and its molecular weight was estimated by SDS-PAGE (33 kDa). The best kinetic parameters of rHsEst were achieved using p-nitrophenyl valerate as substrate (KM = 78 µM, kcat = 0.67 s-1). rHsEst exhibited great stability to most metal ions tested and some solvents (diethyl ether, n-hexane, n-heptane). Purified rHsEst was effectively immobilized using Celite 545. Esterase activities of rHsEst were confirmed by substrate specificity studies. The presence of a serine residue in rHsEst active site was revealed through inhibition with PMSF. The pH for optimal activity of free rHsEst was 8, while for immobilized rHsEst, maximal activity was at a pH range between 8 to 10. Immobilization of rHsEst increased its thermostability, halophilicity and protection against inhibitors such as EDTA, BME and PMSF. Remarkably, immobilized rHsEst was stable and active in NaCl concentrations as high as 5M. These biochemical characteristics of immobilized rHsEst reveal its potential as a biocatalyst for industrial applications.

Electrical Conduction Mechanisms in Ethyl Cellulose Films under DC and AC Electric Fields.

This work reports the dielectric behavior of the biopolymer ethyl cellulose (EC) observed from transient currents experiments under the action of a direct current (DC) electric field (~107 V/m) under vacuum conditions. The viscoelastic response of the EC was evaluated using dynamic mechanical analysis (DMA), observing a mechanical relaxation related to glass transition of around ~402 K. Furthermore, we propose a mathematical framework that describes the transient current in EC using a fractional differential equation, whose solution involves the Mittag-Leffler function. The fractional order, between 0 and 1, is related to the energy dissipation rate and the molecular mobility of the polymer. Subsequently, the conduction mechanisms are considered, on the one hand, the phenomena that occur through the polymer-electrode interface and, on the other hand, those which manifest themselves in the bulk material. Finally, alternating current (AC) conductivity measurements above the glass transition temperature (~402 K) and in a frequency domain from 20 Hz to 2 MHz were carried out, observing electrical conduction described by the segmental movements of the polymeric chains. Its electrical properties also position EC as a potential candidate for electrical, electronics, and mechatronics applications.

Dielectric and Viscoelastic Behavior of Polyvinyl Butyral Films.

Dielectric and thermal properties of polyvinyl butyral (PVB) were studied in this work, using dynamic electrical analysis (DEA) at frequencies from 100 Hz to 1 MHz and temperatures from 293 K to 473 K. Two electrical relaxation processes were investigated: glass transition and interfacial polarization. Above the glass transition temperature (~343 K), interfacial polarization dominates conductive behavior in polyvinyl butyral. The framework of the complex electric modulus was used to obtain information about interfacial polarization. The viscoelastic behavior was analyzed through dynamic mechanical analysis (DMA), where only the mechanical manifestation of the glass transition is observed. The experimental results from dielectric measurements were analyzed with fractional calculus, using a fractional Debye model with one cap-resistor. We were successful in applying the complex electric modulus because we had a good correlation between data and theoretical predictions. The fractional order derivative is an indicator of the energy dissipated in terms of molecular mobility, and the calculated values close to 1 suggest a conductive behavior at temperatures above the glass transition temperature of PVB.

Thermomechanical Characterization and Modeling of NiTi Shape Memory Alloy Coil Spring.

Today, shape memory alloys (SMAs) have important applications in several fields of science and engineering. This work reports the thermomechanical behavior of NiTi SMA coil springs. The thermomechanical characterization is approached starting from mechanical loading-unloading tests under different electric current intensities, from 0 to 2.5 A. In addition, the material is studied using dynamic mechanical analysis (DMA), which is used to evaluate the complex elastic modulus E* = E' - iE″, obtaining a viscoelastic response under isochronal conditions. This work further evaluates the damping capacity of NiTi SMA using tan δ, showing a maximum around 70 °C. These results are interpreted under the framework of fractional calculus, using the Fractional Zener Model (FZM). The fractional orders, between 0 and 1, reflect the atomic mobility of the NiTi SMA in the martensite (low-temperature) and austenite (high-temperature) phases. The present work compares the results obtained from using the FZM with a proposed phenomenological model, which requires few parameters for the description of the temperature-dependent storage modulus E'.

Physicochemical characterization and emulsifying properties of a novel exopolysaccharide produced by haloarchaeon Haloferax mucosum.

The physicochemical characterization and emulsifying functional properties of a novel exopolysaccharide (EPS) produced by haloarchaea Haloferax mucosum (DSM 27191) were investigated. This biopolymer has a high molecular weight of 152 kDa and important protein content of 10%. Different culture media compositions were investigated taking the ATCC 2185 medium as a base and supplementing with varying concentrations of yeast extract and glucose or sucrose as carbon sources to produce the EPS in a liquid medium. The highest EPS production (7.15 ±â€¯0.44 g/L) was obtained at 96 h. EPS aqueous dispersions showed a non-Newtonian rheological behavior which was well fitted to the Cross equation. The EPS (at 0.32% w/w) was capable of stabilizing water-in-oil emulsions with different nonpolar solvents, including n-hexane, kerosene, chloroform, castor oil and mineral oil. EPS retained its emulsifying activity after to be incubated for one hour in a wide range of temperatures (25, 40, 70 and 100 °C), pH (4, 6.5, 7 and 12) and NaCl concentrations (0, 2.0 and 4.0 M). The viscoelastic behavior and stability of hexane-in-water emulsion were examined through oscillatory shear measurements.

Solid State Multicolor Emission in Substitutional Solid Solutions of Metal-Organic Frameworks.

Preparing crystalline materials that produce tunable organic-based multicolor emission is a challenge due to the inherent inability to control the packing of organic molecules in the solid state. Utilizing multivariate, high-symmetry metal-organic frameworks, MOFs, as matrices for organic-based substitutional solid solutions allows for the incorporation of multiple fluorophores with different emission profiles into a single material. By combining nonfluorescent links with dilute mixtures of red, green, and blue fluorescent links, we prepared zirconia-type MOFs and found that the bulk materials exhibit features of solution-like fluorescence. Our study found that MOFs with a fluorophore link concentration of around 1 mol % exhibit fluorescence with decreased inner filtering, demonstrated by changes in spectral profiles, increased quantum yields, and lifetime dynamics expected for excited-state proton-transfer emitters. Our findings enabled us to prepare organic-based substitutional solid solutions with tunable chromaticity regulated only by the initial amounts of fluorophores. These materials emit multicolor and white light with high quantum yields (∼2-14%), high color-rendering indices (>93), long shelf life, and superb hydrolytic stability at ambient conditions.

Enantioselective Synthesis of Indolines, Benzodihydrothiophenes, and Indanes by C-H Insertion of Donor/Donor Carbenes.

We employ a single catalyst/oxidant system to enable the asymmetric syntheses of indolines, benzodihydrothiophenes, and indanes by C-H insertion of donor/donor carbenes. This methodology enables the rapid construction of densely substituted five-membered rings that form the core of many drug targets and natural products. Furthermore, oxidation of hydrazones to the corresponding diazo compounds proceeds in situ, enabling a relatively facile one- or two-pot protocol in which isolation of potentially explosive diazo alkanes is avoided. Regioselectivity studies were performed to determine the impact of sterics and electronics in donor/donor metal carbene C-H insertions to form indolines. This methodology was applied to a variety of substrates in high yield, diastereomeric, and enantiomeric ratios and to the synthesis of a patented indane estrogen receptor agonist with anti-cancer activity.

Optimization of biodiesel synthesis by esterification using a fermented solid produced by Rhizopus microsporus on sugarcane bagasse.

A fermented solid containing lipases was produced by solid-state fermentation of Rhizopus microsporus on sugarcane bagasse enriched with urea, soybean oil, and a mineral solution. The dry fermented solid produced using R. microsporus (RMFS) was used to catalyze the synthesis of alkyl-esters by esterification in a solvent-free system containing ethanol and oleic acid (as a model system) or a mixture of fatty acids obtained from the physical hydrolysis of soybean soapstock acid oil (FA-SSAO) in subcritical water. The conversions were 93.5 and 84.1%, for oleic acid and FA-SSAO, respectively, at 48 h and 40 °C, at a molar ratio (MR) of ethanol to fatty acid of 5:1. A further increase in the MR to 10:1 improved the production of ethylic-esters, giving conversions at 48 h of 98 and 86% for oleic acid and FA-SSAO, respectively. The results obtained in this work foster further studies on scaling-up of an environmentally friendly process to produce biofuels.

Fabrication and Characterization of Isotropic and Anisotropic Magnetorheological Elastomers, Based on Silicone Rubber and Carbonyl Iron Microparticles.

This article focuses on studying the rheological behavior of isotropic and anisotropic magnetorheological elastomers (MREs), made of carbonyl iron microparticles dispersed into a silicone⁻rubber matrix by considering 20 and 30 wt % of microparticles. Sample sets were prepared for each composition, with and without the application of an external magnetic field. Experimental measurements of the material rheology behavior were carried out by a shear oscillatory rheometer at constant temperature, to determine both the shear storage modulus (G') and shear loss modulus (G'') for all characterized samples. Then, experimental data collected from the isotropic and the anisotropic material samples were used to plot the Cole-Cole diagrams to quantify the interfacial adhesion between carbonyl iron microparticles and the silicone-rubber matrix. Furthermore, the Fractional Zener Model (FZM) with two spring-pots in series is used for quantitative analysis of collected experimental data.

Experimental Investigation of the Magnetorheological Behavior of PDMS Elastomer Reinforced with Iron Micro/Nanoparticles.

The aim of this article focuses on identifying how the addition of iron micro- and nanoparticles influences the physical properties of magnetorheological composite materials developed with a polydimethylsiloxane (PDMS) matrix with different contents of silicone oil used as additive. A number of characterization techniques have been performed in order to fully characterize the samples, such as cyclic and uniaxial extension, rheology, swelling, Vibrating sample magnetometer (VSM), X-ray Diffraction (XRD), Scanning electron microscopy (SEM), Fourier-Transform Infrared (FTIR), X-ray photoelectronic spectroscopy (XPS) and Thermogravimetric analysis (TGA). The comparison between two matrices with different shore hardnesses and their mechanical and chemical properties are elucidated by swelling and tensile tests. In fact, swelling tests showed that higher crosslink density leads to increasing elongation at break and tensile strength values for the composite materials. The best mechanical performance in the magnetorheological material was observed for those samples manufactured using a higher silicone oil content in a hard polymeric matrix. Furthermore, it has been found that the magnetic properties are enhanced when nanoparticles are used as fillers instead of microparticles.

Improvement on the productivity of continuous tequila fermentation by Saccharomyces cerevisiae of Agave tequilana juice with supplementation of yeast extract and aeration.

Agave (Agave tequilana Weber var. azul) fermentations are traditionally carried out employing batch systems in the process of tequila manufacturing; nevertheless, continuous cultures could be an attractive technological alternative to increase productivity and efficiency of sugar to ethanol conversion. However, agave juice (used as a culture medium) has nutritional deficiencies that limit the implementation of yeast continuous fermentations, resulting in high residual sugars and low fermentative rates. In this work, fermentations of agave juice using Saccharomyces cerevisiae were put into operation to prove the necessity of supplementing yeast extract, in order to alleviate nutritional deficiencies of agave juice. Furthermore, continuous fermentations were performed at two different aeration flow rates, and feeding sterilized and non-sterilized media. The obtained fermented musts were subsequently distilled to obtain tequila and the preference level was compared against two commercial tequilas, according to a sensorial analysis. The supplementation of agave juice with air and yeast extract augmented the fermentative capacity of S. cerevisiae S1 and the ethanol productivities, compared to those continuous fermentations non supplemented. In fact, aeration improved ethanol production from 37 to 40 g L(-1), reducing sugars consumption from 73 to 88 g L(-1) and ethanol productivity from 3.0 to 3.2 g (Lh)(-1), for non-aerated and aerated (at 0.02 vvm) cultures, respectively. Supplementation of yeast extract allowed an increase in specific growth rate and dilution rates (0.12 h(-1), compared to 0.08 h(-1) of non-supplemented cultures), ethanol production (47 g L(-1)), reducing sugars consumption (93 g L(-1)) and ethanol productivity [5.6 g (Lh)(-1)] were reached. Additionally, the effect of feeding sterilized or non-sterilized medium to the continuous cultures was compared, finding no significant differences between both types of cultures. The overall effect of adding yeast extract and air to the continuous fermentations resulted in 88 % increase in ethanol productivity. For all cultures, pH was not controlled, reaching low pH values (from 2.6 to 3). This feature suggested a reduced probability of contamination for prolonged continuous cultures and explained why no significant differences were found between continuous cultures fed with sterilized or non-sterilized media. Concentrations of volatile compounds quantified in the distillates (tequila) were in the allowed ranges established by the Mexican regulation of tequila (NOM-006-SCFI-2012, Norma Oficial Mexicana: Bebidas alcohólicas-Tequila-specificaciones, 2012). The preference level of the distillates was similar to that of two well-known commercial tequilas. The results suggested the possibility of implementing this innovative technology on an industrial scale, attaining high productivities and using non-sterilized agave juice.

Solid-state fermentation as a potential technique for esterase/lipase production by halophilic archaea.

Halophilic archaea are extremophiles, adapted to high-salt environments, showing a big biotechnological potential as enzyme, lipids and pigments producers. Four inert supports (perlite, vermiculite, polyurethane foam and glass fiber) were employed for solid-state fermentation (SSF) of the halophilic archaeon Natronococcus sp. TC6 to investigate biomass and esterase production. A very low esterase activity and high water activity were observed when perlite, vermiculite and polyurethane were used as supports. When glass fiber was employed, an important moisture loss was observed (8.6%). Moreover, moisture retention was improved by mixing polyurethane and glass fiber, resulting in maximal biomass and esterase production. Three halophilic archaea: Natronococcus sp. TC6, Halobacterium sp. NRC-1 and Haloarcula marismortui were cultured by submerged fermentation (SmF) and by SSF; an improvement of 1.3- to 6.2-fold was observed in the biomass and esterase production when SSF was used. Growth was not homogeneous in the mixture, but was predominant in the glass fiber thus was probably because the glass fiber provides a holder to the cells, while the polyurethane acts as an impregnation medium reservoir. To the best of our knowledge, this work is the first report on haloarchaea cultivation by SSF aiming biomass and esterase/lipase activity production.

A new species of the Rhinella margaritifera species group (Anura, Bufonidae) from the montane forest of the Selva Central, Peru.

We describe a new species of the bufonid toad genus Rhinella from transition montane forest of the buffer zones of the Yanachaga-Chemillén National Park and the Pui Pui Protected Forest (eastern slopes of Andes, Selva Central, Peru). The new species belongs to the Rhinella margaritifera species group (confirmed by mtDNA data) and differs from all its members by the absence of tympanic membrane and tympanic annulus. It is characterized by medium size (SVL 57.5-65.5 mm, n = 5), moderately developed cranial crests, absence of neural crest of vertebrae, absence of bone protrusion at angle of jaw, presence of lateral rows of enlarged tubercles, and absence of subgular vocal sac and vocal slits in males. In addition, based on the molecular phylogenetic analyses of selected Rhinella species we propose the monophylum containing R. chavin, R. festae, R. macrorhina, R. manu, R. nesiotes, R. rostrata, and R. yanachaga as a new species group under the name Rhinella festae species group.

ResumenSe describe una nueva especie de sapo Bufonidae del género Rhinella presente en los bosques montanos transicionales de la zona de amortiguamiento del Parque Nacional Yanachaga Chemillén y el Bosque de Protección Pui Pui (Vertiente Oriental de los Andes, Selva Central, Perú). La nueva especie pertenece al grupo de especies Rhinella margaritifera (confirmado con datos de DNAmt) y difiere de todos sus miembros por la ausencia de membrana timpánica y anillo timpánico. Se caracteriza por su tamaño mediano (SVL 57.5­65.5 mm), crestas craneales moderadamente desarrolladas, ausencia de una cresta neural, ausencia de protuberancia ósea en el ángulo mandibular, presencia de filas laterales de tubérculos agrandados, y ausencia de saco vocal subgular y hendiduras vocales en los machos. Además, basado en los análisis filogenéticos moleculares de especies seleccionadas de Rhinella se propone la monofilia que contiene a R. chavin, R. festae, R. macrorhina, R. manu, R. nesiotes, R. rostrata y R. yanachaga como un nuevo grupo de especies bajo el nombre de grupo de especies Rhinella festae.

Transcriptome analysis identifies genes involved in ethanol response of Saccharomyces cerevisiae in Agave tequilana juice.

During ethanol fermentation, yeast cells are exposed to stress due to the accumulation of ethanol, cell growth is altered and the output of the target product is reduced. For Agave beverages, like tequila, no reports have been published on the global gene expression under ethanol stress. In this work, we used microarray analysis to identify Saccharomyces cerevisiae genes involved in the ethanol response. Gene expression of a tequila yeast strain of S. cerevisiae (AR5) was explored by comparing global gene expression with that of laboratory strain S288C, both after ethanol exposure. Additionally, we used two different culture conditions, cells grown in Agave tequilana juice as a natural fermentation media or grown in yeast-extract peptone dextrose as artificial media. Of the 6368 S. cerevisiae genes in the microarray, 657 genes were identified that had different expression responses to ethanol stress due to strain and/or media. A cluster of 28 genes was found over-expressed specifically in the AR5 tequila strain that could be involved in the adaptation to tequila yeast fermentation, 14 of which are unknown such as yor343c, ylr162w, ygr182c, ymr265c, yer053c-a or ydr415c. These could be the most suitable genes for transforming tequila yeast to increase ethanol tolerance in the tequila fermentation process. Other genes involved in response to stress (RFC4, TSA1, MLH1, PAU3, RAD53) or transport (CYB2, TIP20, QCR9) were expressed in the same cluster. Unknown genes could be good candidates for the development of recombinant yeasts with ethanol tolerance for use in industrial tequila fermentation.

Plant and animal endemism in the eastern Andean slope: challenges to conservation.

BACKGROUND: The Andes-Amazon basin of Peru and Bolivia is one of the most data-poor, biologically rich, and rapidly changing areas of the world. Conservation scientists agree that this area hosts extremely high endemism, perhaps the highest in the world, yet we know little about the geographic distributions of these species and ecosystems within country boundaries. To address this need, we have developed conservation data on endemic biodiversity (~800 species of birds, mammals, amphibians, and plants) and terrestrial ecological systems (~90; groups of vegetation communities resulting from the action of ecological processes, substrates, and/or environmental gradients) with which we conduct a fine scale conservation prioritization across the Amazon watershed of Peru and Bolivia. We modelled the geographic distributions of 435 endemic plants and all 347 endemic vertebrate species, from existing museum and herbaria specimens at a regional conservation practitioner's scale (1:250,000-1:1,000,000), based on the best available tools and geographic data. We mapped ecological systems, endemic species concentrations, and irreplaceable areas with respect to national level protected areas. RESULTS: We found that sizes of endemic species distributions ranged widely (< 20 km2 to > 200,000 km2) across the study area. Bird and mammal endemic species richness was greatest within a narrow 2500-3000 m elevation band along the length of the Andes Mountains. Endemic amphibian richness was highest at 1000-1500 m elevation and concentrated in the southern half of the study area. Geographical distribution of plant endemism was highly taxon-dependent. Irreplaceable areas, defined as locations with the highest number of species with narrow ranges, overlapped slightly with areas of high endemism, yet generally exhibited unique patterns across the study area by species group. We found that many endemic species and ecological systems are lacking national-level protection; a third of endemic species have distributions completely outside of national protected areas. Protected areas cover only 20% of areas of high endemism and 20% of irreplaceable areas. Almost 40% of the 91 ecological systems are in serious need of protection (= < 2% of their ranges protected). CONCLUSIONS: We identify for the first time, areas of high endemic species concentrations and high irreplaceability that have only been roughly indicated in the past at the continental scale. We conclude that new complementary protected areas are needed to safeguard these endemics and ecosystems. An expansion in protected areas will be challenged by geographically isolated micro-endemics, varied endemic patterns among taxa, increasing deforestation, resource extraction, and changes in climate. Relying on pre-existing collections, publically accessible datasets and tools, this working framework is exportable to other regions plagued by incomplete conservation data.

Fermentation of Agave tequilana juice by Kloeckera africana: influence of amino-acid supplementations.

This study aimed to improve the fermentation efficiency of Kloeckera africana K1, in tequila fermentations. We investigated organic and inorganic nitrogen source requirements in continuous K. africana fermentations fed with Agave tequilana juice. The addition of a mixture of 20 amino-acids greatly improved the fermentation efficiency of this yeast, increasing the consumption of reducing sugars and production of ethanol, compared with fermentations supplemented with ammonium sulfate. The preference of K. africana for each of the 20 amino-acids was further determined in batch fermentations and we found that asparagine supplementation increased K. africana biomass production, reducing sugar consumption and ethanol production (by 30, 36.7 and 45%, respectively) over fermentations supplemented with ammonium sulfate. Therefore, asparagine appears to overcome K. africana nutritional limitation in Agave juice. Surprisingly, K. africana produced a high concentration of ethanol. This contrasts to poor ethanol productivities reported for other non-Saccharomyces yeasts indicating a relatively high ethanol tolerance for the K. africana K1 strain. Kloeckera spp. strains are known to synthesize a wide variety of volatile compounds and we have shown that amino-acid supplements influenced the synthesis by K. africana of important metabolites involved in the bouquet of tequila. The findings of this study have revealed important nutritional limitations of non-Saccharomyces yeasts fermenting Agave tequilana juice, and have highlighted the potential of K. africana in tequila production processes.

Divergencia genética en poblaciones peruanas detectada a partir de las frecuencias haplotípicas del mtDNA y del gen nuclear MBL / Peruvian population's genetic differences detected by both mtDNA and MBL nuclear gene haplotypical frequencies

Objetivos: Avanzar en el conocimiento del origen de las poblaciones peruanas estudiadas en un contexto filogeográfico. Diseño: Estudio genético poblacional. Instituciones: Laboratorio de Genética Humana, Facultad de Ciencias Biológicas, Universidad Nacional Mayor de San Marcos, e Instituto de Genética y Biología Molecular, Facultad de Medicina, Universidad San Martín de Porras, Lima, Perú. Participantes: Siete poblaciones peruanas. Metodología: Análisis comparativo de los resultados a partir del estudio del mtDNA y el gen nuclear MBL de siete poblaciones peruanas, procesados de manera separada y luego combinados, utilizando el programa PHYLYP 3.65, para obtener valores FST de diferenciación genética y la construcción de árboles de distancias por aplicación del algorritmo UPGMA y el análisis subsecuente de los agrupamientos (clusters) generados. Principales medidas de resultados: Árboles genéticos generados. Resultados: De manera separada, los árboles generados para cada marcador genético tuvieron topologías propias y diferentes entre sí. Procesados de manera combinada, el árbol resultante demostró que los mayores valores de diferenciación genética se hallaron en las Islas del Lago Titicaca (Puno, Perú) conocidas -Taquile, Amantani y Anapia-, que fue calificada como muy alta, porque mostró valores de FST de 0.3113, 0.2949 y 0.3348 respecto de las poblaciones estudiadas, tanto fuera del Departamento de Puno -como Chachapoyas, Pucallpa y Chiclayo, respectivamente-, así como a la de los Uro del mismo Puno y del mismo Lago Titicaca (0.2837). Fuera de Puno, el par de poblaciones Chachapoyas-Pucallpa fue el menos divergente, al alcanzar entre ellas un valor de FST de 0.0108, calificándosele de pequeña. Conclusiones: El árbol obtenido del procesamiento de los marcadores vía una matriz combinada demostró que las poblaciones que habitan las islas de Taquile, Amantani y Anapia, divergen notablemente de las restantes cuatro procesadas del Perú, incluyendo la más próxima a ellas dentro del mismo Lago Titicaca, como es la de los Uro. Explicar estos hallazgos será el siguiente objetivo de nuestras investigaciones, en principio, mediante la ampliación de los marcadores genéticos empleados y del número de poblaciones analizadas a nivel del Perú.

Objectives: To advance in the knowledge of Peruvian populationsÆ origin in a phylogeographical context. Design: Population genetics study. Setting: Human Genetics Laboratory, Biological Sciences Faculty, Universidad Nacional Mayor de San Marcos, and Genetics and Molecular Biology Institute, Faculty of Medicine, Universidad San Martin de Porras, Lima, Peru. Participants: Seven Peruvian populations. Methods: Comparative analysis of mtDNA and MBL nuclear gene study results in seven Peruvian populations processed separately and then combined using PHYLYP 3.65 Program in order to obtain FST values of genetic differentiation; construction of distance trees by applying UPGMA algorithm and subsequent generated clustersÆ analysis. Main outcome measures: Genetic trees. Results: Trees generated for each genetic marker had proper and distinct topologies among them. Combined processing resulted in a tree with higher values of genetic differentiation in Lago Titicaca Islands (Puno, Peru) Taquile, Amantani y Anapia, graded as very high because they showed 0.3113, 0.2949 y 0.3348 FST values with respect to the populations studied outside of Puno Department -like Chachapoyas, Pucallpa and Chiclayo-, as well as those of both UroÆs in same Puno and Lago TiticacaÆs populations (0.2837). Out of Puno, the pair Chachapoyas-Pucallpa population was the least divergent with 0.0108 FST value between them, classifying as small. Conclusions: The tree obtained from markers by a combined matrix process determined that populations inhabiting in Taquile, Amantani y Anapia islands possess notable genetic divergence respect to the four remainders studied in Peru, including the UroÆs population geographically very close to them and within the same Lago Titicaca. Our next objective will be to explain these findings initially by increasing genetic markers and number of populations analyzed in Peru.

Improvement of growth, fermentative efficiency and ethanol tolerance of Kloeckera africana during the fermentation of Agave tequilana juice by addition of yeast extract.

BACKGROUND: The aim of this work was to improve the productivity and yield of tequila fermentation and to propose the use of a recently isolated non-Saccharomyces yeast in order to obtain a greater diversity of flavour and aroma of the beverage. For that, the effects of the addition of different nitrogen (N) sources to Agave tequilana juice on the growth, fermentative capacity and ethanol tolerance of Kloeckera africana and Saccharomyces cerevisiae were studied and compared. RESULTS: Kloeckera africana K1 and S. cerevisiae S1 were cultured in A. tequilana juice supplemented with ammonium sulfate, diammonium phosphate or yeast extract. Kloeckera africana did not assimilate inorganic N sources, while S. cerevisiae utilised any N source. Yeast extract stimulated the growth, fermentative capacity and alcohol tolerance of K. africana, giving kinetic parameter values similar to those calculated for S. cerevisiae. CONCLUSION: This study revealed the importance of supplementing A. tequilana juice with a convenient N source to achieve fast and complete conversion of sugars in ethanol, particularly in the case of K. africana. This yeast exhibited similar growth and fermentative capacity to S. cerevisiae. The utilisation of K. africana in the tequila industry is promising because of its variety of synthesised aromatic compounds, which would enrich the attributes of this beverage.

Carboxyl ester hydrolases production and growth of a halophilic archaeon, Halobacterium sp. NRC-1.

The capability of Halobacterium sp. NRC-1 to synthesize carboxyl ester hydrolases was investigated, and the effect of physicochemical conditions on the growth rate and production of esterases was evaluated. The haloarchaeon synthesized a carboxyl ester hydrolase, confirming the genomic prediction. This enzymatic activity was intracellularly produced as a growth-associated metabolite. Esterase activity was assayed using different p-nitrophenyl-esters and triacyl-glycerides, which showed a preference for hydrolyzing tributyrin. The archaeal growth rate and esterase production were significantly influenced by the pH and the NaCl concentration. An interaction effect between temperature and NaCl was also seen. The maximal growth rate and esterase production found for Halobacterium sp. NRC-1 were 0.136 h(-1) (at 4.2 M NaCl, pH 6 and 44 degrees C) and 1.64 U/l (at 4.6 M NaCl, pH 6 and 30 degrees C), respectively. Furthermore, the effects of NaCl concentration, pH and temperature on enzyme activity were studied. Two maximal esterase activities were elucidated from the intracellular crude extract when it was incubated at different NaCl concentrations (1 M and 5 M) and at different pHs (6 and 7.5). This is the first report that shows experimentally the synthesis of carboxyl ester hydrolases by Halobacterium sp. NRC-1. This enzyme was found to be extremely halophilic (5 M NaCl) and thermophilic (80 degrees C), making it very interesting for future investigations in non-aqueous biocatalysis.