Evolving a lipase for hydrolysis of natural triglycerides along with enhanced tolerance towards a protease and surfactants

In the accompanying paper, we described evolving a lipase to the point where variants were soluble, stable and capable of degrading C8 TAG and C8 esters. These variants were tested for their ability to survive in an environment that might be encountered in a washing machine. Unfortunately, they were inactivated both by treatment with a protease used in laundry detergents and by very low concentrations of sodium dodecyl sulfate (SDS). In addition, all the variants had very low levels of activity with triglycerides with long aliphatic chains and with naturally occurring oils, like olive oil. Directed evolution was used to select variants with enhanced properties. In the first 10 rounds of evolution, the primary screen was selected for variants capable of hydrolyzing olive oil whereas the secondary screen was selected for enhanced tolerance towards a protease and SDS. In the final six rounds of evolution, the primary and secondary screens identified variants that retained activity after treatment with SDS. Sixteen cycles of evolution gave variants with greatly enhanced lipolytic activity on substrates that had both long (C16 and C18) as well as short (C3 and C8) chains. We found variants that were stable for more than 3 hours in protease concentrations that rapidly degrade the wild-type enzyme. Enhanced tolerance towards SDS was found in variants that could break down naturally occurring lipid and resist protease attack. The amino acid changes that gave enhanced properties were concentrated in the cap domain responsible for substrate binding

Improving on nature's shortcomings: evolving a lipase for increased lipolytic activity, expression and thermostability.

An enzyme must be soluble, stable, active and easy to produce to be useful in industrial applications. Not all enzymes possess these attributes. We set out to determine how many changes are required to convert an enzyme with poor properties into one that has useful properties. Lipase Lip3 from Drosophila melanogaster had been previously optimised for expression in Escherichia coli. The expression levels were good, but Lip3 was mainly insoluble with poor activity. Directed evolution was used to identify variants with enhanced activity along with improved solubility. Five variants and the wild-type (wt) enzyme were purified and characterised. The yield of the wt enzyme was just 2.2 mg/L of culture, while a variant, produced under the same conditions, gave 351 mg. The improvement of activity of the best variant was 200 times higher than that of the wt when the crude lysates were analysed using pNP-C8, but with purified protein, the improvement observed was 1.5 times higher. This means that most of the increase of activity is due to increase in solubility and stability. All the purified variants showed increased thermal stability compared with the wt enzyme that had a T1/2 of 37°C, while the mutant with P291L of 42.2°C and the mutant R7_47D with five mutations had a value of 52.9°C, corresponding to an improvement of 16°C. The improved variants had between five and nine changes compared with the wt enzyme. There were four changes that were found in all 30 final round variants for which sequences were obtained; three of these changes were found in the substrate-binding domain

El orden Carnivora (Mammalia) en el Perú: Estado del conocimiento y prioridades de investigación para su conservación

The high diversity of Peruvian carnivore species may pose problems when deciding to which taxa and topics new research efforts should be directed. In this publication, we evaluated the research effort made on each taxa -by assessing the number of publications per species-, point out the knowledge gaps that are important to the conservation of each species and present the first evaluation of research priorities for this group of animals to the country. We registered 145 publications about Peruvian carnivores made since 1943. The number of publications is significantly different between taxa, between subjects and between ecoregions where the research was conducted. According to the proposed priority scale, the species to be studied with greater priority is Nasua olivacea and the lowest priority is for Leopardus pardalis. The results of our study highlight the urgent need to conduct research on certain species of carnivores about which there are few published data, locally and globally, and which occupy a low number of ecoregions in the country. Both the scale of research priorities and the list of knowledge gaps presented here will be useful to guide logistical and financial efforts, for individual researchers as well as for private or governmental institutions.La alta diversidad de especies de carnívoros del Perú puede generar problemas al momento de decidir los taxa y temas sobre los que deben dirigirse los esfuerzos de investigación. En este trabajo se evalúa el esfuerzo de investigación en base al número de publicaciones realizadas para cada familia y especie de carnívoro en el Perú. Asimismo, se señalan los vacíos de información relevantes para la conservación de cada especie y se presenta la primera evaluación de las prioridades de investigación sobre este grupo animal en el Perú. Se registró 145 publicaciones sobre carnívoros peruanos realizadas desde el año 1943. El número de publicaciones presentó grandes diferencias entre taxa, entre temas estudiados y entre las ecorregiones en las que se realizaron las investigaciones. Según la escala de prioridades propuesta, la especie que debe ser estudiada con mayor prioridad es el coatí andino Nasua olivacea y la de menor prioridad es el ocelote Leopardus pardalis. Los resultados de nuestro trabajo resaltan la urgencia de realizar investigaciones sobre ciertas especies de carnívoros de las que existen pocos datos publicados, tanto a nivel local como global, y que se distribuyen en pocas ecorregiones del Perú. Tanto la escala de prioridades de investigación como la lista de vacíos de información serán de utilidad para guiar esfuerzos logísticos y financieros de investigadores particulares, instituciones privadas y gubernamentales.

Measurement of the radius dependence of charged-particle jet suppression in Pb–Pb collisions at <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" altimg="si1.svg"><mml:msqrt><mml:mrow><mml:msub><mml:mrow><mml:mi>s</mml:mi></mml:mrow><mml:mrow><mml:mi mathvariant="normal">NN</mml:mi></mml:mrow></mml:msub></mml:mrow></mml:msqrt><mml:mo linebreak="goodbreak" linebreakstyle="after">=</mml:mo><mml:mn>5.02</mml:mn><mml:mspace width="0.25em"/><mml:mtext>TeV</mml:mtext></mml:math>

The ALICE Collaboration reports a differential measurement of inclusive jet suppression using pp and Pb–Pb collision data at a center-of-mass energy per nucleon–nucleon collision sNN=5.02 TeV. Charged-particle jets are reconstructed using the anti-kT algorithm with resolution parameters R=0.2, 0.3, 0.4, 0.5, and 0.6 in pp collisions and R=0.2, 0.4, 0.6 in central (0–10%), semi-central (30–50%), and peripheral (60–80%) Pb–Pb collisions. A novel approach based on machine learning is employed to mitigate the influence of jet background. This enables measurements of inclusive jet suppression in new regions of phase space, including down to the lowest jet pT≥40 GeV/c at R=0.6 in central Pb–Pb collisions. This is an important step for discriminating different models of jet quenching in the quark–gluon plasma. The transverse momentum spectra, nuclear modification factors, derived cross section, and nuclear modification factor ratios for different jet resolution parameters of charged-particle jets are presented and compared to model predictions. A mild dependence of the nuclear modification factor ratios on collision centrality and resolution parameter is observed. The results are compared to a variety of jet-quenching models with varying levels of agreement