Exploring the Antarctic soil metagenome as a source of novel cold-adapted enzymes and genetic mobile elements / Explorando el metagenoma del suelo antártico como fuente de nuevas enzimas adaptadas al frío y elementos génicos móviles

Metagenomic library PP1 was obtained from Antarctic soil samples. Both functional and genotypic metagenomic screening were used for the isolation of novel cold-adapted enzymes with potential applications, and for the detection of genetic elements associated with gene mobilization, respectively. Fourteen lipase/esterase-, 14 amylase-, 3 protease-, and 11 cellulase-producing clones were detected by activity-driven screening, with apparent maximum activities around 35 °C for both amylolytic and lipolytic enzymes, and 35-55 °C for cellulases, as observed for other cold-adapted enzymes. However, the behavior of at least one of the studied cellulases is more compatible to that observed for mesophilic enzymes. These enzymes are usually still active at temperatures above 60 °C, probably resulting in a psychrotolerant behavior in Antarctic soils. Metagenomics allows to access novel genes encoding for enzymatic and biophysic properties from almost every environment with potential benefits for biotechnological and industrial applications. Only intI- and tnp-like genes were detected by PCR, encoding for proteins with 58-86 %, and 58-73 % amino acid identity with known entries, respectively. Two clones, BAC 27A-9 and BAC 14A-5, seem to present unique syntenic organizations, suggesting the occurrence of gene rearrangements that were probably due to evolutionary divergences within the genus or facilitated by the association with transposable elements. The evidence for genetic elements related to recruitment and mobilization of genes (transposons/integrons) in an extreme environment like Antarctica reinforces the hypothesis of the origin of some of the genes disseminated by mobile elements among "human-associated" microorganisms.

A partir de muestras de suelo antártico se obtuvo la metagenoteca PP1. Esta fue sometida a análisis funcionales y genotípicos para el aislamiento de nuevas enzimas adaptadas al frío con potenciales aplicaciones, y para la detección de elementos génicos asociados a la movilización de genes, respectivamente. Por tamizaje fenotípico se detectaron 14, 14, 3 y 11 clones productores de lipasas/esterasas, proteasas, amilasas y celulasas, respectivamente, con actividades máximas aparentes de 35 °C para las amilasas y lipasas, y de 35-55 °C para las celulasas, tal como se observó para otras enzimas adaptadas al frío. Sin embargo, una celulasa parece ser compatible con enzimas mesófilas, las que usualmente se mantienen activas hasta por sobre 60 °C. Este hecho probablemente esté asociado a un comportamiento psicrotolerante en los suelos antárticos. La metagenómica permite acceder a una nueva miríada de productos metabólicos con potenciales beneficios para aplicaciones biotecnológicas e industriales. Se detectaron los genes tipo intI y tnp por PCR, y sus productos génicos deducidos tuvieron identidades del 58 al 86 % y del 58 al 73 % con secuencias conocidas, respectivamente. Dos clones, BAC 27A-9 y BAC 14A-5, parecen presentar organizaciones sintéticas únicas, lo cual sugiere la existencia de rearreglos génicos probablemente debidos a divergencias evolutivas dentro del género o facilitados por la asociación de elementos de transposición. La evidencia de elementos génicos relacionados con el reclutamiento y la movilización de genes en ambientes extremos como la Antártida refuerza la hipótesis sobre el origen de algunos genes diseminados por elementos móviles entre los microorganismos asociados al ser humano.

Extração da ascorbato oxidase de Cucurbita maxima por processo descontínuo e contínuo em coluna de discos rotativos perfurados utilizando sistemas de duas fases aquosas / Extraction of ascorbate oxidase from Cucurbita maxima by discontinuous process in perforated rotating disc contactor aqueous two-fase systems

A partição e purificação de ascorbato oxidase de abóbora (Cucurbita maxima) por extração líquido-líquido em sistema de duas fases aquosas (SDFA), pelos processos descontínuos e contínuos, utilizando coluna de discos rotativos perfurados (PRDC), foram estudadas. Foram utilizados planejamentos estatísticos para selecionar as variáveis significativas no processo descontínuo de purificação, e as variáveis estudadas foram massa molar e concentração do polietileno glicol (PEG), concentração de citrato, pH, concentração de NaCl, fator de diluição e massa total do sistema. Os melhores resultados (coeficiente de partição 1,72, recuperação 90,8% e aumento de pureza 3.12) foram obtidos nas seguintes condições: massa molar do PEG 20000 (g/mol), pH 6,0, concentração de PEG 25% (m/m) e concentração de citrato 10% (m/m). No valor de pH 6,0 e temperatura 35ºC, a ascorbato oxidase apresentou seus maiores valores de atividade, e manteve a estabilidade na faixa de pH 5,0 a 9,0 durante 36 horas e a temperaturas de até 40ºC durante 1 hora. Experimentos também foram realizados para estimar as principais propriedades cinéticas e termodinâmicas da atividade e estabilidade da ascorbato oxidase, e esse estudo revelou que a enzima foi estável nas condições testadas. A PRDC mostrou um bom desempenho para extração da ascorbato oxidase em modo contínuo utilizando SDFA. A melhor condição operacional selecionada neste estudo foi selecionada com o auxílio de planejamentos estatísticos, sendo selecionadas as seguintes condições: massa molar do PEG 20000 (g/mol), concentração de PEG 20% (m/m), concentração de citrato 10% (m/m), velocidade de rotação dos discos de 80 rpm e velocidade da fase dispersa de 2 mL/min...

Níveis séricos de enzimas de função hepática em frangos de corte de criação industrial clinicamente saudáveis / Serum levels of hepatic enzyme function in clinically healthy broiler chickens

The values for the main hepatic enzymes included in the profiles of screen clinical biochemistry, alanine-aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (FA), lactate desidrogenase (LDH) and gamaglutamiltransferase (GGT), in samples of serum of broiler chickens in industrial system, clinically healthy, starting from the seventh day of life, until the slaughter (42 days) in weekly intervals were determined. Significant variations were not observed in the analyses in relation to the age of the birds for none of the appraised enzymes.

Characterization of alkaline xylanases from "Bacillus pumilus"

Alkaline xylanases produced by four different strains of "Bacillus pumilus" were characterized. The optimal pH and temperature were pH 9.0 and 60ºC for strain 13(a), and pH 8.0 and 55ºC for strains 5(2), 5(14), and 4(a). Under these conditions the following activities were found after 10 min in the presence of 1(per cent) xylan (birchwood): 328 U.ml(-1), 131 U.ml(-1), 90 U.ml(-1), and 167 U.ml(-1), respectively, for the four strains. The enzymes were stable at 40ºC, with 40(per cent) of the xylanase activity remaining after 2 hours for the enzymes of strain 5(2) and 60(per cent) for the other three strains. Stability at 50ºC was improved by addition of glycerol. Taking into account the conditions under which kraft pulps are bleached during the manufacture of paper xylanases from "B. pumilus" exhibit favorable potential for application to bleaching in the paper making process.

Studies on the lceratinolytic enzymes of thermophilic Actinomycetes II-Partial purification and characterization of thermostable keratinase enzyme from Thermoactinomyces vulgaris C52

The thermostable keratinase enzyme, produced by Thermoacetinomyces vulgaris CS2 in liquid modified. Kosmatchev medium under the optimum condition, was purified 224-fold with an Overall yield of 36.08% of the original activity and specific activity 748.67 units mg [-1] protein by ammonium sulphate precipitation and ion exchange chromatography [DEAF-Cellulose]. Maximal activity of the enzyme was obtained at 55-60 and pH 8.4-85. It was stable at 45-55'in the pH 8.4-8.5. Also the enzyme was slightly activated by CaC12 MgSO4, FeSO4 and CuSO4, but strongly inhibited by KFeCN, KCN, iodine and iodoacedic acid. The partially purified enzyme actively hydrolyzed all keratinaceous waste materials used

Purification and characterization of thermostable xylanases from thermophilic Humicola sp. and their application in pulp improvement

A linhagem termófila Humicola sp., que foi isolada de madeira em decomposiçäo, produz xilanases extracelulares termoestáveis a 50oC. As xilanases foram purificadas e foram encontradas 3 fraçöes de proteínas com atividade de xilanase. As características das 3 xilanases foram estudadas. Verificou-se que a xilanase I é uma endoxilanase; a xilanase II é uma xilosidase (exoxilanase) enquanto que a xilanase III é uma endoxilanase que também apresenta atividade de arabinosidase e CMCase. O tratamento da polpa "Kraft" branqueada obtida de eucalipto com enzima purificadas e bruta aumentou o brilho da polpa quando comparada com a polpa sem este tratamento. Xilanase I e xilanase bruta aumentaram a viscosidade da polpa enquanto xilanase II e III diminuíram a viscosiddade devido à presença de atividade CMCase

In vitro activities of a recombinant foot-and-mouth disease virus replicase expressed in Escherichia coli

The replicase gene of foot-and-mouth disease virus (FMDV) was expressed in Escherichia coli under the control of a tac promoter. The recombinant enzyme was purified by inclusion body precipitation, elution, and poly(U) Sepharose chromatography. The enzyme exhibits poly(A)-dependent oligo(U)-primed poly(U) polymerase activity. The specific activity of the purified replicase is 1.3 x 10 5. The recombinant replicase synthesizes RNA using FMDV RNA as template, as well as heterologous RNAs, such as globin RNA and synthetic RNAs, polyadenylated or not. In all polymerization reactions, RNA products twice the size of the template are formed, both in the presence and absence of an oligo(U) primer. The enzyme is also capable of incorporating [alpha32P]UTP in all RNAs tested except the viral template. This activity does not seem to be related to the primer independent polymerization activity. The products from polymerization reactions were characterized by hybridization. In the absence of primer they consist of the template and a complementary strand covalently attached, while in the presence of primer they consist of two complementary strands synthesized de novo. We propose mechanisms of RNA synthesis by the recombinant FMDV replicase in the absence and presence of primer. These mechanisms are discussed in terms of models for in vitro RNA synthesis of other piconaviruses

Purificación de la enzima de restricción NciI / Purification of NciI restriction endonuclease

La enzima de restricción Nci I producida por el microorganismo Neisseria cinerea, se purificó en nuestro laboratorio utilizando cromatografía de afinidad y de intercambio iónico, lo que permitió obtener una preparación enzimática con una actividad específica de 20 000 U/mg de proteínas y 69,2 % de recobrado global del proceso apta para ser usada en los diferentes trabajos relacionados con la ingeniería genética

Purificación de la enzima de restricción Sma I / Purification of Sma I restriction enzyme

La enzima de retricción Sma I, producida por el microorganismo Serratia marcescens, ha sido purificada en nuestro laboratorio utilizando precipitación con PEG 6000 y cromatografía de intercambio iónico en fosfocelulosa (P-11 Whatman), obteniéndose una preparación final con una actividad específica de 2971 U/mg proteína y 35 % de recobrado total del proceso de purificación apta para ser utilizada en los experimentos de clonación y otras técnicas del ADN recombinante

Producción de enzimas por streptomycoses sp cresciendo con caparazón de camarón como sustrato / Enzyme production of streptomyces up growing on shrimp shell as substrate

Se hizo un estudio comparativo en cuanto a la producción de enzimas por ima ceá de Streptomyces sp, utilizando como substrato caparazón de camarón desmineralizado ó quitina semipurificada. Se encontró que la producción de quitinasas está asociada al crecimiento microbiano cuando se usa como substrato caparazón de camarón, alcanzando una producción máxima de quitinasa, qutitosanasa, carboximetilcelulasa y de proteasa a las 96 h de incubación. Cuando el substrato fue la quitina, se obtuvo un máximo en la producción quitinolítica y quitosanolítica al alcanzar la fase de crecimiento estacionario. El crecimiento de Streptomyces sp fue mayor en caparazón de camarón que en quitina, siendo muy pequeñas las velocidades específicas de crecimiento tanto en caparazón de camarón como en quitina semipurificada