Cloning and high-level expression of Thermus thermophilus RecA in E. coli: purification and novel use in HBV diagnostics

ABSTRACT We studied the role of Thermus thermophilus Recombinase A (RecA) in enhancing the PCR signals of DNA viruses such as Hepatitis B virus (HBV). The RecA gene of a thermophilic eubacterial strain, T. thermophilus, was cloned and hyperexpressed in Escherichia coli. The recombinant RecA protein was purified using a single heat treatment step without the use of any chromatography steps, and the purified protein (>95%) was found to be active. The purified RecA could enhance the polymerase chain reaction (PCR) signals of HBV and improve the detection limit of the HBV diagnosis by real time PCR. The yield of recombinant RecA was ∼35 mg/L, the highest yield reported for a recombinant RecA to date. RecA can be successfully employed to enhance detection sensitivity for the diagnosis of DNA viruses such as HBV, and this methodology could be particularly useful for clinical samples with HBV viral loads of less than 10 IU/mL, which is interesting and novel.

Hydrogen/rydride ion relay - a mechanism for early electron transfer in cytochrome c oxidases / El reléuónico hidrógeno-hidruro: un mecanismo de transferencia temprana de electrones en las citocromo c oxidasas

Cytochrome c oxidase (COX) employs electrons obtained from cytochrome c to bring about the reduction of oxygen to water. It is known that the electrons originate from the haem edge of cytochrome c and enters bovine COX at Trp-104. It is also known that Tyr-105, Glu-198 and Asp-158 of COX subunit II play roles in the enzyme's catalysis but how these roles are linked to electron transfer remain unclear. Recently, we proposed that electrons travel from the haem edge of cytochrome c to CuA, the first metal redox centre of COX, by a hydrogen/hydride ion relay using six residues. Now using a similar computer assisted approach, we investigate the extent to which this hydride/hydrogen ion mechanism is common amongst oxidases. The crystal structures of COX from P denitrificans, R sphaeroides and T thermophilus and quinol oxidase from E coli were downloaded and their binding domains analysed. As with bovine, all four oxidases had only nine amino acid residues in that region and both the sequences and three-dimensional structures were highly conserved. We propose that these residues function as a hydrogen/hydride ion relay, participating directly in electron transfer to CuA. We further suggest that this electron transfer mechanism might be a common feature in oxidases.

La citocromo c oxidasa (COX) emplea electrones obtenidos del citocromo c para producir la reducción del oxígeno a agua. Se sabe que los electrones originan a partir del hemo del citocromo c, y entran en la COX bovina en Trp-104. También se conoce que Tyr-105, Glu-198 y Asp-158 de la subunidad II de COX, desempeñan papeles en la catálisis de la enzima, pero no hay todavía claridad en cuanto a cómo estos papeles se hallan vinculados con la transferencia de electrones. Recientemente, sugerimos que los electrones viajan del borde del hemo del citocromo c al CuA, el primer centro metálico de reacción redox de la COX, por un relé iónico hidrógeno-hidruro, usando seis residuos. Ahora, usando un enfoque similar computarizado, investigamos hasta que punto este mecanismo de iones hidrógeno/hidruro es común entre las oxidasas. Se bajaron y analizaron los dominios de unión de las estructuras cristalinas de la COX de P denitrificans, R sphaeroides, y T thermophilus, y de la quinol oxidasa de la E coli. Como en el caso de la bovina, las cuatro oxidasas tenían sólo nueve residuos de aminoácido en esa región, y tanto las secuencias como las estructuras tridimensionales presentaban un alto grado de conservación. Proponemos que estos residuos funcionan como un relé iónico hidrógeno-hidruro, participando directamente en una transferencia de electrones al CuA. Asimismo, sugerimos que este mecanismo de transferencia de electrones podría ser un rasgo común de las oxidasas.

Structural diversity of eukaryotic 18S rRNA and its impact on alignment and phylogenetic reconstruction

Ribosomal RNAs are important because they catalyze the synthesis of peptides and proteins. Comparative studies of the secondary structure of 18S rRNA have revealed the basic locations of its many length-conserved and length-variable regions. In recent years, many more sequences of 18S rDNA with unusual lengths have been documented in GenBank. These data make it possible to recognize the diversity of the secondary and tertiary structures of 18S rRNAs and to identify the length-conserved parts of 18S rDNAs. The longest 18S rDNA sequences of almost every known eukaryotic phylum were included in this study. We illustrated the bioinformatics-based structure to show that, the regions that are more length-variable, regions that are less length-variable, the splicing sites for introns, and the sites of A-minor interactions are mostly distributed in different parts of the 18S rRNA. Additionally, this study revealed that some length-variable regions or insertion positions could be quite close to the functional part of the 18S rRNA of Foraminifera organisms. The tertiary structure as well as the secondary structure of 18S rRNA can be more diverse than what was previously supposed. Besides revealing how this interesting gene evolves, it can help to remove ambiguity from the alignment of eukaryotic 18S rDNAs and to improve the performance of 18S rDNA in phylogenetic reconstruction. Six nucleotides shared by Archaea and Eukaryota but rarely by Bacteria are also reported here for the first time, which might further support the supposed origin of eukaryote from archaeans.

Expression, purification and enzymatic characterization of adenylate kinase of Thermus thermophilus HB27 in Escherichia coli / 南方医科大学学报

<p><b>OBJECTIVE</b>To clone the gene encoding adenylate kinase of Thermus thermophilus HB27, an extremely thermophilic bacterium, express the protein in Escherichia coil and study the enzymatic characterization.</p><p><b>METHODS</b>The DNA fragment encoding adenylate kinase was obtained by PCR from the total DNA of Thermus thermophilus HB27 and cloned into the vector pET-28a. The recombinant plasmid was identified by PCR, restriction endonuclease digestion and sequence analysis. Enzymatic characterization of the expressed protein was carried out using spectrophotometric assays.</p><p><b>RESULTS</b>The gene coding for adenylate kinase from Thermus thermophilus HB27 was cloned and the protein was overexpressed in Escherichia coli BL21(DE3). The optimum reactive pH and temperature for the enzyme were 8.5 and 90 degrees celsius;, respectively. The Km of the recombinant adenylate kinase for ADP was 68.6 micromol/L, with an V(max)ADP of 0.294 mmol/(L.min). Under the condition of environmental temperature at 70, 80, 90, or 100 degrees celsius; for 7 h, the recombinant adenylate kinase still retained the enzymatic activity with high thermostability. AP5A, a specific adenylate kinase inhibitor, inhibited the enzymatic activity of the protein by 70% at the concentration of 2.0 mmol/L, with a Ki value of 46.39 micromol/L for ADP.</p><p><b>CONCLUSION</b>The gene coding for adenylate kinase of Thermus thermophilus HB27 has been successfully cloned and expressed in Escherichia coil, which provides the basis for potential use of the highly thermostable recombinant HB27 adenylate kinase.</p>

Expression, purification and enzymatic characterization of Thermus thermophilus HB8 aspartate aminotransferase in Escherichia coli / 生物工程学报

To obtain thermostable aspartate aminotransferase, the gene aspC from an extremely thermophilic bacterium, Thermus thermophilus HB8 was cloned, and its product was overexpressed in Escherichia coli BL21 (DE3) and Rosetta (DE3). The expression in Rosetta (DP3) was more efficient. The optimum reactive pH was 7, and the recombinant enzyme activity changed little when incubated in the buffer of pH8 - 10 on 37 degrees C for 1 h. The optimum reactive temprature was 75 degrees C, and the recombinant enzyme was more stable on the temperature of 25 - 55 degrees C. The half life of recombinant enzyme on 65 degrees C was 3.5 h, on 75 degrees C was 2.5 h. KmKG was 7.559 mmol/L, VmaxKG was 0.086 mmol/(L x min), KmAsp was 2.031 mmol/L, VmaxAsp was 0.024 mmol/(L x min). Ca2+, Fe3+, Mn2+ inhibited enzyme activity softly.

Efeito da adição de Streptococus thermophilus como cultura adjunta na maturação e caracterização físico-química e sensorial de queijo prato / Effect of the addition of Streptococcus thermophilus as adjunct culture on ripening and physicochemical and sensory characterization of prato cheese

As culturas lácteas desempenham funções fundamentais na maturação de queijos, tais como produção de ácido lático e de compostos aromatizantes voláteis. Neste trabalho, o objetivo foi avaliar a ação de Streptococcus thermophilus como cultura adjunta, nos índices de proteólise e nas características físico química se sensoriais do queijo prato. As amostras de queijo foram preparadas conforme dois tratamentos: (1) utilização de cultura mesofílica - Lactococcus lactis ssp lactis e Lactococcus lactis ssp cremoris (tratamento 1) e (2) cultura mesofílica acrescida de Streptococcus thermophilus (tratamento 2). Durantea maturação dos queijos, foram realizadas análises físico-químicas (extrato seco total, gordura, gordura no extrato seco - GES, cinzas, nitrogênio, proteína total, índice de extensão da maturação - IEM, índice de profundidade da maturação - IPM, tirosina, triptofano e atividade de água) e sensoriais (odor, aroma,doce, ácido, salgado, amargo, adstringente, picante, elasticidade, firmeza, friabilidade, adesividade, solubilidade e umidade). O queijo fabricado conforme o tratamento 1 foi o mais proteolítico, resultando em características sensoriais mais acentuadas de acidez, amargor, sabor picante, elasticidade e solubilidade, comparado ao produto preparado com adição de Streptococcus thermophilus.

Atenuación térmica de cultivos lácticos destinados a la maduración acelerada de quesos / Attenuation thermophilus of lactic cultivation destinated maturation acelerated of cheese

En el presente trabajo se evalúa el efecto de la atenuación mediante choque térmico a varias temperaturas de cultivos de streptococcus salivarius ssp. thermophilus y lactobacillus delbrueckii ssp. bulgaricus destinados a la maduración acelerada de queso tipo Dambo. El objetivo fue establecer condiciones adecuadas para el tratamiento térmico, suficiente para reducuir la producción de ácido láctico, sin dañar el sistema enzimático proteolítico importante para la maduración del queso. Suspensiones de bacterias se cultivaron a pH constante y se calentaron a 63, 67, 70 y 72ºC. Los cambios en el pH y en la actividad enzimática mediante el método del ácido trinitrobencenosulfónico se evaluaron para cada temperatura de atenuación. Las células calentadas a 70 y 72ºC produjeron ácido lentamente después de un período de retraso de horas, mientras que las células calentadas a 65ºC lo hicieron rápidamente, con un retraso de 2 horas. Los cultivos calentados a 63ºC se comportaron de forma similar al control la actividad proteolítica disminuyó en 30, 70, 83 y 93 por ciento respectivamente, comparada con la de las células sin ningún tratamiento

Elaboración de un yogurt con base en una mezcla de leche y garbanzo (Cicer arietinum) / Elaboration of yogurt made of milk and chickpea (Cicer arietinum) mixture

El presente trabajo, tuvo como objetivo determinar las condiciones experimentales de elaboración de un yogurt extendido con garbanzo (Cicer arietinum L.), inoculado con St.thermophilus y L.bulgaricus, para compararlo fisicoquímica, microbiológica y sensorialmente con un yogurt elaborado con base en leche descremada. Los resultados obtenidos indicaron que de las mezclas obtenidas por el método de calificación química (cálculo de aminoácidos) que cumplieron con los objetivos propuestos fueron la mezcla 70:30 y 80:20 (leche descremada: extracto de garbanzo). Los yogurt elaborados con la mezcla 70:30 adicionados con almidones modificados (ULTRA SPERCE M Y COL-FLO), no eliminaron la sinéresis presente en los productos así como tampoco mejoraron las características sensoriales de los mismos; sin embargo, en le yogurt elaborado con la mezcla 80:20, y el almidón modificado (ULTRA SPERCE M.) se logró eliminar la sinéresis obteniéndose un yogurt <> con características de sabor y textura similar a la de un yogurt elaborado con base en leche, el cuál fue aceptado por el 80 por ciento de los jueces y que cumple además con las especificaciones de la norma oficial mexicana para yogurt

Effect of plant residue additions and temperature on growth criteria of thermophilous cellulolytic fungi from Egyptian soil

The results of this study indicated that incorporation of plant residues into the soil at 50 degrees increased the densities of thermophilic cellulolytic fungi; more prominently with rice or wheat straw. Moreover, irrespective of cellulosic supplements, wheat straw followed by cellulose powder, as a sole C-substrate in the medium, induced the highest counts, whereas alfalfa seemed the least in this regard. The preliminary investigation revealed that Humicola fuscoatra dominated the soil, though Thermomyces lanuginosus followed by Populospora thermophila were high during the middle part of incubation periods, whereas the least was Malbranchea sulfurea which was totally absent after 30 days incubation. The results further showed that the fastest growth rate for all fungi occurred at 50 degrees, however, P. thermophila failed to grow above 52 degrees, whereas the other fungi grew at 60 degrees. In response to 50 degrees, the daily growth rate and the relative growth rate [RGR] of both strains of H. Fuscoatra started by an appreciably high levels then decreased continuously with age whereas both criteria increased with time, reaching maximum during the middle part of the incubation period under the stress of temperature extremes [33, 58 and 60 degrees]