Production of enzymes for the diagnosis of CoVid-19

Os coronavírus representam um grupo de vírus cujos genomas são baseados em RNA fita simples de sentido positivo (+)ssRNA sendo causadores de diversas infecções respiratórias em humanos, incluindo a COVID-19. O diagnóstico rápido e preciso deste vírus é fundamental para nortear o tratamento da doença. Atualmente, os mais importantes testes de diagnóstico deste vírus são baseadas em imunoensaios (ELISA) ou em testes moleculares (PCR). Por se tratar de um genoma de RNA, a detecção do coronavírus se dá pela RT-PCR que envolve o uso de duas enzimas: transcriptase reversa e Taq DNA polimerase

Biochemical and Structural Characterization of Amy1: An Alpha-Amylase from Cryptococcus flavus Expressed in Saccharomyces cerevisiae

An extracellular alpha-amylase (Amy1) whose gene from Cryptococcus flavus was previously expressed in Saccharomyces cerevisiae was purified to homogeneity (67 kDa) by ion-exchange and molecular exclusion chromatography. The enzyme was activated by NH4+ and inhibited by Cu+2 and Hg+2. Significant biochemical and structural discrepancies between wild-type and recombinant α-amylase with respect to Km values, enzyme specificity, and secondary structure content were found. Far-UV CD spectra analysis at pH 7.0 revealed the high thermal stability of both proteins and the difference in folding pattern of Amy1 compared with wild-type amylase from C. flavus, which reflected in decrease (10-fold) of enzymatic activity of recombinant protein. Despite the differences, the highest activity of Amy1 towards soluble starch, amylopectin, and amylase, in contrast with the lowest activity of Amy1w, points to this protein as being of paramount biotechnological importance with many applications ranging from food industry to the production of biofuels

Molecular epidemiology and antimicrobial susceptibility of enterococci recovered from Brazilian intensive care units

ABSTRACT: We studied the antimicrobial resistance and the molecular epidemiology of 99 enterococcal surveillance isolates from two hospitals of Brasília, Brazil. Conventional biochemical tests were used to identify the enterococcal species and the disk diffusion method was used to determine their resistance profiles. Enterococcus faecalis (76%) and E. faecium (9%) were the most prevalent species. No enterococci showed the vanA or vanB vancomycin resistance phenotypes or genotypes. Only the intrinsically resistant species E. gallinarum (n=2) and E. casseliflavus (n=3) harbored the vancomycin-resistance genes vanC1 and vanC2/3, respectively. We found E. faecalis isolates with high-level resistance to gentamicin (22%) and streptomycin (8%) and both E. faecalis and E. faecium isolates with resistance to more than two antimicrobials (84% and 67%, respectively). Nine E. faecalis isolates (12%) were resistant to ampicillin; the minimal inhibitory concentration (MIC) values were 16µg/mL (n=6) and 32µg/mL (n=3). Among these ampicillin-resistant E. faecalis, seven were also resistant to gentamicin, ciprofloxacin, rifampin, penicillin, chloramphenicol, tetracycline and erythromycin. Pulsed-field gel electrophoresis classified those isolates in three different genotypes, suggesting dissemination of genetically related ampicillin-resistant E. faecalis strains among different patients

Engineering Zymomonas mobilis for the production of xylonic acid from sugarcane bagasse hydrolysate

Sugarcane bagasse is an agricultural residue rich in xylose, which may be used as a feedstock for the production of high-value-added chemicals, such as xylonic acid, an organic acid listed as one of the top 30 value-added chemicals on a NREL report. Here, Zymomonas mobilis was engineered for the first time to produce xylonic acid from sugarcane bagasse hydrolysate. Seven coding genes for xylose dehydrogenase (XDH) were tested. The expression of XDH gene from Paraburkholderia xenovorans allowed the highest production of xylonic acid (26.17 ± 0.58 g L−1) from 50 g L−1 xylose in shake flasks, with a productivity of 1.85 ± 0.06 g L−1 h −1 and a yield of 1.04 ± 0.04 gAX/gX. Deletion of the xylose reductase gene further increased the production of xylonic acid to 56.44 ± 1.93 g L−1 from 54.27 ± 0.26 g L−1 xylose in a bioreactor. Strain performance was also evaluated in sugarcane bagasse hydrolysate as a cheap feedstock, which resulted in the production of 11.13 g L−1 xylonic acid from 10 g L−1 xylose. The results show that Z. mobilis may be regarded as a potential platform for the production of organic acids from cheap lignocellulosic biomass in the context of biorefineries

Draft genome sequence of FT9, a novel Bacillus cereus strain isolated from a Brazilian Thermal Spring

O presente trabalho trata das bactérias formadoras da esporos, incluindo um patógeno presente na intoxicação alimentar e sistêmica e em infecções locais, trazendo uma sequência do genoma FT9

Transcriptional profiles of the human pathogenic fungus paracoccidioides brasiliensis in mycelium and yeast cells

This work was supported by MCT, CNPq, CAPES, FUB, UFG, and FUNDECT-MS. PbGenome Network: Alda Maria T. Ferreira, Alessandra Dantas, Alessandra J. Baptista, Alexandre M. Bailão, Ana Lídia Bonato, André C. Amaral, Bruno S. Daher, Camila M. Silva, Christiane S. Costa, Clayton L. Borges, Cléber O. Soares, Cristina M. Junta, Daniel A. S. Anjos, Edans F. O. Sandes, Eduardo A. Donadi, Elza T. Sakamoto-Hojo, Flábio R. Araújo, Flávia C. Albuquerque, Gina C. Oliveira, João Ricardo M. Almeida, Juliana C. Oliveira, Kláudia G. Jorge, Larissa Fernandes, Lorena S. Derengowski, Luís Artur M. Bataus, Marcus A. M. Araújo, Marcus K. Inoue, Marlene T. De-Souza, Mauro F. Almeida, Nádia S. Parachin, Nadya S. Castro, Odair P. Martins, Patrícia L. N. Costa, Paula Sandrin-Garcia, Renata B. A. Soares, Stephano S. Mello, and Viviane C. B. ReisParacoccidioides brasiliensis is the causative agent of paracoccidioidomycosis, a disease that affects 10 million individuals in Latin America. This report depicts the results of the analysis of 6,022 assembled groups from mycelium and yeast phase expressed sequence tags, covering about 80% of the estimated genome of this dimorphic, thermo-regulated fungus. The data provide a comprehensive view of the fungal metabolism, including overexpressed transcripts, stage-specific genes, and also those that are up- or down-regulated as assessed by in silico electronic subtraction and cDNA microarrays. Also, a significant differential expression pattern in mycelium and yeast cells was detected, which was confirmed by Northern blot analysis, providing insights into differential metabolic adaptations. The overall transcriptome analysis provided information about sequences related to the cell cycle, stress response, drug resistance, and signal transduction pathways of the pathogen. Novel P. brasiliensis genes have been identified, probably corresponding to proteins that should be addressed as virulence factor candidates and potential new drug targets

Multicopy plasmid integration in Komagataella phaffii mediated by a defective auxotrophic marker

Background: A commonly used approach to improve recombinant protein production is to increase the levels of expression by providing extra-copies of a heterologous gene. In Komagataella phaffii (Pichia pastoris) this is usually accomplished by transforming cells with an expression vector carrying a drug resistance marker following a screening for multicopy clones on plates with increasingly higher concentrations of an antibiotic. Alternatively, defective auxotrophic markers can be used for the same purpose. These markers are generally transcriptionally impaired genes lacking most of the promoter region. Among the defective markers commonly used in Saccharomyces cerevisiae is leu2-d, an allele of LEU2 which is involved in leucine metabolism. Cells transformed with this marker can recover prototrophy when they carry multiple copies of leu2-d in order to compensate the poor transcription from this defective allele. Results: A K. phaffii strain auxotrophic for leucine (M12) was constructed by disrupting endogenous LEU2. The resulting strain was successfully transformed with a vector carrying leu2-d and an EGFP (enhanced green fluorescent protein) reporter gene. Vector copy numbers were determined from selected clones which grew to different colony sizes on transformation plates. A direct correlation was observed between colony size, number of integrated vectors and EGFP production. By using this approach we were able to isolate genetically stable clones bearing as many as 20 integrated copies of the vector and with no significant effects on cell growth. Conclusions: In this work we have successfully developed a genetic system based on a defective auxotrophic which can be applied to improve heterologous protein production in K. phaffii. The system comprises a K. phaffii leu2 strain and an expression vector carrying the defective leu2-d marker which allowed the isolation of multicopy clones after a single transformation step. Because a linear correlation was observed between copy number and heterologous protein production, this system may provide a simple approach to improve recombinant protein productivity in K. phaffii

Sequecing and analysis of subunits alpha and beta of the follicle stimulating hormone from bovine (Bos taurus indicus)

Este trabalho relata uma clonagem e seqüenciamento das subunidades alfa e beta do hormônio folículo estimulante de Bos taurus indicus. Também apresenta os resultados de comparação realizada das seqüências gênicas dessas cadeias com as seqüências das cadeias alfa e beta do FSH de suínos e da cadeia beta de bovinos Bos taurus taurus já presentes no GenBank. Na comparação das seqüências de nucleotídeos e de aminoácidos predita da cadeia αFSH de Bos taurus indicus com as cadeias αFSH de outras espécies como suínos e búfalo (Bubalis bubalis), observou-se que as seqüências são bastante similares. A comparação da seqüência da subunidade αFSH de Bos taurus indicus com a de suíno demonstrou diferenças em três resíduos de aminoácidos. Na comparação com ßFSH, registrou-se modificação na primeira base do codon que levou à alteração no resíduo do aminóacido 83, que, em Bos taurus indicus, é uma glicina, ao invés da serina presente em Bos taurus taurus. Confirmaram-se essa modificação e todas as outras identificadas na seqüência dos cDNA das cadeias αFSH e βFSH em outra clonagem. A modificação Ser para Gly na posição 83 foi a única que alterou a identidade do resíduo de aminoácido na comparação entre as subunidades beta do FSH de Bos taurus indicus e Bos taurus taurus. Contudo, ela não deve alterar significativamente as propriedades fisiológicas do FSH, uma vez que o resíduo de glicina encontrado nessa posição também é encontrado na cadeia βFSH suína. Trata-se, portanto, de uma modificação particular que distingue as cadeias βFSH de B. taurus taurus e B. taurus indicus