Optimal Conditions for Continuous Immobilization of Pseudozyma hubeiensis (Strain HB85A) Lipase by Adsorption in a Packed-Bed Reactor by Response Surface Methodology

This study aimed to develop an optimal continuous process for lipase immobilization in a bed reactor in order to investigate the possibility of large-scale production. An extracellular lipase of Pseudozyma hubeiensis (strain HB85A) was immobilized by adsorption onto a polystyrene-divinylbenzene support. Furthermore, response surface methodology (RSM) was employed to optimize enzyme immobilization and evaluate the optimum temperature and pH for free and immobilized enzyme. The optimal immobilization conditions observed were 150 min incubation time, pH 4.76, and an enzyme/support ratio of 1282 U/g support. Optimal activity temperature for free and immobilized enzyme was found to be 68°C and 52°C, respectively. Optimal activity pH for free and immobilized lipase was pH 4.6 and 6.0, respectively. Lipase immobilization resulted in improved enzyme stability in the presence of nonionic detergents, at high temperatures, at acidic and neutral pH, and at high concentrations of organic solvents such as 2-propanol, methanol, and acetone

Scanning electron microscopy and machine learning reveal heterogeneity in capsular morphotypes of the human pathogen Cryptococcus spp.

Phenotypic heterogeneity is an important trait for the development and survival of many microorganisms including the yeast Cryptococcus spp., a deadly pathogen spread worldwide. Here, we have applied scanning electron microscopy (SEM) to defne four Cryptococcus spp. capsule morphotypes, namely Regular, Spiky, Bald, and Phantom. These morphotypes were persistently observed in varying proportions among yeast isolates. To assess the distribution of such morphotypes we implemented an automated pipeline capable of (1) identifying potentially cell-associated objects in the SEM-derived images; (2) computing object-level features; and (3) classifying these objects into their corresponding classes. The machine learning approach used a Random Forest (RF) classifer whose overall accuracy reached 85% on the test dataset, with per-class specifcity above 90%, and sensitivity between 66 and 94%. Additionally, the RF model indicates that structural and texture features, e.g., object area, eccentricity, and contrast, are most relevant for classifcation. The RF results agree with the observed variation in these features, consistently also with visual inspection of SEM images. Finally, our work introduces morphological variants of Cryptococcus spp. capsule. These can be promptly identifed and characterized using computational models so that future work may unveil morphological associations with yeast virulence

Isolamento e caracterização de Cryptococcus neoformans a partir de amostras fecais de aves no RS

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Fatty acid methyl esters produced by oleaginous yeast Yarrowia lipolytica QU21: an alternative for vegetable oils

Fatty acid methyl esters produced by oleaginous yeast Yarrowia lipolytica QU21: an alternative for vegetable oils. An alternative for vegetable oil is microbial oil. There is, nowadays, an increasing interest in microbial lipidic compounds, called single cell oils (SCO), due to their several potential biotechnological applications, such as biodiesel production, food ingredients and antimicrobial activity, among others. Oleaginous yeasts are able to accumulate lipids up to 20% of their cellular dry weight, and some species can accumulate up to 70% of lipids. We have screened 86 yeast strains isolated from artisanal cheese using Nile red stain for the detection of intracellular lipid droplets by fluorescence microscopy, and 27% of them were promising for the production of microbial oil. Yarrowia lipolytica QU 21 was selected for comparison of five different methods of cell wall disruption for lipid extraction at a laboratory scale (dry biomass maceration, lysis with vortex and glass beads, ultrasonic bath and glass beads, maceration using liquid nitrogen, and liquid nitrogen followed by sonication). The method which showed the highest oil yield value (36%) was liquid nitrogen with sonication. Oil yield was highly influenced by the method used for cell wall lysis. Fatty acid methyl esters (FAME) composition of strain QU 21 after GC analysis was myristic acid (C14:0), myristoleic acid (C14:1), palmitic acid (C16:0), palmitoleic acid (C16:1), heptadecanoic acid (C17:1), stearic acid (C18:0) and oleic acid (C18:1).(Metil ésteres de ácidos graxos produzidos pela levedura oleaginosa Yarrowia lipolytica QU21: uma alternativa para óleos vegetais). Uma alternativa para óleos vegetais é o óleo microbiano. Atualmente, existe um aumento no interesse em compostos lipídicos de origem microbiana, chamados single cell oils (SCO), devido ao seu grande potencial de aplicação biotecnológica, como a produção de biodiesel, suplementos alimentares, atividade microbiana, entre outros. Leveduras oleaginosas são capazes de acumular mais de 20% do seu peso seco em lipídios e algumas espécies conseguem acumular mais de 70% de lipídios. Foram analisadas 86 linhagens de leveduras isoladas de queijo artesanal, utilizando o corante vermelho de Nilo para a detecção de gotas lipídicas intracelulares em microscópia de fluorescência. Dessas, 27% se mostraram promissoras para a produção de óleo microbiano. Yarrowia lipolytica QU21 foi selecionada para comparar cinco métodos de rompimento da parede celular para a extração de lipídios em escala laboratorial (maceração da biomassa seca, lise com pérolas de vidro em agitador vórtex, lise com pérolas de vidro em banho ultrasônico, maceração utilizando nitrogênio líquido e maceração utilizando nitrogênio líquido seguido por sonicador). O método que apresentou maior rendimento lipídico (36%) foi maceração utilizando nitrogênio líquido, seguido por sonicador. O rendimento lipídico é amplamente influenciado pelo método utilizado na etapa de lise da parede celular. A composição de metil ésteres de ácidos graxos (FAME) da linhagem QU21 utilizando cromatografia gasosa (GC) foi ácido mirístico (C14:0), ácido miristoleico (C14:1), ácido palmítico (C16:0), ácido palmitoleico (C16:1), ácido heptadecanóico (C17:1), ácido esteárico (C18:0) e ácido oleico (C18:1)

Produção e caracterização de celulase para formulação de detergentes biodegradáveis

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Expressão do gene chit1 do fungo entomopatogênico Metharhizium anisopliae em Pichia pastoris

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Produção e caracterização de uma amilase para formulação de detergentes biodegradáveis

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Suscetibilidade in vitro a antifúngicos, de amostras clínicas e ambientais de Cryptococcus neoformans isoladas no sul do Brasil

The purpose of the present study was to compare the susceptibility to four antifungal agents of 69 Cryptococcus neoformans strains isolated from AIDS patients with that of 13 C. neoformans strains isolated from the environment. Based on the NCCLS M27-A methodology the Minimal Inhibitory Concentrations (MICs) obtained for amphotericin B, itraconazole and ketoconazole were very similar for clinical and environmental isolates. Clinical isolates were less susceptible to fluconazole than environmental isolates. The significance of these findings and aspects concerning the importance, role and difficulties of C. neoformans susceptibility testing are also discussed.Comparou-se a suscetibilidade de 69 amostras de C. neoformans isoladas de pacientes com SIDA com 13 amostras de C. neoformans isoladas do meio ambiente, frente a quatro agentes antifúngicos. Com base na metodologia preconizada pelo NCCLS (M27-A) as concentrações inibitórias mínimas (CIMs) obtidas para a anfotericina B, itraconazol e cetoconazol foram muito semelhantes nos dois grupos estudados. Todavia, frente ao fluconazol, os isolados clínicos evidenciaram menor sensibilidade do que os provenientes do meio ambiente. Alguns aspectos envolvendo a importância e dificuldades dos testes de suscetibilidade com Cryptococcus neoformans são também discutidos pelos autores

Análise da variabilidade genética de isolados clínicos e ambientais de Criptococcus neoformans do Rio Grande do Sul

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Purification and ultrastructural localization of a copper–zinc superoxide dismutase (CuZnSOD) from the entomopathogenic and acaricide fungus Metarhizium anisopliae

The entomopathogenic fungus Metarhizium anisopliae contains three superoxide dismutases. One of these enzymes was purified and partially characterized as a CuZnSOD. The enzyme has an estimated molecular mass of 30 690 Da and a specific activity of 3838.89 U mg−1. SDS-PAGE and 2D gels show a single band of protein in the fractions eluted from the gel filtration column with a molecular mass of 20 000 and ∼15 000 Da, respectively, and a pI of 6.0. These results suggest that the native enzyme is a dimer consisting of two subunits. Polyclonal antiserum were raised against purified CuZnSOD and used to determine its subcellular localization by immunoelectron microscopy. M. anisopliae CuZnSOD is present in the cell wall