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The enzyme chitinase from Moniliophthora perniciosa the causative agent of the witches' broom disease in Theobroma cacao, was partially purified with ammonium sulfate and filtration by Sephacryl S-200 using sodium phosphate as an extraction buffer. Response surface methodology (RSM) was used to determine the optimum pH and temperature conditions. Four different isoenzymes were obtained: ChitMp I, ChitMp II, ChitMp III and ChitMp IV. ChitMp I had an optimum temperature at 44-73ºC and an optimum pH at 7.0-8.4. ChitMp II had an optimum temperature at 45-73ºC and an optimum pH at 7.0-8.4. ChitMp III had an optimum temperature at 54-67ºC and an optimum pH at 7.3-8.8. ChitMp IV had an optimum temperature at 60ºC and an optimum pH at 7.0. For the computational biology, the primary sequence was determined in silico from the database of the Genome/Proteome Project of M. perniciosa, yielding a sequence with 564 bp and 188 amino acids that was used for the three-dimensional design in a comparative modeling methodology. The generated models were submitted to validation using Procheck 3.0 and ANOLEA. The model proposed for the chitinase was subjected to a dynamic analysis over a 1 ns interval, resulting in a model with 91.7% of the residues occupying favorable places on the Ramachandran plot and an RMS of 2.68.A enzima quitinase de Moniliophthora perniciosa, o fungo causador da doença vassoura de bruxa em Theobroma cacao, foi parcialmente purificada por precipitação com sulfato de amônio e filtração em coluna de Sephacryl S-200 usando fosfato de sódio como tampão de extração. A metodologia de Superfície de resposta (MSR) foi usada para determinar o pH e temperatura ótimos. Foram isoladas quatro quitinases diferentes: ChitMp I, ChitMp II, ChitMp III e ChitMp IV. A ChitMp I mostrou temperatura ótima de 44-73ºC e pH ótimo de 7,0-8,4. A ChitMp II apresentou temperatura ótima entre 45-73ºC e pH ótimo de 7,0-8,4. ChitMp III apresentou temperatura ótima de 54-67ºC e pH ótimo de 7,3-8,8. ChitMp IV apresentou temperatura ótima de 60ºC e pH ótimo em 7,0. Em relação à biologia computacional a sequência primária foi feita in silico usando banco de dados do Projeto Genoma/Proteoma do M. perniciosa, obtendo-se uma sequência com 564 pb e 188 aminoácidos, que foi utilizada para a obtenção da estrutura tridimensional por metodologia de modelagem comparativa. Os modelos gerados foram submetidos à validação usando Procheck 3.0 and ANOLEA. O modelo proposto para a quitinase foi submetido à análise dinâmica por 1 ns, resultando em um modelo com 91,7% de seus resíduos em locais regiões favoráveis no gráfico de Ramachandran e RMS de 2,68.

The enzyme glucanase from Moniliophthora perniciosa was produced in liquid medium and purified from the culture supernatant. A multivariate statistical approach (Response Surface Methodology - RSM) was employed to evaluate the effect of variables, including inducer (yeast extract) and fermentation time, on secreted glucanase activities M. perniciosa detected in the culture medium. The crude enzyme present in the supernatant was purified in two steps: precipitation with ammonium sulfate (70%) and gel filtration chromatography on Sephacryl S-200. The best inducer and fermentation time for glucanase activities were 5.9 g L-1 and 13 days, respectively. The results revealed three different isoforms (GLUI, GLUII and GLUIII) with purification factors of 4.33, 1.86 and 3.03, respectively. The partially purified enzymatic extract showed an optimum pH of 5.0 and an optimum temperature of 40°C. The enzymatic activity increased in the presence of KCl at all concentrations studied. The glucanase activity was highest in the presence of 0.2 M NaCl. The enzyme showed high thermal stability, losing only 10.20% of its specific activity after 40 minutes of incubation at 90°C. A purified enzyme with relatively good thermostability that is stable at low pH might be used in future industrial applications.

The enzyme glucanase from Moniliophthora perniciosa was produced in liquid medium and purified from the culture supernatant. A multivariate statistical approach (Response Surface Methodology - RSM) was employed to evaluate the effect of variables, including inducer (yeast extract) and fermentation time, on secreted glucanase activities M. perniciosa detected in the culture medium. The crude enzyme present in the supernatant was purified in two steps: precipitation with ammonium sulfate (70%) and gel filtration chromatography on Sephacryl S-200. The best inducer and fermentation time for glucanase activities were 5.9 g L-1 and 13 days, respectively. The results revealed three different isoforms (GLUI, GLUII and GLUIII) with purification factors of 4.33, 1.86 and 3.03, respectively. The partially purified enzymatic extract showed an optimum pH of 5.0 and an optimum temperature of 40°C. The enzymatic activity increased in the presence of KCl at all concentrations studied. The glucanase activity was highest in the presence of 0.2 M NaCl. The enzyme showed high thermal stability, losing only 10.20% of its specific activity after 40 minutes of incubation at 90°C. A purified enzyme with relatively good thermostability that is stable at low pH might be used in future industrial applications.A enzima glucanase de Moniliophthora perniciosa foi produzida em meio líquido e purificada a partir do sobrenadante da cultura. A metodologia de superfície de resposta (MSR) foi usada para avaliar os efeitos das variáveis, incluindo indutor (extrato de levedura) e tempo de fermentação, na atividade da glucanase de M. perniciosa detectada no meio de cultura. A enzima presente no sobrenadante foi purificada em duas etapas: precipitação com sulfato de amônio (70%) e cromatografia de filtração em gel em Sephacryl S-200. A produção da enzima glucanase foi maior na concentração de 5,9 g L-1 de extrato de levedura e 13 dias de fermentação. Os resultados mostraram três diferentes isoformas (GLUI, GLUII e GLUIII) com fatores de purificação de 4,33, 1,86 e 3,03, respectivamente. O extrato enzimático parcialmente purificado mostrou um pH ótimo de 5,0 e uma temperatura ótima de 40°C. A atividade enzimática aumentou na presença de KCl em todas as concentrações estudadas. A atividade da glucanase foi maior na presença de NaCl 0,2 M. A enzima apresentou alta estabilidade térmica, perdendo apenas 10,20% de sua atividade específica após 40 minutos de incubação a 90°C. Os resultados de termoestabilidade e a atividade em baixo pH mostraram que a enzima glucanase de M. perniciosa tem características promissoras para futuras aplicações industriais.

Cardiotonic steroids are used to treat heart failure and arrhythmia and have promising anticancer effects. The prototypic cardiotonic steroid ouabain may also be a hormone that modulates epithelial cell adhesion. Cardiotonic steroids consist of a steroid nucleus and a lactone ring, and their biological effects depend on the binding to their receptor, Na,K-ATPase, through which, they inhibit Na+ and K+ ion transport and activate of several intracellular signaling pathways. In this study, we added a styrene group to the lactone ring of the cardiotonic steroid digoxin, to obtain 21-benzylidene digoxin (21-BD), and investigated the effects of this synthetic cardiotonic steroid in different cell models. Molecular modeling indicates that 21-BD binds to its target Na,K-ATPase with low affinity, adopting a different pharmacophoric conformation when bound to its receptor than digoxin. Accordingly, 21-DB, at relatively high µM amounts inhibits the activity of Na,K-ATPase α1, but not α2 and α3 isoforms. In addition, 21-BD targets other proteins outside the Na,K-ATPase, inhibiting the multidrug exporter Pdr5p. When used on whole cells at low µM concentrations, 21-BD produces several effects, including: 1) up-regulation of Na,K-ATPase expression and activity in HeLa and RKO cancer cells, which is not found for digoxin, 2) cell specific changes in cell viability, reducing it in HeLa and RKO cancer cells, but increasing it in normal epithelial MDCK cells, which is different from the response to digoxin, and 3) changes in cell-cell interaction, altering the molecular composition of tight junctions and elevating transepithelial electrical resistance of MDCK monolayers, an effect previously found for ouabain. These results indicate that modification of the lactone ring of digoxin provides new properties to the compound, and shows that the structural change introduced could be used for the design of cardiotonic steroid with novel functions.