Produção de enzimas quitosanolíticas utilizando Paenibacillus ehimensis e Paenibacillus chitinolyticus para obtenção de quitoologossacarídeos

The acquisition of oligosaccharides from chitosan has been the subject of several studies in the pharmaceutical, biochemical, food and medical due to functional properties of these compounds. This study aimed to boost its production of chitooligosaccharides (COS) through the optimization of production and characterization of chitosanolytic enzymes secreted by microorganisms Paenibacillus chitinolyticus and Paenibacillus ehimensis, and evaluating the antioxidant potential of the products obtained. In the process of optimizing the production of chitosanase were employed strategies Fractional Factorial Experimental Design and Central Composite Rotatable Design. The results identified the chitosan, peptone and yeast extract as the components that influenced the production of chitosanase by these microorganisms. With the optimization of the culture media was possible to obtain an increase of approximately 8.1 times (from 0.043 to 0.35 U.mL U.mL-1) and 7.6 times (from 0.08 U.mL-1 to 0.61 U.mL-1) in the enzymatic activity of chitosanase produced by P. chitinolyticus and P. ehimensis respectively. Enzyme complexes showed high stability in temperature ranges between 30º and 55º C and pH between 5.0 and 9.0. Has seen the share of organic solvents, divalent ions and other chemical agents on the activity of these enzymes, demonstrating high stability of these crude complexes and dependence of Mn2+. The COS generated showed the ability of DPPH radical scavenging activity, reaching a maximum rate of scavenging of 61% and 39% when they were produced with enzymes of P. ehimensis and P. chitinolyticus respectively. The use of these enzymes in raw form might facilitate its use for industrial applications

Análise de fluxos metabólicos para otimização da síntese do antibiótico cosmomicina por Streptomyces olindensis ICB20

Metabolic flux analysis (MFA) is a powerful tool for analyzing cellular metabolism. In order to control the growth conditions of a specific organism, it is important to have a complete understanding of its MFA. This would allowed us to improve the processes for obtaining products of interest to human and also to understand how to manipulate the genome of a cell, allowing optimization process for genetic engineering. Streptomyces olindensis ICB20 is a promising producer of the antibiotic cosmomycin, a powerful antitumor drug. Several Brazilian researchers groups have been developing studies in order to optimize cosmomycin production in bioreactors. However, to the best of our knowledge, nothing has been done on metabolic fluxes analysis field. Therefore, the aim of this work is to identify several factors that can affect the metabolism of Streptomyces olindensis ICB20, through the metabolic flux analysis. As a result, the production of the secondary metabolite, cosmomycin, can be increased. To achieve this goal, a metabolic model was developed which simulates a distribution of internal cellular fluxes based on the knowledge of metabolic pathways, its interconnections, as well as the constraints of microorganism under study. The validity of the proposed model was verified by comparing the computational data obtained by the model with the experimental data obtained from the literature. Based on the analysis of intracellular fluxes, obtained by the model, an optimal culture medium was proposed. In addition, some key points of the metabolism of Streptomyces olindensis were identified, aiming to direct its metabolism to a greater cosmomycin production. In this sense it was found that by increasing the concentration of yeast extract, the culture medium could be optimized. Furthermore, the inhibition of the biosynthesis of fatty acids was found to be a interesting strategy for genetic manipulation. Based on the metabolic model, one of the optimized medium conditions was experimentally tested in order to demonstrate in vitro what was obtained in silico. It was found that by increasing the concentration of yeast extract in the culture medium would induce to an increase of the cosmomycin production

Otimização do meio de cultura para a produção de quitosanase por metarhizium anisopliae em cultivo descontínuo submerso

In this work a Plackett-Burman Design with 8 factors and 12 trials in 2 levels with 3 repetitions at the center point was used in order to investigate the influence of the concentration of chitosan, peptone, yeast extract, NaNO3, K2HPO4, KCl, MgSO4.7H2O and FeSO4 on chitosanase production by Metarhizium anisopliae. Runs were carried out using submerged discontinuous cultivation for enzyme production. The results of the Plackett & Burman Design showed that only two factors, chitosan concentration as well as FeSO4 had influence on chitosanolytic activity, while the increase in concentration of other factors not contributed significantly to the quitosanolítica activity. Cultivation medium optimization for enzyme production was carried out using a Composite Central Design, with the most important factors for chitosanolytic activity (chitosan and FeSO4), in accordance with Plackett & Burman Design, and keeping the other nutrients in their minimum values. On this other design, it was taken the highest limit in Plackett & Burman Design as the lowest limit (-1) to FeSO4 factor. The results showed that the enzyme production was favoured by increasing the chitosan concentration and by decreasing FeSO4. Maximum production for chitosanolytic activity was about 70.0 U/L and was reached in only 18 h of fermentation, a result about twenty-eight times greater than a former study using the same microorganism (about 2.5 U/L at 48 h)

Produção de enzimas quitosanolíticas utilizando Paenibacillus ehimensis e Paenibacillus chitinolyticus para obtenção de quitoologossacarídeos

The acquisition of oligosaccharides from chitosan has been the subject of several studies in the pharmaceutical, biochemical, food and medical due to functional properties of these compounds. This study aimed to boost its production of chitooligosaccharides (COS) through the optimization of production and characterization of chitosanolytic enzymes secreted by microorganisms Paenibacillus chitinolyticus and Paenibacillus ehimensis, and evaluating the antioxidant potential of the products obtained. In the process of optimizing the production of chitosanase were employed strategies Fractional Factorial Experimental Design and Central Composite Rotatable Design. The results identified the chitosan, peptone and yeast extract as the components that influenced the production of chitosanase by these microorganisms. With the optimization of the culture media was possible to obtain an increase of approximately 8.1 times (from 0.043 to 0.35 U.mL U.mL-1) and 7.6 times (from 0.08 U.mL-1 to 0.61 U.mL-1) in the enzymatic activity of chitosanase produced by P. chitinolyticus and P. ehimensis respectively. Enzyme complexes showed high stability in temperature ranges between 30º and 55º C and pH between 5.0 and 9.0. Has seen the share of organic solvents, divalent ions and other chemical agents on the activity of these enzymes, demonstrating high stability of these crude complexes and dependence of Mn2+. The COS generated showed the ability of DPPH radical scavenging activity, reaching a maximum rate of scavenging of 61% and 39% when they were produced with enzymes of P. ehimensis and P. chitinolyticus respectively. The use of these enzymes in raw form might facilitate its use for industrial applications

Características genotípicas e fenotípicas de Candida Albicans isoladas da cavidade bucal de pacientes transplantados renais com ênfase na ação do extrato bruto de Eugenia uniflora em fatores de virulência

Candida albicans is a diploid yeast that in some circumstances may cause oral or oropharyngeal infections. The investigation of natural products is mandatory for the discovery of new targets for antifungal drugs development. This study aimed to determine the genotypes of 48 clinical isolates of C. albicans obtained from the oral cavity of kidney transplant patients from two distinct geographic regions of Brazil. In addition, we investigated three virulence factors in vitro: phospholipase activity, morphogenesis and the ability to evade from polymorphonuclear neutrophils. The expression of these virulence factors in vitro was also investigated in the presence of the crude extract of Eugenia uniflora. The genotype A was the most prevalent (30 isolates; 62.5%), followed by genotype C (15 isolates; 31.5%) and genotype B (3 isolates; 6.25%). When microsatellite technique with primer M13 was applied, 80% of the isolates from the South were placed within the same cluster. All Genotype C strains were grouped together within two different clusters. Genotype C was considered more resistant to PMNs attack than genotypes A and B. Strains isolated from the South of Brazil showed higher ability to combat PMNs phagocytosis. We found a high rate of genotype C strains isolated from the oral cavity of this group of patients. The crude extract of E. uniflora inhibited proper hypha formation and phagocytosis by PMNs, but had no significant effect on phospholipase activity. This study characterized oral C. albicans strains isolated from kidney transplant recipients and will contribute for the better understanding of the pathogenesis and alternative therapeutics for oral candidiasis