RESUMO
Burkholderia contaminans, a species of the Burkholderia cepacia complex-prevalent in certain Latin-American and European countries-can cause chronic pulmonary infection in persons with cystic fibrosis. Our aim was to gain insights into long-term lung infections with a focus on correlating how bacterial phenotypic traits in the chronic infection impact on patients' clinical outcome. Genotypic characteristics of 85 B. contaminans isolates recovered from 70 patients were investigated. For 16 of those patients, the clinical status and bacterial phenotypic characteristics, e.g. several virulence factors, phenotypic variants, and the antimicrobial susceptibility pattern, were evaluated. Two clones were found in the whole bacterial population: (i) the multiresistant ST 872 PCR-recA-RFLP-HaeIII-K-pattern clone, which carries a pathogenic island homologous to BcenGI11 of B. cenocepacia J2315, and (ii) the ST 102 PCR-recA-RFLP-HaeIII-AT-pattern clone. The emergence of certain bacterial phenotypes in the chronic infection such as the nonmucoid phenotype, small colony variants, brownish pigmented colonies, and hypermutators, proved to be, together with coinfection with Pseudomonas aeruginosa, the possible markers of more challenging infections and poor prognosis. The presence of cocolonizers and the bacterial phenotypes that are especially adapted to persist in long-term respiratory tract infections have a crucial role in patients' clinical outcomes.
Assuntos
Infecções por Burkholderia , Complexo Burkholderia cepacia , Fibrose Cística , Pneumonia , Humanos , Infecção Persistente , Fibrose Cística/complicações , Fibrose Cística/microbiologia , Pulmão/microbiologia , Fenótipo , Infecções por Burkholderia/microbiologiaRESUMO
Exoinulinases-enzymes extensively studied in recent decades because of their industrial applications-need to be produced in suitable quantities in order to meet production demands. We describe here the production of an acid-stable recombinant inulinase from Aspergillus kawachii in the Pichia pastoris system and the recombinant enzyme's biochemical characteristics and potential application to industrial processes. After an appropriate cloning strategy, this genetically engineered inulinase was successfully overproduced in fed-batch fermentations, reaching up to 840 U/ml after a 72-h cultivation. The protein, purified to homogeneity by chromatographic techniques, was obtained at a 42% yield. The following biochemical characteristics were determined: the enzyme had an optimal pH of 3, was stable for at least 3â¯hâ¯at 55⯰C, and was inhibited in catalytic activity almost completely by Hg+2. The respective Km and Vmax for the recombinant inulinase with inulin as substrate were 1.35â¯mM and 2673⯵mol/min/mg. The recombinant enzyme is an exoinulinase but also possesses synthetic activity (i. e., fructosyl transferase). The high level of production of this recombinant plus its relevant biochemical properties would argue that the process presented here is a possible recourse for industrial applications in carbohydrate processing.
Assuntos
Aspergillus/enzimologia , Proteínas Fúngicas/metabolismo , Glicosídeo Hidrolases/metabolismo , Proteínas Recombinantes/metabolismo , Aspergillus/genética , Estabilidade Enzimática , Fermentação , Proteínas Fúngicas/genética , Proteínas Fúngicas/isolamento & purificação , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/isolamento & purificação , Concentração de Íons de Hidrogênio , Microbiologia Industrial/métodos , Inulina/metabolismo , Cinética , Pichia/genética , Especificidade por SubstratoRESUMO
The non-acidic polygalacturonases produced by Aspergillus kawachii in a glucose/tryptone medium were adsorbed to a glass fiber microfilter that was used to clarify the fermentation broth. Maximum adsorption occurred at pH 3 under low ionic strength conditions. The adsorbed activity could be readily released with a buffer solution at pH 5. Based upon these observations, a separation process was developed which enabled the broth to be clarified and, at the same time, the non-acidic polygalacturonases to be concentrated 20-fold and purified 100-fold in a unique filtration step. The practical advantage of recovering polygalacturonases by a filtration process lies in the simplicity and efficiency of the operation involved.