Immobilization of Beauveria bassiana Lipase on Silica Gel by Physical Adsorption

Extracellular lipase from Beauveria bassianastrain CG481 was immobilized by using thirteen different immobilization protocols. Silica gel was chosen as the most suitable adsorbent with 94.8% of activity yield. The adsorption on silica gel did not change the optimum pH (8.5) and temperature (45ºC) values of the free lipase (FL) for lipolytic activity, and it showed higher activities in extreme conditions (pH 9.0 to 10.5, 60ºC). The lipase immobilized on silica gel (ILS) showed enhanced stability at pH 7.0 after 120 h incubation (69.0%) when compared to FL (33.3%). The thermal stability was also enhanced by immobilization at 60ºC in aqueous (64.6%) and organic medium (95.1%), while FL showed only 40.6% of residual activity in aqueous medium and exhibited no activity for esterification reaction in n-heptane. The treatment of ILS with 0.8 M NaCl prevented lipase desorption while Triton X-100 (0.1%) resulted the enzyme leakage. The ILS was reused for four times for esterification reaction with 80.8% of initial activity

Evaluation of kinetic parameters of chitinases produced by Beauveria bassiana (Bals.) Vuill.

Beauveria bassiana é um fungo entomopatogênico utilizado no controle biológico de insetos-praga que infestam produtos agrícolas. O mecanismo de infecção envolve a produção de enzimas extracelulares, como proteases e quitinases que degradam a cutícula dos insetos. O objetivo deste trabalho foi avaliar parâmetros cinéticos de pH, temperatura, concentração iônica e tempo de reação sobre a atividade de quitinases. O fungo B. bassiana CG432 foi repicado em broca do café Hypothenemus hampei (Ferrari) e utilizado para preparar o inóculo contendo 108conídios ativados/mL. Estes conídios foram inoculados 1% (v/v) em meio de cultura líquido constituído por D-glicose (10g), extrato de levedura (5g), NaNO3 (1,58g), Na2HPO4.7H2O (1,05g), KCl (1g), MgSO4.7H2O (0,6g) e KH2PO4 (0,36g) por litro. O cultivo foi conduzido a 28°C e 180rpm durante 5 dias. O fluido extracelular do cultivo foi obtido por filtração e centrifugação a 8.000g, e as quitinases foram isoladas e concentradas por ultrafiltração entre membranas de 10 e 100kDa sob pressão de nitrogênio. A atividade das quitinases foi determinada pela quantificação da N-acetilglicosamina liberada pela hidrólise da quitina coloidal durante 60 minutos de incubação a temperaturas de 40 a 60ºC, concentrações iônicas dos tampões acetato de sódio pH 4,0 a 6,0; fosfato de sódio pH 6,0 a 8,0; e glicina-NaOH pH 8,0 a 10,0, a 50, 100 e 200 mM. A atividade das quitinases foi máxima a 45ºC, em pH 5,5, mas também foi elevada em pH 6,0 e 8,5 em concentração iônica de 50mM. A atividade de quitinases aumentou durante uma hora de incubação demonstrando estabilidade das enzimas nas condições ótimas da reação.    Entomopathogenic fungus Beauveria bassiana is currently used as a biocontrol agent for agricultural pests. The infection process involves extracellular enzymes such as proteases and chitinases that degrade the cuticle of the insects. The objective of this work was to evaluate kinetic parameters of pH, temperature, ionic concentration and time of reaction on chitinases activity. The fungus B. bassiana CG432 was cultivated on coffee berry borer Hypothenemus hampei (Ferrari) and the conidia grown on insect were used to prepare the inoculum containing 108conídia/mL. These conidia were inoculated at 1% (v/v) in culture liquid medium containing D-glucose (10g), yeast extract (5g), NaNO3 (1,58g), Na2HPO4.7H2O (1,05g), KCl (1g), MgSO4.7H2O (0,6g) and KH2PO4 (0,36g) per liter. The cultivation was carried at 28°C and 180rpm during 5 days. Culture fluid was obtained by filtration and centrifugation at 8.000g, and the chitinases were isolated and concentrated by ultrafiltration using 10 and 100kDa cut off membranes under nitrogen pressure. Chitinase activity was detected and quantified using N-acetylglucosamine released by hydrolysis of colloidal chitin at 40 to 60ºC, at 50, 100 and 200 mM ionic concentrations of buffers sodium acetate (pH 4.0 to 6.0); sodium phosphate (pH 6.0 to 8.0); and Glycine-NaOH (pH 8.0 to 10.0) during 60 minutes. Maximum chitinase activity was at 45ºC and pH 5.5, and was also high at pH 6.0 and pH 8.5 using 50mM buffer. The chitinase activity increased and was stable during an hour at optimum conditions of the reaction, shown the stable nature of this enzyme

Vancomycin-resistant Enterococcus spp.: clinical characteristics and risk factors

Vancomycin-resistant Enterococci (VRE) have emerged as a relevant multidrug-resistant pathogen and potencially lethal etiology of healthcare associated infections worldwide. This study intends to show the epidemiology and clinical characteristics of patients with VRE in a Hospital in South Brazil. A retrospective study was conducted from January 2005 to November 2007. A total of 122 VRE were identified in this period at the University Hospital of Londrina. All patients with VRE clinical culture have identified and their medical records have reviewed. The presence of colonization was evaluated through rectal swab cultures, and the species identification of clinical samples was performed by automated method MicroScan®. The mean age of patients was 54 years. Urinary tract (68.0%) and blood (23.8%) were the most frequent sites, and ICU was the largest sector of occurrence (49.2%). E. faecium was the predominant species, in 82.8% of cases. The risk factors presents were length of hospitalization (mean 58.2 days), previous use of antimicrobials and invasive procedure, such as use of central venous catheter, urinary catheter and mechanical ventilation. Control barriers and surveillance cultures are essential to prevent the VRE spread. The results obtained in this study contribute to a better understanding of the epidemiological dynamics of infections and the spread of VRE in University Hospital of Londrina