Camila Souza Porto

Healing activity induced by Cramoll 1,4 lectin in healthy and immunocompromised mice.

Cramoll 1,4 is a lectin extracted from seeds of Cratylia mollis Mart. Many assays have shown the cytokine release activity and pro-inflammatory profile of this lectin. Here, we used Cramoll 1,4 in the treatment of cutaneous wounds in normal and immunocompromised mice for available your cicatricial power. Surgical wounds were treated daily with a topical administration of Cramoll 1,4 and parameters as edema, hyperemia, scab, granulation and scar tissues as well as contraction of wounds were analyzed. Cramoll 1,4 wounds showed higher edema and arrival of more polimorphonuclear cells at the site of lesions. Granulation tissue and collagen fiber deposition were observed with higher intensity in all Cramoll 1,4 treated wounds and promoted excellent closing and repair of lesions in less time than other groups. Results showed that Cramoll 1,4 lectin was effective in the repair of experimental lesions in mice and can be used as a future cicatricial compound.

Kinetic and Thermodynamic Investigation on Ascorbate Oxidase Activity and Stability of a Cucurbita maxima Extract

The kinetic and thermodynamic properties of ascorbate oxidase (AO) activity and stability of a Cucurbita maxima extract were investigated. Activity tests performed at 25 °C using initial ascorbic acid concentration in the range 50–750 μM allowed estimating the Michaelis constant for this substrate (Km = 126 μM) and the maximum initial rate of ascorbic acid oxidation (A0,max = 1.57 mM min−1). The main thermodynamic parameters of the enzyme reaction (ΔH* = 10.3 kJ mol−1; ΔG* = 87.2 kJ mol−1; ΔS* = –258 J mol−1 K−1) were estimated through activity tests performed at 25–48 °C. Within such a temperature range, no decrease in the initial reaction rate was detected. The long‐term thermostability of the raw extract was then investigated by means of residual activity tests carried out at 10–70 °C, which allowed estimating the thermodynamic parameters of the irreversible enzyme inactivation as well (ΔH*D = 51.7 kJ mol−1; ΔG*D = 103 kJ mol−1; ΔS*D = –160 J mol−1 K−1). Taking into account the specific rate of AO inactivation determined at different temperatures, we also estimated the enzyme half‐life (1047 min at 10 °C and 21.2 min at 70 °C) and predicted the integral activity of a continuous system using this enzyme preparation. This work should be considered as a preliminary attempt to characterize the AO activity of a C. maxima extract before its concentration by liquid‐liquid extraction techniques.

Purification of a fibrinolytic protease from Mucor subtilissimus UCP 1262 by aqueous two-phase systems (PEG/sulfate)

A fibrinolytic protease from M. subtilissimus UCP 1262 was recovered and partially purified by polyethylene glycol (PEG)/sodium sulfate aqueous two-phase systems (ATPS). The simultaneous influence of PEG molar mass, PEG concentration and sulfate concentration on the enzyme recovery was first investigated using a 2(3) full factorial design, and the Response Surface Methodology used to identify the optimum conditions for enzyme extraction by ATPS. Once the best PEG molar mass for the process had been selected (6000g/mol), a two-factor central composite rotary design was applied to better evaluate the effects of the other two independent variables. The fibrinolytic enzyme was shown to preferentially partition to the bottom phase with a partition coefficient (K) ranging from 0.2 to 0.7. The best results in terms of enzyme purification were obtained with the system formed by 30.0% (w/w) PEG 6000g/mol and 13.2% (w/w) sodium sulfate, which ensured a purification factor of 10.0, K of 0.2 and activity yield of 102.0%. SDS-PAGE and fibrin zymography showed that the purified protease has a molecular mass of 97kDa and an apparent isoelectric point of 5.4. When submitted to assays with different substrates and inhibitors, it showed selectivity for succinyl-l-ala-ala-pro-l-phenylalanine-p-nitroanilide and was almost completely inhibited by phenylmethylsulfonyl fluoride, behaving as a chymotrypsin-like protease. At the optimum temperature of 37°C, the enzyme residual activity was 94 and 68% of the initial one after 120 and 150min of incubation, respectively. This study demonstrated that M. subtilissimus protease has potent fibrinolytic activity compared with similar enzymes produced by solid-state fermentation, therefore it may be used as an agent for the prevention and therapy of thrombosis. Furthermore, it appears to have the advantages of low cost production and simple purification.

Produção e caracterização de protease fibrinolítica de Streptomyces parvulus DPUA 1573

As proteases fibrinoliticas sao capazes de degradar coagulos de fibrina formados dentro dos vasos sanguineos, evitando a trombose intravascular. Em animais, a tromboflebite, que acomete frequentemente os equinos, ocasiona, em seus casos graves, a obstrucao jugular e tambem um edema de laringe, derivando a obstrucao das vias aereas, o que possibilita um edema cerebral, ocorrendo o obito do animal. Devido ao fato de o tratamento ser de custo elevado, faz-se necessaria a investigacao de outras fontesde proteases fibrinoliticas com custos menores e com menos efeitos colaterais. Diante disso, este estudo tem como objetivo produzir e caracterizar proteases fibrinoliticas obtidas de Streptomyces parvulus DPUA 1573. Para producao da enzima, foi utilizado um planejamento fatorial 24 avaliando a concentracao da farinha de soja (0,5, 1,0 e 1,5%) e da glicose (0, 0,5 e 1,0g/L), temperatura (28, 32 e 37oC) e agitacao (150, 200 e 250rpm) sobre a biomassa e a atividade fibrinolitica. Pode-se verificar que a protease fibrinolitica apresentou atividade maxima (835U/mL) nas condicoes de concentracao de 1,5% de soja, 1g/L de glicose, 28°C e 150rpm com 48 horas de fermentacao. A protease fibrinolitica obtida teve temperatura e pH otimos de 55°C e pH 9,0, respectivamente. A atividade enzimatica foi inibida pelo EDTA, pelo ion Fe2+ e pelo SDS, o que indicou a enzima ser uma metaloprotease. A linhagem Streptomyces parvulus DPUA 1573 foi capaz de produzir protease fibrinolitica, possuindo caracteristicas bioquimicas favoraveis a aplicacao na medicina veterinaria e possivelmente humana.