RESUMEN
Cardiovascular diseases, resulting from the deposition of clots in blood vessels, are the leading cause of death worldwide. Fibrinolytic enzymatic activity can catalyze blood clot degradation. Findings show that 36 fungal isolates recovered from Caatinga soils have the potential to produce fibrinolytic protease under submerged conditions. About 58 % of the isolates displayed fibrinolytic activity above 100 U/mL, with Mucor subtilissimus UCP 1262 being the most active. The protease was biochemically and biophysically characterized, showing that the enzyme had a high affinity for SAApNA substrate and was significantly inhibited by fluoride methyl phenyl sulfonyl-C7H7FO2S, suggesting that it is a chymotrypsin-like serine protease. The highest enzyme activity was detected at pH 5.0 and 28 °C. This fibrinolytic protease's far-UV circular dichroism (CD) showed that its secondary structure was primarily α-helical. The purified fibrinolytic enzyme may represent a novel therapeutic agent for treating thrombosis. At temperatures above 65 °C, the enzyme lost all its secondary structure. Its melting temperature was 58.1 °C, the denaturation enthalpy 85.1 kcal/mol, and the denaturation entropy 0.26 kcal/Kâmol.
Asunto(s)
Mucor , Mucor/enzimología , Concentración de Iones de Hidrógeno , Dicroismo Circular , Microbiología del Suelo , Péptido Hidrolasas/química , Péptido Hidrolasas/aislamiento & purificación , Péptido Hidrolasas/metabolismo , Temperatura , Fibrinolíticos/química , FibrinólisisRESUMEN
Copaiba oil is a natural product used by Amazonian populations and recognized for its medicinal properties because it has significant antimicrobial activity for several pathogenic microorganisms. The present work aimed to evaluate and characterize the effect of natural oil produced by copaiba - Copaifera multijuga against multiresistant isolates of bubaline mastitis. The nitrocefin test was performed with isolates of Staphylococcus aureus from bubaline mastitis, which were 100% positive for beta-lactamase enzyme detection. Minimum Bactericidal Concentration (MBC) of 25% to 3.12% was obtained for Enterococcus faecalis and Escherichia coli and 50% and 25% for S. aureus, but Klebsiella pneumoniae and Bacillus subtilis were resistant. MBC with 12.5% and 6.25% oil were obtained for most multiresistant bubaline mastitis isolates from the states of Pernambuco, Ceará, Bahia and Alagoas. The results demonstrated the great potential of using copaiba natural oil in the treatment of buffalo mastitis.
Asunto(s)
Antiinfecciosos , Fabaceae , Mastitis , Brasil , Femenino , Humanos , Mastitis/tratamiento farmacológico , Mastitis/veterinaria , Pruebas de Sensibilidad Microbiana , Staphylococcus aureusRESUMEN
The industrial demand for proteolytic enzymes is stimulating the search for new enzyme sources. Fungal enzymes are preferred over bacterial enzymes, and more effective and easier to extract. The aim of this work was to evaluate the potential of protease production by solid state fermentation (SSF) of Mucor subtilissimus UCP 1262, evaluate different specific activities, purify and partially characterize the enzyme in terms of biochemical as to the optimal pH and temperature. Initially, the enzyme crude extract was screened for 3 different proteolytic activities, collagenolytic (161.4 U/mL), keratinolytic (39.6 U/mL) and fibrinolytic (26.1 U/mL) in addition to conventional proteinase activity. After ammonium sulfate precipitation, the active fractions with fibrinolytic activity were dialyzed in 15 mM Tris-HCl buffer, pH 8, loaded onto DEAE-Sephadex A50 ion-exchange column and gel filtrated through Superdex 75 HR10/300. The enzyme showed a fibrinolytic maximum activity at 40 C and pH 9,0. The purified enzyme showed activity against a chromogenic chymotrypsin substrate, SDS-PAGE showing a molecular mass of approximately 70 kDa and, the specific activity of 25.93 U/mg. These characteristics suggest that the enzyme could be and efficiently produced in a simple and low-cost way using Mucor subtilissimus UCP 1262 in SSF.