Multiplicity of aspartic proteinases from Cynara cardunculus L.

Aspartic proteinases (AP) play major roles in physiologic and pathologic scenarios in a wide range of organisms from vertebrates to plants or viruses. The present work deals with the purification and characterisation of four new APs from the cardoon Cynara cardunculus L., bringing the number of APs that have been isolated, purified and biochemically characterised from this organism to nine. This is, to our knowledge, one of the highest number of APs purified from a single organism, consistent with a specific and important biological function of these protein within C. cardunculus. These enzymes, cardosins E, F, G and H, are dimeric, glycosylated, pepstatin-sensitive APs, active at acidic pH, with a maximum activity around pH 4.3. Their primary structures were partially determined by N- and C-terminal sequence analysis, peptide mass fingerprint analysis on a MALDI-TOF/TOF instrument and by LC-MS/MS analysis on a Q-TRAP instrument. All four enzymes are present on C. cardunculus L. pistils, along with cyprosins and cardosins A and B. Their micro-heterogeneity was detected by 2D-electrophoresis and mass spectrometry. The enzymes resemble cardosin A more than they resemble cardosin B or cyprosin, with cardosin E and cardosin G being more active than cardosin A, towards the synthetic peptide KPAEFF(NO(2))AL. The specificity of these enzymes was investigated and it is shown that cardosin E, although closely related to cardosin A, exhibits different specificity.

In search of synergistic effects in antioxidant capacity of combined edible mushrooms.

The antioxidant activity of different edible mushrooms was evaluated considering the different contribution of individual and combined extracts. The radical scavenging capacity was evaluated through hydrogen atom transfer and single electron transfer reaction-based assays: DPPH radical scavenging activity and reducing power, respectively. The inhibition of lipid peroxidation was studied in lipossomes solutions by the ß-carotene-linoleate system. Three types of interactions (synergistic, additive and negative synergistic effects) were observed, synergism being the most abundant effect. Marasmius oreades is present in the mixtures with higher antioxidant properties and synergistic effects, while Cantharellus cibarius is present in the mixtures with lowest antioxidant properties and negative synergist effects. Two discriminant analyses were performed considering individual species in one case and mushroom mixtures in the other. The five mushroom species were clustered in five individual groups, but a similar result could not be obtained for the combined mushrooms, for which only the cases containing C. cibarius were separated in individual clusters.

Biochemical Characterization of SFC-1, a class A carbapenem-hydrolyzing beta-lactamase.

The carbapenem-hydrolyzing beta-lactamase SFC-1 from Serratia fonticola UTAD54 was overexpressed in Escherichia coli, purified, and characterized. The enzyme exhibited an apparent molecular mass of 30.5 kDa, determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. SFC-1 hydrolyzes penicillins, cephalosporins, aztreonam, and carbapenems and is inhibited by clavulanic acid, sulbactam, and tazobactam.

Purification and characterization of a new peptide antibiotic produced by a thermotolerant Bacillus licheniformis strain.

A Bacillus licheniformis strain, 189, isolated from a hot spring environment in the Azores, Portugal, strongly inhibited growth of Gram-positive bacteria. It produced a peptide antibiotic at 50 degrees C. The antibiotic was purified and biochemically characterized. It was highly resistant to several proteolytic enzymes. Additionally, it retained its antimicrobial activity after incubation at pH values between 3.5 and 8; it was thermostable, retaining about 85% and 20% of its activity after 6 h at 50 degrees C and 100 degrees C, respectively. Its molecular mass determined by mass spectrometry was 3249.7 Da.