Adriano Brandelli

Biochemical features of microbial keratinases and their production and applications

Keratinases are exciting proteolytic enzymes that display the capability to degrade the insoluble protein keratin. These enzymes are produced by diverse microorganisms belonging to the Eucarya, Bacteria, and Archea domains. Keratinases display a great diversity in their biochemical and biophysical properties. Most keratinases are optimally active at neutral to alkaline pH and 40–60°C, but examples of microbial keratinolysis at alkalophilic and thermophilic conditions have been well documented. Several keratinases have been associated to the subtilisin family of serine-type proteases by analysis of their protein sequences. Studies with specific substrates and inhibitors indicated that keratinases are often serine or metalloproteases with preference for hydrophobic and aromatic residues at the P1 position. Keratinolytic enzymes have several current and potential applications in agroindustrial, pharmaceutical, and biomedical fields. Their use in biomass conversion into biofuels may address the increasing concern on energy conservation and recycling.

Feather keratin hydrolysis by a Vibrio sp. strain kr2

The aim of the study was to characterize feather‐degrading bacteria isolated from poultry industry waste. A Vibrio sp. strain kr2 producing a high keratinolytic activity when cultured on native feather‐containing broth was isolated. The bacterium grew with an optimum at pH 6·0 and 30 °C, where maximum feather‐degrading activity was also observed. Keratinase production was similar at both 25 and 30 °C, while the maximum concentration of soluble protein was reached at 30 °C. Reduction of disulphide bridges was also observed, increasing with cultivation time. The keratinase of strain kr2 was active on azokeratin, azocasein, benzoyl‐arginine‐p‐nitroanilide and Ala‐Ala‐p‐nitroanilide as substrates. The amino acid composition of the feather hydrolysate was determined, presenting similarities with that reported for feather lysate, feather meal and raw feathers. A novel feather‐degrading bacterium was isolated and characterized, showing high keratinolytic activity. Complete feather degradation was achieved during cultivation. Strain kr2 shows potential for use for biotechnological processes involving keratin hydrolysis.

Anti-adhesion and antibacterial activity of silver nanoparticles supported on graphene oxide sheets.

This work reports on the preparation, characterization and antibacterial activity of a nanocomposite formed from graphene oxide (GO) sheets decorated with silver nanoparticles (GO-Ag). The GO-Ag nanocomposite was prepared in the presence of AgNO3 and sodium citrate. The physicochemical characterization was performed by UV-vis spectroscopy, X-ray diffraction (XRD), thermogravimetric analysis (TGA), Raman spectroscopy and transmission electron microscopy (TEM). The average size of the silver nanoparticles anchored on the GO surface was 7.5 nm. Oxidation debris fragments (a byproduct adsorbed on the GO surface) were found to be crucial for the nucleation and growth of the silver nanoparticles. The antibacterial activity of the GO and GO-Ag nanocomposite against the microorganism Pseudomonas aeruginosa was investigated using the standard counting plate methodology. The GO dispersion showed no antibacterial activity against P. aeruginosa over the concentration range investigated. On the other hand, the GO-Ag nanocomposite displayed high biocidal activity with a minimum inhibitory concentration ranging from 2.5 to 5.0 μg/mL. The anti-biofilm activity toward P. aeruginosa adhered on stainless steel surfaces was also investigated. The results showed a 100% inhibition rate of the adhered cells after exposure to the GO-Ag nanocomposite for one hour. To the best of our knowledge, this work provides the first direct evidence that GO-Ag nanocomposites can inhibit the growth of microbial adhered cells, thus preventing the process of biofilm formation. These promising results support the idea that GO-Ag nanocomposites may be applied as antibacterial coatings material to prevent the development of biofilms in food packaging and medical devices.

Characterization of a bacteriocin produced by a newly isolated Bacillus sp. Strain 8 A

Aims: The aim of this research was to investigate the production of bacteriocins by Bacillus spp. isolated from native soils of south of Brazil. 
Methods and Results: A bacteriocin produced by the bacterium Bacillus cereus 8 A was identified. The antimicrobial activity was produced starting at the exponential growth phase, although maximum activity was at stationary growth phase. A crude bacteriocin obtained from culture supernatant fluid was inhibitory to a broad range of indicator strains, including Listeria monocytogenes, Clostridium perfringens, and several species of Bacillus. Clinically relevant bacteria such as Streptococcus bovis and Micrococcus luteus were also inhibited. Bacteriocin was stable at 80°C, but the activity was lost when the temperature reached 87°C. It was resistant to the proteolytic action of trypsin and papain, but sensitive to proteinase K and pronase E.Bacteriocin activity was observed in the pH range of 6·0–9·0. 
Conclusions: A bacteriocin produced by Bacillus cereus 8 A was characterized, presenting a broad spectrum of activity and potential for use as biopreservative in food. 
Significance and impact of study: The identification of a bacteriocin with large activity spectrum, including pathogens and spoilage microorganisms, addresses an important aspect of food safety.

Characterization of an antibacterial peptide produced by Brevibacterium linens

Aims: The aim of this research was to investigate the antimicrobial activity produced by Brevibacterium linens ATCC 9175.

Bacteriocin‐like substance production by Bacillus licheniformis strain P40

Aims:  To investigate the production of bacteriocin‐like compounds by Bacillus spp. isolated from the Amazon basin.

Characterization of a protease of a feather-degrading Microbacterium species

Aims:  To characterize a new feather‐degrading bacterium.

In vitro antimicrobial activity of a new series of 1,4-naphthoquinones

The antibacterial activity of a series of 1,4-naphthoquinones was demonstrated. Disk diffusion tests were carried out against several Gram-positive and Gram-negative bacteria. The compound 5-amino-8-hydroxy-1,4-naphthoquinone was the most effective, presenting inhibition zones measuring 20 mm against staphylococci, streptococci and bacilli at 50 microg/ml. Methicillin-resistant Staphylococcus aureus and several clinical isolates of this bacterium were also inhibited. Naphthazarin, 5-acetamido-8-hydroxy-1,4-naphthoquinone, and 2,3-diamino-1,4-naphthoquinone were the next most active compounds. The minimal inhibitory concentration of the active compounds was determined against S. aureus, ranging from 30 to 125 microg/ml. All compounds presented a minimal bactericidal concentration higher than 500 microg/ml, indicating that their effect was bacteriostatic. The EC50, defined as the drug concentration that produces 50% of maximal effect, was 8 microg/ml for 5-amino-8-hydroxy-1,4-naphthoquinone against S. aureus, S. intermedius, and S. epidermidis. These results indicate an effective in vitro activity of 5-amino-8-hydroxy-1,4-naphthoquinone and encourage further studies for its application in antibiotic therapy.

Effect of nanovesicle-encapsulated nisin on growth of Listeria monocytogenes in milk

Commercial nisin was encapsulated in nanovesicles (mean diameter 140 nm) prepared from partially purified soy lecithin. Nisin-loaded liposomes and unencapsulated (free) nisin were initially tested in BHI medium and skim milk inoculated with Listeria monocytogenes and incubated for 48 h at 30 degrees C. At such abuse temperature conditions, free nisin showed better inhibitory than the liposomal counterparts. Subsequently, the effect of encapsulated or free nisin was evaluated in combination with refrigeration (7 +/- 1 degrees C) in both whole (3.25% fat) and skim (0% fat) milk for up to 14 day. A decrease of 3-4 log cycles in L. monocytogenes counts was observed for free and encapsulated nisin at 0.5 mg/ml concentration. Liposome encapsulation of antimicrobial peptides may be important to overcome stability issues and interaction with food components. The utilization of nanovesicle-encapsulated nisin in combination with low temperatures appeared to be effective to control L. monocytogenes in milk, emphasizing the importance of hurdle technology to assure food safety.

Synthesis and spectroscopic characterisation of 2-(2'-hydroxyphenyl)benzazole isothiocyanates as new fluorescent probes for proteins

Abstract Three new benzazole isothiocyanate (BzITC) fluorescent dyes, 2-(5′-isothiocyanate-2′-hydroxyphenyl)benzoxazole, 2-(5′-isothiocyanate-2′-hydroxyphenyl)benzimidazole and 2-(5′-isothiocyanate-2′-hydroxyphenyl)oxazole[4,5-b]pyridine, were synthesised, purified until optical purity grade and characterised by elemental analysis, 1 H NMR , IR, UV–VIS and fluorescence spectroscopy. These dyes exhibit an intense fluorescence emission with a large Stokes shift due to an excited state intramolecular proton transfer (ESIPT) mechanism. The BzITCs were also studied for labelling three proteins (bovine serum albumin (BSA), concanavalin-A (con-A) and rabbit immunoglobulin G (rabbit IgG)) and the resulting conjugates presented good and stable fluorescence. A simple assay for detection of these proteins was reported here. The method is based on the direct fluorescence detection of protein-labelled with BzITC fluorophores after polyacrylamide gel electrophoresis and present potential use as fluorescent probes for proteins.