Alessandro Riffel

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.

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.

Keratinolytic bacteria isolated from feather waste

The aim of this study was to characterize keratinolytic bacteria isolated from feather waste. Four isolates were selected after growth on solid medium with feather meal as sole carbon and nitrogen source and screened for proteolytic activity on milk agar plates. Three isolates were Gram-negative (belonged to the genera Burkholderia, Chryseobacterium and Pseudomonas) and one was Gram-positive (Microbacterium sp.). These bacteria grew on diverse keratin wastes such as feather meal, raw feathers, chicken nails, hair and wool. Keratinase activity was detected during growth, but the complete degradation of these substrates was not always achieved. The proteolytic character of crude enzymes was assessed using azokeratin and azocasein as substrates. The keratinases were active on both substrates and were similar in keratin hydrolysis when compared with commercially available microbial peptidases. These novel keratinolytic isolates have potential biotechnological use in processes involving keratin hydrolysis.

Production of an extracellular keratinase from Chryseobacterium sp. growing on raw feathers

The strain Chryseobacterium sp. kr6 shown to be useful for biotechnological purposes such as hydrolysis of poultry feathers and de-hairing of bovine pelts. The effect of temperature, initial pH and media composition on protease production by this keratinolytic strain was studied. The enzyme was produced between 25 and 37oC, with maximum activity and yield at 30oC. When protease production was tested in media with different initial pH, maximum activity was observed when cultivation was carried out at 30oC and initial pH ranging from 6.0 to 8.0. Higher activity was observed when feathers or feather meal were used as growth substrates, followed by soybean meal. The addition of carbohydrates or surfactants to feather broth resulted in decrease in keratinolytic activity.

Characterisation of cellulose-hydrolysing enzymes from the fungus Bipolaris sorokiniana

Cellulose-hydrolysing enzymes from the phytopathogenic fungus Bipolaris sorokiniana were partially purified and characterised. The enzyme production was variable according to the carbon source. β-Glucosidase and cellobiohydrolase activities were higher by growing the fungus on cellulose than on other carbon sources. Carboxymethyl cellulase production was stimulated by other carbohydrates, mainly lactose. Partial enzyme purification was carried out by liquid chromatography on Sepharose CL4B. The purification was about 17-fold, with a yield of 41% as judged by assay with p-nitrophenyl-β-D-glucopyranoside as substrate. The optimum pH and temperature were 5.0 and 55–60 °C respectively. The enzyme was stable at 28 and 37 °C but lost about 50% of its initial activity after 120 min at 55 °C. Saccharification of cellulosic materials such as crystalline cellulose, filter paper and wheat straws was carried out using the partially purified enzyme, resulting in the production of reducing sugars. © 1999 Society of Chemical Industry

Purification and characterization of ββ‐N‐acetylhexosaminidase from the phytopathogenic fungus Bipolaris sorokiniana

N‐acetylhexosaminidase (HEX) from the phytopathogenic fungus Bipolaris sorokiniana was isolated and characterized. The production of HEX by B. sorokiniana was not altered by growing on different carbon sources. Enzyme purification was carried out by sequential liquid chromatography on Sephacryl S‐200 HR, and p‐aminobenzyl‐2‐acetamido‐2‐deoxy‐β‐d‐thioglucopyranoside agarose. The purification was about 70‐fold, with a yield of 41%, determined with p‐nitrophenyl‐N‐acetylglucosaminide as substrate. The enzyme had pH and temperature optima of 4·5 and 55 °C, respectively. The molecular weight of non‐denatured enzyme was estimated as 120 000 Da by gel filtration chromatography, and about 55 000 Da by SDS‐PAGE. The fungal HEX had glycosylated residues as evidenced by binding to Concanavalin‐A. Bipolaris sorokiniana enzyme was also active with p‐nitrophenyl‐chitobioside and p‐nitrophenyl‐N‐acetylgalactosaminide as substrates.

Peritoneal fluid modulates the sperm acrosomal exocytosis induced by N-acetylglucosaminyl neoglycoprotein

The effect of peritoneal fluid (PF) on the human sperm acrosome reaction (AR) was tested. Sperm was pre-incubated with PF and the AR was induced by calcium ionophore A23187 and a neoglycoprotein bearing N-acetylglycosamine residues (NGP). The AR induced by calcium ionophore was inhibited 40% by PF from controls (PFc) and 50% by PF from the endometriosis (PFe) group, but not by PF from infertile patients without endometriosis (PFi). No significant differences were found in the spontaneous AR. When the AR was induced by NGP, pre-incubation with PFc reduced (60%) the percentage of AR, while PFe and PFi caused no significant differences. The average rates of acrosome reactions obtained in control. NGP- and ionophore-treated sperm showed that NGP-induced exocytosis differed significantly between the PFc (11%) and PFe/PFi groups (17%), and the ionophore-induced AR was higher for PFi (33%) than PFc/PFe (25%). The incidence of the NGP-induced AR was reduced in the first hour of pre-incubation with PFc and remained nearly constant throughout 4 h of incubation. The present data indicate that PF possesses a protective factor which prevents premature AR.