Increased expression of keratinase and other peptidases by Candida parapsilosis mutants.

Keratinases are enzymes of great importance involved in pathogenic processes of some fungi. They also have a widespread ecological role since they are responsible for the degradation and recycling of keratin. On the one hand, studying them furthers our knowledge of pathogenicity mechanisms, which has important implications for human health, and on the other hand, understanding their ecological role in keratin recycling has biotechnological potential. Here, a wild-type keratinolytic Candida parapsilosis strain isolated from a poultry farm was treated with ethyl methanesulfonate in order to generate mutants with increased keratinase activity. Mutants were then cultured on media with keratin extracted from chicken feathers as the sole source of nitrogen and carbon. Approximately 500 mutants were screened and compared with the described keratinolytic wild type. Three strains, H36, I7 and J5, showed enhanced keratinase activity. The wild-type strain produced 80 U/mL of keratinolytic activity, strain H36 produced 110 U/mL, strain I7, 130 U/mL, and strain J5, 140 U/mL. A 70% increase in enzyme activity was recorded for strain J5. Enzymatic activity was evaluated by zymograms with proteic substrates. A peptidase migrating at 100 kDa was detected with keratin, bovine serum albumin and casein. In addition, a peptidase with a molecular mass of 50 kDa was observed with casein in the wild-type strain and in mutants H36 and J5. Gelatinase activity was detected at 60 kDa. A single band of 35 kDa was found in wild-type C. parapsilosis and in mutants with hemoglobin substrate.

Increased expression of keratinase and other peptidases by Candida parapsilosis mutants

Keratinases are enzymes of great importance involved in pathogenic processes of some fungi. They also have a widespread ecological role since they are responsible for the degradation and recycling of keratin. On the one hand, studying them furthers our knowledge of pathogenicity mechanisms, which has important implications for human health, and on the other hand, understanding their ecological role in keratin recycling has biotechnological potential. Here, a wild-type keratinolytic Candida parapsilosis strain isolated from a poultry farm was treated with ethyl methanesulfonate in order to generate mutants with increased keratinase activity. Mutants were then cultured on media with keratin extracted from chicken feathers as the sole source of nitrogen and carbon. Approximately 500 mutants were screened and compared with the described keratinolytic wild type. Three strains, H36, I7 and J5, showed enhanced keratinase activity. The wild-type strain produced 80 U/mL of keratinolytic activity, strain H36 produced 110 U/mL, strain I7, 130 U/mL, and strain J5, 140 U/mL. A 70 percent increase in enzyme activity was recorded for strain J5. Enzymatic activity was evaluated by zymograms with proteic substrates. A peptidase migrating at 100 kDa was detected with keratin, bovine serum albumin and casein. In addition, a peptidase with a molecular mass of 50 kDa was observed with casein in the wild-type strain and in mutants H36 and J5. Gelatinase activity was detected at 60 kDa. A single band of 35 kDa was found in wild-type C. parapsilosis and in mutants with hemoglobin substrate.

Keratinolytic activity of Bacillus subtilis AMR using human hair.

AIMS: To determine the ability of a novel Bacillus subtilis AMR isolated from poultry waste to hydrolyse human hair producing peptidases including keratinases and hair keratin peptides. METHODS AND RESULTS: The Bacillus subtilis AMR was identified using biochemical tests and by analysis of 16S rDNA sequence. The isolate was grown in medium containing human hair as the sole source of carbon and nitrogen. The supplementation of hair medium (HM) with 0.01% yeast extract increased the keratinolytic activity 4.2-fold. B. subtilis AMR presented high keratinase production on the 8th day of fermentation in hair medium (HM) supplemented with 0.01% yeast extract (HMY) at pH 8.0. Keratinase yield was not correlated with increase in biomass. Zymography showed keratin-degrading peptidases migrating at c. 54, 80 and 100 kDa and gelatin-degrading bands at c. 80, 70 63, 54 32 and 15 kDa. Keratinases were optimally active at 50 degrees C and pH 9.0 and was fully inhibited by the serine proteinase inhibitor (PMSF). Scanning electron microscopy showed complete degradation of the hair cuticle after exposure to B. subtilis AMR grown in HMY. MALDI-TOF analysis of culture supernatant containing peptides produced during enzymatic hydrolysis of hair by B. subtilis AMR revealed fragments in a range of 800-2600 Da. CONCLUSIONS: This study showed that B. subtilis AMR was able to hydrolyse human hair producing serine peptidases with keratinase and gelatinase activity as well as hair keratin peptides. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report describing the production and partial characterization of keratinases by a B. subtilis strain grown in a medium containing human hair. These data suggest that peptides obtained from enzymatic hair hydrolysis may be useful for future applications on pharmaceutical and cosmetic formulations.

Increased plasma levels of soluble CD40, together with the decrease of TGF beta 1, as possible differential markers of Alzheimer disease.

Alzheimer's disease (AD) is a progressive neurodegenerative illness and the most frequent cause of dementia in the elderly. The identification of activated microglia within neuritic plaques, coupled with the presence of numerous inflammatory proteins, suggests that inflammation is an integral part of the pathogenetic process in AD. In the present paper we have investigated the levels of circulating inflammatory mediators as potential AD biomarkers concentrating essentially on (a) soluble CD40 (sCD40), a member of the tumor necrosis factor receptor superfamily lacking the membrane-associated endodomain by alternative splicing, and (b) transforming growth factor (TGF)-beta 1, a cytokine deeply involved in AD and playing a protective role on CNS. Decrease of TGF-beta1 in AD patients could enhance the effects of pro-inflammatory cytokines produced by activated microglia as well as the expression of factors, such as the CD40/CD40 ligand complex, by microglia and astrocytes. Total venous blood samples were obtained from 33 patients with clinical diagnosis of possible late-onset AD, 40 healthy age-matched and 11 healthy young individuals. A significant increase of sCD40 levels plasma of AD patients versus healthy controls was measured, concomitantly with a decrease in TGF-beta1 concentration. These variations, however, showed no correlation with the expression of ApoE epsilon 4 allele, which was determined in order to assess the different frequency of this risk factor between AD and control groups. Since no comparable modifications were detected in patients affected by Parkinson's disease or non-AD-based dementia, we propose that sCD40 and TGF-beta1 plasma levels might represent possible differential biomarkers of AD, and be useful pre-mortem to support the clinical diagnosis of late-onset AD.