Keratin Azure Research Articles

High keratinase and other types of hydrolase activity of the new strain of Bacillus paralicheniformis.

Keratinases, a subclass of proteases, are used to degrade keratin thereby forming peptones and free amino acids. Bacillus paralicheniformis strain T7 was isolated from soil and exhibited high keratinase, protease, collagenase, amylase, xylanase, lipase, and phosphatase activities. Keratinases of the strain showed maximum activity at 70°C and pH 9.0 as well as high thermal stability. A mass-spectrometric analysis identified seven peptidases with molecular masses of 26.8-154.8 kDa in the secretory proteome. These peptidases are members of S8 and S41 serine peptidase families and of M14, M42, and M55 metallopeptidase families. Additionally, α-amylase (55.2 kDa), alkaline phosphatase (59.8 kDa), and esterase (26.8 kDa) were detected. The strong keratinolytic properties of the strain were confirmed by degradation of chicken and goose feathers, which got completely hydrolyzed within 4 days. Submerged fermentation by strain B. paralicheniformis T7 was carried out in a pilot bioreactor, where the highest keratinase production was noted after 19 h of cultivation. After the fermentation, in the culture fluid, the keratinase activity toward keratin azure was 63.6 ± 5.8 U/mL. The protease activity against azocasein was 715.7 ± 40.2 U/mL. The possibility of obtaining enzyme preparations in liquid and powder form was demonstrated, and their comparative characteristics are given. In the concentrate, the keratinase, protease, α-amylase, phosphatase, and esterase/lipase activities were 2,656.7 ± 170.4, 29,886.7 ± 642.9, 176.1 ± 16.3, 23.9 ± 1.8, and 510.9 ± 12.2 U/mL, respectively. In the lyophilizate, these activities were 57,733.3 ± 8,911.4, 567,066.7 ± 4,822.2, 2,823.0 ± 266.8, 364.2 ± 74.8, and 17,618.0 ± 610.3 U/g, respectively. In the preparation obtained by air flow drying at 55°C, these activities were 53,466.7 ± 757.2, 585,333.3 ± 4,277.1, 2,395.8 ± 893.7, 416.7 ± 52.4, and 15,328.1 ± 528.6 U/g, respectively. The results show high potential of B. paralicheniformis strain T7 as a producer of keratinases and other enzymes for applications in agricultural raw materials and technologies for processing of keratin-containing animal waste.

Description of Chryseobacterium fluminis sp. nov., a keratinolytic bacterium isolated from a freshwater river.

A Gram-stain-negative, aerobic, rod-shaped bacterial strain, designated MMS21-Ot14T, was isolated from a freshwater river, and shown to represent a novel species of the genus Chryseobacterium on the basis of the results from a polyphasic approach. The 16S rRNA gene sequence analysis revealed that MMS21-Ot14T represented a member of the genus Chryseobacterium of the family Weeksellaceae and was closely related to Chryseobacterium hagamense RHA2-9T (97.52 % sequence similarity), Chryseobacterium gwangjuense THG A18T (97.46 %) and Chryseobacterium gregarium P 461/12T (97.27 %). The optimal growth of MMS21-Ot14T occurred at 25-30 °C, pH 6.0-7.0 and in the absence of NaCl. MMS21-Ot14T was capable of hydrolysing casein, starch, DNA, Tween 20 and tyrosine. The strain also showed keratinolytic activity with keratin azure and decolourising activity with remazol brilliant blue R (RBBR), which indicated potential ability to degrade keratin and lignin. The main polar lipids of MMS21-Ot14T were phosphatidylethanolamine, unidentified aminophospholipids, unidentified aminolipids, an unidentified phospholipid and several unidentified lipids. The predominant fatty acids of MMS21-Ot14T were iso-C15 : 0 and iso-C17 : 0 3-OH, and the major isoprenoid quinone was menaquinone 6 (MK-6). The whole genome of MMS21-Ot14T was 5 062 016 bp in length with a DNA G+C content of 37.7 %. The average nucleotide identity and digital DNA-DNA hybridisation values between MMS21-Ot14T and phylogenetically related members of the genus Chryseobacterium were well below the threshold values for species delineation. It is evident from the results of this study that MMS21-Ot14T should be classified as representing a novel species of the genus Chryseobacterium, for which the name Chryseobacterium fluminis sp. nov. (type strain, MMS21-Ot14T = KCTC 92255T = LMG 32529T) is proposed.

Bioinformatics based discovery of new keratinases in protease family M36

Keratinases are proteases that can catalyze the degradation of insoluble keratinous biomass. Keratinases in protease family M36 (MEROPS database) are endo-acting proteases. In total, 687 proteases are classified in family M36. In the present study, new keratinolytic enzymes were identified in protease family M36 using the bioinformatics tool Conserved Unique Peptide Patterns (CUPP). Via CUPP, M36 family members were classified into 11 groups, with CUPP group 1 containing the three currently known and sequenced family M36 keratinases (derived from the fungi Fusarium oxysporum, Microsporum canis and Onygena corvina) as well as an additional 71 uncharacterized M36 proteases. In order to assess the relevance of CUPP group 1 categorization to keratinolytic function, four uncharacterized M36 proteases and the known keratinase from F. oxysporum (in CUPP group 1) were selected for recombinant expression and keratinolytic activity assessment. The four hitherto unknown M36 proteases were from Phaeosphaeria nodorum, Aspergillus clavatus, Pseudogymnoascus pannorum and Nectria haematococca, and represent four different fungal taxonomical classes. The genes encoding the selected M36 proteases were individually expressed in Pichia pastoris and all proteases displayed keratinase activity on keratin azure. Additionally, the activity on different keratinase substrates, optimal reaction conditions and thermal stability were determined for the two most active new keratinases. The results validate the applicability of CUPP for function-based discovery of non-characterized keratinases and present new robust keratinases for potential use in keratin upgrading.

Isolation of Chrysosporium indicum from poultry soil for keratinase enzyme, its purification and partial characterization

Keratinases are produced by microorganisms as fungi, actinomycetes and bacteria and have the capacity to degrade tough insoluble keratin proteins, including feathers. Feathers are waste produced from the poultry industry worldwide and accumulated as solid waste. Therefore, keratinolytic fungal strains Chrysosporium indicum were isolated by hair baiting method from poultry farm soil of Punjab, India. Isolated C. indicum were screened for proteolytic activity on skimmed milk agar. Field Emission Scanning Electron Microscopy (FeSEM) analysis confirmed morphological characters as C. indicum. Fourier transform infrared spectroscopy analysis was studied for the structural and mechanism analysis of feather degraded during keratinase production. Keratinase enzyme was purified 48.03% recovery by ammonium sulphate precipitation, dialysis for desalting and chromatography. Diethylaminoethyl sepharose (DEAE sepharose) and Sephadex-G75 column were used to perform chromatography and partial characterization of the keratinase for temperature, pH, and substrate. The maximum keratinase activity was observed at 500C, at pH 10. The maximum enzyme activity of 289.1 U/ml was observed with keratin powder as substrate and minimum enzyme activity 67.1 U/ml with keratin azure. This is the first report on the purification and characterization of keratinase by C. indicum using DEAE sepharose as affinity chromatography for the purification of the keratinase enzyme.

IDENTIFICATION LOCAL ISOLATES OF Trichophyton mentagrophytes AND DETECTION OF KERATINASE GENE USING PCR TECHNIQUE

This study was aimed to identify dermatophytic selective isolate using PCR technique as a rapid molecular assay. The results of this study showed among 60 samples of patients suffering from ringworm disease. forty isolates (66%) were Trichophyton mentagrophytes which diagnosed as dermatophytosis according to morphological and cultural methods. In order to investigate the ability of isolates to keratin analyses using solid medium supplemented with keratin azure , the results revealed that 20 isolates appeared best ability to keratin analysis and nine isolates had best ability for keratinase production in submerged culture. According to this results T. mantagrophytes (K3) (had higher activity for keratinase) was chosen for molecular identification. The results of PCR revealed that primer for18S rRNA gene of T. mentagrophytes K1 isolate and specific primer for subtilisin like protease gene were amplified and appeared as single DNA band with a molecular base of 690 bp and 623bp respectively .The blast result of sample sequences of amplified fragment revealed that the isolate were 100% identical to reference sequence of T.mentagrophytes var. interdigtal and depending on data base in NCBI The result of PCR product for enzyme showed new type named GBF60362 (402) subtilisin-like protease related to T. mentagrophytes 1354684064 BFBSOLP00892.

Feather degradation efficiency and hide dehairing ability of a new keratinolytic bacillus halotolerans strain, isolated from a tannery wastewater

Here, we report a new keratinolytic-producing bacterium identified as Bacillus halotolerans 4BC based on 16S rRNA gene sequencing. 4BC strain isolated from a tannery wastewater, showed proteolytic activity when grown on keratin azure, bovine hair and feather meal agar plates. B. halotolerans 4BC degraded almost 88% of chicken feathers after 10 days of cultivation in feather powder broth at pH 8 and 37°C. It was also efficiently able to degrade bovine hair keratin, despite the complexity of this substrate in comparison to feather keratin. The effects of different liquid to substrate ratios, inoculum sizes and incubation times on keratinase production were studied using response surface methodology to find the optimum conditions required for maximum B. halotolerans keratinase yield. The maximum keratinase yield (1059±53 mU/ml) was found under the following conditions: incubation period of 10 days, liquid/solid ratio of 5 and inoculum size of 2.3 % v/v). The crude enzyme exhibited a remarkable activity and stability under high temperature and alkaline conditions (pH 10 at 80°C). Additionally, the touch-visual and histological results demonstrated that the enzyme treated dehaired hides exhibit similar or improved characteristics without damaging the collagen layer, which makes the crude keratinase a potential effective and eco-friendly candidate for application in leather industry to avoid pollution problems associated with the use of chemicals.

Biochemical characterization of a surfactant-stable keratinase purified from Proteus vulgaris EMB-14 grown on low-cost feather meal.

The bioaccumulation of keratinous wastes from poultry and dairy industries poses a danger of instability to the biosphere due to resistance to common proteolysis and as such, microbial- and enzyme-mediated biodegradation are discussed. In submerged fermentation medium, Proteus vulgaris EMB-14 utilized and efficiently degraded feather, fur and scales by secreting exogenous keratinase. The keratinase was purified 14-fold as a monomeric 49kDa by DEAE-Sephadex A-50 anion exchange and Sephadex G-100 size-exclusion chromatography. It exhibited optimum activity at pH 9.0 and 60°C and was alkaline thermostable (pH 7.0-11.0), retaining 87% of initial activity after 1h pre-incubation at 60°C. The Km and Vmax of the keratinase with keratin azure were respectively 0.283mg/mL and 0.241 U/mL/min. Activity of P. vulgaris keratinase was stimulated by Ca2+, Mg2+, Zn2+, Na+ and maintained in the presence of some denaturing agents, except β-mercaptoethanol while Cu2+ and Pb2+ showed competitive and non-competitive inhibition with Ki 6.5mM and 17.5mM, respectively. This purified P. vulgaris keratinase could be surveyed for the biotechnological transformation of bioorganic keratinous wastes into valuable products such as soluble peptides, cosmetics and biodegradable thermoplastics.

Keratinolytic enzyme-mediated biodegradation of recalcitrant poultry feathers waste by newly isolated Bacillus sp. NKSP-7 under submerged fermentation.

Microbial and enzymatic degradation of keratin waste is more preferred over various conventional approaches which are costly and not environmentally suitable. Diverse niches are auspicious for the discovery of new microorganisms. To discover novel keratinolytic bacteria, 60 isolates from different poultry dumping sites were initially screened, and among these found a potent keratinolytic isolate (NKSP-7) that displayed higher feather-degrading ability. The selected isolate was identified as Bacillus sp. NKSP-7 based on 16S rDNA sequencing as well as physiochemical and morphological characteristics. The strain NKSP-7 showed complete hydrolysis of native chicken feathers (10g/L) in nutrient medium after 24h of incubation at 37°C under agitation (150 rev/min) and produced thermostable extracellular keratinase. The crude enzyme displayed maximal keratinolytic activity (34.7U/mL) in phosphate buffer of pH7.0, and at 60°C using keratin azure as a substrate. Keratinolytic enzyme showed stability at 20-65°C for 4h over the pH range of 5.5-8.0. No obvious inhibitory influence was perceived by cations, organic solvents, EDTA, and detergents. Whereas, enzyme activity was enhanced by adding β-mercaptoethanol, Na+, Cd2+, and Mn2+. All these notable features of keratinase make it a promising candidate for various industrial applications especially for dehairing process in leather industry, bioconversion of poultry waste, and in detergents formulations.

Azo dying of α-keratin material improves microbial keratinase screening and standardization.

SummaryMicrobial conversion through enzymatic reactions has received a lot of attention as a cost‐effective and environmentally friendly way to recover amino acids and short peptides from keratin materials. However, accurate assessment of microbial keratinase activity is not straightforward, and current available methods lack sensitivity and standardization. Here, we suggest an optimized Azokeratin assay, with substrate generated directly from azo‐dyed raw keratin material. We introduced supernatant filtration in the protocol for optimal stopping of keratinase reactions instead of the widely used trichloroacetic acid (TCA), as it generated biases and impacted the sensitivity. We furthermore suggest a method for standardization of keratinase activity signals using proteinase K, a well‐known keratinase, as a reference enabling reproducibility between studies. Lastly, we evaluated our developed method with several bacterial isolates through benchmarking against a commercial assay (Keratin Azure). Under different setups, the Azokeratin method was more sensitive than commonly used Keratin Azure‐based assays (3‐fold). We argue that this method could be applied with any type of keratin substrate, enabling more robust and sensitive results which can be used for further comparison with other studies, thus representing an important progress within the field of microbial keratin degradation.

The Role of Reactive Oxygen Species and Nitric Oxide in the Inhibition of Trichophyton rubrum Growth by HaCaT Cells.

Trichophyton rubrum (T. rubrum) is one of the most important agents of dermatophyte infection in humans. The aim of this experiment was to evaluate the effect of HaCaT cells on T. rubrum, investigate the responsible mechanism of action, and explore the role of reactive oxygen species (ROS) and nitric oxide (NO) in the inhibition of T. rubrum growth by HaCaT cells. The viability of fungi treated with HaCaT cells alone and with HaCaT cells combined with pretreatment with the NADPH oxidase inhibitor (DPI) or the nitric oxide synthase (NOS) inhibitor L-NMMA was determined by enumerating the colony-forming units. NOS, ROS, and NO levels were quantified using fluorescent probes. The levels of the NOS inhibitor asymmetric dimethylarginine (ADMA) were determined by enzyme-linked immunosorbent assay (ELISA). Micromorphology was observed using scanning electron microscopy (SEM) and transmission electron microscopy (TEM). In addition, fungal keratinase activity was assessed by measuring dye release from keratin azure. In vitro fungal viability, keratinase activity, and ADMA content decreased after HaCaT cell intervention, whereas the levels of ROS, NO, and NOS increased. The micromorphology was abnormal. Fungi pretreated with DPI and L-NMMA exhibited opposite effects. HaCaT cells inhibited the growth and pathogenicity of T. rubrum in vitro. A suggested mechanism is that ROS and NO play an important role in the inhibition of T. rubrum growth by HaCaT cells.

Two novel S1 peptidases from Amycolatopsis keratinophila subsp. keratinophila D2T degrading keratinous slaughterhouse by-products.

Two proteases, named C- and T-like proteases, respectively, were purified from the culture supernatant of Amycolatopsis keratinophila subsp. keratinophila D2T grown on a keratinous slaughterhouse by-product of pig bristles and nails as sole nitrogen and carbon source. The two proteases belong to peptidase family S1 as identified by mass spectrometric peptide mapping, have low mutual sequence identity (25.8%) and differ in substrate specificity. T-like protease showed maximum activity at 40°C and pH 8-9, and C-like protease at 60°C and pH 8-10. Peptides released from the keratinous by-product were identified by mass spectrometry and indicated P1 specificity for arginine and lysine of T-like and alanine, valine and isoleucine of C-like protease as also supported by the activity of the two proteases towards synthetic peptide and amino acid substrates. The specific activities of the C- and T-like proteases and proteinase K on keratin azure and azokeratin were comparable. However, C- and T-like proteases showed 5-10-fold higher keratin/casein (K/C) activity ratios than that of another S1 and two keratin-degrading S8 peptidases used for comparison. The findings support that the range of peptidase families considered to contain keratinases should be expanded to include S1 peptidases. Furthermore, the results indicated the quite thermostable C-like protease to be a promising candidate for use in industrial degradation of keratinous slaughterhouse by-products.

Keratin hydrolysis by dermatophytes

Dermatophytes are the most common cause of superficial fungal infections (tinea infections) and are a specialized group of filamentous fungi capable of infecting and degrading keratinised tissues, including skin, hair, and nail. Essential to their pathogenicity and virulence is the production of a broad spectrum of proteolytic enzymes and other key proteins involved in keratin biodegradation and utilization of its breakdown products. The initial stage of biodegradation of native keratin is considered to be sulfitolysis, in which the extensive disulfide bridges present in keratin are hydrolyzed, although some secreted subtilisins can degrade dye-impregnated keratin azure without prior reduction (Sub3 and Sub4). Sulfitolysis facilitates the extracellular biodegradation of keratin by the dermatophytes' extensive array of endo- and exoproteases. The importance of dermatophyte proteases in infection is widely recognized, and these enzymes have also been identified as important virulence determinants and allergens. Finally, the short peptide and amino acid breakdown products are taken up by the dermatophytes, using as yet poorly characterised transporters, and utilized for metabolism. In this review, we describe the process of keratin biodegradation by dermatophytes, with an especial focus on recent developments in cutting edge molecular biology and '-omic' studies that are helping to dissect the complex process of keratin breakdown and utilization.

The Role of NADPH Oxidase in the Inhibition of Trichophyton rubrum by 420-nm Intense Pulsed Light

Objectives: To evaluate the effect of intense pulsed light (IPL) on Trichophyton rubrum and investigate its mechanism of action.Methods: The viability of fungi treated with IPL alone and with IPL combined with an NADPH oxidase inhibitor (DPI) pretreatment was determined by MTT assays. The reactive oxygen species (ROS) were quantified with a DCFH-DA fluorescent probe. Malondialdehyde (MDA) content and superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities were determined by commercial kits. The transcription of the Nox gene was quantified using quantitative real-time PCR (qRT-PCR) analysis, and micromorphology was observed using scanning electron microscopy (SEM). In addition, fungal keratinase activity was detected by measuring dye release from keratin azure.Results: The growth declined with statistical significance after 6 h of treatment (P < 0.001). The ROS and MDA content increased after IPL treatment, whereas the SOD and GSH-Px activity decreased. Nox gene expression was upregulated, and the micromorphology was damaged. Keratinase activity decreased. Fungi that received DPI pretreatment exhibited contrasting outcomes.Conclusion: We found that 420-nm IPL significantly inhibited the growth and pathogenicity of T. rubrum in vitro. A suggested mechanism involves Nox as a factor that mediates 420-nm IPL-induced oxidative damage of T. rubrum.

SHG10 keratinolytic alkaline protease from Bacillus licheniformis SHG10 DSM 28096: Robust stability and unusual non-cumbersome purification.

Present study underlines an unusual non-cumbersome-powerful strategy for purification of SHG10 keratinolytic alkaline protease from Bacillus licheniformis SHG10 DSM 28096 with robust stability properties. The enzyme was impressively purified to homogeneity with specific activity, purification fold, and yield of 613.82 U mg-1 , 58.91 and 99%, respectively, via a sequential two-step purification strategy: precipitation with 65% (NH4 )2 SO4 and flow through fractions of DEAE-cellulose DE 53 column. SDS-PAGE conferred a monomeric enzyme with a molecular mass of 30.4 kDa. The enzyme demonstrated optimal activity at pH (10.0-11.0) and at 65 °C. It exhibited full stability at pH (6.0-11.0) over 38 h at 4 °C and at 65 °C for 15 min. Remarkable enhanced enzyme activity (130.15 and 126.37%) was retained in presence of commercial laundry detergents Oxi and Ariel after 1 h, respectively. Organic solvent stability of the enzyme was verified in butanol, ether, acetonitrile, isopropanol, and chloroform. Imposingly, full storage stability (100%) of the enzyme along 1 year in -20 °C was confirmed. Km -Vmax was 0.00174 mM-534.2 mM Sub · min-1 · mg protein-1 and 1.266 mg-28.89 mg Sub · h-1 · mg protein-1 on N-Suc-Ala-Ala-Pro-Phe-pNA and keratin azure, respectively. Robust stability properties of SHG10 keratinolytic alkaline protease along with rapid-efficient purification underpin its potential commercialization for industrial exploitation.

Determining the Pathogenic Potential of Non-sporulating Molds Isolated from Cutaneous Specimens.

Although non-sporulating molds (NSM) are frequently isolated from patients and have been recognized as agents of pulmonary disease, their clinical significance in cutaneous specimens is relatively unknown. Therefore, this study aimed to identify NSM and to determine the keratinolytic activity of isolates from cutaneous sites. NSM isolates from clinical specimens such as skin, nail, and body fluids were identified based on their ribosomal DNA sequences. Of 17 NSM isolates (7 Ascomycota, 10 Basidiomycota), eleven were identified to species level while five were identified to the genus level. These include Schizophyllum commune, a known human pathogen, Phoma multirostrata, a plant pathogen, and Perenniporia tephropora, a saprophyte. To determine fungal pathogenicity, keratinolytic activity, a major virulence factor, was evaluated ex vivo using human nail samples by measuring dye release from keratin azure, for NSM along with pathogens (Trichophyton mentagrophytes, Trichophyton rubrum, Microsporum canis and Fusarium spp.) and nonpathogenic (endophyte) fungi for comparison. This study showed that pathogenic fungi had the highest keratinolytic activity (7.13±0.552 keratinase units) while the nonpathogenic endophytes had the lowest activity (2.37±0.262 keratinase units). Keratinolytic activity of two Ascomycota NSM (Guignardia mangiferae and Hypoxylon sp.) and one Basidiomycota NSM (Fomitopsis cf. meliae) was equivalent to that of pathogenic fungi, while Xylaria feejeensis showed significantly higher activity (p<0.05) than nonpathogenic endophytes. These results indicate that the pathogenic ability of NSM is species dependent; clinical isolates, especially more frequently isolated species, may be involved in disease etiology.

Purification and partial characterization of serine-metallokeratinase from a newly isolated Bacillus pumilus NRC21

A serine metallokeratinase enzyme (30kDa) produced by a newly isolated Bacillus strain (Bacillus pumilus NRC21) cultivated under optimized conditions in medium containing chicken feather meal was purified and characterized in a set of biochemical assays. The purification was carried out using two successive chromatographic steps; cation exchange chromatography on CM-cellulose and gel filtration on sephadex G-100 columns. The purified enzyme showed a specific activity of 2000units/mg protein against 170units/mg protein for crude extract with 12 fold purification. The enzymatic activity of the keratinase stimulated by (Na+, K+, Mg2+), Hg+2 had no effect, and inhibited by entire tested cations, serine and metalloproteinase inhibitors, therefore it can be considered as a serine metalloenzyme. The optimum pH and temperature for the purified enzyme were (7.5, 8.5) and (50, 45°C) when using keratin azure and azocasein as substrates, respectively. The purified enzyme was highly stable at broad pH and temperature ranged (5–10) and (20–60°C), respectively and its thermoactivity and thermostability were enhanced in the presence of 5mMMg+2. These results suggest that the purified keratinase may be used in several industrial applications.

Optimization of Keratinase Production by Amycolatopsis sp. Strain MBRL 40 from a Limestone Habitat

Background: The class Actinobacteria accounts for a high proportion of soil microbial biomass and more than half of the bioactive compounds including antibiotics, immunosuppressive agents, antitumor agents and industrial enzymes such as keratinases. Keratinases are modern proteases that can valorise poultry and leather industry wastes and may find applications in prion degradation and treatment of neurodegenerative diseases. A wide diversity of bacteria, actinomycetes and fungi are known to be keratin degraders. Manipur, being a part of Indo-Burma Hotspot, might harbor a rich diversity of bioactive actinomycetes. The objectives of the study are: a) Isolation of new keratinolytic actinomycetes. b) Medium optimization by statistical approaches for keratinase production. Methods: A protease producing actinomycete strain from Hundung limestone quarry at Ukhrul, Manipur, India, was investigated for its keratinolytic activities. Optimization of keratinase production was done using statistical methods namely, Plackett-Burman Design (PBD) and Response Surface Methodology (RSM). Eleven (11) variables were screened using PBD. Results: The strain showed keratinolytic activity with keratin azure and chicken feather as substrates. Maximum keratinolytic activity was observed at 40°C, pH 7 and 48 h of incubation. Yeast extract, MgSO4, and corn flour were found to affect the response signal positively whereas glucose, CaCO3, K2HPO4, NaCl and soyabean meal had negative effects. Yeast extract, cornflour, and soyabean meal were further studied using RSM. A 2.3 fold increase in keratinase production was achieved in submerged fermentation after the use of statistical optimization methods. Conclusion: On the basis of biochemical properties and 16S rRNA gene sequence analysis, the strain was identified as Amycolatopsis sp. strain MBRL 40. Novelty of the work: There are meagre reports of keratinolytic bacteria from limestone biotopes. In addition, keratinolytic Amycolatopsis species are scanty in the literature. This possibly is the first report of a keratinolytic Amycolatopsis strain from a limestone habitat.

Purification and biochemical characterization of a detergent-stable keratinase from a newly thermophilic actinomycete Actinomadura keratinilytica strain Cpt29 isolated from poultry compost

An extracellular thermostable keratinase (KERAK-29) was purified and biochemically characterized from a thermophilic actinomycete Actinomadura keratinilytica strain Cpt29 newly isolated from Algerian poultry compost. The isolate exhibited high keratinase production when grown in chicken feather meal media (24,000 U/ml). Based on matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF/MS) analysis, the purified enzyme is a monomer with a molecular mass of 29,233.10-Da. The data revealed that the 25 N-terminal residue sequence displayed by KERAK-29 was TQADPPSWGLNNIDRQTAFTKATSI, which showed high homology with those of Streptomyces proteases. This keratinase was completely inhibited by phenylmethanesulfonyl fluoride (PMSF) and diiodopropyl fluorophosphates (DFP), which suggests that it belongs to the serine protease family. Using keratin azure as a substrate, the optimum pH and temperature values for keratinase activity were pH 10 and 70°C, respectively. KERAK-29 was stable between 20 and 60°C and pH 3 and 10 for 5 and 120 h, respectively, and its thermoactivity and thermostability were enhanced in the presence of 5 mM Mn(2+). Its catalytic efficiency was higher than that of the KERAB keratinase from Streptomyces sp. strain AB1. KERAK-29 was also noted to show high keratinolytic activity and significant stability in the presence of detergents, which made it able to accomplish the entire feather-biodegradation process on its own. The ability of the A. keratinilytica strain Cpt29 to grow and produce substantial levels of keratinase using feather as a substrate could open new promising opportunities for the valorization of keratin-containing wastes and reduction of its impacts on the environment.

Production and characterization of thermostable metallo-keratinase from newly isolated Bacillus subtilis NRC 3

Novel keratinolytic enzyme (32kDa) secreted by a newly isolated Bacillus strain (Bacillus subtilis NRC3) cultivated in medium containing chicken feather meal was purified and partially characterized in a set of biochemical assays. The purification was carried out by applying a protocol of two successive chromatographic steps; cation exchange chromatography on CM-cellulose and gel filtration on sephadex G-75 columns. The purified enzyme showed a specific activity of 5233units/mg protein against 169units/mg protein for crude extract with 31 fold purification. The enzymatic activity of the purified keratinolytic enzyme stimulated by Na+, K+, Mg2+, Ba2+, Ca2+, and inhibited by entire tested cations and metalloproteinase inhibitors, indicating that it belongs to metallo-keratinase enzymes. The optimum pH and temperature for the purified enzyme were (7.5, 8.0) and (50, 40°C) when using keratin azure and azocasein as substrates, respectively. The purified enzyme was highly stable at broad pH and temperature ranged (5–10) and (20–60°C), respectively. These results suggest that this keratinase may be a useful alternative and ecofriendly route for handling the abundant amount of waste feathers.

Non-sporulating molds in clinical specimens: agents of infections?

Non-sporulating molds (NSMs) or mycelia sterilia isolated from clinical specimens are assumed to be environmental contaminants and their identification is not usually pursued. Knowing the identity of these isolates may help to determine their importance as pathogens. In this study, internal transcribed spacer (ITS) sequences were used to identify 15 non-sporulating fungi isolated as pure cultures from clinical specimens such as skin, nail, blood and other fluids. Fungal DNA was directly extracted from Whatman FTA® cards inoculated with fungal suspension, amplified using ITS 1 and 2 primers and sequenced. Sequences were matched using BLAST search, with published fungal identities. Of 15 NSMs, one isolate from blood taken from a lymphoma patient was identified as Schizophylum commune, a Basidiomycete fungus which has been reported as a human pathogen causing systemic disease. Four isolates were identified as plant pathogens; Penicillium ochrochloron from bronchoalveolar fluid, Phlebia sp. from pleural fluid, Phoma multirostrata and Inonotus rickii from skin specimens. Two others were identified as saprophytes or endophytes; Xylaria feejeensis from nail and Fomitopsis cf. meliae from skin. All other isolates, including three from blood and two from peritoneal & endotracheal fluid could not be conclusively identified based on the ITS sequences alone. To determine the presence of possible virulence factors in NSMs, the Phlebia sp. isolate was evaluated for keratinase activity in vitro, on human nail and was shown to have high keratin degrading activity which indicates potential pathogenicity. In conclusion, this study has found that non-sporulating fungi may have a role in causing infections in humans; further studies are needed to establish their involvement. Introduction When non-sporulating molds (NSM) are isolated as pure cultures from patients, it raises a question on their ability to cause infection. Although often dismissed as insignificant, environmental organisms, DNA based identification has revealed potential pathogens among NSMs (Pounder et al. 2007). Molecular methods for fungal identification are well established, with the ribosomal DNA internal transcribed spacer (ITS) sequence utilised for species determination. Pathogenic fungi are known to produce proteolytic enzymes, such as keratinase in dermatophytes, which is important for fungal invasion of cutaneous sites (Viani et al. 2001). The keratinase activity level correlates with severity of infection caused. This study aimed to identify clinical NSM isolates and determine their pathogenic capability. Results Figure 1 : NSM isolates identified based on ITS sequences ReferenceS Pounder, J.I., Simmon, K.E., Barton, C.A., Hohmann, S.L., Brandt, M.E. & Petti, C.A. 2007. Discovering Potential Pathogens among Fungi Identified as Nonsporulating Molds. Journal of Clinical Microbiology 45(2): 568-571. Viani, F.C., Santos, J.I.D., Paula, C.R., Larson, C.E. & Gambale, W. 2001. Production of extracellular enzymes by Microsporum canis and their role in its virulence. Medical Mycology 39: 463-468. Dentinger, B.T.M., Margaritescu, S. & Moncalvo J.M. 2010. Rapid and reliable high-throughput methods of DNA extraction for use in barcoding and molecular systematics of mushrooms. Molecular Ecology Resources. 10: 628-633. METHODOLOGY UZ 177/10 Schizophylum commune UZ 517 Penicillium ochrochloron UZ 529/10 Phoma multirostrata UZ 586/10 Phlebia sp. UZ 661/10 Fomitopsis cf. meliae UZ 365/08 Inonotus rickii UZ 634 Xylaria feejeensis Fungal Molecular Identification (Dentinger et al. 2010) Fungal suspension prepared from PDA inoculated onto Whatman FTA® card (200 μL) DNA directly extracted from FTA card punches using Sigma Extract-N-AmpTM Plant PCR Kit Extracted DNA amplified using ITS 1 and ITS 2 primers PCR product purified and sequenced; sequences matched with published data Keratinase Activity Determination (Viani et al. 2001) JADUAL 1. Nilai purata dan sisihan piawai bagi kawalan negatif, kawalan positif dan fungus ujian Fungal isolate Keratinase Unit SD F. solani 5.340 2.097 T. mentagrophytes 4.000 1.572 Non-sporulating mold (Phlebia sp.) 3.060 1.501 F. oxysporum 2.273 1.258 Table 1: Keratinase activity of pathogenic fungi and NSM CONCLUSION Of fifteen NSMs, seven isolates were identified; one (S.commune) as a known pathogen. The Phlebia sp. produced keratinase enzyme at levels similar to dermatophyte fungi. Non-sporulationg molds have pathogenic potential and may be the cause of infections in humans. Fungal suspension incubated with human nail powder and keratin azure in a mineral medium for 21 days. Keratinase activity determined by quantitatively measuring dye release from keratin azure in mineral medium using a spectrophotometre. An increase of 0.01 in the absorbance measurement at 595 nm is equivalent to 1 Keratinase Unit (KU) of enzyme activity.