Keratinase Activity Research Articles

Biochemical Characterization and Application of Thermostable-Alkaline Keratinase From Bacillus halodurans SW-X to Valorize Chicken Feather Wastes

The production, purification, and characterization of thermostable-alkaline keratinase from Bacillus halodurans SW-X (keratinase_SW-X), and its applicability towards the production of white chicken feather hydrolysate and bioplastic starch/keratin production were investigated. A maximal keratinase activity of 16.05 ± 0.25 U/mL was achieved by cultivation of B. halodurans SW-X in 5 L stirred tank bioreactor at 47 °C, 200 rpm, and 2 vvm for 48 h. Keratinase_SW-X was purified as a monomeric protein with the estimated MW of 25 kDa, 14.4-fold, and 20% recovery yield. The protein was classified as thermostable-alkaline keratinase due to its optimal pH (pH 10) and temperature (70 °C), pH stability (pH 9.0–11), thermostability (> 3 h at 50–60 °C), and it showed the highest specificity towards chicken feather keratin with Km of 0.45 mg/mL, and Vmax of 3.51 μmol/min/mg. Crude keratinase_SW-X biodegraded white chicken feathers into two types of low (10–20 kDa) and high (≥ 30 kDa) chicken feather (keratin) hydrolysates, with total recovery yields of 30%. The bioplastic starch/keratin was synthesized using cassava starch (70%, w/w), glycerol (30%, w/w), and keratin hydrolysate (0–0.30%, w/w). The optimal concentration of keratin hydrolysate was 0.10% (w/w), resulting in improved strength and elongation at break, with a good appearance of the bioplastic starch/keratin. Our study suggests that B. halodurans SW-X is a potential strain for thermostable-alkaline keratinase production. Moreover, from environmental perspectives, keratinases_SW-X is a promising candidate enzyme that valorizes low cost chicken feather waste to a high value added product, indicating a novel approach for feather waste treatment and utilization.

Larvae of the Clothing Moth Tineola bisselliella Maintain Gut Bacteria that Secrete Enzyme Cocktails to Facilitate the Digestion of Keratin.

The evolutionary success of insects is promoted by their association with beneficial microbes that enable the utilization of unusual diets. The synanthropic clothing moth Tineola bisselliella provides an intriguing example of this phenomenon. The caterpillars of this species have adapted to feed on keratin-rich diets such as feathers and wool, which cannot be digested by most other animals and are resistant to common digestive enzymes. Inspired by the hypothesis that this ability may be conferred by symbiotic microbes, we utilized a simple assay to detect keratinase activity and a method to screen gut bacteria for candidate enzymes, which were isolated from feather-fed larvae. The isolation of DNA from keratin-degrading bacterial strains followed by de novo genome sequencing resulted in the identification of a novel bacterial strain related to Bacillus sp. FDAARGOS_235. Genome annotation identified 20 genes with keratinase domains. Proteomic analysis of the culture supernatant from this gut bacterium grown in non-nutrient buffer supplemented with feathers revealed several candidate enzymes potentially responsible for keratin degradation, including a thiol-disulfide oxidoreductase and multiple proteases. Our results suggest that the unusual diet of T. bisselliella larvae promotes their association with keratinolytic microorganisms and that the ability of larvae to feed on keratin can at least partially be attributed to bacteria that produce a cocktail of keratin-degrading enzymes.

Biochemical and Molecular Characterization of a Thermostable Alkaline Metallo-Keratinase from Bacillus sp. Nnolim-K1.

Keratinases are considerably gaining momentum in green technology because of their endowed robustness and multifaceted application potentials, such as keratinous agro-wastes valorization. Therefore, the production of novel keratinases from relatively nonpathogenic bacteria grown in agro-wastes formulated medium is cost-effective, and also imperative for the sustainability of thriving bioeconomy. In this study, we optimized keratinase production by Bacillus sp. Nnolim-K1 grown in chicken feather formulated medium. The produced keratinase (KerBNK1) was biochemically characterized and also, the keratinase-encoding gene (kerBNK1) was amplified and sequenced. The optimal physicochemical conditions for extracellular keratinase production determined were 0.8% (w/v) xylose, 1.0% (w/v) feather, and 3.0% (v/v) inoculum size, pH 5.0, temperature (25 °C) and agitation speed (150 rpm). The maximum keratinase activity of 1943.43 ± 0.0 U/mL was achieved after 120 h of fermentation. KerBNK1 was optimally active at pH and temperature of 8.0 and 60 °C, respectively; with remarkable pH and thermal stability. KerBNK1 activity was inhibited by ethylenediamine tetra-acetic acid and 1,10-phenanthroline, suggesting a metallo-keratinase. The amplified kerBNK1 showed a band size of 1104 bp and the nucleotide sequence was submitted to the GenBank with accession number MT268133. Bacillus sp. Nnolim-K1 and the keratinase displayed potentials that demand industrial and biotechnological exploitations.

Exoproduction and characterization of a detergent-stable alkaline keratinase from Arthrobacter sp. KFS-1

Arthrobacter sp. KFS-1 previously isolated from a dump site was used to produce keratinase in basal medium. The physico-chemical conditions were optimized to enhance the keratinase production, and biochemical properties of the enzyme were also evaluated. Arthrobacter sp. KFS-1 optimally produced keratinase in a basal medium that contained 1.0 g/L xylose, 2.5–5.0 g/L chicken feather; with initial pH, incubation temperature and agitation speed of 6.0, 30 °C and 200 rpm, respectively. Maximum keratinase activity of 1559.09 ± 29.57 U/mL was achieved at 96 h of fermentation; while optimal thiol concentration of 665.13 ± 38.73 μM was obtained at 144 h. Furthermore, the enzyme was optimally active at pH 8.0 and 60 °C. The enzyme activity was inhibited by ethylene diamine tetraacetic acid and 1,10-phenanthroline, but not affected by phenylmethylsulfonyl floride. In addition, the crude enzyme retained 55%, 63%, 80%, 81% and 90% of the original activity after respective pretreatment with some commercial detergents (Maq, Omo, Surf, Sunlight and Ariel). Moreso, the enzyme showed remarkable stability in the presence of reducing agents, surfactants, and organic solvents. Arthrobacter sp. KFS-1 significantly produced keratinase which exhibited excellent stability in presence of chemical agents and commercial laundry detergents; hence, suggesting its industrial application potentials especially in detergent formulation.

Response Surface Methodology for the Optimization of Keratinase Production in Culture Medium Containing Feathers by Bacillus sp. UPM-AAG1

Keratinase is a type of proteolytic enzyme with broad application in industry. The main objective of this work is the optimization of keratinase production from Bacillus sp. strain UPM-AAG1 using Plackett-Burman (PB) and central composite design (CCD) for parameters, such as pH, temperature, feather concentration, and inoculum size. The optimum points for temperature, pH, and inoculum and feather concentrations were 31.66 °C, 6.87, 5.01 (w/v), and 4.53 (w/v), respectively, with an optimum keratinase activity of 60.55 U/mL. The keratinase activity was further numerically optimized for commercial application. The best numerical solution recommended a pH of 5.84, temperature of 25 °C, inoculums’ size of 5.0 (v/v), feather concentration of 4.97 (w/v). Optimization resulted an activity of 56.218 U/mL with the desirability value of 0.968. Amino acid analysis profile revealed the presence of essential and non-essential amino acids. These properties make Bacillus sp. UPM-AAG1 a potential bacterium to be used locally for the production of keratinase from feather waste.

Comparative Analysis of Putative Virulence-Associated Factors of Microsporum canis Isolates from Human and Animal Patients.

Microsporum canis is a zoophilic dermatophyte and the most common fungus isolated from dogs and cats worldwide. To invade skin, this pathogen uses different enzymes, which may be associated with virulence, that contribute to the fungal pathogenicity. The aim of this study is to compare the expression of enzymes that may be associated with virulence, and thermotolerance of M. canis strains isolated from dogs, cats, and humans. The in vitro expression of the enzymes keratinase, catalase, urease, hemolysin, and aspartic protease was evaluated in 52M. canis strains recently isolated from 14 human patients, 12 dogs, 15 symptomatic, and 11 asymptomatic cats. In addition, thermotolerance was assessed by comparative analysis of fungal growth at 25°C and 35°C. Keratinase activity was low in 34 and moderate in 18 strains. Aspartic-protease activity was low in 7, moderate in 33, and high in 12 strains. Hemolysin activity was low in 44 and moderate in 8 strains. All strains were classified as low producers of catalase. All but three strains produced urease in vitro, with a broad range of activity. The strains presented in vitro growth at the two studied temperatures were classified as presenting low (36.5%), medium (44.3%), or high (19.2%) thermotolerance. There was no statistically significant difference in the new putative virulence-associated factors studied among the different hosts, which suggests that they may have a similar role on human, cat, and dog infection. Also, no difference was observed between strains isolated from symptomatic and asymptomatic cats. This suggests that these factors have a limited impact on the fate of feline dermatophytosis caused by M. canis.

Optimization for Production of a Plant Growth Promoting Agent from the Degradation of Chicken Feather Using Keratinase Producing Novel Isolate Bacillus pumilus JYL

Feather is a good source of nitrogen but the highly rigid structure limits its utilization and leads to environmental pollution and resource wastage. Traditional feather degradation processes such as alkali hydrolysis and steam pressure cooking not only destroy the amino acids but also consume large amounts of energy. Therefore, searching for feather degrading bacteria to transform feather waste to nitrogen fertilizer to improve soil and plant growth is needed. And this also will solve the soil nitrogen deficiency problem in China. In this study, a bacteria that could degrade feather and promote plant growth was isolated from soil sample using dilution plate method. And the isolated bacteria was identified as Bacillus pumilus JYL according to its morphology, biochemical characteristics and 16S rRNA sequence. And the maximum keratinase activity (57.14 U/mL) was obtained under optimal conditions of 0.036% FeSO4, 0.025% MgSO4, 0.073% K2HPO4, 0.5% feather with 2% inoculant at pH 6.5 fermented for 36 h at 35 ℃ after optimization using (response surface methodology) RSM. The effect of hydrolyzate on wheat seed germination and growth was studied. The results showed that germination engergy and germination index with 3% feather hydrolyzate addition group was 94.22% and 87.52%, significantly higher than that of the control group (p < 0.05), respectively. The plant height, root length, fresh weight and dry weight in the T2 treatment (added 5 mL feather hydrolysate in 100 g soil) at 60th day were 1.10, 1.16, 1.77 and 1.73 times than that in the CK treatment (without addition of hydrolysate), respectively. And the soil enzyme activities in all treatment groups (T1, T2, T3) significantly increased compared with control group (CK) (p < 0.05). This result will provide a potential utilization method for feather valoration.

Keratinase Activity of A Newly Keratinolytic Bacteria, Azotobacter chroococcum B4

Keratinase Activity of A Newly Keratinolytic Bacteria, Azotobacter chroococcum B4

Isolation of Bacillus Strains with Keratinolitic Activity

Environmental safety and economic feasibility determine the search for new ways of processing waste in poultry farms. Most of this waste is down and feathers, which are 90% β-keratin. Feathers can be a valuable source of amino acids and peptones when properly processed. The most effective is enzymatic treatment of feather keratin. The search for new strains producing keratinolytic enzymes seems to be a promising direction. On the territory of the poultry farm, 4 strains were isolated from the places of accumulation of feathers. They are able to use chicken feathers as their sole source of organic matter. Based on morphological, genomic, and proteomic analyzes, the isolated strains were identified as Bacillus sp. It was found that the strains secrete proteolytic enzymes that hydrolyze collagen, casein, β-keratin and do not hydrolyze bovine serum albumin. Feather hydrolysis experiments showed that the Bacillus sp. A5.3 possesses maximum keratinolytic activity, and on the second day, the destruction of the second order barbs is observed. The keratinase activity of the strain on azokeratin after an hour of incubation on feather medium was 27.4 U/ml. The optimal conditions for the complex of secreted proteolytic enzymes are pH 7.0-8.0 and temperature 35-40 °C. The isolated Bacillus sp. A5.3 strain is a promising source of proteases and keratinases.

Proteolytic bacteria isolated from agro-waste dumpsites produced keratinolytic enzymes

Microbial bioconversion of carbonoclastic materials is an efficient tool for the exploitation and valorization of underutilized agro-industrial wastes. The agro-industrial sector accumulates tones of keratinous wastes biomass which may be valorized into high value products. Consequently, the keratinolytic potentials of some bacteria isolated from terrestrial milieu was evaluated. Soil samples were collected from dumpsites, keratinase producing bacteria were isolated. Bacterial species were identified through 16S rRNA gene sequences. The keratinase activity was assessed in relation to thiol formation, percentage feather degradation and quantitation of keratinase produced. Keratinolytic bacteria were identified as Bacillus spp. (accession numbers: MG214989 – MG214992, MG214997, MG214998, MG215000, MG215002–MG215005) and Arthrobacter sp. (accession numbers; MG215001). The degree of chicken feather degradation ranged from 61.5 ± 0.71 % to 85.0 ± 1.41 %. Similarly, the activity of keratinase, total protein and thiol group ranged from 198.18 ± 15.43–731.83 ± 14.14 U/mL; 0.09 ± 0.01–0.87 ± 0.05 mg/mL; and 0.69 ± 0.12–2.89 ± 0.11 mM respectively. Notably, Bacillus sp. Nnolim-K1 displayed the best keratinolytic potential with extracellular keratinase activity and feather degradation of 731.83 ± 14.14 U/mL and 85.0 ± 1.41 % respectively, and that is an indication of a potential relevance biotechnologically.

Purification of Keratinase from Bacillus sp. MD24 using Ammonium Sulfate Fractionation

A keratin degrading bacterium, Bacillus sp MD24, was isolated from soil. The crude keratinase produced by the bacterium has been reported to dehair goat skin. However, the dehairing process took 72 hours. In order to shorten the dehairing time it is necessary to increase the keratinase concentration. This could be done by optimizing keratinase production either finding the best fermentation media or optimizing fermentation condition. Another way to increase the concentration could be done by partially purifying the enzyme. Keratinase from Bacillus sp MD24 had been produced under submerged fermentation, however, it produced a relatively low amount of enzyme. Although an effort to increase enzyme production had been reported by solid state fermentation, the enzyme concentration was not enough for industrial purposes. This work aimed to increase enzyme concentration by partial purification through enzyme precipitation using ammonium sulphate. The research was conducted in three stages: (1) regeneration the bacterium, (2) production of keratinase, and (3) purification of keratinase with ammonium sulfate fractionation. Keratinase activity was measured by Anson method and protein concentration was measured by Lowry method. Enzyme purity was clarified using a combination of specific activity, purity level, and SDS-PAGE analysis. Based on the analysis result, ammonium sulphate did not act as a good precipitation agent for the keratinase. Two major bands were suggested as keratinase with an estimated molecular weight of 25 and 66 kDa as monomer and dimer form, respectively.

Biotransformation of keratin waste to amino acids and active peptides based on cell-free catalysis

BackgroundKeratin is the primary constituent of the vertebrate epidermis and epidermal appendages, as well as the main waste product generated during poultry processing from feathers, hair, scales, nails, etc. Keratin is generally hard, stubborn and difficult to hydrolyze; however, it is also inexpensive and contains more than 85% protein. Currently, tens of millions of tons of keratin waste are produced each year worldwide; however, no effective methods for the recovery of keratin waste have been reported thus far, making such research urgent. Keratinase has been reported to be useful for keratin waste recovery; however, nearly all keratinases are unable to hydrolyze keratin after they are detached from living cell systems. This may be due to low keratinase activity and lack of synergistic factors.ResultsHerein, the keratinase gene from Bacillus licheniformis BBE11-1 was successfully expressed in Bacillus subtilis WB600, allowing for improved activity of the recombinant keratinase KerZ1 to 45.14 KU/mL via promoter substitution and screening of the ribosome-binding sites. Further, real-time control of temperature, pH, dissolved oxygen, and feed strategy allowed the activity of KerZ1 to reach 426.60 KU/mL in a 15-L fermenter, accounting for a 3552-fold increase compared to the wild-type keratinase (120.1 U/mL). Most importantly, we proposed a method based on the synergistic action of keratinase KerZ1 and sodium sulfite, to hydrolyze feathers into amino acids. In specific, 100 g/L of feather waste can be successfully converted into 56.6% amino acids within 12 h, while supporting the production of dozens of bioactive peptides.ConclusionsThe activity of recombinant keratinase can be greatly enhanced via transcription and translational regulation in Bacillus subtilis. The synergistic action of keratinase and sulfite can rapidly degrade feather waste and produce amino acids and polypeptides.

Bacillus sp. FPF-1 Produced Keratinase with High Potential for Chicken Feather Degradation.

Chicken feathers are predominantly composed of keratin; hence, valorizing the wastes becomes an imperative. In view of this, we isolated keratinase-producing bacteria and identified them through the 16S rDNA sequence. The process condition for keratinase activity was optimized, and electron micrography of the degradation timelines was determined. Keratinolytic bacteria were isolated and identified as Bacillus sp. FPF-1, Chryseobacterium sp. FPF-8, Brevibacillus sp. Nnolim-K2, Brevibacillus sp. FPF-12 and Brevibacillus sp. FSS-1; and their respective nucleotide sequences were deposited in GenBank, with the accession numbers MG214993, MG214994, MG214995, MG214996 and MG214999. The degree of feather degradation and keratinase concentration among the isolates ranged from 62.5 ± 2.12 to 86.0 ± 1.41(%) and 214.55 ± 5.14 to 440.01 ± 20.57 (U/mL), respectively. In the same vein, 0.1% (w/v) xylose, 0.5% (w/v) chicken feather, an initial fermentation pH of 5.0, fermentation temperature of 25 °C and an agitation speed of 150 rpm, respectively, served as the optimal physicochemical conditions for keratinase activity by Bacillus sp. FPF-1. The time course showed that Bacillus sp. FPF-1 yielded a keratinase concentration of 1698.18 ± 53.99(U/mL) at 120 h. The electron microscopic imaging showed completely structural dismemberment of intact chicken feather. Bacillus sp. FPF-1 holds great potential in the valorization of recalcitrant keratinous biomass from the agro sector into useful products.

Efficient keratinase expression via promoter engineering strategies for degradation of feather wastes

Keratinases are promising alternatives over ordinary proteases in several industrial applications due to their unique properties compared with their counterparts in the protease categories. However, their large-scale industrial application is limited by the low expression and poor fermentation efficiency of keratinase. Here, we demonstrate that the expression level of keratinase can be improved by constructing a more efficient enzyme expression system hereby enables the highest production titer as regarding recombinant keratinase production to date. Specially, ten promoters were evaluated and the aprE promoter exhibits a significant promotion of keratinase (kerBv) titer from 165 U/mL to 2605 U/mL in Bacillus subtilis. The batch fermentation mode resulted in a maximum keratinase activity of 7176 U/mL at 36 h in a 5-L fermenter. Furthermore, the extracellular keratinase activity attained up to 16,860 U/mL via fed-batch fermentation within 30 h. The combination of keratinase with l-cysteine brings about 66.4 % degree of degradation of feather. Our work provides a new insight into the development of efficient keratinase fermentation processes with B. subtilis cell factory.

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.

Microbial Bioremediation of Feather Waste for Keratinase Production: An Outstanding Solution for Leather Dehairing in Tanneries.

In leather industries and tanneries, large amount of wastes has been disposed; which polluting water, soil, and atmosphere and causing serious human health problems. In particular, chemical dehairing process of leather industries produces fair amount of toxic wastes. It is, thus, urgently needed to use alternative processes free from pollution. As more than 90% of keratin is contained in feather, it is desirable to develop bioremediation process using keratinolytic microorganisms. In the present investigation, therefore, we first identified Bacillus cereus and Pseudomonas sp. to be able to produce keratinase. Then, the optimization was performed to maximize the keratinase activity with respect to cultivation temperature, pH, and incubation time. Moreover, the effects of metal ions and various substrates on keratinase activity were also investigated. The result indicates that keratinase activity became maximum at 50°C for both strains, whereas the optimal pH was 10.0 for B. cereus and 7.0 for Pseudomonas sp. The highest keratinase activity of 74.66 ± 1.52 U/mL was attained by B. cereus, whereas 57.66 ± 2.52 U/mL was attained by Pseudomonas sp. Enzymatic dehairing efficiency of leathers was also compared with chemical dehairing (Na2S and CaO), where complete dehairing was achieved by treating them with crude keratinase. Partial enzyme purification was performed by acetone precipitation. Batch cultivation of B. cereus using 1 L fermentor indicates a potential candidate for large-scale keratinase production. Thus, keratinase enzyme by degrading poultry wastes (feather) can be an alternative approach to chemical dehairing in leather industries, thus preventing environmental pollution through bioremediation.

Fermented chicken feathers using Bacillus subtilis to improve the quality of nutrition as a fish feed material

Chicken feather meals constitutes 80-85% protein, the main component being betakeratin, a fibrous and insoluble structural protein extensively cross linked by disulfide bonds. The keratins can not be absorbed directly in the digestive system, therefore a processing technique is required to make it more absorbable. Processing technique can be fermentation by microorganism Bacillus subtilis to degrade keratin by secretion of keratinase. The aim of this study was to examine fermentation of chicken feather meal with B. subtilis to improve the quality of fish feed ingredients. The treatments were: no-fermented; fermentation of chicken feather meals: 6, 8, 10, 12 and 14 ml inoculum fermentation using B. subtilis, respectively for chicken feather meal as much as 2 g. The results showed that the processing fermentation with 10 ml inoculum B. subtilis gives the best results in the highest keratinase activity (273.33 U/ml), increased the protein content of chicken feather meals (74.16 to 85.20%), but decreated of lipid content (2.44 to 1.42%) and carbohydrate content (7.86 to 2.05%) with a change in the physical properties of white -yellow (color), soft (texture), and less typical sting (smell).

Inhibition of Trichophyton rubrum by 420-nm Intense Pulsed Light: In Vitro Activity and the Role of Nitric Oxide in Fungal Death.

Trichophyton rubrum is a common dermatophyte of the skin. The aim of this experiment was to explore the role of nitric oxide (NO) in the inhibition of T. rubrum growth induced by 420-nm intense pulsed light (IPL). This study found that nitric oxide synthase (NOS) and NO levels were increased, whereas asymmetric dimethylarginine (ADMA) level, keratinase activity, and fungal viability were decreased after IPL treatment compared with the control condition in vitro. Moreover, micromorphology was damaged by IPL treatment. Fungal viability was increased, and the damage to the fungal structure was reduced after pretreatment with an NOS inhibitor (L-NMMA) compared with IPL treatment alone. Compared with IPL alone, pretreatment with L-NMMA decreased NOS expression and NO level and increased keratinase activity. We found that 420-nm IPL treatment can inhibit the growth of T. rubrum by regulating NO in vitro.

ANTIFUNGAL EFFECT OF GOLD NANOPARTICLES ON FUNGI ISOLATED FROM ONYCHOMYCOSIS PATIENTS

Onychomycosis is a widely distributed fungal nail infection which can be caused by dermatophytes, yeasts or non-dermatophytic fungi. In our study a total of 7 species related to 4 genera were isolated from 50 patients suspected with the fungal nail infection (onychomycosis) these namely Aspergillus flavus (8 isolates), Aspergillus niger (13 isolates), Candida albicans (8 isolates), C. tropicalis (5 isolates), Epidermophyton floccosum (1 isolate), Trichophyton mentagrophytes (2 isolates) and Trichophyton rubrum (1 isolate). Nowadays the field of nanotechnology becomes one of the most topics of interest. The impact of gold nanoparticles on the isolated fungal species was evaluated by agar well diffusion method and micro-dilution method. Best antifungal activity of gold nanoparticles was observed by using 100 µl of AuNps containing 20 µg of gold nanoparticles with the greatest zone of inhibition (19 mm) against C. albicans. AuNps showed variable MIC (minimum inhibitory concentration). MIC50 and MIC90 values of AuNPs ranged from 3.125 to 25.0 μg/ml and from 12.5 to100 μg/ml respectively. All isolated fungi could grow on keratin agar medium but with variable degrees indicating their ability to hydrolyze keratin. Keratinase activity in presence and absence of AuNPs was determined for dermatophytes and Aspergillus species. AuNPs had an inhibitory effect causing reduction in keratinase enzyme activity reaching 52.17 %, 40 % and 37.5 % was attained in case of E. floccosum, A. flavus and A. niger (the most susceptible isolates) respectively, by application of 20 µg/ml of AuNPs.