Investigating the Effects of Aqueous and Alcoholic Extracts of Allium Hirtifolium and Allium Jesdianum on Keratinase Activity of Trichophyton Mentagrophytes

Background: Trichophyton spp., as pathogenic species to humans and animals, cause different forms of dermatophytosis through the production of particular enzymes, playing an essential role in tissue invasion. Among these, herein, keratinase was investigated, for the specific case of Trichophyton mentagrophytes, as a target of the effects of Allium hirtifolium and Allium jesdianum extracts, thus pharmacological potential of these plants was studied against keratinase activity. Methodology: Sampling was carried out on 20 bald patients from medical diagnostic laboratories and mycology centers, with suspected dermatophytosis of scalp. For confirming the presence of Trichophyton mentagrophytes in the specimens, different laboratory procedures were applied. Trichophyton mentagrophytes isolates were cultured on a screening medium containing keratin to verify production of the keratinase enzyme. The best enzyme-producing isolate was selected by measuring diameter of transparent halo around colony to be used in subsequent stages. Afterwards, the optimized conditions maximizing enzyme production and activity were determined. Finally, the inhibitory effect of different dilutions of aqueous and alcoholic extracts of Allium jesdianum and Allium hirtifolium on extracellular keratinase activity was studied. Results : Sixteen out of 20 fungal isolates were identified as the Trichophyton mentagrophytes. The most desirable reduction on keratinase activity was reported for dilution values of 50 and 100 mg/ml of both aqueous and ethanolic extracts of A.jesdianum, though much more significant decrease belonged to the latter, and for dilution values of 25 and 100 mg/ ml of both aqueous and ethanolic extracts of A.hirtifolium. Conclusion : Concerning our results, it is suggested that paying special attention to these natural compounds for the treatment of dermatophytosis could be remarkably effective, considering significant production of keratinase observed in T. mentagrophytes, and they are beneficial, as they have no side effects and offer an alternative to currently available medications, which are under the restriction of drug resistance.

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.

Feather Biodegradation For Feather Protein Lysate/ Feather Meal Mutual Formations: Optimization Using Crude YNDH Protease/Keratinase; Enzyme Partial Purification and Characterization

Incubation parameters used for the production of a protein lysate from enzymatically degraded waste feather using crude keratinase produced by laceyella sacchari YNDH was optimized using RSM, amino acids quantification was estimated as well. The optimization resulted in total soluble protein 2089.5µg/ml. The optimal conditions were, time 20.2h, feather concentration 3g% and keratinase activity 24.5U/100ml at pH, 10; and cultivation temperature 50oC. The FPL was found enriched with essential and rare amino acids.In parallel, this YNDH enzyme group had been partially purified and some characteristics of it were studied. Crude enzymes were first concentrated with Amicon Ultra 10k centrifugal filter, and then concentrated proteins were applied to "QFF" strong anion column chromatography. The partially purified enzyme has an estimated molecular masses ranged 6 to 10 kDa. The maximum enzyme activity was observed at 70°C and pH 10.4 when measured by both casein and keratin azure as substrates. Interestingly, keratinolytic activity of this group was not affect by EDTA, PMSF and DTT. Generally the overall characters of this group protease/keratinase nearly the same when its activity was measured with both substrates suggesting that all these 3 protein bands working together as a group of keratinases.

A study on optimization of biomass of Bacillus pumilus for feather degradation

Soil samples were collected from the feather dumped area, and they were screened for the presence of keratinolytic bacteria Bacillus pumilus. Based on its growth on Bacillus isolation agar, Skim milk agar, and Starch agar, it was conformed as Bacillus pumilus. The growth of bacteria was estimated by biomass estimation. In the optimization study, the optimum incubation period observed for feather degradation was 48hrs, pH 7, and temperature 40°C. Purified Keratinase enzyme was used for the feather degradation study. The maximum degradation observed was 29% at the temperature of 400C. The size of kerinase produced was estimated as 52KDa.

Optimization of Keratinase Production Using Pseudomonas aeruginosa SU-1 Having Feather as Substrate

In this optimization study, Pseudomonas aeruginosa SU-1 was producing keratinase at optimal condition of 4 days, pH – 7 and temperature 37 0C, where it was producing 23.7 U/mL After the one factor at a time, RSM was performed to understand the combination of the physical parameter that ends up for the maximum production of keratinase enzyme and the degradation percentage. The study involved in three variables (pH(A), temperature(B) and Incubation Design (C)) in three ranges (-1,0,+1) using Box-Behnken Design (BBD). The results of the analysis of variance and regression analysis of the second order model showed that the factorial effect if the degradation. The optima of the variables pH - 7, temperature - 30 and incubation time – 4 days. The isolated Pseudomonas species was subjected to feather degradation for 4 days and it was degrading 55.26 %. Keratinase was to be size of 56KDa.

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.