الفهرس 
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Abstract
The goal of this work was to generate microbial keratinase enzymes and use them as a potent biodegradation agent for wastes containing keratin. Thirteen indicated microbial strains (five bacteria —six fungi and two actinomycetes) were evaluated for keratinolytic activity. The NRC’s Culture Collection Center in Egypt was the source of all the screened microorganisms, whereas Pseudomonas aeruginosa C11, Fervidobacterium pennavorans, Trichoderma polysporum HZ-31, and Penicillium sp. were obtained from the National Center for Agricultural Utilization Research located in Peoria, Illinois 61604, USA. Aspergillus niger and Trichoderma atroviride F6 were procured from the Microbiology Department of the Faculty of Science at Ain Shams University located in Egypt.
The present study could be summarized as follows:
1. Thirteen microbial strains were screened to see if they could produce keratinase enzymes. Out of the thirteen microorganisms that were tested, Trichoderma polysporum HZ-31 produced the highest amount of keratinase enzymes (56.8 U/mL) after five days of shaking incubation. This organism was selected as the most potent one, generating extremely active keratinases, and it was used in all succeeding studies.
2. The basal production medium was formulated as follows (%,w/v) whole chicken feather, 0.5; glucose, 0.2; peptone, 0.5; yeast extract, 0.5; K2HPO4, 0.1; KH2PO4, 0.3; CaCl2, 0.1; MgSO4, 0.1 and pH was adjusted to 7.0.
3. The production medium’s initial pH of 9.0 resulted in the highest keratinase productivity (63.12 UmL-1); in the presence of feathers, beef extract was the appropriate organic and inorganic nitrogen source for the highest keratinase productivity, followed by sodium nitrate; in the absence of feathers, sodium nitrate was the most suitable and led to the highest keratinase production (52.52 U/mL), followed by amm.molybdate (52.43 U/mL). Using sucrose in the production medium as a carbon source allowed Trichoderma polysporum HZ-31 to produce keratinolytic enzymes with the highest productivity (64.59 U/mL) of any carbon source examined. The greatest keratinase productivity (65.23 U/mL) was obtained with an inoculum size of 15% (w/v), additionally, the maximum keratinase output by a 5-day-old Trichoderma polysporum HZ-31 shaken culture was achieved at an inoculum age of 48 hours For Trichoderma polysporum HZ-31 to produce the maximum keratinase production, the ideal agitation speed and incubation temperature were 180 rpm and 28°C, respectively.
4. White Duck feathers were the most utilizable by Trichoderma polysporum HZ-31 and led to the highest keratinolytic activity (66.45 U/mL), followed by white chicken feathers, which afforded 66.01 U/mL keratinase activity and those represent the abundant keratinaceous wastes applicable for the production of keratinase enzymes by the Trichoderma polysporum HZ-31.
5. Certain additives increased the activity of the fungal keratinolytic enzyme. CuSO4 produced keratinase with the highest productivity (67.37 U/mL), followed by CaCl2 (66.50 U/mL).
6. A fractional precipitation of the crude enzyme using various precipitating agents, such as ammonium sulphate, ethanol or acetone was required for the partial purification of the crude Trichoderma polysporum HZ-31 enzyme preparation. The three precipitating agents produced seventeen fractions, two of which precipitated at acetone concentrations of 80 and 90% showed a high degree of similarity in terms of protein recovery and total activity. As a result, the two fractions were well combined and utilized as partially purified keratinase enzyme (80-90% acetone).
7. At an enzyme protein concentration of 1200 μg/mL, substrate concentration of 1.4% (w/v), pH 9.2, and reaction temperature of 50ºC, the crude Trichoderma polysporum HZ-31 keratinase preparation attained its maximal activity, while At protein concentration of 1400 μg/mL, 1.4% (w/v) substrate concentration, pH 9.2, and 50°C, the partially purified Trichoderma polysporum HZ-31 keratinase enzyme preparation demonstrated its maximal activity.
8. After just 30 minutes of heating at 40ºC and pH 10.7, the crude enzyme form showed the greatest stability and retained 99.12% of its activity, while the PPE form was more stable and retained 98.91, 98.81, 98.80% after 30, 45 and 60 min, respectively at 40ºC and pH 9.2. PPE exhibited high stability after 30 min incubation alone at 45 ºC and retained 99.90%.
9. Applying the recommended Woolf plot, each of Michaelis constant (Km) and maximum velocity constant (Vmax) were 2 mg/reaction and 57.1Umg-1 protein, respectively.
10. Both the crude and PPE keratinase forms reached their maximum activity after 120 min reaction time.
11. Among all the substances tested in different concentrations, K+1, Mg+2 & Mn+2 at 100 mM concentration were enzyme activators and led to considerable activating effects on the partially purified enzyme form afforded relative activity amounted to 107.33, 105.82& 104.93%, respectively. Also, 1 mM Cu+2 led to relative activity amounted to 102.2%. On the other hand, 1 and 10 mM Ca+2 little activated the enzyme to 101.89 & 102.52%, respectively and above this concentration 100 mM afforded 102.15%. Furthermore, higher concentrations (100 mM) of EDTA or heavy metals (Zn & Hg) inhibited the PPE activity to 28.94, 23.72 and 2.93%, respectively.
12. The partially purified keratinase preparation was successfully applied in callus treatment, blood stains removal and cow leather unhairing under specified conditions.