Keratinase is an enzyme produced by microbes that breaks down tough keratin found in feathers, hair, and similar materials. In the food industry, it is mainly used to enhance the nutritional value of animal feed by converting keratin-rich waste into easily digestible proteins. Among the 28 isolates, GHADAH-Y3 exhibited the highest bioconversion potential for keratinaceous waste as unexploited protein reserve into high-value products. It gave the highest keratinase activity (897.98 ± 9.34 unit per gram of dry solid; U/gds), feather degradation efficiency (89.35 ± 7.24%), IAA production (974.15 ± 15.63 µg/gds), ammonia release (159.23 ± 8.51 µg/gds), and phosphate solubilization (872.06 ± 19.37 µg/gds) under solid-state fermentation using a feather medium. Based on its phenotypic and genotypic characteristics, it was identified and designated as Bacillus sp. GHADAH-Y3. Notably, the fermented feather extract showed negligible cytotoxicity, with an IC50 value exceeding 200 and 125 μg/mL, against normal cells Vero and the hepatocellular carcinoma cells, respectively. The purification process resulted in a 20.37-fold purified enzyme with a specific activity of 54.79 U/mg protein and a recovery yield of 10.20%. The purified GHADAH-Y3 keratinase showed a single protein band with an approximate molecular mass of ∼28 kDa. The keratinase from Bacillus sp. GHADAH-Y3 exhibited optimal activity and stability at 50–60 °C and pH 8.0. Notably, it retained 68.31% of its original activity at 100 °C and 86.34% at pH 11.0, demonstrating its remarkable thermal and alkaline stability. However, serine-protease and metallo-protease inhibitors suppressed its activity, suggesting that it functions as a serine–metalloprotease. Furthermore, Na+, K+, Mg2+, Ca2+, Ba2+, Co2+, Mn2+, Cu2+, Zn2+, and Fe3+ activate the GHADAH-Y3 keratinase. The most sensitive keratin-containing raw materials for hydrolysis by the purified keratinase from Bacillus sp. GHADAH-Y3 was chicken feathers that was hydrolyzed by 84.00 ± 4.28. Scanning electron microscopy revealed extensive structural alterations, including disintegration, deterioration, severe damage, and significant lysis in feathers, horns, and hooves after treatment with the purified GHADAH-Y3 keratinase. These findings highlight the enzyme’s potent keratin-degrading ability, suggesting its potential applications in diverse industries such as bioenergy, pharmaceuticals, animal feed production, and fertilizer enhancement.

Production, Purification and Characterization of Alkaline Thermo-Tolerant Keratinase from Bacillus sp. GHADAH-Y3 Under Solid State Fermentation Using Keratinous Wastes

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发表日期：

2026年1月11日

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Ghadah Khaled Yousuf

摘要

Keratinase is an enzyme produced by microbes that breaks down tough keratin found in feathers, hair, and similar materials. In the food industry, it is mainly used to enhance the nutritional value of animal feed by converting keratin-rich waste into easily digestible proteins. Among the 28 isolates, GHADAH-Y3 exhibited the highest bioconversion potential for keratinaceous waste as unexploited protein reserve into high-value products. It gave the highest keratinase activity (897.98 ± 9.34 unit per gram of dry solid; U/gds), feather degradation efficiency (89.35 ± 7.24%), IAA production (974.15 ± 15.63 µg/gds), ammonia release (159.23 ± 8.51 µg/gds), and phosphate solubilization (872.06 ± 19.37 µg/gds) under solid-state fermentation using a feather medium. Based on its phenotypic and genotypic characteristics, it was identified and designated as Bacillus sp. GHADAH-Y3. Notably, the fermented feather extract showed negligible cytotoxicity, with an IC₅₀ value exceeding 200 and 125 μg/mL, against normal cells Vero and the hepatocellular carcinoma cells, respectively. The purification process resulted in a 20.37-fold purified enzyme with a specific activity of 54.79 U/mg protein and a recovery yield of 10.20%. The purified GHADAH-Y3 keratinase showed a single protein band with an approximate molecular mass of ∼28 kDa. The keratinase from Bacillus sp. GHADAH-Y3 exhibited optimal activity and stability at 50–60 °C and pH 8.0. Notably, it retained 68.31% of its original activity at 100 °C and 86.34% at pH 11.0, demonstrating its remarkable thermal and alkaline stability. However, serine-protease and metallo-protease inhibitors suppressed its activity, suggesting that it functions as a serine–metalloprotease. Furthermore, Na⁺, K⁺, Mg²⁺, Ca²⁺, Ba²⁺, Co²⁺, Mn²⁺, Cu²⁺, Zn²⁺, and Fe³⁺ activate the GHADAH-Y3 keratinase. The most sensitive keratin-containing raw materials for hydrolysis by the purified keratinase from Bacillus sp. GHADAH-Y3 was chicken feathers that was hydrolyzed by 84.00 ± 4.28. Scanning electron microscopy revealed extensive structural alterations, including disintegration, deterioration, severe damage, and significant lysis in feathers, horns, and hooves after treatment with the purified GHADAH-Y3 keratinase. These findings highlight the enzyme’s potent keratin-degrading ability, suggesting its potential applications in diverse industries such as bioenergy, pharmaceuticals, animal feed production, and fertilizer enhancement.