RESUMEN
The high demand for keratinolytic enzymes and the modest presentation of fungal keratinase diversity studies in scientific sources cause a significant interest in identifying new fungal strains of keratinase producers, isolating new enzymes and studying their properties. Four out of the 32 cultures showed a promising target activity on protein-containing agar plates-Aspergillus amstelodami A6, A. clavatus VKPM F-1593, A. ochraceus 247, and Cladosporium sphaerospermum 1779. The highest values of keratinolytic activity were demonstrated by extracellular proteins synthesized by Aspergillus clavatus VKPM F-1593 cultivated under submerged conditions on a medium containing milled chicken feathers. The enzyme complex preparation was obtained by protein precipitation from the culture liquid with ammonium sulfate, subsequent dialysis, and lyophilization. The fraction of a pure enzyme with keratinolytic activity (pI 9.3) was isolated by separating the extracellular proteins of A. clavatus VKPM F-1593 via isoelectric focusing. The studied keratinase was an alkaline subtilisin-like non-glycosylated protease active over a wide pH range with optimum keratinolysis at pH 8 and 50 °C.
Asunto(s)
Plumas , Queratinas , Animales , Queratinas/metabolismo , Biodegradación Ambiental , Hongos/metabolismo , Concentración de Iones de Hidrógeno , TemperaturaRESUMEN
Microbial keratinases exhibit a momentous role in converting keratin biowastes into exceedingly valuable protein supplements. This study reports a novel, highly stable keratinase from Bacillus pacificus RSA27 for the production of pure peptides rich in essential amino acids from chicken feathers. Purified keratinase showed a specific activity of 38.73 U/mg, 2.58-fold purification, and molecular weight of 36 kDa. Kinetic studies using a chicken feather as substrate report K m and V max values of 5.69 mg/ml and 142.40 µg/ml/min, respectively, suggesting significant enzyme-substrate affinity/biocatalysis. Identification and in silico structural-functional analysis of keratinase discovered the presence of distinct amino acid residues and their positions. Besides, keratinase possesses a high-affinity calcium-binding site (Asp128, Leu162, Asn164, Ile166, and Val168) and a catalytic triad of Asp119, His151, and Ser308, known attributes of serine protease (subtilisin family). Furthermore, a scale-up to 5 L fermenter revealed complete feather hydrolysis (94.5%) within 24 h with high activity (789 U/ml) and total amino acid of 153.97 µmol/ml. Finally, cytotoxicity evaluation of protein hydrolysate resulted in negligible cytotoxic effects (1.02%) on the mammalian hepatoblastoma cell line, signifying its potential biotechnological applications.
RESUMEN
Currently, the growth of the global population leads to an increase in demand for agricultural products. Expanding the obtaining and consumption of food products results in a scale up in the amount of by-products formed, the development of processing methods for which is becoming an urgent task of modern science. Collagen and keratin make up a significant part of the animal origin protein waste, and the potential for their biotechnological application is almost inexhaustible. The specific fibrillar structure allows collagen and keratin to be in demand in bioengineering in various forms and formats, as a basis for obtaining hydrogels, nanoparticles and scaffolds for regenerative medicine and targeted drug delivery, films for the development of biodegradable packaging materials, etc. This review describes the variety of sustainable sources of collagen and keratin and the beneficial application multiformity of these proteins.