RESUMEN
Products produced from waste are a relatively recent innovation. Feather substrates are abundant in keratin content and improper disposal can cause ecosystem contamination. However, these pollutants can be transformed into value-added products for industrial application. Physical, chemical and cutting-edge microbiological methods were utilized for decomposing keratin and aid in the identification and estimation of amino acids from poultry feather wastes. These beneficial approaches are receiving more attention due to their retrieval of harmless and value added byproducts. These keratin-based compounds are used widely in pharmaceutical, livestock feed, fertilizer, and a variety of other industrial sectors. Since keratin is primarily consisting of amino acids, it can be utilized to affirm and estimate the amino acids in these feather substrates. This study primarily highlights the various methodologies employed for the qualitative estimation of amino acids in feather waste samples and the inhibitory activity of keratinase enzyme by EDTA and pepstatin in order to accumulate amino acids for drug delivery purpose and their importance in various pharmaceutical industries. In addition to that, molecular docking studies of cysteine with many standard pharmaceutical drugs like acetaminophen, pethidine, methylphenidate, carbamazepine, cillin and amlodipine were performed using autodock to demonstrate how cysteine greatly reduces conventional drug toxicity and its side effects.
RESUMEN
Keratinase has shown great significance and application potentials in the biodegradation and recycle of keratin waste due to its unique and efficient hydrolysis ability. However, the inherent instability of the enzyme limits its practical utilization. Herein, we obtained a thermostability-enhanced keratinase based on a combination of bioinformatics analysis and rational design strategies for the efficient biodegradation of feathers. A systematical in silico analysis combined with filtering of virtual libraries derived a smart library for experimental validation. Synergistic mutations around the highly flexible loop, the calcium binding site and the non-consensus amino acids generated a dominant mutant which increased the optimal temperature of keratinase from 40 °C to 60 °C, and the half-life at 60 °C was increased from 17.3 min to 66.1 min. The mutant could achieve more than 66% biodegradation of 50 g/L feathers to high-valued keratin product with a major molecular weight of 36 kDa. Collectively, this work provided a promising keratinase variant with enhanced thermostability for efficient conversion of keratin wastes to valuable products. It also generated a general strategy to facilitate enzyme thermostability design which is more targeted and predictable.
Asunto(s)
Biología Computacional , Plumas , Animales , Plumas/química , Queratinas/química , Péptido Hidrolasas/química , Péptido Hidrolasas/genética , Péptido Hidrolasas/metabolismo , TemperaturaRESUMEN
Keratinases can specifically degrade keratins, which widely exist in hair, horns, claws and human skin. There is a great interest in developing keratinase to manage keratin waste generated by the poultry industry and reusing keratin products in agriculture, medical treatment and feed industries. Degradation of keratin waste by keratinase is more environmentally friendly and more sustainable compared with chemical and physical methods. However, the wild-type keratinase-producing strains usually cannot meet the requirements of industrial production, and some are pathogenic, limiting their development and utilization. The main purpose of this study is to improve the catalytic performance of keratinase via directed evolution technology for the degradation of feathers. We first constructed a mutant library through error-prone PCR and screened variants with enhanced enzyme activity. The keratinase activity was further improved through fermentation conditions optimization and fed-batch strategies in a 7-L bioreactor. As a result, nine mutants with enhanced activity were identified and the highest enzyme activity was improved from 1150 to 8448 U/mL finally. The mutant achieved efficient biodegradation of feathers, increasing the degradation rate from 49 to 88%. Moreover, a large number of amino acids and soluble peptides were obtained as degradation products, which were excellent protein resources to feed. Therefore, the study provided a keratinase mutant with application potential in the management of feather waste and preparation of protein feed additive.
RESUMEN
Sustainable development in the bio-treatment of large-scale biomass bulks requires high performance enzymes adapted to extreme conditions. An extracellular keratinolytic extract was obtained from the culture broth of a halotolerant strain of Salicola marasensis. Keratin hydrolyzing activity of the concentrated enzyme extract was observed on a 100 mg of pretreated feather waste. The concentrated enzyme was able to hydrolyze the poultry feathers by 25% after 12 h incubation. The bio-waste material was optimally hydrolyzed at pH 9 and temperature of 40 °C. Among reductants, 1,4-dithiothreitol, L-cysteine, 2-mercaptoethanol, glutathione, and sodium sulfate showed the most remarkable effect on the bio-waste keratinolysis, while the tested surfactants and urea had no significant effect on the keratinolytic activity. Hexane and hexadecane indicated strong effect on keratinase activity and bio-treatment in the presence of 1-butyl-3-methylimidazolium hexafluorophosphate ([BMIM][PF6]) as a hydrophobic ionic liquid resulted in a maximal of 80% extraction yield of soluble proteins from feathers. Considering the stability of the extracellular keratinolytic content in [BMIM][PF6], the observed keratinase activity was noteworthy suggesting that the secreted enzyme may contribute to the bioconversion of feather wastes.