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Feather waste produced by livestock and poultry breeding industry is a kind of biomass resource with great application prospect. Current study aims to obtain a feather-degradation bacteria that could efficiently biodegrade feather waste and maintain environmental friendliness. By using the casein agar medium, a novel keratinase production strain Stenotrophomonas sp. Yang-5 was isolated and then identified by subsequent 16S rRNA sequence. Further keratinase production medium was optimized by single factor and orthogonal experiments. Also, crude keratinase was characterized. Consequently, the highest enzyme activity of 161.95 ± 3.65 U/mL was obtained after 48 h of incubation with optimized conditions consisting of reaction temperature 32.5 °C, initial pH 7.5, 0 g/L CaCl2, 50 g/L fructose, and 15 g/L feather. Yang-5 keratinase demonstrated a broad temperature range of 25 °C − 95 °C and optimal catalytic efficiency at pH 9.0. The keratinase had a half-life of more than 120 min at 70° C, indicating satisfactory thermal stability. Furthermore, 0.25 mol/L Mg2+, Mn2+, K+, and Ca2+ could enhance the enzyme activity to some extent and assigned as the metallo-class of keratinase, among of which Mn2+ resulted in the highest improvement by 2-fold activity enhancement. In addition, 5% (v/v) reducing agents including DMSO, DTT and 2-mercaptoethanol all reinforced the enzyme activity by 3–5 folds. To sum up, thermostable alkaline metallo-keratinase from Stenotrophomonas sp. Yang-5 exhibited favorable stability and could serve as a promising candidate for feather waste degradation as well as environmental protection and feed additive industry. Graphical Abst ract: [ABSTRACT FROM AUTHOR]

Considering the growing environmental concerns, bioutilization of keratin waste with keratinase has emerged as a potential solution to alleviate the significant burden of poultry processing waste. The main objectives of the study were to isolate and screen keratinase-producing bacteria and to optimize the production conditions of the enzyme using a statistical approach. A promising bacterium, Bacillus subtilis GH2, was isolated from a traditional leather tanning pond. This bacterium was identified using 16 S rDNA gene sequencing. By optimizing the medium components and culture conditions, a significant increase in keratinase activity of 421.65 U was obtained at a medium pH of 7.5 and temperature of 38 °C. This corresponds to a 1.96-fold increase compared to the first screening stage. The optimal activity of the enzyme was obtained at a pH of 7 and a temperature of 45 °C. Ca+2 showed the highest improved keratinase activity. Furthermore, the bacterium showed promising potential for feather biodegradation. [ABSTRACT FROM AUTHOR]

The accumulation of feather waste is a critical problem in solid waste management; therefore, microbial management of waste is highly significant. Consequently, the keratinolytic capacity of some bacteria isolated from the keratinous waste area was evaluated. The most promising isolate coded as L10 showed the highest feather degradation efficiency 72.7% was identified as Bacillus cereus L10 using 16S rRNA gene sequencing. The effect and contribution of independent process variables were studied by Plackett–Burman design. The optimal values of the independent variables obtained from the Plackett–Burman design were chicken feather, 1%; K2HPO4, 0.3%; KH2PO4, 0.05%; MgSO4 7H2O, 0.01%; yeast extract, 0.05%; inoculum size 4% with pH 7 and incubation period 5 days at 35 °C. The optimal parameters were validated resulting in the enhancement of keratinase production (9.602 U/ml), protein concentration (521.17 µg/ml), and feather degradation (94%) along with a 4.56-fold increased of keratinase production. The enzyme is relatively thermostable, completely stable between 30 and 80 °C for 1 h and retained 97.66% of its activity at 100 °C. B. cereus L10 was able to degrade different types of keratins. Interestingly, the feather hydrolysate from optimized keratinase production medium showed plant growth promoting activity by producing indole-3-acetic acid (IAA). The feathers hydrolysate produced a great benefit by its use as a soil biofertilizer through enhancing the germination of Triticum aestivum. The important implication of keratinase in laundry detergent and leather industry was examined. Results revealed that the enzyme achieved great efficiency in removing protein and blood-rich stains within 30 min at 36 °C in addition to its thoroughly dehairing of cow-hide after 40 h incubation without any damage. The dehaired pelt exhibited a smooth, velvety, and white color surface, good flexibility. Therefore, our study presents an effective method to turn waste into wealth. [ABSTRACT FROM AUTHOR]

The production of keratinases was evaluated in submerged fermentation with Aspergillus niger and by pigs' swine hair in a batch bioreactor. Experimental planning was performed to assess the interaction between different variables. The enzyme extract produced was characterized at various pH and temperatures and subjected to enzyme concentration using a biphasic aqueous system and salt/solvent precipitation techniques. In addition, the substrate's potential in reducing hexavalent chromium from synthetic potassium dichromate effluent with an initial concentration of 20 mg L−1 of chromium was evaluated. The resulting enzyme extract showed 89 ± 2 U mL−1 of keratinase. The enzyme concentration resulted in a purification factor of 1.3, while sodium chloride/acetone and ammonium sulfate/acetone resulted in a purification factor of 1.9 and 1.4, respectively. Still using the residual substrate of swine hair from the fermentation, a 94% reduction of hexavalent chromium concentration occurred after 9 h of reaction. Thus, the study proved relevant for producing keratinases, with further environmental applicability and the possibility of concentrating the extract via low-cost processes. [ABSTRACT FROM AUTHOR]

Keratinases are enzymes that catalyze the degradation of keratin, a resilient protein found in various biological materials such as: feathers, hair, and fish scales. Two isolates identified from previous work as Alcaligenes faecalis (SCA A1) and Pseudomonas aeruginosa (FSA A2) was used for keratinase production. The enzyme produced were purified using dialysis, ion exchange chromatography and gel filtration chromatography and the purified enzyme was characterized and used to check for keratin substrate specificity. Fermentation studies revealed that A. faecalis (SCA A1) displayed optimal growth at pH 9.5 and 10.5, particularly at higher temperatures (48°C, 58°C, and 68°C) when using Mackerel fish scales as the substrate. In contrast, P. aeruginosa (FSA A2) demonstrated better growth at pH 7.5 and 8.5, with the highest growth observed at 28°C and 38°C using both Mackerel and Elephant fish scales. Enzyme activity profiles showed that A. faecalis exhibited higher activity with Mackerel scales (65.7 U/ml), while P. aeruginosa showed greater activity using Elephant scales (68.1 U/ml). Purification of the keratinase enzymes from both isolates resulted in a 7-fold increase in specific activity, reaching 35.16 U/mg for A. faecalis and 37.80 U/mg for P. aeruginosa. Characterization of the purified enzymes revealed that both exhibited optimal activity at 70°C, with P. aeruginosa displaying slightly higher activity. The enzymes also shows good activity over a wide pH range, with optima at pH 7.3 and 6.3 for A. faecalis and P. aeruginosa, respectively. Substrate specificity analysis indicated that both enzymes had the highest activity towards Mackerel fish scales, followed by Casein, Human Hair, Chicken feathers, and Elephant fish scales. These findings highlight the potential of A. faecalis and P. aeruginosa for the biodegradation of fish scale waste, offering a sustainable solution for waste management and the production of valuable bioproducts. These results suggest that these keratinolytic bacteria could be utilized in the development of bioremediation strategies for fish processing waste. Additionally, the keratinase enzymes produced by these isolates could be utilized in various industrial applications, such as the production of animal feed supplements, leather processing, and the development of cosmetic and pharmaceutical products. [ABSTRACT FROM AUTHOR]

The gene editing and synthetic biological tools have led to the implementation of diverse metabolic engineering approaches to enhance the production of specific enzymes. Microbial keratinases can convert keratin wastes into valuable compounds for mankind. Since the market for keratinases cannot be satisfied by production from wild hosts, it is obligatory to develop hosts with high keratinase yields. The intention of this review is to evaluate microbial keratinase advancement through protein engineering, breeding techniques, and fermentation optimization. The main aim of protein engineering is to improve the heat resistance ability and catalytic activity of keratinases by employing mutagenesis methods. Moreover, modifying the expression elements and host engineering are also two unique ways to augment the keratinase yield. Intending to accelerate the production of modified keratinase, this review attempts to highlight the optimization of expression elements, such as promoter engineering, UTR, signal peptide, and codon optimization. Moreover, the approaches of host engineering including strengthening precursor supply, membrane surface engineering, and optimization of secretion pathways were also explained here. Furthermore, it is also essential to optimize the medium composition and fermentation condition for high keratinase yield. This review also addressed the present advancements, difficulties, and tendencies in the field of microbial keratinase production, along with its potential.

The gene editing and synthetic biological tools have led to the implementation of diverse metabolic engineering approaches to enhance the production of specific enzymes. Microbial keratinases can convert keratin wastes into valuable compounds for mankind. Since the market for keratinases cannot be satisfied by production from wild hosts, it is obligatory to develop hosts with high keratinase yields. The intention of this review is to evaluate microbial keratinase advancement through protein engineering, breeding techniques, and fermentation optimization. The main aim of protein engineering is to improve the heat resistance ability and catalytic activity of keratinases by employing mutagenesis methods. Moreover, modifying the expression elements and host engineering are also two unique ways to augment the keratinase yield. Intending to accelerate the production of modified keratinase, this review attempts to highlight the optimization of expression elements, such as promoter engineering, UTR, signal peptide, and codon optimization. Moreover, the approaches of host engineering including strengthening precursor supply, membrane surface engineering, and optimization of secretion pathways were also explained here. Furthermore, it is also essential to optimize the medium composition and fermentation condition for high keratinase yield. This review also addressed the present advancements, difficulties, and tendencies in the field of microbial keratinase production, along with its potential. [ABSTRACT FROM AUTHOR]

The recalcitrant, fibrous protein keratin is found in the outermost layer of vertebrate skin, feathers, hair, horn, and hooves. Approximately, 10 million tons of keratin wastes are produced annually worldwide, of which around 8.5 million tons are from feather wastes. The biodegradation of keratin has been a challenge due to the lack of understanding of biological parameters that modulate the process. Few soil-borne microbes are capable of producing keratinase enzyme which has the potential to degrade the hard keratin. However, various pesticides are abundantly used for the management of poultry farms and reports suggest the presence of the pesticide residues in feather. Hence, it was hypothesized that pesticides would interact with the substrate-binding or allosteric sites of the keratinase enzyme and interferes with the keratin-degradation process. In the present study, molecular interactions of 20 selected pesticides with the keratinase enzyme were analyzed by performing molecular docking. In blind docking, 14 out of 20 pesticides showed higher inhibitory potential than the known inhibitor phenylmethylsulfonyl flouride, all of which exhibited higher inhibitory potential in site-specific docking. The stability and strength of the protein complexes formed by the top best potential pesticides namely fluralaner, teflubenzuron, cyhalothrin, and cyfluthrin has been further validated by molecular dynamic simulation studies. The present study is the first report for the preliminary investigation of the keratinase-inhibitory potential of pesticides and highlights the plausible role of these pesticides in hindering the biological process of keratin degradation and thereby their contribution in environmental pollution. Illustration depicting the hypothesis, experimental procedure, and the resultant keratinase-inhibitory potential of selected pesticides. [ABSTRACT FROM AUTHOR]

The application of biotechnology through the utilization of enzymes is considered an easy and inexpensive method of producing valuable products from waste resources. The present study describes the production of a keratinolytic enzyme from Pseudomonas aeruginosa S-04, which showed efficiency for feather biodegradation. The production of extracellular keratinase was improved 1.3-fold by optimizing various parameters through the one factor at a time (OFAT) approach. The keratinase was purified to homogeneity by ammonium sulfate precipitation and DEAE-cellulose anion exchange chromatography. The purified keratinase (35.5 kDa) displayed optimal activity at 60 °C and pH 9.5 and was stable between pH 7 and 9.5 and temperatures ranging from 4 to 40 °C for 2 h. The catalytic activity of keratinase was enhanced in the presence of Fe3+ and Mn2+ ions, Triton X-100, Tween 20, DMSO, isopropyl alcohol and ethanol, but reduced activity was recorded in the presence of methanol and acetone. Furthermore, the enzyme activity was deactivated by EDTA, suggesting that this keratinase belongs to a metalloprotease family. The Km and Vmax of the purified keratinase were 7.62 mg/ml and 200 U/mg protein, respectively. The partially purified keratinase displayed remarkable feather degradation capabilities (93% after 24 h), and the nutritional properties of the resulting feather hydrolysate make it a promising candidate for use as a poultry feed ingredient. Thus, thermostable alkaline keratinase from P. aeruginosa S-04 is a promising candidate that converts low-cost chicken feather waste into a value-added product, indicating a novel approach to feather waste treatment and utilization. [ABSTRACT FROM AUTHOR]

Feather waste is a highly prevalent form of keratinous waste that is generated by the poultry industry. The global daily production of feather waste has been shown to approach 5 million tons, typically being disposed of through methods such as dumping, landfilling, or incineration which contribute significantly to environmental pollutions. The proper management of these keratinous wastes is crucial to avoid environmental contamination. The study was carried out to isolate the keratinolytic fungi from the poultry disposal sites of different region of North-East India to evaluate its potential in bioremediation of the feathers wastes. Out of 12 fungal strains isolated from the sites, the fungus showing the highest zone of hydrolysis on both the skim milk and keratin agar medium was selected for the study and the molecular identification of the isolate was performed through DNA sequence analysis by amplifying the internal transcribed spacer (ITS) region. The sequence results showed higher similarity (above 95%) with Aspergillus spp. and was named Aspergillus sp. Iro-1. The strain was further analyzed for its feather degrading potential which was performed in submerged conditions under optimized conditions. The study showed that the strain could effectively degrade the feathers validated through weight loss method, and the structural deformations in the feathers were visualized through scanning electron microscopy (SEM). Aspergillus sp. Iro-1 was obtained from the southern region of Assam. It would be of great importance as the implementation of this sp. can help in the bioremediation of feathers wastes in this region. This is the first study of identification of feather degrading fungus from southern part of Assam (Barak). [ABSTRACT FROM AUTHOR]

BACKGROUND: This work aimed to study different methodologies for extracting enzymes produced by the endophytic fungi isolated from mangrove Neofusicoccum parvum, Penicillium glabra series and Bjerkandera sp. grown on organic rice. The enzymatic activities of amylase, cellulase, laccase, lipase, peroxidase, protease and keratinase were evaluated for future applications in biotechnology and enzymology. The evaluation of the extraction was carried out using the design of experiments methodology, where variations were made in pH (4–8), temperature (20–36 °C) and agitation (150–200 rpm), in times of 1, 6 and 12 h. RESULTS: Among the enzymes analyzed, promising activities were found for amylase, cellulase, lipase, protease, peroxidase and especially keratinase, presenting activities of up to 7049.47 U g−1 in the fermentation carried out with the fungus Neofusicoccum parvum, 3538.52 U g−1 with the fungus Penicillium glabra series and 5068.20 U g−1 with the fungus Bjerkandera sp. This indicates that the fungi are promising for the production of multienzyme cocktails. CONCLUSION: It should be emphasized that the microorganisms and extraction conditions evaluated resulted in a series of promising results, that is, in the case of aiming to obtain different enzymes by other extraction conditions, after fermentation of various microorganisms on rice, several options of situations are presented and validated in this work, which allows the choice of the same, depending on the future application potential of these biocatalysts. © 2023 Society of Chemical Industry (SCI). [ABSTRACT FROM AUTHOR]

Keratinases are proteolytic enzymes that have the capability of degrading insoluble keratin substrates. LS4, LS5, and KS3 were newly isolated strains showing ability to hydrolyze keratins and keratin based products. In this study, different bacterial strains were isolated from soil, screened for protease and keratinase production, and the best isolates were identified. Serial dilution and plating method were used for isolation of pure bacterial culture. Isolated, 48 strains were screened, and their feather degrading ability on feather meal agar plates and keratin azure-based agar. The proteolytic activity of the isolates was also screened on casein agar. Three strains; LS4, LS5, and KS3 shows keratinolytic activity that reveal the keratinase enzyme on medium; feather meal agar, keratin azure agar, and casein agar. Among all the three strains (LS4, LS5, and KS3), KS3 show largest inhibition zone that show maximum enzymatic activity. For its characterization 16S RNA was performed, sequence KS3 was submitted to GenBank and was identified as Bacillus tropicus KS3. Newly isolated strain B. tropicus KS3 showed the keratinase production and highest keratinase activity (35.06 ± 2.5 U/ml). B. tropicus KS3 also show the highest degradation of feather efficiently showed 84% of whole chicken feather biodegradation at 37 °C within 15 days among all the isolated strains. Keratinase enzyme possesses a wide range of potential applications in the bioremediation of feather waste and bio-fertilizer in agricultural land. The industry demands a better source of this enzyme due to its multivariate application and huge market demand. In the present study, a novel keratinase-producing bacteria was isolated and characterized as Bacillus tropicus KS3 from poultry waste contamination soil. [ABSTRACT FROM AUTHOR]

Background: Tinea of vellus hair is a dermatophyte infection with or without localised infection of adjacent skin. The epidemiology and pathogenesis of this disorder have insufficiently been established. Objectives: To gain insight into the epidemiology and analysis dissection of patterns of vellus hair infection. Methods: A comprehensive examination of our series of cases and existing literature, clinical information was performed. Microsporum species were assayed for levels of keratinase and SUB1‐3 level after different keratin‐induced cultures. Results: The infection affects both children and adults, with a male‐to‐female ratio of 1:1.3. The most afflicted areas are the face, neck and upper limbs. Both typical (ringworm‐like) and atypical (tinea incognito) tinea lesions can be observed. The disorder is caused primarily by the zoophilic Microsporum canis (40.39%). Topical antifungal drugs alone are ineffective; mycological cure is achieved in combination with systemic antifungal therapy for 4–8 weeks. In vitro, M. canis exhibited significantly elevated levels of secreted protease and SUB1 and SUB3 transcripts in cat hair cultures. Conversely, the findings of anthropophilic dermatophytes M. audouinii and M. ferrugineum demonstrated the opposite trend, suggesting that there are associations between keratinase activity and expression of SUB1 and SUB3 induced by different keratin substrates among M. canis complex members. Conclusions: The epidemiological characteristics of tinea of vellus hair help us to understand its transmission patterns and develop effective prevention strategies. There are associations between keratinase activity and expression of SUB1‐3 induced by different substrates, which may explain the host shift and adaptation from pet animals to particular micro‐habitats on the human host. [ABSTRACT FROM AUTHOR]

The growing animal protein production results in a high generation of waste considered environmental pollutants, such as swine hair, which is composed of keratin. This protein has characteristics such as rigidity, making it a natural polymer. Given this, alternatives for this waste to be disposed of without harming the environment are desirable, such as biological pre-treatment that makes the waste less rigid, facilitating access to nutrients present in its structure when disposed of in the soil and also enabling the production of keratinase, one an enzyme with high specificity for residues composed of keratin. In this context, this study aimed to evaluate the final disposition of swine hair pre-treated with Trichoderma sp. in the soil and its effect on the development of tomato seedlings and to evaluate the production of keratinase from the pre-treatment process. As a result, the final disposal of these pre-treated residues was effective since there were no significant changes in soil characteristics but increased nutrients such as nitrogen, potassium, and calcium in tomato seedlings. Also, keratinolytic activity values of 326.61 ± 42.33 and 403.3 ± 71.65 U/g were obtained within 24 h of the process. Finally, it can be concluded that in addition to not negatively affecting the environment and the development of tomato seedlings, after biologically pre-treated, swine hair can act as an adsorbent of heavy metals, reducing their concentrations in the soil. So, the significance of this study could be synthesized in biological pre-treatment enabled the use of swine hair for multivariate purposes; the fermentation process resulted in elevated keratinolytic activity; the treated swine hair decreased concentrations of heavy metals in the soil; and swine hair pre-treated biologically provide the increment of nutrients in the soil. [ABSTRACT FROM AUTHOR]

Keratin, a versatile bioprotein, has seen a tremendous industrial exploitation due to its outstanding biodegradability and biocompatibility nature. Keratin is primarily derived from substrates such as feathers, nails, horns, beaks, hair and bovine hoofs. The generation of chicken and pigeon feather wastes has risen over the years in Vellore. When it is not discarded properly, this acts as a major root cause for the dissemination of various diseases to humans. The primary aim of this research is to remediate these wastes by extracting the keratin and to prove that beta-mercaptoethanol enhances keratinase activity by in-silico approach for degradation of these wastes. In light of this, the feathers from the dumped sites of Vellore were exploited and the keratin was extracted from country chicken and pigeon feathers which yields a concentration of 3.44 mg/L and 2.8 mg/L of keratin. This extraction procedure is significantly more cost-effective and yields more keratin than earlier approaches. The crystallographic structures of keratin were examined by X-ray diffraction studies. The functional group of the keratin was analyzed by using Fourier-transform infrared spectroscopy and confirmed the presence of cysteinecystine, glutamic acid and tryptophan. The mode of action and degree of effectiveness of keratin were studied using Lipinski’s rule of 5. In addition, the Protein Data Bank was used to retrieve the keratinase structure of Meiothermus taiwanensis WR-220, which has the National Center for Biotechnology Information (NCBI) and Protein Data Bank (PDB) accession ID-5WSL and the enzyme’s tertiary structure was studied with the SwissModel tool. Using Autodock, beta-mercaptoethanol was docked with keratinase and enzyme-ligand in-silico interactions prove that ligand enhances the activity of microbial keratinase. [ABSTRACT FROM AUTHOR]

Feather, horn, hoof, and other keratin waste are protein-rich but limited by natural keratinase synthesis, activity, pH, and temperature stability. It is challenging to realize its large-scale application in industries. Bacillus subtilis spores are a safe, efficient, and highly resistant immobilized carrier, which can improve target proteins' resistance. In this research, KERQ7, the keratinase gene of Bacillus tequilensis strain Q7, was fused to the Bacillus subtilis genes coding for the coat proteins CotG and CotB, respectively, and displayed on the surface of B. subtilis spores. Compared with the free KERQ7, the immobilized KERQ7 showed a greater pH tolerance and heat resistance on the spore surface. The activity of CotG-KERQ7 is 1.25 times that of CotB-KERQ7, and CotG-KERQ7 is more stable. When the flexible linker peptide L3 was used to connect CotG and KERQ7, the activity was increased to 131.2 ± 3.4%, and the residual enzyme activity was still 62.5 ± 2.2% after being kept at 60 ℃ for 4 h. These findings indicate that the flexible linker and CotG were more effective for the spore surface display of keratinase to improve stress resistance and promote its wide application in feed, tanning, washing, and other industries. [ABSTRACT FROM AUTHOR]

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The huge amounts of keratin-rich waste generated daily by various industries, slaughterhouses, and processing plants need to be properly managed. Most keratin degradation-related research focuses on keratin from bird feathers, but a vast minority focuses on keratin from sheep wool, which also presents a serious environmental problem. In this article, we describe the isolation, identification, and characterization of new keratinolytic microorganisms capable of sheep wool degradation from sheep wool and soil enriched with wool keratin. We isolated four bacterial species from the genus Bacillus (B. subtilis, B. altitudinis, B. mycoides, and B. wiedmannii), one streptomycete species Streptomyces coelicoflavus identified by whole genome sequencing, and a fungal species Aphanoascus reticulisporus. In some of the isolated microorganisms, we detected keratinolytic activity for the first time, and for most of them, the ability to degrade sheep wool has not been previously demonstrated. The keratinases of the new isolates are active in a wide range of temperatures (25–85 °C) and pH (6.0–10.0), so all isolates show great potential for further biotechnological use in industry and in various environmental and agricultural applications to reduce and recycle keratin-rich wastes such as sheep wool and waste woollen textiles. [ABSTRACT FROM AUTHOR]

Keratin waste has become an increasingly serious environmental and health hazard. Keratin waste is mainly composed of keratin protein, which is one of the most difficult polymers to break down in nature and is resistant to many physical, chemical, and biological agents. With physical and chemical methods being environment damaging and costly, microbial degradation of keratin using keratinase enzyme is of great significance as it is both environment friendly and cost-effective. The aim of this study was to extract and purify keratinase from bacterial species isolated from the soil. Among the organisms, an isolate of Bacillus velezensis, coded as MAMA could break down chicken feathers within 72 hours (h). The isolated strain produced significant levels of keratinase in mineral salt medium by supplying chicken feathers as the sole source of nitrogen and carbon. Feather deterioration was observed with the naked eye, and enzyme activity was evaluated using a spectrophotometric assay. Sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and zymography results revealed that the keratinase protein produced by Bacillus velezensis had a molecular weight between 40 and 55 kilodalton (kDa).

Chicken feathers represent an abundant and sustainable resource that can be harnessed for multiple value-added products. Bioplastic reinforced with bacterial nanocellulose was synthesized using enzymatically digested chicken feathers. A highly efficient keratinolytic bacterium, identified as Bacillus sp. DRS4 through biochemical characterization and 16S rRNA gene sequence analysis, was isolated from deposit soils of Lake Chitu in Ethiopia. Bacillus sp. DRS4 was able to completely degrade chicken feathers within 48 h. Optimization of the physicochemical parameters increased the enzyme yield from Bacillus sp. DRS4 by 30%. The enzyme showed optimal keratinolytic activity at 37 °C and pH 11, hydrolyzing white chicken feathers in 72 h and providing hydrolysates with a total protein content of 251.145 mg/mL. Further, the mechanical and thermal properties of a bioplastic made from hydrolysates and reinforced with bacterial nanocellulose were assessed. The bioplastic exhibited a remarkable tensile strength of 5.769 MPa and reached a melting temperature of 127.5 °C, suggesting that bacterial nanocellulose acts as an effective stabilizer. Fourier Transform Infrared spectroscopy (FTIR) analysis revealed additional peaks in BNC-reinforced plastic films, indicating a binding interaction that enhanced the bioplastic properties. Overall, Bacillus sp. DRS4 is a potential strain for alkaline keratinase production and a promising candidate for upgrading chicken feathers into high-value-added products.

ABSTRACT: The tons of keratin waste are produced by the poultry and meat industry which is an insoluble and protein-rich material found in hair, feathers, wool, and some epidermal wastes. These waste products could be degraded and recycled to recover protein, which can save our environment. One of the potential strategy to achieve this target is use of microbial biotreatment which is more convenient, cost-effective, and environment-friendly by formulating hydrolysate complexes that could be administered as protein supplements, bioactive peptides, or animal feed ingredients. Keratin degradation shows great promise for long-term protein and amino acid recycling. According to the MEROPS database, known keratinolytic enzymes currently belong to at least 14 different protease families, including S1, S8, S9, S10, S16, M3, M4, M14, M16, M28, M32, M36, M38, and M55. In addition to exogenous attack (proteases from families S9, S10, M14, M28, M38, and M55), the various keratinolytic enzymes also function via endo-attack (proteases from families S1, S8, S16, M4, M16, and M36). Biotechnological methods have shown great promise for enhancing keratinase expression in different strains of microbes and different protein engineering techniques in genetically modified microbes such as bacteria and some fungi to enhance keratinase production and activity. Some microbes produce specific keratinolytic enzymes that can effectively degrade keratin substrates. Keratinases have been successfully used in the leather, textile, and pharmaceutical industries. However, the production and efficiency of existing enzymes need to be optimized before they can be used more widely in other processes, such as the cost-effective pretreatment of chicken waste. These can be improved more effectively by using various biotechnological applications which could serve as the best and novel approach for recycling and degrading biomass. This paper provides practical insights about molecular strategies to enhance keratinase expression to effectively utilize various poultry wastes like feathers and feed ingredients like soybean pulp. Furthermore, it describes the future implications of engineered keratinases for environment friendly utilization of wastes and crop byproducts for their better use in the poultry feed industry.

Background: Chicken feathers contribute to large quantities of keratinaceous wastes that pose serious environmental problems and must be catered to properly. Chicken feathers are also a potential source of vital proteins, peptides, and amino acids, which could be used as low-cost animal feeds. Therefore, there has been increasing interest in keratinase-producing microbes for reprocessing and using keratinous biomaterials. Methods: Among the five isolated keratinolytic microorganisms, one microbe, Bacillus XT 01, produced a significant amount of enzyme activity, which was partially characterized. The potential of this protease-producing microbe was investigated for converting feather keratin waste to valuable protein hydrolysate. Results: Maximum keratinase production was observed after 5 days of incubating Bacillus XT 01 at an optimum temperature of 45 °C and pH 8.5. Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) and zymogram of ammonium sulfate precipitated culture supernatant showed the presence of several proteolytic enzymes with molecular weights between 30 and 60 kDa. The Bacillus strain caused almost complete feather degradation (98%) after 7 days of incubation at 45 °C in a shake culture medium. Antioxidant and reducing activities of the feather protein hydrolysate (FPH) elevated with increased cultivation time. Investigation of the effect of feather protein hydrolysate on plants indicated improved plant growth regarding the agronomic parameters, such as plant height, number of trifoliate leaves, number of pods, pod length, number of seeds per pod, and root length, which increased by 30.84%, 49.32%, 70.90%, 53.27%, 60.03%, and 54.71%, respectively. Conclusions: The prospective of Bacillus XT 01 for degrading feather waste keratin to highly valued hydrolyzed feather protein offers effectiveness in the poultry industry and ultimately decreases environmental pollution hazards.

In recent decades, there has been growing concern and negativity from the environmental impact of industrial development, which produces lots of technological changes. The significant contribution of the leather industry has a good impact on the economic sector; however, it is faced with challenges caused by pollution to the environment. Special attention is required for disposal of solid wastes because of the large amounts that are generated and legal restrictions. Environmental problems are caused by the large amount of wastes originating from chemicals during processing, especially in dehairing process. Conventional dehairing by lime sulfide process causes high chemical oxygen demand emission, biological oxygen demand, and total suspended solids. The dehairing mechanism is vaguely understood at present from the point of view of the enzyme specificity, which is needed for consistent and satisfactory hair removal without deleterious effect on the leather quality. It is known that the use of enzymes has potential as an alternative that can be used to reduce the harm caused by toxic chemicals including for waste management. Recently, there has been increasing research on application of enzymes in various leather production processes. The use of protease and keratinase enzymes in leather processing industry has the most promising potential to improve the surrounding environment. Therefore, this study aimed to review relevant literature in terms of dehairing processes without adverse effects in order to make good quality leather. Further scientific discussion is needed for understanding the gap of these critical issues in this area. [ABSTRACT FROM AUTHOR]

Feathers become hazardous pollutants when deposited directly into the environment. The rapid expansion of the poultry industry has significantly increased feather waste, necessitating the development of new ways to degrade and utilize feathers. This study investigated the ability of Bacillus licheniformis WHU to digest intact chicken feathers in water. The results indicated that yields of free amino acids, bioactive peptides, and keratin-derived nano-/micro-particles were improved in bacteria- versus purified keratinase–derived feather hydrolysate. Bacteria-derived feather hydrolysate supplementation induced health benefits in mice, including significantly increased intestinal villus height and zonula occludens-1 protein expression, as well as increased secretory immunoglobulin A levels in the intestinal mucosa and superoxide dismutase activity in serum. Additionally, feather hydrolysate supplementation modulated the mouse gut microbiota, reflected by increased relative abundance of probiotics such as Lactobacillus spp., decreased relative abundance of Proteobacteria at the phylum level and pathogens such as Staphylococcus spp., and increased Bacteroidota/Firmicutes ratio. This study developed a simple, cost-effective method to degrade feathers by B. licheniformis WHU digestion, yielding a hydrolysate that can be directly used as a bioactive nutrient resource. The study findings have applications in the livestock, poultry, and aquaculture industries, which have high demands for cheap protein. Key points: • Bacillus licheniformis could degrade intact feather in water. • The resulting feather hydrolysate shows prebiotic effects on mouse. [ABSTRACT FROM AUTHOR]

Daily accumulation of feather waste due to the development of the poultry industry is a critical issue, considering the environmental and health hazards. Among feather waste disposal methods, biodegradation is a practical, cheap, and environment-friendly method. This study aimed to enhance the keratinolytic activity of FUM-2 isolate, a native keratinolytic bacterium, using mutations and culture improvement. The isolate was identified as Bacillus paralicheniformis based on biochemical, morphological, and molecular analysis. Using the one factor at a time (OFAT) method, the isolate produced more keratinase at 45 °C, pH 11, 2% carbon substrate concentration, 75% aeration rate, and 48 hours incubation. Also, it was able to degrade both α and β keratin. Culture improvement increased enzyme production from 507.6 U/mL to 1706.4 U/mL. In random mutation using ultraviolet radiation, three mutants were isolated, and a 10-minute UV-treated mutant of UVF2-D showed a 36.3% increase in keratinolytic activity compared to its parent. In the next round of mutagenesis by ethidium bromide, seven double mutants were isolated from the UVF2-D mutant, among which the EUF2-D2 double mutant showed a 79% increase in keratinolytic activity. No viable mutants were isolated from the wild strain using ethidium bromide. Our results show that many organisms can potentially produce beneficial products that could be improved using random mutagenesis and optimization methods, which are more useful for strain improvement than complex and costly molecular techniques. [ABSTRACT FROM AUTHOR]

Keratin is a fibrous, insoluble structural protein that is highly cross-linked with hydrophobic, hydrogen, and disulfide bonds. Keratinases are enzymes that belong to the category of serine hydrolases that are capable of breaking down keratin. The results of the determination of the better fermentation system showed that the production of keratinase from local A.terreus A13 isolate by submerged fermentation (SmF) system was the best system to give the highest specific activity (113.4 U/mg) of keratinase compared with solid-state fermentation (SSF). The optimum conditions for keratinase production by SmF, were determined via cultivation conditions, including carbon source, nitrogen source, temperature, pH of the medium, and time of incubation were optimized to enhance the production of total keratinase production in a culture of A.terreus A13 with incubator shaker. The highest product of total keratinase was achieved in feather broth with 2 % sucrose, and 0.5 % soya bean, with a pH of 5.5 at 28 °C for 8 days. Separation and purification of keratinase from a local isolate of A.terreus A13 was done by precipitating with 0-75 % saturated ammonium sulfate, then by ion-exchange chromatography on DEAE-Cellulose column and sephadex G-150 gel. Partially purified keratinase gave an activity of 5.1 U/ml, protein concentration of 0.004 mg/ml, and specific activity of 1275 U/mg with purification fold of 4.96 and 49 % as yield. The aim of the present study was to optimize the production of keratinase from A. terreus A13, cultivated using optimum conditions, and its use for the biodegradation of feathers. [ABSTRACT FROM AUTHOR]

Abstract In this study, 25 actinomyces isolates were obtained from 10 different poultry farms and tested for their keratinase activity. The isolate with the highest keratinase activity was identified through molecular identification by PCR and sequencing of the 16S rRNA gene to be Streptomyces spp. and was named Streptomyces werraensis KN23 with an accession number of OK086273 in the NCBI database. Sequential mutagenesis was then applied to this strain using UV, H2O2, and SA, resulting in several mutants. The best keratinolytic efficiency mutant was designated as SA-27 and exhibited a keratinase activity of 106.92 U/ml. To optimize the keratinase expression of mutant SA-27, the Response Surface Methodology was applied using different parameters such as incubation time, pH, carbon, and nitrogen sources. The optimized culture conditions resulted in a maximum keratinase specific activity of 129.60 U/ml. The genetic diversity of Streptomyces werraensis KN23 wild type compared with five mutants was studied using Inter-simple sequence repeat (ISSR). The highest total and polymorphic unique bands were revealed in the S. werraensis KN23 and SA-18 mutant, with 51 and 41 bands, respectively. The dendrogram based on combined molecular data grouped the Streptomyces werraensis and mutants into two clusters. Cluster I included SA-31 only, while cluster II contained two sub-clusters. Sub-cluster one included SA-27, and sub-cluster two included SA-26. The sub-cluster two divided into two sub-sub clusters. Sub-sub cluster one included SA-18, while sub-sub cluster two included one group (SA-14 and S. werraensis KN23).

The specifics of the processing of livestock and poultry products is that in the process of obtaining the main marketable products, about half the feedstock at various stages of the technological process turns into waste that pollutes the environment. These by-products contain large amounts of the hard-to-digest keratin protein. The use of specific enzymes capable of degrading this protein helps not only to reduce the negative anthropogenic impact on nature but also to obtain valuable hydrolysates that can be used as a fertilizer for plants or a feed additive. The aim of this work was to study the ability of Bacillus megaterium UCM B-5710 to split various keratin-containing substrates: black and white chicken feathers, white turkey feathers, parrot feathers of various colors, sheep wool, pig bristles, and baby hair and nails. Methods. The culture was grown under conditions of submerged cultivation at 40 °C, with a nutrient medium stirring rate of 201 rpm for 6 days. For growth, a basic nutrient medium containing 0.5% defatted chicken feathers or other keratin-containing substrates as sole sources of carbon and nitrogen were used. Keratinase activity was assessed by UV absorption at 280 nm of hydrolysis products of keratin-containing raw materials. Protein was determined by the Lowry method, caseinolytic (total proteolytic) activity was determined by the Anson method modified by Petrova, and amino acid content was determined by the ninhydrin method. The degree of hydrolysis of the substrates was evaluated by the ratio of the initial and final weight of the substrate. Results. It was shown that the synthesis of keratinase by the culture of B. megaterium UCM B-5710 begins from the 6th hour of cultivation. The level of protein and proteolytic activity and the content of amino acids increased throughout the entire period of culture growth. The supernatant of the culture liquid of B. megaterium UCM B-5710 was most effective in splitting white chicken's and turkey's feathers, a little slower -- feathers of black chicken and blue parrots, as well as wool of white sheep. According to the degree of splitting, the substrates used can be arranged in the following order: white turkey feathers > white chicken feathers > black chicken feathers > blue parrot feathers > white sheep wool > baby nails > pig bristle > baby hair. The study of the effect of feather color on the resistance to decomposition showed that black, blue, and red feathers are more resistant, which coincides with the literature data. Conclusions. B. megaterium UCM B-5710 produces keratinase capable of splitting both α- and β-keratins, however, with different efficiencies and rates. [ABSTRACT FROM AUTHOR]

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Keratinases are enzymes belonging to the serine hydrolases group which are capable of degradation of keratin, an insoluble and fibrous structural protein widely cross-linked with hydrogen, disulfide, and hydrophobic bonds. Attempts to find new sources of enzymes and amino acids for fundamental knowledge of enzyme evolution, structure‐function relationships, catalysis mechanisms of enzymes, and even for the identification of novel protein folds. In this study, seventy-nine samples were collected from different places in the University of Baghdad, and the best isolate for amino acid production by feathers degradation was by using Streptomyces venezuelae AZ15. The best fermentation system and the optimum culture conditions for amino acid production from feathers as substrate utilizing S. venezuelae AZ15 were elucidated. Using feathers as a substrate, the results showed that the submerged fermentation (SmF) system was the best to give the highest yield of amino acid. Furthermore, the highest yield of total amino acid was gained in feather broth supplemented with 2% glucose and 0.5 % ammonium nitrate, with a pH of 6.0 at 37 °C for 8 days. [ABSTRACT FROM AUTHOR]

The disposal of keratinous wastes produced by several leather industries is evolving into a global problem. Around 1 billion tonnes of keratin waste are released into the environment each year. In the breakdown of tannery waste, certain enzymes, such as keratinases produced from microorganisms, might be a better substitute for synthetic enzymes. Keratinase enzymes are able to hydrolyze gelatin, casein, bovine serum albumin and insoluble protein present in wool, feather. Therefore, in this study, bacterial strains from tannery effluent-contaminated soil and bovine tannery hide were isolated and assessed for their ability to produce the keratinolytic enzyme. Among the six isolates, the strain NS1P showed the highest keratinase activity (298 U/ml) and was identified as Comamonas testosterone through biochemical and molecular characterization. Several bioprocess parameters such as pH, temperature, inoculum size, carbon sources, and nitrogen sources were optimized in order to maximize crude enzyme production. The optimized media were used for inoculum preparation and subsequent biodegradation of hide hairs. The degradation efficacy of the keratinase enzyme produced by Comamonas testosterone was examined by degrading bovine tannery hide hairs, and it was found to be 73.6% after 30 days. The morphology of the deteriorated hair was examined using a field emission scanning electron microscope (FE-SEM), which revealed significant degradation. Thus, our research work has led to the conclusion that Comamonas testosterone may be a promising keratinolytic strain for the biodegradation of tannery bovine hide hair waste and the industrial production of keratinases. [ABSTRACT FROM AUTHOR]

To augment the keratinolytic ability of Bacillus halotolerans L2EN1 isolated from the poultry farm (Nahan, District Sirmour) of Himachal Pradesh, different cultural conditions were optimised using One Variable at a Time (OVAT) approach accompanied by Response Surface Methodology (RSM). Optimisation (OVAT) results revealed that after 3rd day of incubation, maximum enzyme activity was attained at 45 °C, pH 11.0 with 12.5% of inoculum size in the presence of Mn2 + and EDTA in the production medium. Sucrose (1.5%) and yeast extract (2.0%) were observed to be best carbon and nitrogen sources, respectively. A significant increase of 73.19 per cent in the keratinase activity was observed using Central Composite Design (CCD) of RSM. The SDS-PAGE results revealed that crude keratinase is a heterotetramer made up of four polypeptide chains with molecular weights of 17, 37, 40 and 60 kDa. Partial purification by 90 − 100 percent ammonium sulphate gave maximum keratinase production of 22.66 U/mL with purification of 1.68 and yield of 11.47 per cent. The enzyme showed compatibility with different commercial detergents and retained its activity in the order: Reshma (97.77%) > Speed (93.44%) > Tide (79.93%) >Ariel (70.18%) > Surf excel (67.98%) at 50 °C after 1 h of incubation. Wash performance analysis demonstrated that washing with tap water at 18, 28, 35 and 45 °C for 30, 45 and 60 min removed some amount of blood stains from the cotton cloth pieces. However, replacement of detergent's enzyme (Reshma) with crude keratinase achieved complete blood stain removal under same conditions, suggesting its suitability as a potential cleaning additive in detergents for the removal of blood (proteinaceous) stains for long washing cycles (1 h). [ABSTRACT FROM AUTHOR]

A distinctive isolate was discovered and visually recognized as a member of the genus Didymella during a routine examination of Coelomycetes isolated from diverse fruit juices. Based on sequencing of the internal transcribed spacer (ITS), the fungus was identified as Didymella keratinophila since it showed a 100% identity to the type strain. The strain thrived and produced keratinase and collagenase enzymes by hydrolyzing native chicken feathers in submerged fermentation (SmF). After 10 days of fermentation at 30 °C, pH 9 using sodium nitrate as a nitrogen supply produced the highest keratinase activity of 8780 ± 620 U/mL/min, while pH 6 and beef extract produced the maximum collagenase activity of 11,230 ± 1290 U/mL/min. The partially-purified keratinase enzyme worked best at pH 7.0 and 45 °C, exhibiting a specific activity of 44,903 ± 1555 U/mg protein. The activity of the partially-purified collagenase enzyme was excellent at pH 6.0 at 35 °C, generating 15,753 ± 110 U/mg enzyme-specific activity. Mn2+ and K+ were the most efficient inhibitors of keratinases and collagenase, respectively. Both EDTA and metal ions significantly decreased the activity of keratinase and collagenase. This report identified a workable supplier of collagenase and keratinase enzymes derived from chicken feathers, offering a reliable way to exploit and manage these wastes for obtaining high-value products. [ABSTRACT FROM AUTHOR]

In this study, 25 actinomyces isolates were obtained from 10 different poultry farms and tested for their keratinase activity. The isolate with the highest keratinase activity was identified through molecular identification by PCR and sequencing of the 16S rRNA gene to be Streptomyces spp. and was named Streptomyces werraensis KN23 with an accession number of OK086273 in the NCBI database. Sequential mutagenesis was then applied to this strain using UV, H2O2, and SA, resulting in several mutants. The best keratinolytic efficiency mutant was designated as SA-27 and exhibited a keratinase activity of 106.92 U/ml. To optimize the keratinase expression of mutant SA-27, the Response Surface Methodology was applied using different parameters such as incubation time, pH, carbon, and nitrogen sources. The optimized culture conditions resulted in a maximum keratinase specific activity of 129.60 U/ml. The genetic diversity of Streptomyces werraensis KN23 wild type compared with five mutants was studied using Inter-simple sequence repeat (ISSR). The highest total and polymorphic unique bands were revealed in the S. werraensis KN23 and SA-18 mutant, with 51 and 41 bands, respectively. The dendrogram based on combined molecular data grouped the Streptomyces werraensis and mutants into two clusters. Cluster I included SA-31 only, while cluster II contained two sub-clusters. Sub-cluster one included SA-27, and sub-cluster two included SA-26. The sub-cluster two divided into two sub-sub clusters. Sub-sub cluster one included SA-18, while sub-sub cluster two included one group (SA-14 and S. werraensis KN23). [ABSTRACT FROM AUTHOR]

The keratin-degrading bacterium Bacillus licheniformis secretes a keratinase with potential industrial interest. Here, the Keratinase gene was intracellularly expressed in Escherichia coli BL21(DE3) using pET-21b (+) vector. Phylogenetic tree analysis showed that KRLr1 is closely related to Bacillus licheniformis keratinase that belongs to the serine peptidase/subtilisin-like S8 family. Recombinant keratinase appeared on the SDS-PAGE gel with a band of about 38 kDa and was confirmed by western blotting. Expressed KRLr1 was purified by Ni-NTA affinity chromatography with a yield of 85.96% and then refolded. It was found that this enzyme has optimum activity at pH 6 and 37°C. PMSF inhibited the KRLr1 activity and Ca2+ and Mg2+ increased the KRLr1 activity. Using keratin 1% as the substrate, the thermodynamic values were determined as Km 14.54 mM, kcat 912.7 × 10-3 (S-1), and kcat/Km 62.77 (M-1 S-1). Feather digestion by recombinant enzyme using HPLC method, showed that the amino acids cysteine, phenylalanine, tyrosine and lysine had the highest amount compared to other amino acids obtained from digestion. Molecular dynamics (MD) simulation of HADDOCK docking results exhibited that KRLr1 enzyme was able to interact strongly with chicken feather keratine 4 (FK4) compared to chicken feather keratine 12 (FK12). These properties make keratinase KRLr1 a potential candidate for various biotechnological applications. [ABSTRACT FROM AUTHOR]

Keratin is an insoluble fibrous protein found in hair, wool, feather, nail, horns and other epithelial covering. The high resistance of keratin to proteases is due to its densely packed and strongly stabilized by several hydrogen bonds and hydrophobic interactions, in addition to several disulfide bonds. A keratinase enzyme is useful for biotechnological purposes, and also for hydrolysis of de-hairing of bovine hides, poultry feathers, and leather surplus. In the present study, B. cereus KJ72442 was isolated from deteriorated leather samples using feather meal as a substrate and identified by 16SrDNA sequencing and with accession number B. cereus Lr3/2 KP015746). The colony showing the highest zone of keratin hydrolysis on feather meal agar was selected for keratinase production. B. cereus showed efficient keratinase production 41.06±2U/ml at optimum temperature of 37° C and pH 7 after 72h using 1% feather meal as a substrate. B. cereus keratinolytic activity was found to be metalloprotease, as inhibited by 1mM EDTA but not by PMSF.In addition to keratinase, B. cereus KP015746 (Lr3/2) was also proficiently found to produce collagenase, caseinase and gelatinase. On the lab scale, the efficiency of B. cereus Lr3/2 KP015746 keratinase to degrade leather was analyzed and observed excellent results. Hence, the experimental outcome revealed that B.cereus KP015746 (Lr3/2) can be effectively used to treat keratin, collagen rich agro industrial waste ecofriendly. [ABSTRACT FROM AUTHOR]

In this study, we describe the characterization of three efficient chicken feather-degrading Streptomyces bacteria isolated from honeybee samples and assess the impact of their co-cultivation on this activity and antistaphylococcal activity. Streptomyces griseoaurantiacus AD2 was the strain showing the highest keratinolytic activity (4000 U × mL−1), followed by Streptomyces albidoflavus AN1 and Streptomyces drozdowiczii AD1, which both generated approximately 3000 U × mL−1. Moreover, a consortium constituted of these three strains was able to use chicken feathers as its sole nutrient source and growth in such conditions led to a significant increase in antibiotic production. S. griseoaurantiacus AD2 was the only strain that exhibited weak antimicrobial activity against Staphylococcus aureus. UPLC analyses revealed that a significant number of peaks detected in the extracts of co-cultures of the three strains were missing in the extracts of individual cultures. In addition, the production of specialized metabolites, such as undecylprodigiosin and manumycin A, was clearly enhanced in co-culture conditions, in agreement with the results of the antimicrobial bioassays against S. aureus. Our results revealed the benefits of co-cultivation of these bacterial species in terms of metabolic wealth and antibiotic production. Our work could thus contribute to the development of novel microbial-based strategies to valorize keratin waste. [ABSTRACT FROM AUTHOR]

Keratinolytic is a microorganism ability to degrade keratin substrates. Fungi are known to produce the enzyme keratinase. This research was carried out aiming to know the keratinolytic fungi potential in inhibiting Ganoderma boninense fungi. Five isolates of keratinolytic fungi coded A 31, A 18, A 12, K 18, and A 29 have antagonistic ability against G. boninense. A 12 and A 18 had the largest inhibitory diameter of 25.23 mm and 24 mm, respectively, after 7 days of incubation. The ability of A 12 and A 18 antagonisms was observed by testing the supernatant of keratinolytic fungi incubated in a feather meal broth medium. An 18 has the largest antagonism percentage of inhibition at 84.72% against G. boninense, followed by A12 (77.78%) and combination treatment of A 12 + A 18 (73.55%) after 7 days of incubation. Keratinolytic fungi can also grow on minimum salt chitin medium (MSCM) and produced a hydrolysis zone after 3 days of incubation. G. boninense hyphae abnormalities were observed by using a 100x magnification microscope. Moreover, hyphae lysis and dwarf, curved, rolled, and curly hyphae were also observed after giving treatment on A 12, A 18, and the combination treatment of both isolates. These results showed that both keratinolytic fungi isolates and the supernatant are powerful biocontrol agents against G. boninense. There is no previous research report on endophytic fungi inhibiting the growth of Ganoderma boninense. In the future, keratinolytic fungi can be applied in agriculture technology. [ABSTRACT FROM AUTHOR]

Keratinolytic microorganisms are highly valuable for decomposition of poultry waste. This study aimed to isolate keratin-decomposing actinobacteria from poultry farm soils and examine their capacity to decompose feathers. Soil samples were placed in a basal medium with feather meal, which is a deposit of carbon and nitrogen. Nine actinobacterial strains were isolated. Actinobacteria were cultured in the media to show clear feather-decomposing potential. Actinobacterial strains were identified using 16S ribosomal RNA (rRNA) sequencing as being related to Streptomyces rochei AM8. Thus, the supernatant of S. rochei AM8 exhibited keratinolytic enzyme activity. Increased biodecomposition of feathers was recorded in a keratinase assay (0.782 U/mL) for separated cultures. The ability of the selected microorganisms to decompose feathers may be an effective biotechnological solution for managing feather waste from poultry.

Abstract Background With the growing concern for the environment, there are trends that bio-utilization of keratinous waste by keratinases could ease the heavy burden of keratinous waste from the poultry processing and leather industry. Especially surfactant-stable keratinases are beneficial for the detergent industry. Therefore, the production of keratinase by Bacillus cereus YQ15 was improved; the characterization and use of keratinase in detergent were also studied. Results A novel alkaline keratinase-producing bacterium YQ15 was isolated from feather keratin-rich soil and was identified as Bacillus cereus. Based on the improvement of medium components and culture conditions, the maximum keratinase activity (925 U/mL) was obtained after 36 h of cultivation under conditions of 35 °C and 160 rpm. Moreover, it was observed that the optimal reacting temperature and pH of the keratinase are 60 °C and 10.0, respectively; the activity was severely inhibited by PMSF and EDTA. On the contrary, the keratinase showed remarkable stability in the existence of the various surfactants, including SDS, Tween 20, Tween 60, Tween 80, and Triton X-100. Especially, 5% of Tween 20 and Tween 60 increased the activity by 100% and 60%, respectively. Furtherly, the keratinase revealed high efficiency in removing blood stains. Conclusion The excellent compatibility with commercial detergents and the high washing efficiency of removing blood stains suggested its suitability for potential application as a bio-detergent additive.

The keratin-degrading bacterium Bacillus licheniformis secretes a keratinase with potential industrial interest. Here, the Keratinase gene was intracellularly expressed in Escherichia coli BL21(DE3) using pET-21b (+) vector. Phylogenetic tree analysis showed that KRLr1 is closely related to Bacillus licheniformis keratinase that belongs to the serine peptidase/subtilisin-like S8 family. Recombinant keratinase appeared on the SDS-PAGE gel with a band of about 38 kDa and was confirmed by western blotting. Expressed KRLr1 was purified by Ni-NTA affinity chromatography with a yield of 85.96% and then refolded. It was found that this enzyme has optimum activity at pH 6 and 37°C. PMSF inhibited the KRLr1 activity and Ca2+ and Mg2+ increased the KRLr1 activity. Using keratin 1% as the substrate, the thermodynamic values were determined as Km 14.54 mM, kcat 912.7 × 10−3 (S−1), and kcat/Km 62.77 (M−1 S−1). Feather digestion by recombinant enzyme using HPLC method, showed that the amino acids cysteine, phenylalanine, tyrosine and lysine had the highest amount compared to other amino acids obtained from digestion. Molecular dynamics (MD) simulation of HADDOCK docking results exhibited that KRLr1 enzyme was able to interact strongly with chicken feather keratine 4 (FK4) compared to chicken feather keratine 12 (FK12). These properties make keratinase KRLr1 a potential candidate for various biotechnological applications.

Biotransformation of wasted feathers via feather-degrading enzyme has gained immense popularity, low conversion efficiency hinders its scale application, and the main purpose of this study is to improve feather-degrading enzyme production in Bacillus licheniformis. Firstly, keratinase from Bacillus amyloliquefaciens K11 was attained with the best performance for feather hydrolysis, via screening several extracellular proteases from Bacillus; also, feather powder was proven as the most suitable substrate for determination of feather-degrading enzyme activity. Then, expression elements, including signal peptides and promoters, were optimized, and the combination of signal peptide SPSacC with promoter Pdual3 owned the best performance, keratinase activity aggrandized by 6.21-fold. According to amino acid compositions of keratinase and feeding assays, Ala, Val, and Ser were proven as critical precursors, and strengthening these precursors' supplies via metabolic pathway optimization resulted in a 33.59% increase in the keratinase activity. Furthermore, keratinase activity reached 2210.66 U/mL, up to 56.74-fold from the original activity under the optimized fermentation condition in 3-L fermentor. Finally, the biotransformation process of discarded feathers by the fermented keratinase was optimized, and our results indicated that 90.94% of discarded feathers (16%, w/v) were decomposed in 12 h. Our results suggested that strengthening precursor amino acids' supplies was an efficient strategy for enhanced production of keratinase, and this research provided an efficient strain as well as the biotransformation process for discarded feather re-utilization. [ABSTRACT FROM AUTHOR]