Alkaline keratinase from Bacillus sp. DRS4 efficiently biodegrades chicken feathers to synthesize improved keratin/bacterial nanocellulose-based bioplastics.

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.

Microbial production of keratinase from Bacillus velezensis strain MAMA: A novel enzyme for eco-friendly degradation of keratin waste.

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).

Identification of molecular interactions of pesticides with keratinase for their potential to inhibit keratin biodegradation.

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. Graphical abstract: Illustration depicting the hypothesis, experimental procedure, and the resultant keratinase-inhibitory potential of selected pesticides.

Perspective on Agricultural Industrialization: Modification Strategies for Enhancing the Catalytic Capacity of Keratinase.

Keratinases is a special hydrolytic enzyme produced by microorganisms, which has the ability to catalyze the degradation of keratin. Currently, keratinases show great potential for application in many agricultural and industrial fields, such as biofermented feed, leather tanning, hair removal, and fertilizer production. However, these potentials have not yet been fully unleashed on an industrial scale. This paper reviews the sources, properties, and catalytic mechanisms of keratinases. Strategies for the molecular modification of keratinases are summarized and discussed in terms of improving the substrate specificity, thermostability, and pH tolerance of keratinases. The modification strategies are also enriched by the introduction of immobilized enzymes and directed evolution. In addition, the selection of modification strategies when facing specific industrial applications is discussed and prospects are provided. We believe that this review serves as a reference for the future quest to extend the application of keratinases from the laboratory to industry.

Effects of various substances on the binding of keratin monomers to S. maltophilia DHHJ cells for the induction of keratinase production.

Biodegradation effectiveness of S. maltophilia DHHJ is determined by its ability to attach to the hydrolyzed feather keratin monomers. This binding capacity can be influenced by many components in the culture medium. Keratin monomers from feathers or those produced by gene overexpression can induce keratinase production in S. maltophilia DHHJ, and several proteases lack the ability to degrade keratin fragments and cysteines. In this study, we co-incubated FITC-labelled keratin monomers with S. maltophilia DHHJ cells in the presence of BSA, DNA, ATP, and several metal ions, and measured fluorescence values and keratinase activity. BSA was found to compete with keratins for cell binding sites, resulting in less keratinase production. DNA did not interfere with cellular binding to keratins revealing unchanged keratinase level. ATP, along with metal ions, enhanced the cellular binding capacity to keratins and increased the production of keratinase by S. maltophilia DHHJ. Fragments of keratin monomers degraded by proteases reduced the ability of cells to bind to keratin and affected enzyme production. Cysteine, a characteristic amino acid of feather keratin, did not have an effect on cellular binding to keratin monomer or on keratinase production. This study will facilitate the tweaking of catalytic parameters to improve feather biodegradation by S. maltophilia DHHJ.

Rational engineering S1' substrate binding pocket to enhance substrate specificity and catalytic activity of thermal-stable keratinase for efficient keratin degradation.

This study reports the rational engineering of the S1' substrate-binding pocket of a thermally-stable keratinase from Pseudomonas aeruginosa 4-3 (4-3Ker) to improve substrate specificity to typical keratinase (K/C > 0.5) and catalytic activity without compromising thermal stability for efficient keratin degradation. Of 10 chosen mutation hotspots in the S1' substrate-binding pocket, the top three mutations M128R, A138V, and V142I showing the best catalytic activity and substrate specificity were identified. Their double and triple combinatorial mutants synergistically overcame limitations of single mutants, fabricating an excellent M128R/A138V/V142I triple mutant which displayed a 1.21-fold increase in keratin catalytic activity, 1.10-fold enhancement in keratin/casein activity ratio, and a 3.13 °C increase in half-inactivation temperature compared to 4-3Ker. Molecular dynamics simulations revealed enhanced flexibility of critical amino acid residues at the substrate access tunnel, improved global protein rigidity, and heightened hydrophobicity within the active site likely underpinned the increased catalytic activity and substrate specificity. Additionally, the triple mutant improved the feather degradation rate by 32.86 % over the wild-type, far exceeding commercial keratinase in substrate specificity and thermal stability. This study exemplified engineering a typical keratinase with enhanced substrate specificity, catalytic activity, and thermal stability from thermally-stable 4-3Ker, providing a more robust tool for feather degradation.

Molecular strategies to enhance the keratinase gene expression and its potential implications in poultry feed industry.

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.

Isolation, identification, and molecular characterization of potential keratinolytic fungus sp. from Southern Assam: relevance to poultry wastes and its biological management.

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).

Improvement in organic solvent resistance of keratinase BLk by directed evolution.

Keratinase, a vital enzyme in hair degradation, requires enhanced stability for industrial applications in the harsh reaction environment used for keratin hydrolysis. Previous studies have focused on improving keratinase thermostability. In this study, directed evolution was applied to enhance the organic solvent stability of the keratinase BLk from Bacillus licheniformis. Three mutants were identified, exhibiting significant enhanced stability in various solvents, although no similar improvements were observed in terms of thermostability. The identified mutations were located on the enzyme surface. The half-lives of the D41A, A24E, and A24Q mutants increased by 47-, 63-, and 61-fold, respectively, in the presence of 50% (v/v) acetonitrile compared to that of the wild type (WT). Similarly, in the presence of 50% (v/v) acetone, the half-lives of these mutants increased by 22-, 27-, and 27-fold compared to that of the WT enzyme. Notably, the proteolytic activity of all the selected mutants was similar to that of the WT enzyme. Furthermore, molecular dynamics simulation was used to assess the possible reasons for enhanced solvent stability. These results suggest that heightened intramolecular interactions, such as hydrogen bonding and hydrophobic interactions, contribute to improved solvent tolerance. The mutants obtained in this study hold significant potential for industrial applications.

Structural and enzymatic characterization of a novel metallo-serine keratinase KerJY-23.

KerJY-23 was a novel keratinase from feather-degrading Ectobacillus sp. JY-23, but its enzymatic characterization and structure are still unclear. In this study, the KerJY-23 was obtained by heterologous expression in Escherichia coli BL21(DE3), and enzymatic properties indicated that KerJY-23 was optimal at 60 °C and pH 9.0 and could be promoted by divalent metal ions or reducing agents. Furthermore, KerJY-23 had a broad substrate specificity towards casein, soluble keratin, and expanded feather powder, but its in vitro degradation against chicken feathers required an additional reducing agent. Homology modeling indicated that KerJY-23 contained a highly conserved zinc-binding HELTH motif and a His-Asp-Ser catalytic triad that belonged to the typical characteristics of M4-family metallo-keratinase and serine-keratinase, respectively. Molecular docking revealed that KerJY-23 achieved a reinforced binding on feather keratin via abundant hydrogen bonding interactions. This work not only deepened understanding of the novel and interesting metallo-serine keratinase KerJY-23, but also provided a theoretical basis for realizing the efficient use of waste feather keratin.

Sustainable valorizing high-protein feather waste utilization through solid-state fermentation by keratinase-enhanced Streptomyces sp. SCUT-3 using a novel promoter.

Feather waste, a rich source of proteins, has traditionally been processed through high-temperature puffing and acid-base hydrolysis, contributing to generation of greenhouse gases and H2S. To address this issue, we employed circular economy techniques to recover the nutritional value of feather waste. Streptomyces sp. SCUT-3, an efficient proteolytic and chitinolytic bacterium, was isolated for feather degradation previously. This study aimed to valorize feather waste for feed purposes by enhancing its feather transformation ability through promoter optimization. Seven promoters were identified through omics analysis and compared to a common Streptomyces promoter ermE*p. The strongest promoter, p24880, effectively enhanced the expression of three candidate keratinases (Sep39, Sep40, and Sep53). The expression efficiency of double-, triple-p24880 and sandwich p24880-sep39-p24880 promoters were further verified. The co-overexpression strain SCUT-3-p24880-sep39-p24880-sep40 exhibited a 16.21-fold increase in keratinase activity compared to the wild-type. Using this strain, a solid-state fermentation process was established that increased the feather/water ratio (w/w) to 1:1.5, shortened the fermentation time to 2.5 days, and increased soluble peptide and free amino acid yields to 0.41 g/g and 0.14 g/g, respectively. The resulting has high protein content (90.49 %), with high in vitro digestibility (94.20 %). This method has the potential to revolutionize the feather waste processing industry.

Identification and characterization of a versatile keratinase, KerZJ, from Stenotrophomonas sp. LMY.

Keratinases have drawn increasing attention in recent decades owing to their catalytic versatility and broad applications from agriculture to medicine. In the present study, we isolated a highly keratinolytic and fibrinolytic bacterium from the campus soil and named it Stenotrophomonas sp. LMY based on genetic information. To identify the potential keratinase genes, the genome sequence of the strain was obtained and analyzed. Sequence alignment and comparison revealed that the protein 1_737 (KerZJ) had the highest sequence homology to a reported keratinase KerBL. We recombinantly expressed KerZJ in Escherichia coli Origami™ (DE) pLysS and purified it to homogeneity. KerZJ showed the highest activity at 40 °C and pH 9.0, and metal ions exhibited no significant effects on its activity. Although reducing agents would break the disulfide bonds in KerZJ and reduce its activity, KerZJ still exhibited the ability to hydrolyze feather keratin in the presence of ß-ME. KerZJ could efficiently digest human prion proteins. In addition, KerZJ showed fibrinolytic activity on fibrin plates and effectively eliminated blood clots in a thrombosis mouse model without side effects. Our results suggest that KerZJ is a versatile keratinase with significant potential for keratin treatment, decontamination of prions, and fibrinolytic therapy.

The flexible linker and CotG were more effective for the spore surface display of keratinase KERQ7.

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.

Effective biodegradation on chicken feather by the recombinant KerJY-23 Bacillus subtilis WB600: A synergistic process coupled by disulfide reductase and keratinase.

Keratin wastes are abundantly available but rich in hard-degrading fibrous proteins, and the keratinase-producing microorganisms have gained significant attention due to their biodegradation ability against keratinous materials. In order to improve the degradation efficiency of feather keratins, the keratinase gene (kerJY-23) from our previously isolated feather-degrading Ectobacillus sp. JY-23 was overexpressed in Bacillus subtilis WB600 strain. The recombinant KerJY-23 strain degraded chicken feathers rapidly within 48 h, during which the activities of disulfide reductase and keratinase KerJY-23 were sharply increased, and the free amino acids especially the essential phenylalanine and tyrosine were significantly accumulated in feather hydrolysate. The results of structural characterizations including scanning electron microscopy, Fourier transform infrared spectrum, X-ray diffraction, and X-ray photoelectron spectroscopy, demonstrated that the feather microstructure together with the polypeptide bonds and SS bonds in feather keratins were attacked and destroyed by the recombinant KerJY-23 strain. Therefore, the recombinant KerJY-23 strain contributed to feather degradation through the synergistic action of the secreted disulfide reductase to break the SS bonds and keratinase (KerJY-23) to hydrolyze the polypeptide bonds in keratins. This study offers a new insight into the underlying mechanism of keratin degradation, and provides a potential recombinant strain for the valorization of keratin wastes.

Reducing Cellular Autolysis of Bacillus subtilis to Improve Keratinase Production.

Bacillus subtilis has been shown to be an excellent expression host for keratinases due to its powerful secretion system. However, cellular autolysis limits its production capacity. Here, we selected seven genes with significantly upregulated transcript levels from 15 genes associated with cellular autolysis as knockout targets by qRT-PCR and constructed a total of 127 strains to reduce cellular autolysis. Among them, the biomass of B. subtilis BSΔXLPC-ker deficient in xpf, lytC, pcfA, and cwlC increased by 57%. This was confirmed by cell staining, green fluorescent protein imaging, and extracellular nucleic acid leakage assay. Keratinase activity was increased by 1.46-fold in the 5 L fermenter. In addition, the activities of nattokinase and subtilisin E were also increased by 1.50-fold and 1.43-fold, respectively, in the modified chassis cells, which further confirms the generalizability of the strategy. Thus, reducing cellular autolysis to increase the ability of B. subtilis to produce subtilisins is promising.

Response surface methodology based optimization of keratinase from Bacillus velezensis strain ZBE1 and nanoparticle synthesis, biological and molecular characterization.

The increasing demands of keratinases for biodegradation of recalcitrant keratinaceous waste like chicken feathers has lead to research on newer potential bacterial keratinases to produce high-value products with biological activities. The present study reports a novel keratinolytic bacterium Bacillus velezensis strain ZBE1 isolated from deep forest soil of Western Ghats of Karnataka, which possessed efficient feather keratin degradation capability and induced keratinase production. Production kinetics depicts maximum keratinase production (11.65 U/mL) on 4th day with protein concentration of 0.61 mg/mL. Effect of various physico-chemical factors such as, inoculum size, metal ions, carbon and nitrogen sources, pH and temperature influencing keratinase production were optimized and 3.74 folds enhancement was evidenced through response surface methodology. Silver (AgNP) and zinc oxide (ZnONP) nanoparticles with keratin hydrolysate produced from chicken feathers by the action of keratinase were synthesized and verified with UV-Visible spectroscopy that revealed biological activities like, antibacterial action against Bacillus cereus and Escherichia coli. AgNP and ZnONP also showed potential antioxidant activities through radical scavenging activities by ABTS and DPPH. AgNP and ZnONP revealed cytotoxic effect against MCF-7 breast cancer cell lines with IC50 of 5.47 µg/ml and 62.26 µg/ml respectively. Characterizations of nanoparticles were carried out by Fourier transform infrared spectroscopy, scanning electron microscopy with energy dispersive X-ray, X-ray diffraction, thermogravimetric analysis and atomic force microscopy analysis to elucidate the thermostability, structure and surface attributes. The study suggests the prospective applications of keratinase to trigger the production of bioactive value-added products and significant application in nanotechnology in biomedicine.

Biodegradation and valorization of feather waste using the keratinase-producing bacteria and their application in environmentally hazardous industrial processes.

Poultry feathers are widely discarded as waste worldwide and are considered an environmental pollutant and a reservoir of pathogenic bacteria. Therefore, developing sustainable and environmentally friendly methods for managing feather waste is one of the important environmental protection requirements. In this study, we investigated a rapid and eco-friendly method for the degradation and valorization of feather waste using keratinase-producing Pseudomonas geniculata H10, and evaluated the applicability of keratinase in environmentally hazardous chemical processes. Strain H10 completely degraded chicken feathers within 48 h by producing keratinase using them as sources of carbon, nitrogen, and sulfur. The culture contained a total of 402.8 µM amino acids, including 8 essential amino acids, which was higher than the chemical treatment. Keratinase was a serine-type metalloprotease with optimal temperature and pH of 30 °C and 9, respectively, and showed relatively high stability at 10-40 °C and pH 3-10. Keratinase was also able to degrade various insoluble keratins such as duck feathers, wool, human hair, and nails. Furthermore, keratinase exhibited more efficient depilation and wool modification than chemical treatment, as well as novel functionalities such as nematicidal and exfoliating activities. This suggests that strain H10 is a promising candidate for the efficient degradation and valorization of feather waste, as well as the improvement of current industrial processes that use hazardous chemicals.

Bioconversion of feather waste into bioactive nutrients in water by Bacillus licheniformis WHU.

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.

Characterization of the Keratinolytic Activity of Three Streptomyces Strains and Impact of Their Co-Cultivation on This Activity.

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.

Isolation of a novel feather-degrading Ectobacillus sp. JY-23 strain and characterization of a new keratinase in the M4 metalloprotease family.

Microbial keratinases have prominent potential in biotransformation of recalcitrant keratin substrates to value-added products which has made keratinases a research focus in the past decades. In this study, an efficient feather-degrading bacterium was isolated and identified as a novel species in Ectobacillus genus and designated as Ectobacillus sp. JY-23. The degradation characteristics analysis revealed that Ectobacillus sp. JY-23 could utilize chicken feathers (0.4% w/v) as the sole nutrient source and degraded 92.95% of feathers in 72 h. A significant increase in sulfite and free sulfydryl group content detected in the feather hydrolysate (culture supernatant) indicated efficient reduction of disulfide bonds, which inferred that the degradation mechanism of isolated strain was a synergetic action of sulfitolysis and proteolysis. Moreover, abundant amino acids were also detected, among which proline and glycine were the predominant free amino acids. Then, the keratinase of Ectobacillus sp. JY-23 was mined and Y1_15990 was identified as the keratinase encoding gene of Ectobacillus sp. JY-23 and designated as kerJY-23. Escherichia coli strain overexpressing kerJY-23 degraded chicken feathers in 48 h. Finally, bioinformatics prediction of KerJY-23 demonstrated that it belonged to the M4 metalloprotease family, which was a third keratinase member in this family. KerJY-23 showed low sequence identity to the other two keratinase members, indicating the novelty of KerJY-23. Overall, this study presents a novel feather-degrading bacterium and a new keratinase in the M4 metalloprotease family with remarkable potential in feather keratin valorization.