Keratinous Materials Research Articles

Methods to Synthesize and Assemble Recombinant Keratins.

Keratin, as one of the most abundant and underexploited protein sources, is a ubiquitous biological material that commonly exists in epithelial cells. Due to the excellent biocompatibility and biodegradability, keratin is widely used in biomedical applications. Previously, these biomaterials were prepared by dissolving and extracting the keratinous materials. However, the keratins obtained by direct extraction is not pure and contain many by-products. Moreover, natural keratins suffer from limited sequence tenability. In comparison, the recombinant keratin proteins produced by recombinant technology can overcome these drawbacks while maintaining the desired chemical and physical characteristics of natural keratins. Accordingly, this chapter mainly introduces the experimental protocols of the recombination of keratin. As these recombinant keratins are often used for assembly of intermediate filaments (IFs) in vitro, assembly protocols are also introduced in this chapter.

Degradation of Temminck's pangolin (Smutsia temminckii) scales with a keratinase for extraction of reproductive steroid hormones

Hormone monitoring in keratinous tissues has become increasingly popular. The insoluble keratin materials are generally pulverised before hormone extraction; however, this is difficult for thicker keratin structures like baleen plates or hooves. A new method, involving the use of keratinase, allows enzymatic digestion of keratin and hormone analysis in the resulting suspension. Pangolins are unique mammals covered in keratinous scales, which are one of the reasons these animals are extensively trafficked. This study aimed to investigate the suitability of Temminck's pangolin scales as hormone matrix for quantifying reproductive steroids. A protocol was developed to digest scales with a keratinase before measuring hormone concentrations. This method can be used to investigate the reproductive endocrinology of Temminck's pangolins but may also be extended to the other extant pangolin species.•Keratinase digests Temminck's pangolin scales and reproductive steroid metabolite concentrations are measurable in the resulting suspension.•Isopropanol is an ideal washing solvent for scales to remove surface contaminants and scale sample mass should be standardised to allow comparisons.•Any section of a scale and scales from any pangolin body region can be used as samples for hormone quantification.

Microbial Keratinase: Next Generation Green Catalyst and Prospective Applications.

The search for novel renewable products over synthetics hallmarked this decade and those of the recent past. Most economies that are prospecting on biodiversity for improved bio-economy favor renewable resources over synthetics for the potential opportunity they hold. However, this field is still nascent as the bulk of the available resources are non-renewable based. Microbial metabolites, emphasis on secondary metabolites, are viable alternatives; nonetheless, vast microbial resources remain under-exploited; thus, the need for a continuum in the search for new products or bio-modifying existing products for novel functions through an efficient approach. Environmental distress syndrome has been identified as a factor that influences the emergence of genetic diversity in prokaryotes. Still, the process of how the change comes about is poorly understood. The emergence of new traits may present a high prospect for the industrially viable organism. Microbial enzymes have prominence in the bio-economic space, and proteases account for about sixty percent of all enzyme market. Microbial keratinases are versatile proteases which are continuously gaining momentum in biotechnology owing to their effective bio-conversion of recalcitrant keratin-rich wastes and sustainable implementation of cleaner production. Keratinase-assisted biodegradation of keratinous materials has revitalized the prospects for the utilization of cost-effective agro-industrial wastes, as readily available substrates, for the production of high-value products including amino acids and bioactive peptides. This review presented an overview of keratin structural complexity, the potential mechanism of keratin biodegradation, and the environmental impact of keratinous wastes. Equally, it discussed microbial keratinase; vis-à-vis sources, production, and functional properties with considerable emphasis on the ecological implication of microbial producers and catalytic tendency improvement strategies. Keratinase applications and prospective high-end use, including animal hide processing, detergent formulation, cosmetics, livestock feed, and organic fertilizer production, were also articulated.

Additive manufacturing of green composites: Poly (lactic acid) reinforced with keratin materials obtained from Angora rabbit hair

AbstractIn this research, additive manufacturing of polylactic acid (PLA) reinforced with keratin was studied. Keratin was obtained from Angora rabbit hair and modified with NaOH. Scanning electron microscopy (SEM) images showed that the modified surfaces were rougher than untreated surfaces. Furthermore, SEM images in the composites' fracture regions showed surface changes, associated with the nature of the reinforcement. Likewise, thermomechanical properties of the composites were attributed to the nature of the reinforcement and the type of keratin. Besides, the 3D printed composites showed higher thermal conductivity values than PLA with the addition of keratin. Cytotoxicity tests revealed an improvement in cell growth compared to the control and PLA. These results are meaningful toward the development of high thermal conductors and biocompatible composites with applications in different fields, where the use of only natural polymers is necessary.

Experimental approximation of the sound absorption coefficient (∝) for 3D printed reentrant auxetic structures of poly lactic acid reinforced with chicken keratin materials

In this research, auxetic composites of polylactic acid (PLA) reinforced by keratin materials (barbs and rachis), were developed through 3D printing manufacture to induce diverse acoustic properties. To evaluate the coefficient of sound absorption, an electro-acoustic experiment was implemented. The capacity of sound absorption for the composites was superior to conventional (honeycomb) and auxetic PLA geometries without reinforcement. Reduction of the free space in cavities (achieved by keratin materials) triggered an increment in the resistance in air flow, improving the sound absorption. Also, increasing the interconnectivity of the cavities provides sound waves with irregular transmission routes, which reduce the wave’s energy. Thus, in this work, acoustic properties are associated with 3D geometry of auxetic composites, shape, and concentration of reinforcements; considering the synergistic effect due to this combination, a range of metamaterials with acoustic capabilities were generated.

A comparative study of materials assembled from recombinant K31 and K81 and extracted human hair keratins

Natural biopolymers have found success in tissue engineering and regenerative medicine applications. Their intrinsic biocompatibility and biological activity make them well suited for biomaterials development. Specifically, keratin-based biomaterials have demonstrated utility in regenerative medicine applications including bone regeneration, wound healing, and nerve regeneration. However, studies of structure-function relationships in keratin biomaterials have been hindered by the lack of homogeneous preparations of materials extracted and isolated from natural sources such as wool and hair fibers. Here we present a side-by-side comparison of natural and recombinant human hair keratin proteins K31 and K81. When combined, the recombinant proteins (i.e. rhK31 and rhK81) assemble into characteristic intermediate filament-like fibers. Coatings made from natural and recombinant dimers were compared side-by-side and investigated for coating characteristics and cell adhesion. In comparison to control substrates, the recombinant keratin materials show a higher propensity for inducing involucrin and hence, maturation in terms of potential skin cell differentiation.

Transferring feather wastes to ductile keratin filaments towards a sustainable poultry industry

Technology for the transformation of waste feathers to quality regenerated filaments has been developed. Regardless of superior properties of natural keratin materials, previously developed regenerated materials from keratin had tensile properties much lower than their natural counterparts due to backbone hydrolysis and inefficient reconstruction of disulfide crosslinkages. In this work, tough keratin filaments have been regenerated from white duck feathers via efficient restoration of disulfide crosslinkages using a dithiol reducing agent. Dithiol substantially reserves free thiol groups in the extraction and formed lengthy intermolecular crosslinkages in regenerated keratin filaments. Due to the high degree of intermolecular reconstruction of disulfide bonds and formation of lengthy crosslinkages via dithiol chain-extension, the keratin filaments exhibited considerable improvements in mechanical properties, especially for ductility and water stability. The tenacity and elongation at break were 160.7 MPa and 14%, respectively. The filaments retained about 80% of the tenacity of natural feathers at either dry or wet conditions and demonstrated stretchability 150% higher than natural feathers. The fiber regeneration technology makes it possible to substitute primary fiber sources by renewable poultry feathers. Successful filament substitution or addition can bring more than 88-billion-dollar revenue. The technology not only contributes to a sustainable fiber and poultry industry but adds substantial values to poultry feathers.

Biochemical Properties of a Partially Purified Protease from Bacillus sp. CL18 and Its Use to Obtain Bioactive Soy Protein Hydrolysates.

Microbial proteases are relevant biocatalysts with diverse applications. Production of protein hydrolysates is recently focused, since they might display biological activities. Therefore, the extracellular protease from Bacillus sp. CL18 was partially purified through ammonium sulfate precipitation (25-50% saturation) and gel filtration chromatography, with a 60.7-fold purification (40,593 U/mg protein) and 21.3% recovery. The partially purified protease (PPP) was characterized as a serine protease, with optimal activity at 51-59 °C and pH 7.4-8.8 and low thermal stability. Thermal inactivation followed first-order kinetics. PPP depended on Ca2+ for higher thermal stability, depicted by increases in half-lives (t1/2), activation energy (Ea), and free energy (ΔG#) for kinetic inactivation. PPP preferentially hydrolyzed casein > soy protein isolate (SPI) >>> keratinous materials. SPI hydrolysis by PPP was further investigated, and the obtained hydrolysates exhibited increased in vitro bioactivities. Hydrolysates displayed antioxidant capacities through the scavenging of synthetic organic radicals and Fe3+-reducing ability. In addition, hydrolysates inhibited the activities of dipeptidyl peptidase IV (DPP IV) and angiotensin-converting enzyme (ACE), suggesting antidiabetic and antihypertensive potentials, respectively. From its biochemical properties, PPP might be used to produce protein hydrolysates with multifunctional bioactivities. Both PPP and SPI hydrolysates can find applications in food biotechnology.

Valorization of refractory keratinous waste using a new and sustainable bio-catalysis

Keratinous materials, the third most abundant polymer in nature after cellulose and chitin, are resistant to degradation by chemical and biological hydrolysis. Conventional biocatalytic conversion of refractory keratinous waste has its intrinsic limitations and disadvantages, such as low-activity and high cost of biocatalysts, being time-consuming, and low yield of value-added bio-based products. In the present work, a novel and simple route for high-efficiency biocatalysis of feather wastes is proposed, using proteolysis with HDSF-assisted conventional protease. This route effectively eliminates the need for substrate-specific keratinases, as it only uses cheap proteases (conventional proteases) to degrade 95% of the feather substrate within 2 h. Thereby, more than 0.8 g of high-value nutrients and bioactive molecules, such as peptides with antidiabetic, anti-hypertension, and antioxidant activities, were produced from 1 g of feather wastes. In addition, different hydrolysis models and degradation mechanisms of keratinolysis and proteolysis of keratins were investigated to gain further insight into the hard-to-degrade biomass-valorization technology. This method can efficiently convert cheap feather wastes into value-added bioproducts in an industrial scale.

Development of an eco-friendly antibacterial textile: lysozyme immobilization on wool fabric.

Lysozyme, a type of natural enzyme, has been widely used for bacteriostatic functionalization of various materials due to its efficient and selective antibacterial properties. Herein, we report the preparation and characterization of an eco-friendly antibacterial textile based on the immobilization of lysozyme from chicken egg white onto wool fibers. Tris(hydroxymethyl)phosphine (THP) was employed as the cross-linker for the immobilization of lysozyme on the surface of wool fiber. The mechanism of THP cross-linking was investigated via phosphorus test, energy-dispersive spectroscopy (EDX) and Fourier transform infrared spectroscopy (FT-IR). The surface staining, optimization of immobilization parameters, morphology, antibacterial properties, and durability of wool fibers with immobilized lysozyme were also assessed. The results show that hydroxymethyl groups of THP reacted with amino groups of wool fiber and lysozyme through Mannich reaction, which successfully immobilized lysozyme on the wool fiber. The wool fibers incorporated with lysozyme had better antibacterial properties and durability compared with the untreated wool fabric. This facile immobilization approach of lysozyme provides an effective strategy for environmentally benign modification and functionalization of keratin and keratin-containing materials.

Fabrication of mechanical robust keratin film by mesoscopic molecular network reconstruction strategy

Regenerated keratin materials have attracted much attention recently. However, practical application of keratin materials is still a challenging issue due to its weak mechanical property. In this study, mechanical robust keratin composite films were designed and engineered at mesoscopic scale by molecular network reconstruction strategy. FTIR and XRD study showed that the β-crystallites structure of silk fibroin template could induce the transformation of free unfolded molecular chains of keratin to β-sheet conformation in keratin composite films and further resulted the controllable manipulating of the mechanical properties of keratin film. The facile mesoscopic molecular network reconstruction strategy is both straightforward and effective for fabricating mechanical robust keratin materials for further application.

Study of Mechanisms of Recombinant Keratin Solubilization with Enhanced Wound Healing Capability

Despite the long-term use and recent development of keratin materials, the mechanism of their solubilization is still not well understood beyond the breakage of disulfide bonds due to the complexit...

Valorisation of waste chicken feathers: Optimisation of keratin extraction from waste chicken feathers by sodium bisulphite, sodium dodecyl sulphate and urea

Extraction of keratin from keratinous waste materials, such as chicken feathers, has been identified as the favourable approach in beneficiation of this biomass. The chemical extractions of keratin by reducing agents are usually preferred because the process is much faster than its counterpart, oxidation extraction. One such reduction extraction is the use of a mixture of sodium bisulphite, sodium dodecyl sulphate and urea. There are at least five factors that may affect the keratin extraction process and its final properties when using this extraction. Even though this extraction method is often used, the effects of its independent variables have not been studied; as a result, the effects of independent variables cannot be fully linked to the extraction process and final keratin properties. Therefore, this study aimed to optimise the extraction of keratin from waste chicken feathers using sodium bisulphite, sodium dodecyl sulphate and urea. The optimisation was statistically performed using Response Surface Methodology (RSM) linked with Box-Behnken Design. After screening the independent variable using one factor at a time method, the concentration of sodium bisulphite, concentration of sodium dodecyl sulphate, reaction temperature and reaction time were chosen for the study. Twenty-nine experiments were statistically designed and executed, and their results were used to analyse the effects of all the independent variables in order to optimise the extraction process. The reaction temperature was found to be the most significant factor, while the concentration of sodium dodecyl sulphate was the most insignificant factor of this extraction process. Independent variables significance order was reaction temperature > reaction time > concentration of NaHSO3 > concentration of NaC12H25SO4. The designed reduced cubic model was significant and was used to predict the protein yield from the keratin extraction using sodium bisulphite.

Azo dying of α-keratin material improves microbial keratinase screening and standardization.

SummaryMicrobial conversion through enzymatic reactions has received a lot of attention as a cost‐effective and environmentally friendly way to recover amino acids and short peptides from keratin materials. However, accurate assessment of microbial keratinase activity is not straightforward, and current available methods lack sensitivity and standardization. Here, we suggest an optimized Azokeratin assay, with substrate generated directly from azo‐dyed raw keratin material. We introduced supernatant filtration in the protocol for optimal stopping of keratinase reactions instead of the widely used trichloroacetic acid (TCA), as it generated biases and impacted the sensitivity. We furthermore suggest a method for standardization of keratinase activity signals using proteinase K, a well‐known keratinase, as a reference enabling reproducibility between studies. Lastly, we evaluated our developed method with several bacterial isolates through benchmarking against a commercial assay (Keratin Azure). Under different setups, the Azokeratin method was more sensitive than commonly used Keratin Azure‐based assays (3‐fold). We argue that this method could be applied with any type of keratin substrate, enabling more robust and sensitive results which can be used for further comparison with other studies, thus representing an important progress within the field of microbial keratin degradation.

Challenges and Opportunities in Identifying and Characterising Keratinases for Value-Added Peptide Production

Keratins are important structural proteins produced by mammals, birds and reptiles. Keratins usually act as a protective barrier or a mechanical support. Millions of tonnes of keratin wastes and low value co-products are generated every year in the poultry, meat processing, leather and wool industries. Keratinases are proteases able to breakdown keratin providing a unique opportunity of hydrolysing keratin materials like mammalian hair, wool and feathers under mild conditions. These mild conditions ameliorate the problem of unwanted amino acid modification that usually occurs with thermochemical alternatives. Keratinase hydrolysis addresses the waste problem by producing valuable peptide mixes. Identifying keratinases is an inherent problem associated with the search for new enzymes due to the challenge of predicting protease substrate specificity. Here, we present a comprehensive review of twenty sequenced peptidases with keratinolytic activity from the serine protease and metalloprotease families. The review compares their biochemical activities and highlights the difficulties associated with the interpretation of these data. Potential applications of keratinases and keratin hydrolysates generated with these enzymes are also discussed. The review concludes with a critical discussion of the need for standardized assays and increased number of sequenced keratinases, which would allow a meaningful comparison of the biochemical traits, phylogeny and keratinase sequences. This deeper understanding would facilitate the search of the vast peptidase family sequence space for novel keratinases with industrial potential.

Construction of Simplified Microbial Consortia to Degrade Recalcitrant Materials Based on Enrichment and Dilution-to-Extinction Cultures.

The capacity of microbes to degrade recalcitrant materials has been extensively explored for environmental remediation and industrial production. Significant achievements have been made with single strains, but focus is now going toward the use of microbial consortia owning to their functional stability and efficiency. However, assembly of simplified microbial consortia (SMC) from complex environmental communities is still far from trivial due to large diversity and the effect of biotic interactions. Here we propose a strategy, based on enrichment and dilution-to-extinction cultures, to construct SMC with reduced diversity for degradation of keratinous materials. Serial dilutions were performed on a keratinolytic microbial consortium pre-enriched from a soil sample, monitoring the dilution effect on community growth and enzymatic activities. An appropriate dilution regime (10–9) was selected to construct a SMC library from the enriched microbial consortium. Further sequencing analysis and keratinolytic activity assays demonstrated that obtained SMC displayed actual reduced microbial diversity, together with various taxonomic composition, and biodegradation capabilities. More importantly, several SMC possessed equivalent levels of keratinolytic efficiency compared to the initial consortium, showing that simplification can be achieved without loss of function and efficiency. This methodology is also applicable to other types of recalcitrant material degradation involving microbial consortia, thus considerably broadening its application scope.

Keratinous materials: Structures and functions in biomedical applications.

Keratins are a family of fibrous proteins anticipated to possess wide-ranging biomedical applications due to their abundance, physicochemical properties and intrinsic biological activity. This review mainly focuses on the biomaterials derived from three major sources of keratins; namely human hair, wool and feather, that have effective applications in tissue engineering, wound healing and drug delivery. This article offers five viewpoints regarding keratin i) an introduction to keratin protein extraction and keratin-based scaffold fabrication methods ii) applications in nerve and bone tissue engineering iii) a review on the keratin dressings applied to different types of wounds to facilitate wound healing and thereby repair the skin iv) the utilization of keratinous materials as a carrier system for therapeutics with a controlled manner v) a discussion regarding the main challenges for using keratin in biomedical applications as well as its future prospects.

Dermatophytosis a worldwide contiguous fungal infection: Growing challenge and few solutions

Dermatophytes are filamentous fungi that survive on keratinous materials. There is a broad prevalence of dermatophytes infection among the world. Dermatophytosis is the disease that mainly caused by different species of dermatophytes within the cutaneous layer of the skin. It has contagious properties to spread from person to another and also from the animal to the human. The skin, hair, and nail of all types of mammalian, including human, are under the risk to develop dermatophytosis. A total of 2530 articles have been investigated in PubMed (n = 1525), Medline (n = 705), and Google Scholar (n = 300). From 2530 articles, just 48 studies were included in this review. This study aimed to review available data and current literature as well as information from personal experiments regarding dermatophytes infection to focus on the dermatophytes general features, dermatophytosis, pathogenesis presented by enzymes production, most common factors associated with infection, prevalence, and treatment of dermatophytes infection, and also to yield a clear vision to other researches about this worldwide predominant contiguous fungal disease.

On the Strength of Hair across Species

Summary The hair of mammals varies in diameter across species from ∼60 μm in humans to over ∼400 μm in giraffes and elephants. Here, we establish that the tensile strength of hair generally decreases with increasing diameter. Although there is a commonality in the morphology of the hair in such differing species as they all possess a cortex surrounded by cuticles, there are also significant differences in the hierarchical structure that contribute to the observed differences in strength. In this work, the internal structure of the hair from different species is examined, with some unique peculiarities in javelina and capybara hair uncovered. The dimensional dependence of hair strength is compared using Weibull predictions. The good correlation of the strength of hair using such Weibull analysis suggests that the failure strength is dictated by the probability distribution of flaws within the cortex.

The Potential of Keratinolytic Fungi for Biotechnological Applications in Leather Manufacture

Keratinophilic fungi are present in soil as decomposers of keratinous substrates, while keratinolytic fungi have the capacity to decompose native keratin, the insoluble fibrous proteins from living organism. Keratin materials, especially by-products from food industry and animal husbandry must be harnessed through innovative, non-polluting and low-cost solutions. The nonpathogenic keratinolytic fungal species produce extracellular keratinases which have many and various applications, one being in leather industry where dehairing process of skin and hides require keratinolytic activity. The present study investigates the biodegradative potential of selected keratinolytic fungal microorganisms expressed towards different types of animal skins. The ability of Fusarium sp. 1 A strain to produce keratinase with a good activity towards animal skins was confirmed. These results suggest that after further studies, Fusarium sp.1A could play an important role in processing of animal wastes.