Impact of solvent dry down, phase change, vehicle pH and slowly reversible keratin binding on skin penetration of cosmetic relevant compounds: II. Solids.

We extended a mechanistic, physics-based framework of the dry down process, previously developed for liquids and electrolytes, to solids and coded it into the latest UB/UC/P&G skin permeation model, herein renamed DigiSkin. The framework accounts for the phase change of the permeant from dissolved in a solvent (liquid) to precipitated on the skin surface (solid). The evaporation rate for the solid is reduced due to lower vapor pressure for the solid state versus subcooled liquid. These vapor pressures may differ by two orders of magnitude. The solid may gradually redissolve and penetrate the skin. The framework was tested by simulating the in vitro human skin permeation of the 38 cosmetically relevant solid compounds reported by Hewitt et al., J. Appl. Toxicol. 2019, 1-13. The more detailed handling of the evaporation process greatly improved DigiSkin evaporation predictions (r2 = 0.89). Further, we developed a model reliability prediction score classification using diverse protein reactivity data and identified that 15 of 38 compounds are out of model scope. Dermal delivery predictions for the remaining chemicals have excellent agreement with experimental data. The analysis highlighted the sensitivity of water solubility and equilibrium vapor pressure values on the DigiSkin predictions outcomes influencing agreement with the experimental observations.

Exploring keratin composition variability for sustainable thermal insulator design.

In pursuing sustainable thermal insulation solutions, this study explores the integration of human hair and feather keratin with alginate. The aim is to assess its potential in thermal insulation materials, focusing on the resultant composites' thermal and mechanical characteristics. The investigation uncovers that the type and proportion of keratin significantly influence the composites' porosity and thermal conductivity. Specifically, higher feather keratin content is associated with lesser sulfur and reduced crosslinking due to shorter amino acids, leading to increased porosity and pore sizes. This, in turn, results in a decrease in ß-structured hydrogen bond networks, raising non-ordered protein structures and diminishing thermal conductivity from 0.044 W/(m·K) for pure alginate matrices to between 0.033 and 0.038 W/(m·K) for keratin-alginate composites, contingent upon the specific ratio of feather to hair keratin used. Mechanical evaluations further indicate that composites with a higher ratio of hair keratin exhibit an enhanced compressive modulus, ranging from 60 to 77 kPa, demonstrating the potential for tailored mechanical properties to suit various applications. The research underscores the critical role of sulfur content and the crosslinking index within keratin's structures, significantly impacting the thermal and mechanical properties of the matrices. The findings position keratin-based composites as environmentally friendly alternatives to traditional insulation materials.

The dark side of the wool? From wool wastes to keratin microfilaments through the solution blow spinning process.

The valorization of discarded wool from dairy sheep breeding is a challenging issue. The most proposed strategies lie in the processing of keratin extracted from wool without reducing the molecular weight of the protein chains (the high molecular weight-HMW keratin). Here, the HMW keratin has been spun for the first time by solution blow spinning. A screening study of the process carried out with a 2-level full factorial design revealed that keratin filaments can be obtained by using the polyethylene oxide at 900 kDa, a 2 bar air pressure, and a 30 cm needle-collector distance. An annealing at 80 °C for 15 min, at pH 3.5 with citric acid contributes to increasing the viscosity of the keratin solutions thereby allowing the production of defect-free and water-stable filaments having diameters from 1 to 6 µm. A negligible toxic effect was observed after 24 and 48 h on HT29 epithelial cells and normal blood cells displayed behavior similar to the control demonstrating that the patches are hemocompatible. Therefore, the developed SBS process of keratin aqueous solutions could represent a valuable platform for developing patches that need to be blood-contacting and deposited in-situ.

Structural determinants of intermediate filament mechanics.

Intermediate filaments (IFs) are integral to the cell cytoskeleton, supporting cellular mechanical stability. Unlike other cytoskeletal components, the detailed structure of assembled IFs has yet to be resolved. This review highlights new insights, linking the complex IF hierarchical assembly to their mechanical properties and impact on cellular functions. While we focus on vimentin IFs, we draw comparisons to keratins, showcasing the distinctive structural and mechanical features that underlie their unique mechanical responses.

Tannic acid modified keratin/sodium alginate/carboxymethyl chitosan biocomposite hydrogels with good mechanical properties and swelling behavior.

Natural polymer-based hydrogels have demonstrated great potential as wound-healing dressings. They help to maintain a moist wound environment as well as promote faster healing. In this work, a multifunctional hydrogel was prepared using keratin, sodium alginate, and carboxymethyl chitosan with tannic acid modification. Micro-morphology of hydrogels has been performed by scanning electron microscopy. Fourier Transform Infrared Spectroscopy reveals the presence of hydrogen bonding. The mechanical properties of the hydrogels were examined using a universal testing machine. Furthermore, we investigated several properties of the modified hydrogel. These properties include swelling rate, water retention, anti-freezing properties, antimicrobial and antioxidant properties, hemocompatibility evaluation and cell viability test in vitro. The modified hydrogel has a three-dimensional microporous structure, the swelling rate was 1541.7%, the elastic modulus was 589.74 kPa, the toughness was 211.74 kJ/m3, and the elongation at break was 75.39%, which was similar to the human skin modulus. The modified hydrogel also showed inhibition of S. aureus and E. coli, as well as a DPPH scavenging rate of 95%. In addition, the modified hydrogels have good biological characteristics. Based on these findings, the K/SA/CCS hydrogel holds promise for applications in biomedical engineering.

Lacticaseibacillus rhamnosus encapsulated cross-linked Keratin-Chitosan hydrogel for removal of patulin from apple juice.

In this study, we developed a hydrogel from cross-linked keratin and chitosan (KC) to remove patulin (PAT) from apple juice. We explored the potential of incorporating Lactobacillus rhamnoses into the KC hydrogel (KC-LR) and tested its effectiveness in removing PAT from simulated juice solutions and real apple juice. The KC hydrogel was developed through a dynamic disulfide cross-linking reaction. This cross-linked hydrogel network provided excellent stability for the probiotic cells, achieving 99.9 % immobilization efficiency. In simulated juice with 25 mg/L PAT, the KC and KC-LR hydrogels showed removal efficiencies of 85.2 % and 97.68 %, respectively, using 15 mg mL-1 of the prepared hydrogel at a temperature of 25 °C for 6 h. The KC and KC-LR hydrogels achieved 76.3 % and 83.6 % removal efficiencies in real apple juice systems, respectively. Notably, the encapsulated probiotics did not negatively impact the juice quality and demonstrated reusability for up to five cycles of the PAT removal process.

[Identification of horn-derived animal medicines based on detection of keratin-derived characteristic peptides].

Due to the highly stable structure of keratin, the extraction and dissolution steps of animal medicines rich in keratin are complex, which seriously restricts the detection efficiency and flux. Therefore, this study simplified the pre-treatment steps of horn samples and optimized the detection methods of characteristic peptides to improve the efficiency of identifying the specificity of horn-derived animal medicines. For detection of the characteristic peptides in horn-derived animal medicines treated with/without iodoace-tamide(IAA), the ion pair conditions of the characteristic peptides were optimized, and the retention time, intensity and other data of the specific peptides were compared between the samples treated with/without IAA. Two pre-treatment methods, direct enzymatic hydrolysis and total protein extraction followed by enzymatic hydrolysis, were used to prepare horn-derived animal medicine samples. The effects of different methods on the detection of specific peptides in the samples of Saiga antelope horn, water buffalo horn, goat horn, and yak horn were compared regarding the retention time of specific peptides and ion intensity. The results indicated that after direct enzymatic hydrolysis, the specific peptides in the samples without IAA treatment can be detected. Compared with the characteristic peptides in the samples treated with IAA, their retention time shifted back and the mass spectrometry response slightly decreased. The specific peptides of the samples without IAA treatment had good specificity and did not affect the specificity identification of horn-derived animal medicines. Overall, the process of direct enzymatic hydrolysis can be used to treat horn samples, omitting the steps of protein extraction and dithiothreitol and IAA treatment, significantly improving the pre-treatment efficiency without affecting the specificity identification of horn-derived animal medicines. This study provides ideas for quality research and standard improvement of horn-derived animal medicines.

Additive-free Absorbable Keratin Sponge With Procoagulant Activity for Noncompressible Hemostasis.

The development of a natural, additive-free, absorbable sponge with procoagulant activity for noncompressible hemostasis remains a challenging task. In this study, we extracted high molecular weight keratin (HK) from human hair and transformed it into a hemostatic sponge with a well-interconnected pore structure using a foaming technique, freeze-drying, and oxidation cross-linking. By controlling the cross-linking degree, the resulting sponge demonstrated excellent liquid absorption ability, shape recovery characteristics, and robust mechanical properties. The HK10 sponge exhibited rapid liquid absorption, expanding up to 600% within 5 s. Moreover, the HK sponge showed superior platelet activation and blood cell adhesion capabilities. In SD rat liver defect models, the sponges demonstrated excellent hemostatic performance by sealing the wound and expediting coagulation, reducing the hemostatic time from 825 to 297 s. Furthermore, HK sponges have excellent biosafety, positioning them as a promising absorbable sponge with the potential for the treatment of noncompressible hemostasis.

Design, fabrication, and evaluation of keratin and pectin incorporated supramolecular structured zero-oxidation state selenium nanogel blended 3D printed transdermal patch.

This study investigates the synthesis of selenium nanoparticles (SeNPs), owing to the low cost and abundance of selenium. However, the toxicity of SeNP prompts the development of a selenium nanocomposite (SeNC) containing pectin, keratin, and ferulic acid to improve the bioactivity of Se[0]. Further, incorporating the SeNC in a suitable formulation for drug delivery as a transdermal patch was worth studying. Accordingly, various analytical techniques were used to characterize the SeNPs and the SeNC, confirming successful synthesis and encapsulation. The SeNC exhibited notable particle size of 448.2 ± 50.2 nm, high encapsulation efficiency (98.90 % ± 2.4 %), 28.1 ± 0.45 drug loading, and sustained drug release at pH 5.5. Zeta potential and XPS confirmed the zero-oxidation state. The supramolecular structure was evident from spectral analysis endorsing the semi-crystalline nature of the SeNC and SEM images showcasing flower-shaped structures. Further, the SeNC demonstrated sustained drug release (approx. 22 % at 48 h) and wound-healing potential in L929 fibroblast cells. Subsequently, the SeNC loaded into a gelling agent exhibited shear thinning properties and improved drug release by nearly 58 %. A 3D printed reservoir-type transdermal patch was developed utilizing the SeNC-loaded gel, surpassing commercially available patches in characteristics such as % moisture uptake, tensile strength, and hydrophobicity. The patch, evaluated through permeation studies and CAM assay, exhibited controlled drug release and angiogenic properties for enhanced wound healing. The study concludes that this patch can serve as a smart dressing with tailored functionality for different wound stages, offering a promising novel drug delivery system for wound healing.

Using highly water-stable wool keratin/CsPbBr3 nanocrystals as a portable amine-responsive fluorescent test strip for onsite visual detection of food freshness.

Perovskite nanocrystals (PNCs) have emerged as promising candidates for fluorescent probes owing to their outstanding photoelectric properties. However, the conventional CsPbBr3 (CPB) NCs are extremely unstable in water, which has seriously limited their sensing applications in water environment. Herein, we present a powerful ligand engineering strategy for fabricating highly water-stable CPB NCs by using a biopolymer of wool keratin (WK) as the passivator and the polyaryl polymethylene isocyanate (PAPI) as the cross-linking agent. In particular, WK with multi-functional groups can serve as a polydentate ligand to firmly passivate CPB NCs by the ligand exchange process in hot toluene; and then the addition of PAPI can further encapsulate CPB NCs by the crosslinking reaction between PAPI and WK. Consequently, the as-prepared CPB/WK-PAPI NCs can maintain âˆ¼ 80 % of their relative photoluminescence (PL) intensity after 60 days in water, and they still maintain âˆ¼ 40 % of their relative PL intensity even after 512 days in the same environment, which is one of the best water stabilities compared previously reported polymer passivation methods. As a proof-of their application, the portable CPB/WK-PAPI NCs-based test strips are further developed as a fluorescent nanoprobe for real-time and visual monitoring amines and food freshness. Among various amine analytes, the as-prepared test strips exhibit higher sensitivity towards conjugated amines, achieving a remarkable detection limit of 18.3 nM for pyrrole. Our research not only introduces an innovative strategy involving natural biopolymers to enhance the water stability of PNCs, but also highlights the promising potential of PNCs for visually and portably detecting amines and assessing food freshness.

Curcumin-loaded keratin-chitosan hydrogels for enhanced peripheral nerve regeneration.

Peripheral nerve injury often leads to symptoms of motor and sensory impairment, and slow recovery of nerves after injury and limited treatment methods will aggravate symptoms or even lead to lifelong disability. Curcumin can promote peripheral nerve regeneration, but how to accurately deliver the appropriate concentration of curcumin in the local peripheral nerve remains to be solved. In this study, we designed a human hair keratin/chitosan (C/K) hydrogel with sodium tripolyphosphate ions crosslinked to deliver curcumin topically. Chitosan improves the mechanical properties of hydrogels and keratin improves the biocompatibility of hydrogels. C/K hydrogel showed good cytocompatibility, histocompatibility and degradability. In vitro experiments showed that hydrogels can continuously release curcumin for up to 10 days. In addition, a comprehensive analysis of behavioral, electrophysiological, histology, and target organ recovery results in animal experiments showed that locally delivered curcumin can enhance nerve regeneration in addition to hydrogels. In short, we provide a new method that combines the advantages of human hair keratin, chitosan, and curcumin for nerve damage repair.

Unraveling UVB effects: Catalase activity and molecular alterations in the stratum corneum.

AIM: Ultraviolet B (UVB) radiation can compromise the functionality of the skin barrier through various mechanisms. We hypothesize that UVB induce photochemical alterations in the components of the outermost layer of the skin, known as the stratum corneum (SC), and modulate its antioxidative defense mechanisms. Catalase is a well-known antioxidative enzyme found in the SC where it acts to scavenge reactive oxygen species. However, a detailed characterization of acute UVB exposure on the activity of native catalase in the SC is lacking. Moreover, the effects of UVB irradiation on the molecular dynamics and organization of the SC keratin and lipid components remain unclear. Thus, the aim of this work is to characterize consequences of UVB exposure on the structural and antioxidative properties of catalase, as well as on the molecular and global properties of the SC matrix surrounding the enzyme. EXPERIMENTS: The effect of UVB irradiation on the catalase function is investigated by chronoamperometry with a skin covered oxygen electrode, which probes the activity of native catalase in the SC matrix. Circular dichroism is used to explore changes of the catalase secondary structure, and gel electrophoresis is used to detect fragmentation of the enzyme following the UVB exposure. UVB induced alterations of the SC molecular dynamics and structural features of the SC barrier, as well as its water sorption behavior, are investigated by a complementary set of techniques, including natural abundance 13C polarization transfer solid-state NMR, wide-angle X-ray diffraction, Fourier transform infrared (FTIR) spectroscopy, and dynamic vapor sorption microbalance. FINDINGS: The findings show that UVB exposure impairs the antioxidative function of catalase by deactivating both native catalase in the SC matrix and lyophilized catalase. However, UVB radiation does not alter the secondary structure of the catalase nor induce any observable enzyme fragmentation, which otherwise could explain deactivation of its function. NMR measurements on SC samples show a subtle increase in the molecular mobility of the terminal segments of the SC lipids, accompanied by a decrease in the mobility of lipid chain trans-gauche conformers after high doses of UVB exposure. At the same time, the NMR data suggest increased rigidity of the polypeptide backbone of the keratin filaments, while the molecular mobility of amino acid residues in random coil domains of keratin remain unaffected by UVB irradiation. The FTIR data show a consistent decrease in absorbance associated with lipid bond vibrations, relative to the main protein bands. Collectively, the NMR and FTIR data suggest a small modification in the composition of fluid and solid phases of the SC lipid and protein components after UVB exposure, unrelated to the hydration capacity of the SC tissue. To conclude, UVB deactivation of catalase is anticipated to elevate oxidative stress of the SC, which, when coupled with subtle changes in the molecular characteristics of the SC, may compromise the overall skin health and elevate the likelihood of developing skin disorders.

The effects of keratin-coated titanium on osteoblast function and bone regeneration.

Wool derived keratin, due to its demonstrated ability to promote bone formation, has been suggested as a potential bioactive material for implant surfaces. The aim of this study was to assess the effects of keratin-coated titanium on osteoblast functionin vitroand bone healingin vivo. Keratin-coated titanium surfaces were fabricated via solvent casting and molecular grafting. The effect of these surfaces on the attachment, osteogenic gene, and osteogenic protein expression of MG-63 osteoblast-like cells were quantifiedin vitro. The effect of these keratin-modified surfaces on bone healing over three weeks using an intraosseous calvaria defect was assessed in rodents. Keratin coating did not affect MG-63 proliferation or viability, but enhanced osteopontin, osteocalcin and bone morphogenetic expressionin vitro. Histological analysis of recovered calvaria specimens showed osseous defects covered with keratin-coated titanium had a higher percentage of new bone area two weeks after implantation compared to that in defects covered with titanium alone. The keratin-coated surfaces were biocompatible and stimulated osteogenic expression in adherent MG-63 osteoblasts. Furthermore, a pilot preclinical study in rodents suggested keratin may stimulate earlier intraosseous calvaria bone healing.

Fabrication of silver nanoparticles decorated on sodium alginate microbeads enriched with keratin and investigation of its catalytic and antioxidant activity.

In this study, an environmentally friendly, effective, easily synthesizable and recoverable nano-sized catalyst system (Ag@NaAlg-keratin) was designed by decorating Ag nanoparticles on microbeads containing sodium alginate (NaAlg) and keratin obtained from goose feathers. The structure, morphology and crystallinity of the Ag@NaAlg-keratin nanocatalyst were evaluated by XRD, FT-IR, FE-SEM, EDS/EDS mapping and TEM analyses. Catalytic ability of designed Ag@NaAlg-keratin nanocatalyst was then investigated against 4-nitrophenol (4-NP) and methyl orange (MO) reductions. Ag@NaAlg-keratin nanocatalyst effectively reduced 4-NP in 6 min and MO in 5 min, with rate constants of 0.17 min-1 and 0.16 min-1, respectively. Additionally, activation energies (Ea) were found as 39.8 kJ/mol for 4-NP and 37.9 kJ/mol for MO. Performed recyclability tests showed that the Ag@NaAlg-keratin nanocatalyst was easily recovered due to its microbead form and successfully reused five times, maintaining both its activity and structure. Furthermore, antioxidant activity of Ag@NaAlg-keratin nanocatalyst was the highest (73.16 %).

Keratin-based bioplastics extracted from chicken feathers: Effect of chitosan concentration on the structural, chemical bonding, and mechanical properties of bioplastics.

Keratin was synthesized by alkaline hydrolysis from chicken feathers and then continue by casting method for producing bioplastics with additional various amounts of chitosan as a filler, polyvinyl alcohol (PVA) and glycerol as a plasticizer. The main purpose is analysis the effect of chitosan on the structural properties using quantitative analysis of X-ray diffraction (XRD) spectra, chemical bonding by Fourier transforms infrared (FTIR) spectra, and mechanical properties by texture analyser to the keratin-based bioplastics. Biodegradation of bioplastics was analysed from the loss of weight by burying in the soil. It's found that, the additional of chitosan (0 %, 2 %, 5 %, and 8 %) increased the crystallinity of bioplastics by 11.83 %, 11.12 %, 18.99 %, and 17.03 %, respectively, but decreasing tensile strength and elasticity of bioplastics. Degradation of bioplastic keratin-based shows that the addition of chitosan can reduce the degradation time which is directly proportional to the loss of CO bonds. The highest degradation rate is 89.29 % in 49 days for keratin-based bioplastics with 8 % chitosan, indicated that high potential for future production.

Keratin-containing scaffolds for tissue engineering applications: a review.

In tissue engineering and regenerative medicine applications, the utilization of bioactive materials has become a routine tool. The goal of tissue engineering is to create new organs and tissues by combining cell biology, materials science, reactor engineering, and clinical research. As part of the growth pattern for primary cells in an organ, backing material is frequently used as a supporting material. A porous three-dimensional (3D) scaffold can provide cells with optimal conditions for proliferating, migrating, differentiating, and functioning as a framework. Optimizing the scaffolds' structure and altering their surface may improve cell adhesion and proliferation. A keratin-based biomaterials platform has been developed as a result of discoveries made over the past century in the extraction, purification, and characterization of keratin proteins from hair and wool fibers. Biocompatibility, biodegradability, intrinsic biological activity, and cellular binding motifs make keratin an attractive biomaterial for tissue engineering scaffolds. Scaffolds for tissue engineering have been developed from extracted keratin proteins because of their capacity to self-assemble and polymerize into intricate 3D structures. In this review article, applications of keratin-based scaffolds in different tissues including bone, skin, nerve, and vascular are explained based on common methods of fabrication such as electrospinning, freeze-drying process, and sponge replication method.

Defining a timeline of colon pathologies after keratin 8 loss: rapid crypt elongation and diarrhea are followed by epithelial erosion and cell exfoliation.

Keratins are epithelial intermediate filament proteins that play a crucial role in cellular stress protection, with K8 being the most abundant in the colon. The intestinal epithelial-specific K8-deficient mouse model (K8flox/flox;Villin-Cre) exhibits characteristics of inflammatory bowel disease, including diarrhea, crypt erosion, hyperproliferation, and decreased barrier function. Nevertheless, the order in which these events occur and whether they are a direct cause of K8 loss or a consequence of one event inducing another remains unexplored. Increased knowledge about early events in the disruption of colon epithelial integrity would help to understand the early pathology of inflammatory and functional colon disorders and develop preclinical models and diagnostics of colonic diseases. Here, we aimed to characterize the order of physiological events after Krt8 loss by utilizing K8flox/flox;Villin-CreERt2 mice with tamoxifen-inducible Krt8 deletion in intestinal epithelial cells, and assess stool analysis as a noninvasive method to monitor real-time gene expression changes following Krt8 loss. K8 protein was significantly decreased within a day after induction, followed by its binding partners, K18 and K19 from day 4 onward. The sequential colonic K8 downregulation in adult mice leads to immediate diarrhea and crypt elongation with activation of proliferation signaling, followed by crypt loss and increased neutrophil activity within 6-8 days, highlighting impaired water balance and crypt elongation as the earliest colonic changes upon Krt8 loss. Furthermore, epithelial gene expression patterns were comparable between colon tissue and stool samples, demonstrating the feasibility of noninvasive monitoring of gut epithelia in preclinical research utilizing Cre-LoxP-based intestinal disease models.NEW & NOTEWORTHY Understanding the order in which physiological and molecular events occur helps to recognize the onset of diseases and improve their preclinical models. We utilized Cre-Lox-based inducible keratin 8 deletion in mouse intestinal epithelium to characterize the earliest events after keratin 8 loss leading to colitis. These include diarrhea and crypt elongation, followed by erosion and neutrophil activity. Our results also support noninvasive methodology for monitoring colon diseases in preclinical models.

Green syntheses of silk fibroin/wool keratin-protected AuAg nanoclusters with enhanced fluorescence for multicolor and patterned anti-counterfeiting.

Counterfeiting is a serious worldwide issue that threatens human health and economic security. How to apply anti-counterfeiting techniques to textile materials remains a great challenge. Herein, we report bimetallic AuAg nanoclusters (NCs) synthesized by one-step reduction of chloroauric acid (HAuCl4) and silver nitrate (AgNO3) with wool keratin (WK) as reducer and silk fibroin (SF) as stabilizer. The strongest orange-red fluorescence under ultraviolet light as well as the highest zeta potential absolute values of -27.97 mV were simultaneously realized in the optimal proportion Au-AgNCs2 (WK/SF is 3/2), which was further processed to a series of anti-counterfeiting films by blending with SF, silk sericin (SS), and polyvinyl alcohol (PVA). After successfully being numbered into fifteen colors, a dark blue-orange-dark red-dark blue cyclic fluorescent anti-counterfeiting color chart was designed. In addition, a two-Maxwell-unit model was constructed to assist with the microstructure analysis, which found that the formation of hydrogen bonds and the secondary structure transition from α-helices to ß-sheets during stretching were responsible for improving the mechanical properties and the two-staged fracture curves of films, respectively. Finally, a patterned and multicolor fluorescence anti-counterfeiting fabric application was demonstrated by combining the color chart and screen printing, indicating the great potential in textile anti-counterfeiting.

Ovine KRTAP36-2: A New Keratin-Associated Protein Gene Related to Variation in Wool Yield.

Keratin-associated proteins (KAPs) are structural components of wool fibres. High-glycine/tyrosine (HGT)-KAPs are a subset of the KAP family, and their abundance in fibres varies. In this study, we report the discovery of an ovine HGT-KAP gene to which we assigned the name KRTAP36-2. Polymerase chain reaction and single-strand conformation polymorphism (PCR-SSCP) analyses revealed four variants of this gene in a screening population of 170 sheep from a variety of breeds. The DNA sequencing of the variants revealed four single-nucleotide polymorphisms (SNPs) and a dinucleotide deletion. Three of these SNPs were in the coding region, and one of these was non-synonymous and potentially led to the amino acid substitution p.Cys27Gly near the middle of the protein. The remaining SNP was located near the putative TATA box, and the di-nucleotide deletion was near the putative transcription initiation site. The effect of this variation in KRTAP36-2 was investigated in 274 Southdown × Merino lambs that were the progeny of five sires. Variation was only found to be associated with wool yield, that is, the proportion of the greasy fleece that remained as clean fleece upon scouring (expressed as a percentage). This may have some value in increasing wool production.

Fusion of Substrate-Binding Domains Enhances the Catalytic Capacity of Keratinases and Promotes Enzymatic Conversion of Feather Waste.

The unique role of keratinases in keratin hydrolysis has garnered huge interest in the recovery of feather waste. However, owing to the high hydrophobicity of feather keratins, the catalytic capacity of keratinases for hydrolyzing feathers is typically low. In this study, we aimed to improve the keratinase feather hydrolysis efficiency by fusing a substrate-binding domain into the enzyme. We screened several carbohydrate-binding modules (CBMs) and linking peptides. We selected the most promising candidates to construct, clone, and express a fusion keratinase enzyme KerZ1/CBM-L8 with a feather hydrolysis efficiency of 7.8 × 10-8 g/U. Compared with those of KerZ1, KerZ1/CBM-L8 has a feather hydrolysis efficiency that is 2.71 times higher, a kcat value that is 179% higher, which translates to higher catalytic efficiency, and Km and binding constant (K) values that are lower, which indicate a higher KerZ1/CBM-L8-keratin binding affinity. Moreover, the number of binding sites to the substrate (N), determined using isothermal titration calorimetry, was 24.1 times higher than that of KerZ1. Thus, the fusion of the substrate-binding domain improved the binding ability of the keratinase enzyme to the hydrophobic substrate, which improved its feather hydrolysis efficiency. Therefore, using the fusion keratinase would significantly improve the recovery of feather waste.