Keratinase and its diverse applications

Abstract

Keratinase is a proteolytic enzyme specialized in the degradation of keratin-rich materials and has garnered significant attention for its potential in various biotechnological applications. This review provides an overview of keratinase, focusing on its structure, classification, function, biochemical properties, mechanisms of action and diverse applications. Keratinase plays an important role in bioremediation and stands out prominently, as it facilitates the eco-friendly degradation of keratinaceous waste materials addressing environmental concerns by reducing pollution and waste accumulation. Moreover, in the textile industry, keratinase plays a pivotal role in bio-pretreatment processes, enhancing the dyeing and finishing properties of animal fibers such as wool and silk. Beyond textiles, this enzyme contributes significantly to animal feed production by hydrolyzing keratin-rich byproducts into digestible components, thereby fostering the creation of high-protein feeds. Its impact extends to the cosmetic and pharmaceutical realms, where keratinase finds use in skincare formulations and in treating certain dermatological conditions owing to its ability to modify and break down keratin structures. By assisting in the removal of dead tissue, it demonstrates potential in biological applications for wound healing. Additionally, the challenges and future perspectives on the commercial scalability of keratinase production and its integration into various sectors are discussed.

Introduction

Keratin is derived from the Greek word “keras” which means horn and was first identified and named by Friedrich Wilhelm Heinrich Alexander von Wolff in the nineteenth century. This fibrous structural protein belongs to the family of scleroproteins, characterized by its toughness and insolubility in water. Structurally, keratins are predominantly composed of long, filamentous polypeptide chains, rich in sulfur-containing amino acids which form disulfide bonds such as cysteine, fostering its stability and insolubility. These chains intertwine to form a highly stable and cross-linked matrix, imparting resilience and strength to the tissues they comprise. This structural integrity allows keratin to withstand various external challenges, making it a crucial component in evolutionary adaptations and ecological specialization (Shah et al. 2019). The primary role of keratin lies in offering protection and support, forming resilient coverings and structures that shield organisms from environmental stresses and physical damage (Salleh and Rashid 2024). In avian species, keratin in feathers enables flight, while in mammals, it provides insulation, protection and structural support for skin and fur (Terril and Shultz 2023). Furthermore, the presence of keratin in reptilian scales, amphibian skin and the outer layers of marine animals exemplifies its adaptive significance across diverse taxa. However, its resistance to degradation poses challenges in waste management, as keratinaceous materials accumulate in various industries, necessitating innovative strategies for recycling or disposal. Understanding the multifaceted roles and properties of keratin not only illuminates the mechanisms behind biological resilience and adaptation but also opens avenues for sustainable utilization and management of this abundant protein in nature.

Keratinase stands at the forefront of biological processes involved in the degradation of keratin which is exceptionally tough in nature (Vidmar and Vodovnik 2018). Its specialized function revolves around the breakdown of the highly cross-linked and insoluble keratin molecules, a task that conventional proteases or enzymes commonly found in living organisms struggle to accomplish efficiently. On the other hand, it possesses a unique set of enzymatic properties tailored for this purpose, making it an indispensable catalyst in the degradation of keratin-rich substrates. It can be derived from a range of microbes, including fungus and bacteria and it exhibits specificity towards keratin substrates, cleaving peptide bonds within the keratin structure. This enzymatic action results in the degradation of keratin into smaller, soluble components like peptides and amino acids, which are more easily utilized by organisms. The remarkable ability of keratinase to break down this notoriously resilient protein has garnered immense interest and attention in biotechnological, environmental, industrial and biomedical sectors due to its potential implications in waste management, textile processing, animal feed production, pharmaceuticals and bioremediation.

The demand for keratinase across diverse applications stems from the inherent resilience of keratin protein and other keratinaceous substrates (Nnolim et al. 2020a, b, c). Traditional methods or common proteases often struggle to efficiently degrade or hydrolyze these keratin-rich materials due to their highly cross-linked structure, which poses challenges in terms of their biodegradability (Zhang et al. 2021). Keratinase ability to specifically target and break down keratin substrates is paramount in several key applications. In bioremediation, the enzymatic action of keratinase aids in mitigating environmental pollution by efficiently degrading keratin-based waste materials. In the textile industry, it facilitates the bio-pretreatment of animal fibers, improving their dyeing and finishing properties (Anbesaw 2022). Moreover, in animal feed production, the conversion of keratin-rich byproducts into digestible components for high-protein feeds relies on the action of keratinase. The enzyme also finds significance in pharmaceuticals and cosmetics, contributing to skincare formulations and wound healing by modifying keratin structure (Gomes et al. 2020). Its role in biotechnological research and protein engineering further underscores its importance, paving the way for innovations in enzyme technology. The multifaceted applications of keratinase offer sustainable solutions in waste management, industrial processes, healthcare and scientific advancements (Rajabi et al. 2020).

Classification, structure and function of keratinolytic proteases

The only class of proteases with a broad pH and temperature range that can effectively break down complex and recalcitrant proteins is keratinases (EC 3.4.-.-) (Gupta et al. 2006). EC 3.4.4.25 was the previous classification for keratinase; however, as seen in Fig. 1, this classification was dropped in the year 1972 and keratinases no longer have a 3-digit or 4-digit EC number (Qiu et al. 2020).

Fig. 1.

EC tree of keratinase enzyme

Since keratinases only catalyze the hydrolysis of peptide bonds in keratinous material effectively, they are not considered to have a defined function, therefore there is no comprehensive EC categorization for them. The active sites of keratinases are serine and metalloproteases and are made up of many conserved residues. Their type as metalloproteases is suggested by their inhibition by 1,10-phenanthroline or EDTA, whereas their serine protease catalytic mechanism is indicated by their inhibition by di-isopropyl fluorophosphate (DFP) or phenylmethanesulfonylfluoride (PMSF). The metalloprotease class of keratinase is inhibited by EDTA by chelating metal ions that catalyze enzymatic reactions (Riffel et al. 2003). Based on similarity in their amino acid sequences, the keratinases were categorized into distinct families. The keratinolytic enzymes are classified into 14 protease families by this family classification system, which includes both serine and metalloproteases: The serine proteases include members of the S1, S9, S8, S10 and S16 families; the metalloproteases include members of the M3, M4, M14, M16, M28, M32, M36, M38 and M55 families. The breakdown of keratin is facilitated by these 14 protease families and are further divided into exo-protease and endo-protease families as shown in Fig. 2. To completely degrade the keratin into single amino acids, these keratinolytic proteases are required. Exo-proteases are enzymes that facilitate the breakage at the terminal end of polypeptides and belong to the M14, M28, M38, M55, S9 and S10 families in contrast the endo-proteases are the enzymes that facilitate the internal breaking of peptide bonds in polypeptides and are members of M36, M16, M4, S16, S8 and S1 families. Families S16, S8 and S1 comprise endo-keratinolytic serine proteases, while families M36, M16 and M4 comprise metalloproteases. Exo-keratinolytic enzymes belong to the metalloprotease families M55, M38, M28 and M14 as well as serine protease families S10 and S9.

Fig. 2.

Classification of keratinolytic proteases based on their structural and catalytic properties

Due to variations in their active sites, these enzymes may have distinct functions in the degradation process of keratin (Qiu et al. 2020). There are crystal structures of several keratinases that show the structural underpinnings of their action and offer direction for maintaining a higher level of stability for use in industries. The crystal structure of keratinase KP2 is shown in Fig. 3.

Fig. 3.

Keratinase KP2 showing a Crystal structure b Enzyme hydrophobicity surface (PDB ID: 6FZX), (Nwofor et al. 2022)

They are crucial for the degradation of keratin substrates and exhibit a complex and specific structural organization that enables their catalytic function in the degradation of both α- keratins and β-keratins. Keratinases comprise a distinctive arrangement of amino acids that form the active sites (Li 2021). These active sites consist of residues that play pivotal roles in substrate recognition and catalysis (Gupta et al. 2017). The catalytic mechanism of keratinase involves the recognition and binding of keratin substrates, particularly at sites vulnerable to enzymatic cleavage. Once bound to the substrate, keratinase employs hydrolysis as its primary catalytic mechanism, wherein certain amino acid residues within the enzyme active site facilitate the cleavage of peptide bonds. Keratinases are classified primarily into serine, metallo and cysteine proteases, each with distinct structural and functional characteristics. Serine keratinases, contain a serine residue in their active site, forming a catalytic triad (serine, histidine, and aspartate) crucial for their proteolytic activity (Sundus et al. 2016). Their structure allows them to hydrolyze peptide bonds with high specificity, making them effective in breaking down the rigid keratin structure. Metalloproteases, on the other hand, rely on a metal ion, typically zinc, for their catalytic mechanism. Their active sites contain a metal-binding motif that stabilizes the enzyme–substrate complex, facilitating the cleavage of keratin disulfide bonds (Martinez et al. 2020). The flexibility of metalloprotease active sites enables them to function under a wide range of environmental conditions, such as varying pH and temperature. Cysteine proteases, which feature a cysteine residue in their active site, operate through a nucleophilic attack mechanism, making them particularly effective in reducing the disulfide bonds that confer keratin its resistance to degradation (Giteru et al. 2023).

These active sites contain amino acid residues, like serine, cysteine and histidine, which act as catalysts during hydrolysis (Lai et al. 2023). This enzymatic action results in the breakdown of the highly cross-linked keratin structure into smaller, soluble components, including peptides and amino acids. The structural configuration and catalytic mechanisms of keratinase enable it to efficiently target and hydrolyze the tough keratin substrates (Sypka et al. 2021).

Structure of keratin

The large quantities of nitrogen, sulfur and carbon make up keratin, which may be altered to create a variety of natural chemicals (Wang et al. 2016). Keratin breakdown is impeded by a large concentration of disulfide bonds (Ma et al. 2024). Some additional enzymes are also required to disrupt the disulfide bonds and to lower the pressure for keratin packaging to allow the proteases to access the proteins (David et al. 2022). Breakage of disulfide bonds and proteolysis are the two mechanisms that contribute to keratin degradation. Disulfide reductase involvement, inorganic sulfite production and mechanical destruction have been identified as factors involved in the disulfide bond breakdown (Kornillowicz and Bohacz 2011). After the breakdown of disulfide bonds, the individual keratin proteins are more accessible to enzymatic degradation by proteolytic enzymes. Proteolysis involves the cleavage of peptide bonds within the keratin protein, resulting in the fragmentation of the protein into smaller peptides and amino acids.

Based on the amount of sulfide or cysteine-containing amino acids present in keratin biomass, the keratins are classified as hard and soft keratins. Keratin is firmly arranged in an α-helix and β-sheet to form a super-coiled polypeptide chain which provides high mechanical stability, resistance to proteolytic hydrolysis and cysteine bridge cross-linking of the type I (acidic) and type II (basic/neutral) protein chains. Hydrophobic interactions, hydrogen bonds and cysteine residues generate disulfide bonds that firmly pack the polypeptide chains into the final structure. The biomass composed of keratin can be divided into two categories: α-keratin and β-keratin based on the secondary protein structure (Fig. 4).

Fig. 4.

Structural composition of keratin consisting of α-keratin and β-keratin

α-keratins

The head domain (also known as the N-terminal domain), the rod domain and the tail (also known as the C-terminal domain) make up the monomeric molecular structure of α-keratin. The globular nonhelical head and tail domains present at the end of intermediate filament proteins have a significant impact on the construction, organization and control of intermediate filaments. The central rod domain plays a primary role in supporting the self-assembly of filaments. The rod domain is composed of four right-handed α-helical subdomains: 1A, 1B, 2A and 2B, each consisting of seven amino acid repetitions. The alpha-helical polypeptide chains twist around each other to form protofibrils, which further assemble into intermediate filaments. These intermediate filaments are arranged hierarchically, ultimately form the insoluble keratin fibers. The majority of keratin-containing material contains both α- and β-keratins. Alpha-keratins provide strength and protection to the skin, hair and nails, resist abrasion and external mechanical stresses and offer waterproofing properties. The α-keratin present in the feathers (41–67%) while wool, hair and bristle typically composed of 50% α-keratin, epidermis between reptile scales control the epithelial cells adhesiveness and ability to change shape when they expand (Banasaz et al. 2024; Tantamacharik 2020).

β-keratins

These are the structural proteins found in the hard tissues of reptiles and birds. They are named for their rich content of stacked beta sheets. They add rigidity to the beaks, claws, and feathers and prevent desiccation. Interestingly, phylogenetic studies suggest that β-keratins of feathers evolved from scale β-keratins, indicating a fascinating evolutionary link between the scales of reptiles and the feathers of birds, however, it has been reported that the pangolin, a mammal and a peculiar scaly anteater, α-keratin and β-keratin both are present in the scales covering its skin. On the contrary, mammalian, avian and reptilian keratin generally contain α-keratin. The β-keratin present in the scales of reptiles has a protective role in nature and shows low extensibility, significant microbiological resistance and hydrophobicity (Perța-Crișan et al. 2021). Additionally, β-keratins are renamed as corneous β-proteins because some individuals believe that the evolutionary relationships between α- αkeratin and β-keratins are completely unrelated.

Sources of keratinase

Microorganisms that produce keratinase are found in nature in large quantities and are easily isolated from the environment. Keratin serves as the source of nitrogen and carbon to bacteria, fungi and actinobacteria, all of which can synthesize keratinases. Keratins containing wastes, like feathers, are typically included in media used for the isolation and production of keratinase-producing microbes, suggesting that keratinase production by microbes is an induced mechanism (Brandelli et al. 2009). It is known that a large variety of actinomycetes, fungi and bacteria may break down keratin. Enzymes that are capable of keratin degradation are widely spread in the natural world and are generated by a group of microbes primarily isolated from the waste of poultry farms. The microbial sources of keratinase are shown in Fig. 5.

Fig. 5.

Microbial sources of keratinase

The ability to produce keratinolytic enzymes is exhibited by members of numerous taxonomic groups of both conditionally pathogenic and non-pathogenic bacteria; however, Bacillus species, such as B. licheniformis and B. subtilis are most frequently found to possess this ability (Shestakova et al. 2021) The bacterial genera such as Vibrio, Stenotrophomonas, Brevibacillus, Pseudomonas, Fervidobacterium, Serratia, Microbacterium, Geobacillus Amycolatopsis, Bacillus, Burkholderia, Chryseobacterium, Rhodococcus, Meiothermus and Paenibacillus have all been shown to have the capacity to produce keratinase (Sangali and Brandelli 2000; Yamamura et al. 2002; Riffel and Brandelli 2006; Bach et al. 2011; Kuo et al. 2012; Fang et al. 2013; Jaouadi et al. 2013; Paul et al. 2014b; Stiborova et al. 2016; Abdel-Fattah et al. 2018; Falco et al. 2019; Alahyaribeik et al. 2020; Nnolim et al. 2020a). Actinobacteria specifically Arthrobacter, Streptomyces, Nocardiopsis and Bevibacterium have been shown to produce keratinase (Mitsuiki et al. 2006; Jaouadi et al. 2010; Pereira et al. 2014; Gong et al. 2015; Barman et al. 2017; Thankaswamy et al. 2018).

According to some reports, fungi that break down keratin are important for the hydrolysis of keratin-rich tissues and are the organisms that naturally inhabit keratinous substrates (Bohacz et al. 2020). In the domain Eukarya, keratinases synthesizing fungi have been identified. Reports regarding this capability in certain insects have appeared from time to time, however further research has demonstrated that the presence of keratin-degrading microbes within the gut microbiome is linked to the assimilation of keratin. The ability of both conditionally pathogenic and non-pathogenic fungi, specifically micromycetes of the genera Doratomyces, Aspergillus, Penicillium, Myrothecium, Scopulariopsis and Purpureocillium to produce keratinolytic enzymes is currently the most researched (Hassan et al. 2020). The keratin-degrading fungi that have been reported include Aspergillus, Doratomyces, Paecilomyces, Fusarium, Trichoderma, Onygena, Acremonium, Microsporum, Cladosporium, Lichtheimia, Aphanoascus, Chrysosporium, Scopulariopsis and Trichophyton (Gradisar et al. 2000; Sharma et al. 2011; Paul et al. 2014a; Sousa et al. 2015; Bagewadi et al. 2018; Abirami et al. 2020). Keratinolytic activity has been widely documented in fungal strains that are found inside dermatophytes. This group has been associated with both human and animal skin mycosis, and its keratinolytic feature may represent their pathogenic potential.

Some microorganisms possess the genetic machinery and metabolic pathways to synthesize and secrete keratinase enzymes as part of their survival strategy in environments rich in keratinaceous materials. The isolation and application of microbes that produce keratinase have garnered significant interest in biotechnological applications, offering potential solutions for waste management, industrial processes and environmental remediation by harnessing the enzymatic capabilities of these organisms to degrade keratin-based waste materials. The various sources of keratin are shown in Fig. 6.

Fig. 6.

Various sources of keratin

Purification of keratinase

The purification of keratinase is a multi-step process. Initially, keratinase-producing microorganisms are cultivated and centrifuged to release the enzyme, followed by precipitation to separate and concentrate the enzyme. Numerous studies standardized the protocol for the purification of keratinases that includes precipitation of enzyme by ammonium sulfate and purification is achieved through various chromatography techniques like gel filtration, ion exchange, or affinity chromatography. The keratinase enzyme from Aspergillus flavus strain K-03 has been purified using DEAE-Sephadex ion exchange chromatography and ammonium sulfate precipitation. The 32.36% yield and 11.53-fold purification were obtained from the ammonium sulfate precipitation of keratinase at 80% saturation (Kim 2007). Keratinase purification from A. aneurinilyticus was accomplished using salt precipitation, dialysis, gel filtration, SephadexG-100 and DEAE cellulose with a 25.43% yield and 20.84-fold purification (Sujata et al. 2022). Maximum keratinase production of 22.66 U/ml was obtained from Bacillus halotolerans L2EN1 with 11.47% yield and 1.68-fold purity by 90 − 100% ammonium sulfate precipitation (Devi et al. 2022) (Table 1). The keratinase purification from Bacillus licheniformis enzyme exhibits a specific activity of 218U/mg with 86-fold purification and 25% yield by ammonium sulfate precipitation and ion exchange chromatography (Suntornsuk et al. 2005).

Table 1.

Keratinase obtained from different organisms by different purification methods

Organism	Purification technique	Molecular weight (kDa)	Specific activity(U/mg)	References
Aspergillus flavus K-03	DEAE-Sephadex ion-exchange chromatography and ammonium sulfate precipitation	31	243.2	Kim 2007
Aneurinibacillus aneurinilyticus	Salt precipitation, gel filtration, sephadex G-100 and DEAE cellulose	34	203.87	Sujata et al. 2022
Bacillus halotolerans L2EN1	Ammonium sulfate precipitation	Heterotetramer of 17, 37, 40, 60	–	Devi et al. 2022
Actinomadura viridilutea DZ50	Ammonium sulfate precipitation	20	26,533 ± 40	Ben Elhoul et al. 2021
Bacillus licheniformis	Ammonium sulfate precipitation and column chromatography	35	218	Suntornsuk et al. 2005
Recombinant P. pastoris (pPZK3)	Ammonium sulfate precipitation and affinity chromatography	39	365.7	Radha and Gunasekaran 2009
Recombinant B. megaterium MS941 (pWAK3)	Ammonium sulfate precipitation and affinity chromatography	30	1277.7	Radha and Gunasekaran 2009

Additionally, amino acid sequencing and structure analysis using techniques like X-ray crystallography or NMR spectroscopy provide insight into keratinase primary and tertiary structure. By understanding these properties, researchers can elucidate keratinase kinetics and keratin degradation mechanisms and explore their potential applications in various fields.

Kinetics and mechanism of keratinase

Keratinases can break down keratin by breaking the peptide links in keratin. Metalloproteases and serine proteases are the keratinases that have the ability to hydrophobically recognize substrates, disulfide bonds and can disrupt peptide bonds within peptide chains. Keratin degradation involves two processes: peptide degradation and keratin peptide release. When an organism is grown in a medium containing keratin, the microbial keratinases are typically released into it (Friedrich et al. 1996). When keratin or substrates containing keratin are present in the media, the majority of microbial keratinases are released into the extracellular matrix (Nnolim and Nwodo 2021). Certain microbes can concurrently produce intracellular and external keratinases. There are some cell-bound keratinases, which could be highly fascinating for industrial use because their immobilization on the cell surface is possible and can be easily applied in waste treatment. A recent study that meticulously and precisely categorized the available keratinases offers insight into their mode of action and clarifies why numerous enzymes are needed for the breakdown of keratinous wastes or keratin (Dahiya et al. 2024).

Keratin wastes are often disposed of improperly either by heating, burning or acid and alkali treatment or by treating the residues with high pressure or high temperature and using them to make substandard feed for animals (Tesfaye et al. 2017). However, chemical treatments can damage certain amino acids, like tryptophan and other essential amino acids (Sinkiewicz et al. 2017; Rajabinejad et al. 2018), and the use of acid and alkaline solutions can cause additional pollution issues (Sharma and Devi 2018). As microbial keratinases are widely studied in terms of their application these enzymes offer a better option to the harsh procedures for treating keratin residues (Sypka et al. 2021). The enzymatic breakdown of these keratin residues and proteins turned out beneficial in terms of economic development and environmental sustainability (Nnolim and Nwodo 2021). The difficulty in breaking down keratin is caused due to a high disulfide bond content. Since the majority of keratinases are proteases and break peptide bonds, additional enzymes are also required to modify disulfide bonding and reduce the strain involved in keratin packaging, which exposes proteins to the proteases (Kasperova et al. 2013). Bacteria and fungi use similar pathways in the degradation of keratin but the only difference is the mechanical destruction in the case of fungi (Korniłłowicz-Kowalska and Bohacz 2011). Keratinases break the polypeptides into amino acids in keratin degradation and the several protease families are able to degrade keratin. Because of the differences in their active sites, capacity to detect amino acid length and preferred substrate cleavage sites the different families of keratinolytic enzymes may play different roles in the breakdown of keratin (Qiu et al. 2020).

Substrate specificity

Substrate specificity plays a crucial role in the diverse applications of keratinase across various industries. The ability of an enzyme to recognize and bind to particular substrates, such as keratin-rich compounds is referred to as enzymatic specificity. Substrate specificity in bioremediation and waste management guarantees effective keratinaceous pollutant breakdown, enabling contaminated environments to be cleaned up more easily. The specificity of the enzyme allows keratin to be hydrolyzed into digestible peptides and amino acids during the manufacturing of animal feed, hence increasing the nutritional content of feed ingredients. Furthermore, substrate specificity in the textile sector enables focused alteration of textile fibers, improving the texture, look and functionality of final goods. Similarly, keratinases can precisely recognize substrates in biomedical and cosmetic applications and allow customization of medication delivery, skincare formulations and wound healing treatments. Substrate specificity makes keratinase a useful enzyme in a variety of fields by ensuring its optimal performance and efficacy.

Both soluble and insoluble substrates for keratinase proteolytic activity were investigated in various studies (Table 2). Keratin, fibrin and nail keratin, and were examples of some complex substrates used by different types of keratinases based on the structure of their active sites such as serine keratinases active sites typically prefer substrates with hydrophobic or bulky residues at the P1 position. Their substrate specificity is determined by the interactions between the P1 residue of the substrate and the enzymes active site. The presence of a metal ion in the active site of metalloproteases influences the substrate with polar or charged residues and cysteine keratinases use cysteine residue in their active site for catalysis. The substrate specificity of cysteine proteases is typically broader, with preferences for substrates that form disulfide bonds, such as those found in keratin (Ashraf et al 2023).

Table 2.

Substrates used to assay keratinase activity

Substrates	Absorbance	References
Keratin azure	At 595 nm	Letourneau et al. (1998)
Azo-keratin	At 360 nm	Herzog et al. (2015)
Feather	At 280 nm	Bohacz et al. (2020)
Feather powder	At 280 nm	Qu et al. (2018)
Keratin powder and wool keratin	At 280 nm	Iglesias et al. (2017)

Applications of keratinase

Though microbial keratinases were initially proposed for use in biotechnological processes involving the hydrolysis of waste keratin, keratinolytic enzymes showed potential in other areas also. Numerous potential biotechnological applications of keratinases (Fig. 7) have been proposed as a result of advances in our understanding of how these enzymes affect keratolysis.

Fig. 7.

Biotechnological applications of keratinase

In agriculture

The demand for food on a worldwide scale is driving up the usage of synthetic fertilizers, mostly to increase the amount of nitrogen (N), the primary component that limits plant development in the soil. Synthetic fertilizers, despite their role in enhancing crop yields, can lead to several detrimental effects on agriculture. Over-reliance on these fertilizers can result in soil degradation, including soil acidification and compaction, reducing its natural fertility and causing dependency on external inputs. This can further disrupt the balance of soil nutrients, leading to imbalances in essential macronutrients and micronutrients critical for plant growth. Moreover, the excessive application of synthetic fertilizers contributes to environmental pollution, with nitrogen and phosphorus runoff causing water pollution and contributing to the eutrophication of water bodies. Poultry waste, such as feathers, can be utilized as biofertilizers to replace synthetic fertilizers and reduce the overuse of traditional inorganic fertilizers in agricultural activities. Feathers are excellent suppliers of nitrogen (Silva 2018). More than 90 percent of proteins in feathers are made up of β-keratin, which is heavily cross-linked by hydrogen, disulfide and hydrophobic connections. Nitrogen makes up about 15% (w/w) of feathers, however using them directly as a fertilizer is challenging due to their recalcitrance, which causes nitrogen in the soil to degrade and mineralize slowly (Jain et al. 2016). Concentrated feather meal is high in protein can be used for chicken feed and can also be applied to organic farming as a slow-releasing nitrogen fertilizer (Hadas and Kautsky 1994; Choi and Nelson 1996). The hydrolysates produced after the enzymatic degradation of feathers are abundant in amino acids, peptides and other macro and micronutrients such as calcium, iron, phosphorus, potassium, manganese and zinc as they are useful as organic fertilizers for plants. (Nurdiawati et al. 2017). The soil bacteria may mineralize the protein hydrolysates, releasing nitrogen that the plants can take (Nurdiawati et al. 2019). Additional plant tissues can absorb peptides and amino acids directly through their roots and leaves, where they are then translocated and function as growth promoters. The production of indole acetic acid (IAA), a hormone that has crucial roles in plant growth, requires tryptophan as a basic amino acid (Kshetri et al. 2019). According to the study by Nafady et al. (2018), feathers could be broken down by the bacterium B. licheniformis, which could then be utilized to make IAA from L-tryptophan. During the breakdown of feathers, other amino acids such as valine, serine, tryptophan, proline, isoleucine, alanine, glycine and asparagine, have been identified. Figure 8 depicts the process of conversion of feather waste into biofertilizer. The tryptophan in the feather hydrolysate alone was sufficient for the synthesis of the phytohormone IAA, even in a medium devoid of L-tryptophan supplementation. A potential biofertilizer was discovered by evaluating the germination, growth and development of Cicer arietinum seeds with feather hydrolysate. Soil enriched with feather culture lysate showed earlier seed germination and higher plant development than the control experiment (Menezes et al. 2021).

Fig. 8.

Process of conversion of feather waste into biofertilizer by keratinase enzyme

In environmental remediation

One of the main byproducts of the chicken, abattoir, leather and fur industries is keratinous waste. Due to the growth in the poultry industry, the most frequent keratinous waste produced by slaughterhouses and poultry farms is feathers (Intagun and Kanoksilapatham 2017). Waste containing keratin can now be remediated more effectively and sustainably with the use of keratinase. Keratinase can be used to break down trash that is rich in keratin and to make feather feed additives, organic fertilizers, films, coatings, glues and other commercial goods. It is significant to note that the degradation process in natural environments could take many weeks to finish. The increasing demand of the poultry industry generates a significant amount of trash that needs to be managed properly. Poultry waste has been treated using a variety of waste management techniques, such as chemical treatments, mechanical treatments and biological treatments. The incineration (chemical treatment) process uses a high amount of energy and produces a significant amount of unburnt hydrocarbons and carbon dioxide. Additionally, the heat and alkaline treatment breaks down several important amino acids and produces toxic, non-nutritive amino acids (Das et al. 2024).

There are a variety of microorganisms that are capable of bioremediation of the keratin-rich waste and remediate the environment from this waste. Using microorganisms for biodegradation has a variety of benefits, including safety, cost-effectiveness and reduction in the production of carbon dioxide. The feathers are fully broken down to soluble form in 48 h by the Bacillus S-2 protease (Baid et al. 2016). Feathers were hydrolyzed in two stages using 5% enzyme and hot potassium hydroxide at 70 °C, which shows approximately 91% breakdown (Mokrejs et al. 2011). Meiothermus ruber, a moderate thermophile, was used to investigate an aerobic acidulocomposting process in a waste treatment device of the bio type (BioClean BC-02). In a study within 14 days, almost 70% of the 30 g of chicken feathers were metabolized (Shigeri et al. 2009). Bio-hydrogen production from keratin-rich waste was successfully tested using a two-stage fermentation system that used the anaerobic hyperthermophilic archaeon, Thermococcus litoralis and the keratinolytic, mesophilic Bacillus licheniformis KK1 bacterium. In this instance, Bacillus licheniformis KK1 was employed to ferment keratin-containing trash into a product rich in amino acids and peptides (Bálint et al. 2005). Extensive feather degradation has been identified in thermophilic and highly thermophilic species of Fervidobacterium, including F. islandicum AW-1, F. pennivorans, Fervidobacterium sp. FA004 and F. thailandense FC2004, (Intagun and Kanoksilapatham 2017).

In textile and leather industry

The abundance of pollutants and hazardous materials like sulfides, found in the wastewater released from the tanneries and leather industry produced by traditional leather processing methods raised concerns regarding environmental pollution. Among the many processes involved in leather processing technologies, pre-tanning accounts for the majority of pollutants (about 70%). The primary causes of increased chemical oxygen demand (COD), biochemical oxygen demand (BOD) and total dissolved solids (TDS) are solid wastes produced during pre-tanning, lime and sodium sulfide (Thanikaivelan et al. 2004). Enzymatic dehairing (Fig. 9) can be an alternative to the use of hazardous chemicals such as strong acids that are traditionally used for the dehairing process. As keratinases work effectively in hair removal and provide a cost-effective substitute for the dangerous chemicals that are commonly employed, they are identified as efficient enzymes to solve this issue (Akram et al. 2020). To preserve the qualities of the leather, such as its softness, flexibility and fullness, excessive collagen hydrolysis must be prevented during the leather-making process (Su et al. 2020). As a result, an enzyme that is approved for use as a depilatory agent needs to be highly active in removing hair while having little to no activity in breaking down collagen and elastin. According to a few reports, keratinases may be effective depilating agents (Letourneau et al. 1998; Bressollier et al. 1999; Allpress et al. 2002; Friedrich and Kern 2003). It has been stated that the keratinase from B. subtilis S14 (Macedo et al. 2005) eliminates the requirement for harmful sodium sulfide. Keratinases have been shown to be useful in the processing of leather in many articles. The production of a keratinase from Bacillus sp. LCB12 in Bacillus subtilis SCK6 was studied as a possible substitute for sulfide used in the hair removal process on goat skin. (Tian et al. 2019).

Fig. 9.

Application of keratinolytic protease in the dehairing of animal hides and as an additive in detergents

In research conducted by Akhter et al. (2020), enzymatic and chemical dehairing methods for cow leather were examined and it was observed that keratinase from Bacillus cereus showed ideal outcomes when it comes to hair removal (Table 3). Studies have also been done on the application of keratinases to reduce the toxicity of wastewater from the leather and textile industry (Jaouadi et al. 2015; Kalaikumari et al. 2019). Keratinases-producing microorganisms can be used to clean wastewater and clear obstructions from sewage systems (Brandelli et al. 2009 and Zahara et al. 2020).

Table 3.

Keratinase-producing microorganisms and their application as an additive in detergents and in leather industry

Microorganism	Application	References
Brevibacterium luteolum	Leather industry	Thankaswamy et al. (2018)
Coriolopsis byrsina	Additive in detergents	Dufeck et al. (2020b)
Citrobacter diversus	Additive in detergents	Dufeck et al. (2020a)
Arthrobacter sp. KFS-1	Additive in detergents	Nnolim et al. (2020a, b, c)
Streptomyces aureofaciens	Additive in detergents	Gong et al. (2015)
Bacillus aerius NSMk2	Additive in detergents and Dehairing of leather	Bhari et al. (2019)
Bacillus cereus	Dehairing of leather	Akhter et al. (2020)

As an additive in detergents

The detergent business is always looking for novel enzymes with antibacterial, tissue-care, and cleaning capabilities. Appropriate proteases in the detergents are needed to fulfill number of requirements, such as being able to efficiently remove stains, being active and stable at alkaline pH, and being resistant to surfactants, oxidizing agents and bleaching agents. Proteolytic enzymes that adhere to these biochemical characteristics have been found and engineered through research efforts. Proteinaceous stains can be effectively removed from fabric by enzyme-mediated detergent washing, a non-toxic, environmentally friendly and biodegradable method (Sen et al. 2021). Since keratinases are active alkaline enzymes with broad selectivity for both soluble and insoluble proteins, they are desirable detergent agents. This explains the plethora of research that has examined and documented the presence of these enzymes in commercial detergents (Akram et al. 2020; Dufeck et al. 2020a, b; Emon et al. 2020; Nnolim et al. 2020a, b, c). The stained fabric when washed merely with tap water showed the lowest reflectivity (%) but the cloth that was washed with detergent (7 mg/ml) and enzyme (90 ± 2 U/ml) and incubated for 10 min at 60 °C had a greater reflectivity (Reddy et al. 2017).

In Cosmetics and pharmaceuticals

Keratinase is being studied for its applications in cosmetics for developing formulations for treating hair, skin acne, skin infections and also in hair removal creams. The main cause of acne is believed to be excess keratin production at the hair surface, which clogs the pores and prevents sebum from coming out of the skin which causes acne. Keratinase is a necessary ingredient of the products used to remove hair and it is included in facial scrubs to treat acne because dead cells can clog pores and foster an environment that is beneficial to acne (Adelere and Lateef 2016). Based on the study by Sanghvi et al. (2016), keratinase from Bacillus subtilis DP1 was shown to be compatible with the composition of the cream, leading to effective hair removal. In another cosmetic examination, the ability of Bacillus subtilis AMR keratinase to produce a keratin hydrolysate for use in hydrating shampoo formulations was assessed (Villa et al. 2013). Keratinases may also be useful in treating corn, acne and removing calluses.

Keratinases have been explored in pharmaceutical proposals to break down keratinous material to increase drug penetration and may be used to treat fungal diseases (Sarma 2022). Dermatophytosis, often known as ringworm, is a clinical illness caused by a fungal infection of the skin that affects humans and domesticated animals. The fungus that causes the parasitic illness (dermatophytosis) feeds on keratin, which is present in the outer layer of the skin, hair and nails. Thus, there is increased interest in keratinase, also known as keratinolytic protease, as a potential treatment for dermatophytosis. As a vaccination for dermatophytosis, keratinases are commonly employed (Gupta and Ramnani 2006).

According to Rai et al., (2020), β-keratinase immobilized silver nanoparticles (AgNPs) can boost AgNPs bactericidal action. To remove biofilm, protease enzymes have been studied, particularly those that have a wide specificity spectrum. Stenotrophomonas maltophilia Kb2 keratinase has been shown to have both the biofilm dispersal activity and antibiofilm effect against pathogenic bacteria, including Staphylococcus aureus MTCC96 and Escherichia coli MTCC-739 (Bhange et al. 2015). Keratinolytic enzymes (keratinases) can also be utilized to extract keratin, which in turn can be utilized to produce biomaterials for use in biomedical applications. For example, the potential of enzymatically extracted keratin to produce a hydrogel for use in wound healing applications was assessed (Su et al. 2020b). According to Mitsuiki et al., (2006) and Okoroma et al., (2013), there was also a proposal that recommends using keratinases for the decontamination of prions (Table 4), which may help prevent the burning of medical samples and equipment. The use of microbial keratinase for in vitro destruction of β-amyloid fibrils has also been described (Ningthoujam et al. 2019).

Table 4.

Commercially used products containing keratinase (Shestakova et al. 2021)

Product Name	Source	Area of Application	Functions	Manufacturer
FEED-0001	B. licheniformis	Feed additive for animals	Insoluble keratin degradation	Creative Enzymes
Versazyme	B. licheniformis	Feed additive for animals	Animal feed preparation, keratin and collagen degradation	BioResource International Inc
Prionzyme TM	B. licheniformis	Prion degradation	Engineered keratinase with increased activity, thermostability, and broader specificity for effective decontamination of medical instruments from prion proteins	Genencor International Inc
Keratoclean HYDRA PB	B. licheniformis	Cosmetology	Anti-aging, anti-wrinkle, exfoliation, hair removal	Proteos Biotech
Keratopeel PB	B. licheniformis	Cosmetology	Enzymatic peeling, skin cell renewal	Proteos Biotech
Esperase (protease P5860)	Bacillus sp.	Scientific research	Used to break down proteins by hydrolyzing peptide bonds	Creative Enzymes
Proteinase K	Tritirachium album	Scientific research	To digest proteins and enzymes	Sigma
PURE100 KERATINASE	Bacillus sp. and recombinant strains	Cosmetology and prion degradation	Regulation of keratin concentration in pores for blisters and keratinized skin treatment, scars, dermatophytic and nail diseases treatment, as well as epithelial regeneration	Proteos Biotech

Dead cell removal from the nail and skin surfaces is facilitated by keratinase action on keratins in the nails and skin. Acne and hyperkeratosis, which are skin-thickening conditions brought on by the accumulation of excess keratin, can be effectively treated with keratinase, a topical drug (Brown et al. 2024). Keratinase is now found in face scrubs, anti-dandruff shampoos and other personal care products for hair removal and youthful-looking skin. (Gupta et al. 2013).

In animal feed production

Keratinase effectively breaks down the complex protein structures in feathers, yielding short peptides and amino acids that are more digestible for animals (Saranya et al. 2021). In the past, feathers were baked under high pressure and temperatures to produce feather meal, which was added to animal feed. The hydrothermal process is costly and also destroys certain important amino acids, such as tryptophan, lysine and methionine, resulting in a product with inconsistent nutrient quality and poor digestibility. Wang et al. (2011) investigated the effects of feeding weaned piglets a corn-soybean diet supplemented with 0.05% (w/w) keratinase concentration. The tests employed the Bacillus licheniformis PWD-1 enzyme Cibenza DP100TM keratinase by Novus International, Shanghai, China, which has shown the ability to hydrolyze soybean protein in the past. Only 11.81% of glycine and 24.20% of β-conglycinin were hydrolyzed in the absence of keratinase, however, in the presence of keratinase, the hydrolysis percentage values reached 94.74% and 88.89%, respectively. Previous studies indicate that keratinase-treated feather meal can replace up to 7% of dietary protein for growing chicks, significantly improving amino acid digestibility (Kumar 2021). The addition of keratinase to animal feed resulted in the production of hydrolysates, which improved the intestinal architecture and ecology of the piglets. In the piglet’s intestines, there was a significant reduction in E. coli count, total aerobic counts, ammonia, nitrogen concentration and branched-chain volatile fatty acid content. Keratinase and products containing keratinase are used as additives in animal feed (Table 4).

Additionally, reports exist about the usage of versazyme keratinase (BioResource International) as a meal supplement to enhance the digestibility of animals. This enzyme is derived from Bacillus licheniformis PWD-1. Versazyme supplementation increased body weight, breast meat output and feed conversion ratio in broiler chickens. Additionally, the presence of the enzyme enhanced the intestinal growth and digestibility of feed components (such as starch) in chickens (Wang et al. 2016). Compound enzymatic hydrolysis (CEH) from B. amyloliquefaciens treated the feather meal, increasing the concentration of soluble peptides and free amino acids. In vitro digestibility and protein solubility increased by 10.27 and 20.75 times, respectively. These findings imply that using CEH could be an effective way to increase the nutritional content of feather waste (Zhou et al. 2020).

In Biogas production

Biogas production utilizing keratinase presents an innovative approach with promising potential. The most feasible biological process for the production of renewable energy by using a variety of substrates is anaerobic digestion. The anaerobic digestion of keratin-rich waste is also possible where the substrates used are feathers, hairs and other keratin-rich substances such as hooves, horns, nails, etc. Feathers normally decompose anaerobically in thermophilic or mesophilic environments, typically with a combination of other waste materials. Ammonia, carbon dioxide, hydrogen, amino acids, organic acids and trace amounts of sulfur compounds are produced during the digestion of these substances (Onwosi et al. 2020). Among these, the synthesis of methane requires acetic acid, carbon dioxide and hydrogen. The feathers have been pretreated with the recombinant strain of Bacillus megaterium which contains keratinolytic enzyme-producing gene from Bacillus licheniformis and then comes the anaerobic digestion which results in the production of methane with increased yield as compared to the methane production without pretreatment of feathers (Forgcács et al. 2011). A related research demonstrated that methane can be produced from chicken feathers that had been pretreated with C4 bacterium of Bacillus sp. that produces α-keratinase and β-keratinases. Compared to the anaerobic digestion of untreated feathers, methane outputs increased by 292% and 105%, when feather hydrolysate was utilized as a substrate in an anaerobic culture with sludge or bacterial granules respectively. (Patinvoh et al. 2016). A study by Bálint et al. (2005) demonstrates that the biological breakdown of waste, high in keratin can also yield hydrogen, in addition to methane. Feathers were fermented into a rich peptide and amino acid broth using the bacterium Bacillus licheniformis KK1, which was then further broken down by Thermococcus litoralis. Biohydrogen was a by-product of the fermentation process, produced as a result of Bacillus licheniformis KK1 growing in the keratin hydrolysate (Bálint et al. 2005).

Challenges and future directions

There are several restrictions and challenges while using keratinase enzymes to break down keratin. One important drawback is that most keratinases have minimal efficacy against β-keratin, which is found in reptile scales and claws and predominantly target α-keratin found in hair and wool. The complex composition and insoluble nature of keratin substrates could impede the enzymatic process, resulting in slower response rates and partial breakdown. Moreover, the large-scale industrial uses are hampered by the high expense of producing and purifying enzymes. Despite these difficulties, there are still plenty of opportunities for research and development which include the use of protein engineering techniques to increase the substrate specificity and activity of keratinases, looking into the new microbial sources for keratinase production, improving fermentation conditions for higher enzyme yield and looking into the use of immobilized enzyme systems for more effective keratin degradation in a variety of industrial processes like waste management, production of animal feed and textile processing.

Conclusion

The review emphasizes the pivotal role of keratinases across diverse applications, emphasizing its versatility and importance. Notably, keratinase exhibits significant potential in waste management, efficiently converting keratin-rich waste materials into valuable resources, thereby promoting sustainability. Its applications extend to bioremediation, where it aids in the cleanup of keratin-contaminated environments, highlighting its environmental remediation capabilities. Moreover, in industries such as animal feed production and textiles, keratinase enhances processes by improving digestibility, nutritional value and quality while reducing environmental impact. Additionally, its relevance in biomedical and pharmaceutical sectors is noteworthy, contributing to wound healing, drug delivery and bioactive peptide production. The versatility of enzyme also extends to cosmetics and personal care, offering solutions for skin and hair care products. Overall, the review focusses on the diverse and significant applications of keratinase, positioning it as a key player in various industries and environmental endeavors.

Funding

The authors declare that no funds, grants or other support were received during the preparation of this manuscript.

Data availability

There is no data available as it is a review article.

Declarations

Conflict of interests

On behalf of all authors, the corresponding author states that there is no conflict of interest.