Microorganisms can be found everywhere and are the primary source of proteases, which have vast biotechnological implications. Additionally, protease can efficiently degrade various proteins. Keratin is a fibrous and resistant protein found in feathers, nails, horns, hooves, and the epidermis of the skin, among other tissues (Figure 1). ^1-5 The durability and rigidity of these proteins is due to the high degree of disulfide bonds, hydrogen bonds, and hydrophobic interactions.^4,6,7 Fibrous proteins are stable and resistant to degradation by proteolytic enzymes such as trypsin, pepsin, and papain due to their compact disulfide and intra-molecular-bond-based structural organization.⁸ Due to their slow biodegradation, keratin-based wastes wreak havoc on the environment. The poultry industry is one of the emerging industries due of the rise in global meat consumption, as chicken meat is a good protein sources.⁹ The major producers of these keratin-based solid wastes that are difficult to degrade are the poultry, leather, and textile industries.¹⁰ It is estimated that the annual production of keratinous wastes from chicken processing farms is in the millions of tons.⁶ They have become harmful contaminants in the environment because of their rigidity, high mechanical strength, recalcitrant property, and poor deposition of wastes.¹¹ Traditional methods of keratin decomposition, such as incineration, burial, composting, and mechanical grinding, produce toxic gases that harm terrestrial and aquatic life. Chemical treatment of keratin wastes (acid/alkaline hydrolysis or treatment with oxidizing/reducing agents) is a costly and energy-intensive process that reduces the nutritional value of the amino acids obtained through keratin decomposition.^10,12–14 The accumulation of solid waste reduces the land’s surface and contributes to land pollution. One of the major problem caused by the poultry industries in the solid waste problem originated from the slow degradation of hard keratin protein feather. Due to the presence of pathogenic microorganisms, improper disposal of poultry feathers has been linked to a number of human health issues, such as chlorosis and fowl cholera.¹⁵ Particulate matter (PM), greenhouse gas emissions (such as methane and sulfur dioxide), nitrogenous compounds (NH₃, N₂O, NO_x), and volatile organic compounds (VOCs) are just some of the reported hazardous substances produced by the poultry industry. Ecosystems (via processes like soil acidification following atmospheric deposition) as well as human health are negatively affected by these threats. Bronchitis, asthma in youngsters, heart disease, lung illness, and cancer are some of the ailments that might develop in poultry farm workers.^16–18 Poultry manure can also contain microorganisms and pharmaceuticals (such as antibiotics) used in poultry production, which can contaminate soil and water, leading to antimicrobial resistance, including multidrug resistance.¹⁷

The microorganisms have the potential to produce proteases. Keratinase is a protease produced by microorganisms that can break down the protein keratin to release amino acids. The degradation of keratin by microbial keratinases maintains the original structure of the final products, including short peptides, amino acids, and organic nitrogen.¹¹ This method is cost-effective and environmentally friendly. The products resulting from the hydrolysis of feathers are suitable for use in fertilizers and as a source of valuable amino acids such as serine, cysteine, and proline.¹⁹ The ability of keratinase to withstand extreme conditions is advantageous for the development of green technology and sustainability. On the basis of the enzyme’s catalytic site, keratinases can be classified as serine or metallo-serine proteases.²⁰

Keratinase-producing bacteria consist of Chryseobacterium sp. strain kr6²¹, Stenotrophomonas maltophilia DHHj²², Bacillus subtilis SLC²³, Bacillus megaterium SN1²⁴, Bacillus pumilus GRK²⁵, Bacillus pumilus FH9¹⁹, Bacillus licheniformis ALW1¹², Bacillus sp. Nnolim-K1¹⁴, Ochrobactrum intermedium²⁶, Deinococcus geothermalis²⁷,  Bacillus aerius NSMk2²⁸, Chryseobacterium cucumeris FHN1⁶ etc and keratinase producing fungi includes Aspergillus oryzae²⁹, Trichoderma atroviride F6³⁰, Aspergillus parasiticus³¹, Coriolopsis byrsina³², Aspergillus sp. DHE7³³. Since keratin is used in so many different industries (including textiles, leather, detergents, cosmetics, and bio fertilizer)^6,34, it has become the prominent areas of research.

Structure and classification of keratin
Keratins are intermediate superfamily proteins that are a major component of epidermal appendages such as nails, hair, feathers, and horn.^35–37 Keratins are highly resistant proteins because of their compact structure. The structural classification of keratin is determined by its amino acid composition and polypeptide chain arrangement. The fibrous keratin is classified into α-keratin which is composed of α-helix and β-keratin is defined by β-sheets shown in Figure 2. Mammalian cells contain α-keratin, whereas avian and reptilian tissues contain β-keratin, with the exception of the pangolin, which contains both types of keratin in its skin^8,36. Around 91 percent of the keratin in feathers is a β-keratin. α-helical structures are the coiled structures formed by α-helical-coils types I and II (basic/neutral) that form a helix filament and a fibril held together by an interchain linkage. α-helix structure is rigid due to the disulfide bond of cysteine and the hydrogen bond. The α-keratin is distinguished by its low sulfur content. The diameters of α- and β-keratin were determined to be 7-10 nm and 3-4 nm, respectively, using transmission electron microscopy. Molecular analyses of α-keratin revealed the helix chain, which causes the chain to twist and display a helical shape due to the presence of a disulphide bond. Stability in β-keratin is provided by intermolecular hydrogen bonds between the protein’s β-sheets, which are arranged in a parallel or antiparallel orientation.³⁷ α-keratin has a size of 40–68 kDa, while β-keratin is 20–40 kDa (10-22 kDa). Additionally, keratins have been categorized into distinct groups, such as reptilian keratin, mammalian keratin, and avian keratin.³⁸ Due to its insolubility in inorganic solvents and water, beta-keratin is resistant to the proteolytic effects of common enzymes such as trypsin and pepsin.⁹ γ-keratin, also known as, is a globular protein with a molecular weight of 15 kDa. It provides structural integrity via disulfide bond and is a structural component of the epidermis, fibril cortex, and globular fiber matrix. Based on the sulfur content, keratin are further classified into soft and hard keratin of which epidermal cells of skin constitute the soft keratin while feathers, nail, horn, hooves etc., are hard keratin.³⁶

Mechanism of keratin degradation
Keratin is a highly resistant and indigestible protein due to its complex structure. The mechanism of its decomposition is still unknown. Numerous hypotheses have been developed to illustrate the microbial breakdown of keratin.  The biochemical mechanism of keratin disruption found that the degradation begins only after the denaturation of disulfide bond in the keratin followed by the action of extracellular proteases³⁹. Similarly,  Rajak et al. hypothesized that keratin degrades via sulfitolysis and keratinolysis.⁴⁰ The mechanism begins with the cleavage of the keratin disulfide bond, followed by the unfolding of the active sites for microbial keratinases, which subsequently degrade the keratin into amino acids and peptides.⁴¹ Numerous theories illustrate the initial phrase denaturation or the destruction of the disulfide bond. The biomembrane potential theory illustrates the ability of the cells to produce sulfite or reducing substance in the medium which disintegrate the disulfide bond. The reduction of disulfide bond in feather Streptomyces pactum and found the production of reducing agent that disintegrate the disulfide bond and makes the protein chains available for cleavage by proteases.⁴² Similarly, the presence of disulfide reductase, which is responsible for the dissociation of disulfide linkages, was detected during the degradation of feathers by a bacterial cell, as inferred by using glutathione.⁴³ Mycelia invading the structure’s epidermal layer and destroying the surface keratin protein are central to the mechanical pressure theory.⁴⁰ Thiolysis theory explains how denaturation occurs when a microbe secretes sulfite, disulfide reductase, or enzymes, which then facilitates further hydrolysis of the protein by proteases.⁴¹ The keratinolytic enzyme aids in the further breakdown of resistant keratin, as proposed by the theory of enzyme hydrolysis. According to the theories, the dissolution of the disulfide bond is a prominent feature of keratin degradation. Along with provided theories of keratin degradation, the cooperative action of disulfide reductase and proteases in the breakdown of feather keratin. The rigid complex cysteine disulfide bond is shattered by disulfide reductase, and the action of keratinases loosens the keratin structure in conjunction with the formation of amino acids.⁴⁴ To facilitate the breakdown of keratin in microorganisms, Peng et al. proposed a cascade of reactions involving cysteine catabolism. The microbial cell can convert cysteine to sulfite, which exits the cell and breaks the disulfide bond in feather keratin. Cysteine, a byproduct of feather keratin hydrolysis, enters the cell and the cycle is repeated.⁴⁵ Keratin degradation begins with the hydrolysis of a cysteine bond, which is then catalyzed by microbial keratinases. The possible mechanism of keratin degradation by microbial keratinases is depicted in Figure 3, which depicts the initial phase, i.e. denaturation, and the subsequent phase, hydrolysis, which produces amino acids.

Characterization of keratinases
Different factors have a significant impact on the keratinases’ activity and output. The pH and the temperature stand out the most. According to the measured physical parameters, such as pH, keratinase activity increases from an acidic to an alkaline medium. Most of the keratinases from Bacillus megaterium SN1 are active at a pH of 3²⁴. Researchers found that keratinase activity was greatest between the pH range of 7 and 9, which is neutral to alkaline. The bacterial and fungal keratinases showing maximum activity under alkaline pH includes keratinases from Streptomyces gulbargensis,⁴⁶ Bacillus pumilus FH9¹⁹, Scopulariopsis brevicaulis,⁴⁷ Paecilomyces marquandii and Doratomyces microspores,⁴⁸ Aspergillus sp. DHE7³³, Bacillus  licheniformis ALW1¹², Scopulariopsis brevicaulis⁴⁷, Proteus vulgaris EMB-14⁴⁹, Aspergillus sp. DHE7³³, Trichophyton sp. HA-2³⁰ etc. The alkaline medium facilitated the severing of the disulfide bond, resulting in the highest keratinase activity at alkaline pH. However, the keratinases from thermophilic bacterium Fervidobacterium islandicum AW-1 showed maximum activity in the range from 90 to 100°C.⁵⁰ The optimal temperature range for most keratinases was 40°C to 60°C. According to a survey of published data on microbial keratinolytic proteases, their molecular weights can vary anywhere from 17 to 240 kDa^{12,14,31–33,48,51–53,19,22–26,29,30} Keratinases from S. maltophilia BBE11-1⁵⁴ and S. albidoflavus⁵⁵ have low molecular weights of 17 and 18 kDa, respectively. In contrast, the keratinase produced by K. rosea was discovered to have a molecular weight of up to 240 kDa.⁵⁶ The various biochemical characterizations of keratinases has been described in Table 1 and Table 2.

Table (1):
Biochemical characterization of keratinases from some fungal species.

Abbreviations: EDTA- Ethylenediaminetetraacetic acid, SDS-sodium dodecyl sulfate, PMSF-phenylmethylsulfonyl fluoride, BSA- Bovine serum albumin DDT-Dichlorodiphenyltrichloroethane, β-ME-β- mercaptoethanol

Table (2):
Production and characterization of keratinases from bacterial species.

Abbreviations: EDTA- Ethylenediaminetetraacetic acid, SDS-sodium dodecyl sulfate, PMSF-phenylmethylsulfonyl fluoride, BSA-bovine serum albumin, DDT-Dichlorodiphenyltrichloroethane, β-ME-β- mercaptoethanol.

Strategies to improve keratinases for keratinases production
Protein Engineering Techniques
There are numerous applications for microbial keratinases in biotechnological industries, but their poor tolerance to temperature, pH, organic salts, and detergent, as well as their low catalytic activity, precludes their use. Because the processes of degrading feathers and dehairing leather took place in an alkaline environment, the high performance of alkaline keratinase may be especially significant. One of the most important characteristics of an enzyme for its potential use in industrial processes is its resistance to salt, also known as halotolerance. Protein engineering methods were used to develop superior enzymes with enhanced catalytic efficiency, thermostability, and substrate specificity.³⁴ To achieve the desired yield, scientists use protein engineering techniques like signal pro-peptide engineering, domain swapping, truncation, etc. to improve the protein’s activity, thermostability, and catalytic efficiency. Analysis of keratinase amino acid sequences reveals that the different domains of the enzymes are largely responsible for substrate specificity, thermostability, alkalinity, feather degrading efficiency, etc., as well as other effects of organic compounds and detergents. Keratinases from Stenotrophomonas maltophilia BBE11-1 have been deduced to have three distinct domains based on their amino acid sequences: an N-propeptide domain, a catalytic domain, and a PPC (Pre-peptidase C-terminal) domain.⁵⁷ When each domain was altered, specific results were observed. Substrate specificity was found to be controlled by a C-terminal domain. Changes in the N-terminal domain exchange between the isolated keratinases KerSMF and KerSMD formed a mutant FDD with enhanced catalytic and feather degradation activity.⁵⁸

The variant V355 from Keratinase KerSMD of Stenotrophomonas maltophilia was created by partial truncation of the PPC domain, and it exhibited improved secretion of keratinase, enzyme stability, salt tolerance, surfactant stability, and other properties that make it prominent for use in laundry detergents and leather treatments.^59,99 The enhancement of protein or the enzymes can be achieved by two approaches which are as follows:

Rational protein engineering
The rational protein design can effectively predict and modify the residues responsible for the enzyme’s substrate and co-factor binding sites, thermostability, and other specific functional properties by comparing the target sequence (predicted structure) to fully characterized enzymes.⁶⁰ Site directed mutagenesis which is one of rational approach in protein engineering is used for modifying enzyme substrate specificity.  Site directed mutagenesis was studied in mutant FDD keratinases from by the N-terminal domain exchange between the isolated keratinases KerSMF and KerSMD from Stenotrophomonas sp.⁵⁷ The keratinase variant FDD thus engineered through site directed mutagenesis and the two variants (Y94F and Y215F) showed higher extracellular keratinolytic activity (about 3-fold increase).⁵⁸

Directed evolution
Directed evolution tries to accelerate up the natural evolution of biological molecules and it is based on random mutagenesis and/or gene recombination to generate a library of variants and screening/selection of variants with enhanced phenotype.⁶¹ This strategies has been used to make enzymes with a wide range of traits, such as substrate specificity, resistance to organic solvents, stability at high temperatures, and the best working pH.^61–63 The method was used to engineered keratinases (KerPA) from Pseudomonas aeruginosa CCTCC AB2013184 and found that the variant of keratinases (Y21pBpF,Y70pBpF and Y114pBpF)  thus obtained showed enhanced activity and thermostability.⁶⁴ Similarly, Zhang et al., found that keratinase mutant (KerBp)  obtained from using directed evolution technology showed enhanced enzyme activity from 1150 to 8448 U/mL and feather degradation rate increases from 49 to 88% when trypsin was added.⁶⁵

The outcomes of the various protein engineering techniques used to improve enzyme performance are summarized in the table 3 that follows.

Table (3):
Different molecular Strategies to improve keratinases production and its possible outcomes.

Mutagenesis
UV mutagenesis
The effect of non-ionizing radiation on mutagenesis is due to the excitation of DNA molecules, which results in the formation of thymine base pairs between adjacent bases in the DNA molecule. The strain’s keratinase production is changed by UV light incubation. Lateef et al. discovered that the mutant (89.2 U/mL) from Bacillus safensis LAU 13 produced more keratinase than that of the wild type (57.4 U/mL) and, when the strain was pre-incubated with UV light of 254 nm, the production increased further. It was observed that the resulting mutant possessed dehairing capabilities.⁶⁶ The  Bacillus cereus group with isolates (S1, S13, S15, S26, and S39) were introduced with UV radiation and found the mutants S13uv and S26uv showed high keratinase activity showed higher keratinase production of after 72 hours of incubation.⁶⁷ Similarly, the purified proteases obtained from the mutant RS 1 obtained through UV irradiation of Bacillus sp. resulted in 44-fold increase in specific activity compared with the crude protease of the wild type Bacillus sp. RS1⁶⁸.

Chemical Mutagenesis
Enzyme production is significantly impacted by the presence of chemical agents like ethyl methyl sulfonate (EMS), N-methyl-N-nitro-N-nitrosoguanidine (MNNG), and ethidium bromide in the culture medium. A 2011 study by Duarte et al. found that incubating the Candida parapsilosis strain in 3% ethyl methanesulfonate (EMS) resulted in greater keratinase activity in the mutant strain J5(140 U/mL) than in the wild type strain (80 U/mL).⁶⁹ The bacterial cereus isolates (S1, S13, S15, S26, and S39) were treated with EMS and the mutant thus formed showed increased keratinase activity from 1.5–3.7-fold compared with the wild isolates.⁶⁷

Site-Directed mutagenesis
Site-directed mutagenesis entails the modification of a gene via substitutions, targeted base insertions, or deletions. The process is primarily concerned with increasing the activity, yield, efficiency, and thermostability of enzymes, as well as their resistance to acidic to alkaline environments.⁷⁰ Various molecular strategies applied for obtaining higher keratinases yield along with the possible outcomes were tabulated in table 3.

Biotechnological applications of keratinases
Keratinases are essential enzymes for the degradation of numerous keratin-based substances. Keratinases provide numerous environmental and agricultural benefits. The eco-friendly nature of the microbial degradation of resistant hard keratin by keratinases is of utmost importance, as it prevents the loss of nutritionally significant amino acids that can be damaged by chemical treatment. Several applications of keratinases are described below: –

Animal feed additives
Keratin can be broken down by microbial keratinases, which can then be used by the microbes as a source of carbon and nitrogen. Essential amino acids like cysteine, threonine, phenylalanine, leucine, valine, and isoleucine are produced as a byproduct of keratinous waste and can be used to strengthen animal feed.^71,72 Odetallah et al. investigated the effects of adding keratinases to the chickens’ diet on their overall body weight. Supplementing corn-soy based broiler starter diets with a keratinase was found to boost chick growth performance and increase body weight. It was deduced that the young chickens’ increased body mass was the result of their consuming the crude protein, which served as a substrate for the enzymes and released the free amino acids that the chicks then consumed. When added to low protein diets on day one, keratinase increased chicken body weight by day 21.⁷³ Aina et al. conducted a study in which Wickerhanomyces Anomalus keratinase was introduced into chicken feed. It was found that when enzymes were introduced into the feed, the total protein content and nitrogen content both increased.⁷⁴ The study done by Xu et al. in 2022 showed that when keratinase (200,000 U/kg) was added to a corn-soybean-feather meal-based diet (BD) for 6 weeks, the body weight of broiler chicken increases from by 3.6% to 4.3% and also improves the meat quality of the chicken.⁷⁵ Using Ross 308 chicks, Ayanwale et al. did an experiment to compare the growth performance and meat quality of broiler chickens fed on keratinase-treated and untreated feather meal-based diets. The broiler chickens fed a diet with 16% enzyme-treated feather meal (T6) had the highest average final body weight (2113.00 g), compared to the untreated feather meal-based diets that had average final body weight (1935.70g).⁷⁶ Therefore, the supplementation of keratinase could be used as feed additives to enhance growth performance, total protein content and meat quality in the poultry industries.

Leather Industry
In developing nations, the leather trade is seen as a promising new sector. Chemicals like sodium sulfite, lime, and calcium carbonate are used in the dehairing process, and they lower the quality of the leather and pollute the air with their byproducts. When compared to chemical dehairing, which damages fibers and yields low-quality leather, the use of microbial proteases as an alternative is both environmentally friendly and cost-effective. Keratinolytic proteases are more effective for depilation and show no damage to leather when the process is optimized, in comparison to common alkaline proteases.^70,77 Depilation requires an extremely alkaline environment and the use of detergents like sodium dodecyl sulfonate, inactivate most of the proteases. In contrast, keratinases are recalcitrant enzymes that can withstand high concentrations of alkali and detergents like SDS etc. Keratinases are used in the dehairing process to remove hair follicles, leaving the skin smooth and hairless which infers good quality leather.⁷⁰ Chaturveti et al. found that all of the hairs were removed when goat hairs were incubated with 40 U/ml of crude keratinase for 20 hours. Skin treated with keratinase was soft, swollen, and white in color after removal of hairs, while skin treated with sodium sulfite and calcium carbonate was hard, dark, and brown in color after shrinking.⁷⁸ SEM analysis confirmed that both the crude and purified keratinases from Bacillus sp. NKSP-7  were able to completely dehair goat skin while leaving it otherwise undamaged.⁷⁹ Goat, rabbit, bovine, and sheep skins were completely dehaired when treated with the recombinant keratinase rKERDZ isolated from Actinomadura viridilutea DZ50. Scanning electron microscopy (SEM) analyses corroborated the findings, showing that the leather’s quality was unaltered from the control sample. Leather processing using chemicals raises concentrations of harmful byproducts like COD and BOD, which is bad for the environment. As a result, enzymatic techniques may have biotechnological and ecological significance. Bioremediation with the help of the microbial product has significant economic value and advantages over chemical treatment in the leather industry. Utilizing purified or recombinant keratinase may ameliorate the process more effectively than would be possible with crude enzymes.⁸⁰

Detergent industry
Some keratinases are considered thermoactive, which provides them with a high degree of tolerance against any solvent, surfactant, or detergent. In addition to aiding in the removal of scarves and blood stains from cotton garments in hospitals, they have extensive application in the detergent industry.⁸¹ Researchers have analyzed keratinases for their potential use in the detergent industry due to their enzymatic washing properties. On various fabrics stained with blood, egg yolk, and chocolate, the crude enzyme (76 U/ml) supplemented with inactive detergent was used to observe its cleansing effects. Protein stains like blood and egg yolk were found to be easily removed by the enzyme, while the chocolate stain was not completely removed.⁸² Both the wild type (57.4 U/mL) and the mutant strain (89.2 U/mL) created by UV mutagenesis were able to efficiently remove the blood stain from the fabric without damaging the fibers. Keratinases from Bacillus sp. NKSP-7 were found to be stable and detergent-compatible. When incubated with blood-stained fabrics, both the purified enzyme and the crude enzyme demonstrated cleaning efficacy.⁷⁹ Another cleansing properties was observed when the cloth stained with coffee and blood was incubated with keratinases from Stenotrophomonas maltophillia.⁸³ The washing capacity of the egg stained was shown by Citrobacter diversus-derived keratinases which was tolerant to detergent mixture which can implemented in detergent formulations.⁸⁴ Therefore, the use of keratinases plays a significant role in the detergent industry, as it can give a natural means of cleaning without compromising the fabric quality, which is negatively affected by the use of detergents.

Other Applications
Keratinases can be utilized as both biopesticides and plant growth factor. Root knot nematodes (Meloidogyne incognita), which cause damage to plants, can be destroyed by treating with purified alkaline keratinases obtained from Bacilllus sp., which digest the nematode’s keratin and collagen cuticle. Keratinases’ ability to degrade the cuticle layer of nematodes lead to the dead of the nematodes.⁸⁵ The role of keratinases in growth is investigated in Vigna radiate. The plant treated with feather hydrolysate obtained from Stenotrophomonas maltophilia showed higher growth in plant compared to untreated one.⁸³ The application of keratinases can also be observed in transdermal drug delivery systems. In mice infected with Staphylococcus aureus, keratinases from Bacillus cereus were used as a drug delivery agent alongside fusidic acid by Shalaby et al. When a keratinases/fusidic acid mixture was used, rather than fusidic acid alone, S.aureus growth was greatly reduced. It was hypothesized that keratinase could break down the stratum corneum, allowing the drug to penetrate the skin and speed up the mice’s recovery. The research laid the groundwork for future investigations into the medical application of keratinases as delivery systems.⁸⁶

The current scope and future challenges in keratinases research
Assessment of research publications accumulated over a while on a topic of interest is a very reliable method to decipher the relevance, viability, aspects, national/ international cooperation, funding pattern by the different funding agencies, and existing lacunae on the concerned research topic. Such studies not only give an insight into the researcher’s involvement but also inherently depicts and dissect the focal themes and trends of research.⁸⁷ A recent study conducted by Nnolim and Nwodo based on bibliometric data of three decades (1990 – 2019) on keratinase research by 20 top most researchers in this area highlighted that the focus of research has largely been associated with different aspects of keratinase production, characterization, and keratinous biomass degradation. The study also revealed that India scaled the summit among the top twenty most productive countries on keratinase research, ahead of China and Brazil, by achieving not only ~ 26% of the total research publications but also the highest number of citations (1533 times with average article citation of 18.04) documented during this 3-decade period.⁸⁸

The ultimate aim needs to focus on collective efforts through national/ international collaborations for addressing the unsolved and most challenging research areas on keratinases to make the study more profound and innovative that in turn would contribute more to the discovery sciences rather than settling for just mundane findings. International collaborations not only bring about cross-pollination of ideas to enhance productivity and innovation but also foster scientific capacity.⁸⁹ While some of the seminal research work conducted during the past 3-decades has now eventually established excellent platforms to cement the academia-industry linkages and collaborations for harnessing the laboratory-based keratinase research to accrue economic benefits out of the same, there remains considerable scope to explore and exploit new vistas in this arena. One of the major limitations encountered by numerous researchers involved in keratinase research is towards identifying the peptidases possessing keratinase activity. The impediment to the identification of the potential keratinase is compounded by the challenge of predicting the substrate specificity of the peptidase with keratinolytic activity.⁹⁰ The establishment of efficient analytical methods is fundamental, both for the production as well as its application to environmental waste management.⁹¹

Although it is imperative to have the stimulus of appropriate S&T infrastructure, adequate research funding, and sound collaborative partnerships for qualitative and quantitative research outputs, it is even more crucial to define the research niches on keratinases and the expected outputs from those researches before framing any research proposal. This assumes even greater significance as currently, due to overall constraints of R&D funding globally, efforts are directed towards resource optimization and restrictions in funding proposals that project only incremental advances in domain knowledge in any particular area of S&T research. Hence, to secure grants from different funding agencies, the different aspects of research on Keratinases that needs to be conceptualized may revolve around at least one or more factors such as: i) research that contributes to value addition to discovery sciences, ii) research that caters to generating more academia-industry link-ups ensuring economic returns, iii) research having a significantly high societal impact, and iv) research that addresses grand challenges by aligning with one or more of the National Missions related to sustainable habitats and green energy.