Qualitative methods for keratinolytic activity detection on solid matrix are usually appropriate for the initial assessment of the samples either when screening for keratinolytic strains or enzyme fractions. Commonly known assays for protease activity detection apply agar/agarose plates, with incorporated target substrate (26). A commonly applied variation of this method is the radial diffusion test, where the proteolytic activity is proportional to hydrolysis zones around small holes in agar plate containing immobilized substrate (27). This type of screening media used different substrates, including e.g. 0.1% wool (28), feather meal (25), hair (29, 30), keratin powder, soy flour, etc. The applied substrates typically also act as enzyme inducers, however, it is essential that the screening medium be free of repressors and inhibitors of target enzymes such as pepstatin, high concentrations of dextrose or glucose (31).

Zymography is used as a semi-quantitative method for keratinase detection on solid phase, since it has several advantages over other methods (32). This highly sensitive method allows the identification of specific active proteases in complex enzyme mixtures. While the rest of the qualitative and semi-quantitative methods serve exclusively for screening tests, zymography offers the information on molecular masses of the separate enzymes in the extracts (33).

After initial isolation of keratinolytic strain or specific enzyme(s), quantification of keratinolytic activity under different conditions is usually of interest as a key part of further target characterization. Quantitative methods allow exact determination of proteolytic activity in a liquid phase (spectrophotometry, fluorimetry, radiometry, chromatography, capillary electrophoresis and enzyme-linked immunosorbent assays are generally used) (32). Keratinolytic activity in protein extracts may be determined using a variety of insoluble chromogenic substrates (examples are keratin azure, azocasein, azokeratin, etc.) (34, 35). Enzyme extract is typically mixed with the substrate suspension and incubated at the appropriate temperature. Afterwards, centrifugation of samples removes the insoluble substrate and the absorbance at the appropriate wavelength detects the release of the product (dye) (32).

Metagenomics appeared at the end of the last century as an alternative approach to conventional microbial screening of environmental samples (82, 83). Metagenomic sequence analyses enable the reconstruction of the global enzyme potential in the environmental sample – an information on total consortium of enzymes encoded in examined microbial community (84, 85).

Research strategies for accessing novel biocatalysts include (optional) pretreatment of genes of interest, nucleic acid extraction, vector and host selection and the screening of metagenomic libraries. Extracting and purification of the sample DNA is a critical step in the process, since the construction of a metagenomic library requires a sufficient amount of high-quality DNA. The next step in building a metagenomic library is the selection of an appropriate vector and host systems, which depends on the characteristics of the environmental samples and intended purpose of the constructed database (82).

A selection of proper vector determines whether heterologous DNA will successfully enter the host cells and be actively expressed (the latter is important for functional metagenomics, which is an experimental approach for studying gene function). The choice of a vector system depends on the quality of the extracted DNA and research goals, taking into account the size of inserted fragments, the required number of copies of the vector, type of the used host strain and potential methods of screening. Plasmids, bacterial artificial chromosomes (BACs), cosmids and fosmids are commonly in use (82).

Currently, the most widely used cloning and expression host is Escherichia coli. However, many eukaryotic genes cannot be functionally expressed in E. coli, due to the lack of appropriate mechanisms of post-translational modifications, which makes the development and establishment of new, alternative host systems extremely important (86). So far there have been reports of microorganisms such as Streptomyces sp. and Pseudomonas sp. applied as host strains for the construction of metagenomic libraries (87).

An unbiased manner to mine natural microbial communities for new keratinases is by screening expression library for the desired enzymatic activity. To allow for an efficient route for mining new enzymes, high-throughput approaches have to be adapted that will allow to rapidly screen 10⁶–10⁸ samples (88). The enzymatic activity is usually tested on solid matrices supplemented with keratinous substrate. By screening metagenomic library on the plates, positive clones can be identified through a visual examination of discolouration or staining zones around enzyme-producing colonies (89). This type of screening does not necessarily require extra devices and can be performed at high-throughput level, but the signals are often poor, which may be the reason for low hit rate. Some alternative approaches can improve the sensitivity of agar-based screening. One strategy with improved sensitivity involves the growth of cell library in microtiter plates, followed by chemical or physical lysis and activity detection (90–92). If agar plates are used, targeted activities can be linked to the survival of host organisms, which makes screening or selection sensitive and high-throughput. Another strategy is screening of large libraries by multiple cycles of enrichment using flow cytometry or selective immobilization of active clones (88).

The genes encoding target enzymes represent a very small portion of the (meta)genome and in some cases even less than 0.01% of total sample of nucleic acids derives from environmental sources. Low abundance of target genes in the library plays a key role in the selection of mining strategy (93). Metagenomic approaches therefore sometimes involve custom-designed oligonucleotide hybridization probes, which hybridize to specific sequences in metagenomic libraries. For example, catalytic triplet Asp191, His252 and Ser441 are highly conserved regions with a strong preservation in oxyanion region in all keratinases. These regions can be used for designing probes for identification of new keratinases in unexplored sources (19).

Another type of target metagenomic enrichment method is stable-isotope probing (SIP) (94), in which microorganisms grow in the presence of isotopically labelled substrates. Due to metabolic activity, the isotopes (usually ¹³C or ¹⁵N) are incorporated exclusively in macromolecules of the microbes that metabolize the target substrate (keratin). This increases the target organism’s DNA or RNA density that can later be separated from unlabelled molecules (94). High density DNA then serves as a template to amplify the functional genes involved in the selected metabolic pathways by PCR, which allows the study of active microorganisms involved in the processes (95). Combining pyrosequencing with SIP allows metagenomics of high resolution that targets specific enzymes (75).

A major disadvantage of approaches based on (meta)genomic library sources of novel enzymes is the high percentage of non-coding and/or non-target DNA regions, which unnecessarily increase the size of the library and (in the case of eukaryotic DNA) need to be removed in order to produce functional enzymes. In addition to the problems related to the incorrect gene positioning with respect to its promoters, non-excised introns and the differential codons usage, post-translational modifications may also represent a problem when expressing these genes in heterologous hosts (96). The advantage of using cDNA libraries is that most of the non-encoding DNA elements are removed, reducing the size of libraries to the genes that are actively expressed in certain conditions (97).

Non-ionizing radiation (for example by UV light) results in the excitation of electrons in DNA molecules, which typically results in mutagenic effect. Random mutations in structural gene or regulatory elements may have positive or negative effect on the production and properties of target enzymes. According to Zeng et al. (111), UV irradiation of Deinococcus ficus CC-ZG207 mutants caused a 2-fold increase in keratinase production, in comparison to the wild type strain (101). Chemical agents, such as ethyl methylsulfonate (EMS), N-methyl-N’-nitro-N-nitroso-guanidine (MNNG) or ethidium bromide, can also induce random mutations in DNA molecules. Exposing B. subtilis to MNNG, for example, resulted in 2,5-fold higher keratinase activity in mutated strain than in the wild-type (109). Brevibacillus sp. AS-S10-II treatment with EMS resulted in mutants having a higher growth rate and keratinase production (101).

Molecular methods for improvement of biocatalysts (or regulatory elements affecting their production) is usually performed by one of the following approaches: the rational design or directed evolution.

Rational protein design is an effective strategy, which requires the information on the relationship between the sequence, structure and function of target enzymes. It is based on the comparison of the target sequence (predicted structure) to the already characterized enzymes, which allows the prediction and modification of the residues responsible for specific functional properties of the enzyme, substrate and co-factor binding sites, thermostability or enantioselectivity. Novel binding sites for metal ions, active sites, etc. can also be introduced into the protein (106). Site-directed mutagenesis is a method that applies target changes in double-stranded DNA, resulting in a modified amino acid sequence and tertiary structure of the enzyme (112). It is typically used to improve the thermostability, efficiency, activity of keratinases or enzyme yield (promotor mutagenesis) (103). Thermostability of B. licheniformis BBE11-1 keratinase has been improved by using a computer design (algorithm PoPMuSiC), which was applied for predicting change of free energy of amino acid substitutions. Afterwards, four amino acid substitutions (N122Y, N217S, A193P and N160C) were introduced into the enzyme, using site-directed mutagenesis and mutated genes were later on expressed in B. subtilis WB600. The amino acid replacement N122Y caused 5,6-fold increase in the catalytic efficiency, in comparison to wild-type keratinase (101, 104).

Specific nucleases PstI and KpnI were used for improving stability of serine proteases from Bacillus sp., for which oxidation-sensitive methionine 222 was removed and replaced by non-oxidizing amino acid (Ser, Ala or Leu). Consequently, protease mutants, containing modified amino acids, were more resistant to chemical oxidation (105).

The relatively rapid PCR-based method used in rational design involves splicing by overhang extension (SOE) (106) that generates improved protein variants containing modified (specific) amino acid. This method offers several advantages – it is simple, fast and highly repeatable and does not require restriction enzymes or DNA ligases to generate site-directed mutations (106).

Engineering of enzyme immobilization enables the impro- vement of the efficiency, thermostability and other enzyme characteristics. According to Wang et al. (113) immobilization of keratinases of genus Bacillus sp. can be achieved by fusion of two genes – kerA, encoding keratinase, and stp, encoding streptavidin, which binds to biotin. Fusion construct kerA-stp allowed formation of a recombinant protein keratinase-streptavidin, which was secreted into the medium. Fusion protein was then isolated from the medium using biotinylated beads. This method of immobilization substantially improved the thermostability and pH tolerance of the enzyme (101).

Directed evolution is one of the most effective approaches to tailor biocatalysts for industrial purposes (106). Fundamentally, it is an imitation of Darwinian evolution on a laboratory scale, based on sequential random mutagenesis and selection steps (114). This approach has already been used to improve a whole series of enzymes for which the structure-function relationship was not known (115).

The first step in directed evolution is the introduction of numerous random point mutations by PCR and/or recombination, resulting in the emergence of many molecular variants of a wild-type gene. Point mutations can be introduced using error-prone PCR, for example by substituting Mg²⁺ with other type of cations (e.g. Mn²⁺) or by the application of highly error-prone polymerase. Another possible way of generating novel gene variants is DNA shuffling, which is based on recombination of related genes and also allows the generation of proteins with improved properties. Gene shuffling results in different combinations of hybrid genes, originating from different origin cells or even species (106).

Successful selection of engineered enzymes requires a “high-throughput” screening method, which considerably increases the chance of obtaining desired properties and reduces the time and cost of the analysis (116). Emerging new technologies that provide sufficient performance are necessary to support the testing of millions of samples in a highly reproducible manner. The most convenient strategy for the detection of enzymes involves the use of chromogenic or fluorogenic substrates (107). This strategy is based on the ability of bacteria harbouring the target genes to selectively convert substrates to detectable products. A high-throughput method for single cell screening can be applied by microfluidic droplet sorting yielding improved production hosts for secreted enzymes, which results from an improvement of the cellular functions of the production host. Enzymes producing single cells are encapsulated in 20-pL droplets with a fluorogenic reporter substrate. The platform enables small reagent consumption for a screening experiment, which greatly reduces the costs of screening and engineering of production strains (107).

Recent progress in high-throughput enzyme assays has included analytical instruments for parallel screening, thermistor arrays, new fluorogenic and chromogenic substrates, as well as novel applications of pH indicator methods (117).

Leather industry is one of the oldest, fast-growing industries in the world and plays an important role in today’s economy. At the same time, it is considered as one of the world’s biggest sources of pollution, as leather processing includes use of toxic substances, causing hazardous effects on the environment and workers in industrial plants (60).

The treatment of hides involves a series of processes, among which pre-tanning is considered as a major source of pollution (8). Chemicals such as sodium sulfide, lime and solid wastes, arising as a result of the preliminary tanning, are the main reason for the increase in biochemical oxygen demand (BOD), chemical oxygen demand (COD) and total quantity of dissolved solids (TDS) in wastewater produced in these plants (8). The enzyme practice is a green alternative leading to reduction in environmental pollution and improves the leathers (120). Proteolytic enzymes are nowadays increasingly used for hide softening to improve pliability of the hides and prepare them for tanning process. Various keratinolytic enzyme preparations for the removal of animal hair without damaging the skin (collagen) are also in use. They selectively degrade the soft keratin tissue in the follicle, thereby pulling out intact hair without affecting tensile strength of the leather (121). Enzymes, mainly originating from Bacillus sp. (122–126), Pseudomonas stutzeri (127), Caldicoprobacter algeriensis (128), Acinetobacter sp. (129), Paenibacillus woosongensis (130), Vibrio metschnikovii (131) and different fungi from species Aspergillus tamarii (132), Penicillium chrysogenum and Trichoderma harzianum have been reported to be used in leather industry (133, 134). The application of keratinases in leather industry improves the quality of the final product, and reduces environmental pollution by chemicals, providing a safer working environment (60, 135).

Alkaline proteases represent 89% of proteolytic enzymes used in detergent industry (8). Enzymes suitable for use in detergents need to be compatible with other components of the washing agents, and exhibit activity and stability at higher pH values and temperatures. The alkaline keratinase from Paenibacillus woosongensis TKB2 is one of the enzymes with potential application in the laundry industry for removing the composite stains without affecting the fabric texture, fibres and strength of clothes (130).

Textile industry uses keratinases for processing wool fibres. Wool is composed of structural proteins with high degree of cross-linked disulfide bridges that give fibres mechanical strength and resistance to degradation. This is attributed to the overlapping layers of the epidermis (epi-, exo- and endocuticle) (136). Epicuticle is rich in lipids, while the exo- and endocuticle are composed of keratin. The epidermis plays a key role in fibre contraction during washing and dyeing processes. Shrinkage has traditionally been controlled using absorbable organic chlorides. The disadvantages of this process include loss of natural wool character, yellowing of material, pollution of waste water with AOX (adsorbable organically bound halogens), affected biodegradability of fabric (caused by limited bioavailability) and high energy and time consumption (137). Environmentally safe alternatives to the chemicals used in this process are enzyme preparations, typically composed of proteases and lipases. The role of proteases is to remove an outer layer of coarse fibre, reducing the rough feeling of the wool. It is necessary to carefully dose the enzymes, as some proteases penetrate deep into fibres, causing their damage, loss of mass and tensile strength (138). This is also the reason why currently no process is exclusively based on enzymes. To some extent, penetration can be limited by increasing the molecular mass of proteases aimed for this application by chemical crosslinking, attachment of synthetic polymers, etc. (33). A better alternative is the use of specific keratinases that selectively act only on keratinous layers of the wool, without adverse effects on the other fibre parts (137).

Prions are very robust, compact proteins that cause serious neurodegenerative diseases, such as TSEs (transmissible spongiform encephalopathies) (19). Due to increased number of prion horizontal transmissions from animals to humans (including iatrogenic transmissions via contaminated surgical tools), effective methods for decontamination of material infected with prions are of vital importance. Since conventional methods are based on aggressive and energetically demanding processes, enzymatic degradation of prions may be a promising approach for destruction of these highly aggregated proteins with structural similarities to keratin. So far there have been reports of only two keratinases used for prion decontamination purposes – one isolated from B. licheniformis PWD-1 (137) and the other from Streptomyces sp. (139). Nevertheless, in addition to enzymatic treatment, further treatment of infected material with alkaline reagents, detergents and high temperatures is required.

Keratinases are also used in cosmetic industry for treatment of acne, calluses, keratinized and dry skin removal, treatment of psoriasis, etc. (140, 141). Applications of keratinases in pharmaceutical industry are mainly associated with improving the passage of fungicidal drugs through the keratinous nail surface. Nail disorders, mainly associated with fungal infections, are ranging from relatively harmless (pigmentation), to painful conditions such as nail dystrophy (142). Treatment of fungal infections of nails (onychomycosis) is extremely challenging and traditionally involves long-term consumption of antifungal medicines and repeated monthly injections with corticosteroids, causing many side effects, such as rashes and liver damage (143). An alternative form of treatment is a topical application of antimycotic drugs directly to the affected area. The main drawback of this approach is impermeability of the nail surface which affects drug penetration and treatment efficiency. A variety of mechanical (nail abrasions and separation), physical (etching, laser treatment, hydration and fast closing of nails) and chemical methods (keratinolytic agents, such as urea, thioglycolic acid, salicylic acid, N-acetyl cysteine, mercaptoethanol) are being used for improving the medicine transfer to the site of action (144). However, these chemicals are only effective in high concentrations and may have a pungent smell (137). Keratinases, on the other hand, may be very effective in loosening the nail plates already in low concentrations, which was first demonstrated by keratinase from Paecilomyces marquandii (137, 143).

According to regulation (EC) 1774/2002 (145) of the European parliament, keratinous waste is classified as the third category of animal products, meaning: (i) it is obtained from animal carcases, (ii) is not intended for human consumption, and (iii) should not transmit diseases to humans or animals (145). Large amounts of waste produced by animal processing plants can be used as a substrate for bioenergy and value-added products if properly treated prior to their use. Traditional treatment of keratinous waste includes the use of alkaline and acidic hydrolysis, processing at high pressure and high temperatures (up to 150 °C) (146). Despite the aforementioned advantages, these methods are expensive, consume large amounts of energy and lead to loss of some essential amino acids important for subsequent production of value-added products or bioenergy (33, 147).

The main waste in poultry industry are feathers, representing 7–10% of chicken mass. In 2012, around 8.5 billion tonnes of poultry feather were generated worldwide (148). Feathers consist of about 90% keratin (majority being β-keratin) and contain large amounts of serine, glutamic acid, proline and small amounts of methionine, histidine and lysine (145). Several million tonnes of feathers per year represent one of the biggest waste byproducts of poultry industry, and an important source of environmental pollution (8, 149). One of the options for valorization of feather waste involves their transformation to the feather meal, which can be used as a raw material in biodiesel production, an ingredient in bioplastics or as animal feed (150). Traditional processing of feathers includes high temperature and pressure, which are the cause of high costs and destruction of some essential amino acids (methionine, lysine and tryptophan). Consequently, final products may be poorly digestible and have a variable nutritional value (8). Such shortcomings can be avoided by enzymatic hydrolysis of feathers by keratinases, resulting in production of higher quality amino acids that can be supplemented to poultry, pig, ruminant and fish feeds (8, 150).

Hydrolyzed feathers can also be converted to bio-hydrogen (119), or used as a fertilizer for organic farming that allows slow release of nitrogen, improves plant growth, promotes soil microbial activity, structures the soil and increases its water retention capacity (8).

Anaerobic digestion is a promising process for microbial production of renewable energy from different types of waste substrates. During this process, substrates are hydrolysed to amino acids, which are later converted to a variety of organic acids, ammonia, carbon dioxide, hydrogen and minor amounts of sulphur compounds. These are the substrates for methanogenesis – a microbial process in which acetate, hydrogen and carbon dioxide are used to produce methane and carbon dioxide (151). Anaerobic degradation of feathers runs under thermophilic (151) or mesophilic (151, 152) conditions and usually includes different types of waste (manure, mixed bone fractions, leftovers and offal).

Theoretical methane yield from proteins is 0.496 m³/kg of volatile solids (VS) (153, 154), but only around 0.21 m³/kg was reported to have been produced from waste feathers (155). Low yield is the result of low accessibility of nutrients due to compact keratin ultrastructure in feathers and can be increased by pretreatment (156). In recent years different chemical, physical and enzymatic methods of pretreatment are being explored (155, 157). Development of some recombinant keratinolytic strains with increased production of enzymes aims to increase and improve keratinous waste decomposition before/during anaerobic digestion (155). A recombinant B. megaterium strain carrying B. licheniformis keratinase gene was developed in order to increase hydrolysis of chicken feathers. The recombinant strain effectively degraded feathers prior to biogas production, which resulted in production of up to 80% of the theoretical methane yield originating from protein digestion. The positive effect is due to the fact that a strong inducible promotor controls the keratinase production in B. megaterium, resulting in a prompt and efficient turnout of enzymes without lag phase (158).

Keratinases are also used in a variety of other applications, including for bioaugmentation of composting of keratin-rich waste (159), for modifications of basic structure of fibres in wool or silk (8), in different cosmetic products and processing of edible bird’s nests. One of the unconventional applications is in pearl bleaching. At the time of bead formation, organic impurities such as free cells, mucus cells and necrotic tissue may be present in the layer of mounting. Therefore it is necessary to improve the quality of beads prior to sale. The pearls are traditionally treated by gentle bleaches (hydrogen peroxide) that can provide soft lightening, although they can offset their colouring and may affect colour irregularity. Zhang et al. (160) reported using keratinases as an alternative for the traditional-type bleaching process.