Systematic functional analysis and application of a cold-active serine protease from a novel Chryseobacterium sp. 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). (47) and Kocuria sp. Other expression hosts are also being used for heterologous keratinase production, for example yeast Pichia pastoris, which provides a good environment for the appropriate post-translational modifications and protein folding of eukaryotic keratinases. Purification and characterization of a protease produced by Bacillus megaterium RRM2: Application in detergent and dehairing industries. Different approaches used for identification, production and characterization of novel keratinases and optimization of their performance. Zymography is used as a semi-quantitative method for keratinase detection on solid phase, since it has several advantages over other methods (32). Friedrich J, Gradiar H, Vrecl M, Poganik A. Li Q, Gao KQ, Meng Q, Clarke JA, Shawkey MD, DAlba L, et al. Keratinase of an anaerobic thermophilic bacterium Thermoanaerobacter sp. KH3 for reducing waste activated sludge and biofilm formation. Zhang S, Long L, Yin H, Xiao Z, Li Q, Zhang S, et al. The treatment of hides involves a series of processes, among which pre-tanning is considered as a major source of pollution (8). Enzyme extract is typically mixed with the substrate suspension and incubated at the appropriate temperature. Structure and functions of keratin proteins in simple, stratified, keratinized and cornified epithelia. Microbial production of proteolytic enzymes is a complex and highly regulated process, dependent on the microbial growth stage (39, 40). Community proteogenomics highlights microbial strain-variant protein expression within activated sludge performing enhanced biological phosphorus removal. Engineering of enzyme immobilization enables the impro- vement of the efficiency, thermostability and other enzyme characteristics. These include cloning of genes encoding novel enzymes directly from the metagenomes and modifications of the existent enzymes by protein engineering (Fig. The platform enables small reagent consumption for a screening experiment, which greatly reduces the costs of screening and engineering of production strains (107). Comparison of different Bacillus subtilis expression systems. 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). Most of them are monomeric, although there are descriptions of some multimeric enzymes (25). Transungual drug delivery: A promising route to treat nail disorders. Keratinases are also used in cosmetic industry for treatment of acne, calluses, keratinized and dry skin removal, treatment of psoriasis, etc. Isolation of three novel Antarctic psychrotolerant feather-degrading bacteria and partial purification of keratinolytic enzyme from Lysobacter sp. The intermediate filament architecture as determined by X-ray diffraction modeling of hard alpha-keratin. HHS Vulnerability Disclosure, Help Novel molecular methods for discovery and engineering of biocatalysts from uncultured marine microorganisms. Metaproteomics provides functional insight into activated sludge wastewater treatment. Sturmberger L, Wallace PW, Glieder A, Birner-Gruenberger R. Synergism of proteomics and mRNA sequencing for enzyme discovery. In addition, this unicellular eukaryote is simple for culturing and manipulations and has so far been successfully used for expression of many proteases from bacteria, fungi and mammals (102). However, current mainstream strategies of keratinous waste valorization involve physical/chemical (pre)treatment of the substrates that are not environmentally friendly. This increases the target organisms DNA or RNA density that can later be separated from unlabelled molecules (94). Recombinant environmental libraries provide access to microbial diversity for drug discovery from natural products. Fig. 
High-throughput screening for biocatalysts. However, it is estimated that more than 99% of microorganisms from natural environments have not yet been isolated, which greatly reduces the number of available microbiological sources (73, 74). Keratins typically consist of three domains with different secondary structures: head domain, central helical domain and tail domain (6). Leather industry is one of the oldest, fast-growing industries in the world and plays an important role in todays economy. One strategy with improved sensitivity involves the growth of cell library in microtiter plates, followed by chemical or physical lysis and activity detection (9092). Brandelli et al. Isolation of novel strains producing significant yields of well performing target enzymes is a prerequisite for the development of a viable industrial fermentation process. The https:// ensures that you are connecting to the Biotechnological applications and prospective market of microbial keratinases. Metagenomic approaches therefore sometimes involve custom-designed oligonucleotide hybridization probes, which hybridize to specific sequences in metagenomic libraries. 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). This is an open-access article distributed under the terms of the Creative Commons Attribution No Derivatives (CC BY-ND) 4.0 License. The three-dimensional structure of the first keratin molecule was first described in 1959, but the number of studies in this area increased extensively after 1990 (5). 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. nov. Low abundance of target genes in the library plays a key role in the selection of mining strategy (93). bacillus strain td5k td5b enzyme keratinase yogyakarta strains According to Wang et al. A03. Park GS, Hong SJ, Jung BK, Khan AR, Park YJ, Park CE, et al. (51), Stenotrophomonas sp. Effective dehairing properties of keratinase from Paenibacillus woosongensis TKB2 obtained under solid state fermentation. Blint B, Bagi Z, Tth A, Rkhely G, Perei K, Kovcs KL. Improving protein extraction and separation methods for investigating the metaproteome of anaerobic benzene communities within sediments. Novel binding sites for metal ions, active sites, etc. Majority of currently described and applied keratinases originate from isolated microbial strains. Keratinase production and keratin degradation by a mutant strain of Bacillus subtilis. Enhancement of the thermostability and the catalytic efficiency of Bacillus pumilus CBS protease by site-directed mutagenesis. radiant vit renewal complex pbs Revisiting microbial keratinases: Next generation proteases for sustainable biotechnology. Demir T, Gbe , Ycel M, Hame Kocaba EE. Consequently, protease mutants, containing modified amino acids, were more resistant to chemical oxidation (105). Anaerobic carboxydotrophic bacteria in geothermal springs identified using stable isotope probing. 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. 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. A recombinant B. megaterium strain carrying B. licheniformis keratinase gene was developed in order to increase hydrolysis of chicken feathers. A promising alternative approach that might solve this issue would be the application of cost-effective proteolytic enzymes, specialized for the degradation of these substrates (3). degradation microbial substrate keratinase Enzymatic degradation of keratin is a multistage process that requires the following steps: (i) adsorption of the keratinases to the surface of macromolecule by electrostatic and hydrophobic interactions, followed by (ii) catalytic action. This method of immobilization substantially improved the thermostability and pH tolerance of the enzyme (101). Biotechnological and industrial applications of keratinases. 
nov., an extremely thermophilic bacterium isolated from a hot spring. 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). Moreira FG, de Souza CGM, Costa MAF, Reis S, Peralta RM. in B. subtilis DB104 and B. licheniformis T399D) (100). 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). 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. Kublanov IV, Tsiroulnikov KB, Kaliberda EN, Rumsh LD, Haertl T, Bonch-Osmolovskaya EA. Pearl albefaction method mediated by keratinase and combined with redox. Gousterova A, Braikova D, Goshev I, Christov P, Tishinov K, Vasileva-Tonkova E, et al. Innovative pretreatment strategies for biogas production. sharing sensitive information, make sure youre on a federal Approximately 294 000 metallopeptidases have been identified so far and classified into 73 families and 15 clans (MEROPS, edition 12.0, 2018) (24). It is typically used to improve the thermostability, efficiency, activity of keratinases or enzyme yield (promotor mutagenesis) (103). They selectively degrade the soft keratin tissue in the follicle, thereby pulling out intact hair without affecting tensile strength of the leather (121). Functional metagenomics for enzyme discovery: Challenges to efficient screening. Keratinolytic fungi represent dermatophytes (Microsporum, Epidermophyton and Trichophyton) and related species of the genus Chrysosporium. 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). and Pseudomonas sp. At the same time, it is considered as one of the worlds biggest sources of pollution, as leather processing includes use of toxic substances, causing hazardous effects on the environment and workers in industrial plants (60). Microbiology and industrial biotechnology of food-grade proteases: A perspective. Zhang RX, Gong JS, Zhang DD, Su C, Hou YS, Li H, et al. Novel microbial-mediated modifications of wool. 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). 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. Kalyuzhnaya MG, Lapidus A, Ivanova N, Copeland AC, McHardy AC, Szeto E, et al. proteolyticus, S. griseus, S. moderatus NRRL 3150, Streptomyces sp. JB 99. Molecular mass of bacterial keratinases ranges from 18 (enzyme SK1-02 from Streptomyces albidoflavus) to 200 kDa (enzymes from Kocuria rosea and Fervidobacterium islandicum), while the enzymes of pathogenic fungi can reach up to 440 kDa (for example keratinase II of Trichophyton mentagrophytes) (8, 24, 25). The domains and subdomains of one keratin molecule interact with those of adjacent keratin molecules forming heterodimers, tetramers and, finally, keratin fibres. 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). Nevertheless, it is important to note that constitutive expression of proteases is mostly associated with caseinolytic and not keratinolytic activity, and it is suggested that the keratinolytic enzymes are mainly inducible (8). Bethesda, MD 20894, Web Policies Usually, peculiar microecosystems in which microorganisms have to adapt to the lack of nitrogen/carbon sources other than keratin represent the best sources of these enzymes (33, 68, 69). 
Keratinases are ubiquitous in nature and can be found in representatives from the three domains of life: Eukarya, Bacteria and Archaea. Current developments in biotechnology and bioengineering. Manczinger L, Rozs M, Vgvlgyi C, Kevei F. Isolation and characterization of a new keratinolytic Bacillus licheniformis strain. Ismail AMS, Housseiny MM, Abo-Almagd HI, El-Sayed NH, Habib M. Novel keratinase from Trichoderma harzianum MH-20 exhibiting remarkable 24 dehairing capabilities. 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). These constantly contribute to the recycling of the carbon, nitrogen and sulphur by the degradation of vast amounts of keratinous waste (hair and birds feathers, etc.) Courtois S, Cappellano CM, Ball M, Francou FX, Normand P, Helynck G, et al. for the keratin degradation. 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). To allow for an efficient route for mining new enzymes, high-throughput approaches have to be adapted that will allow to rapidly screen 106108 samples (88). Degradation of keratinous materials by the plant pathogenic fungus Myrothecium verrucaria. Oxford, UK: Elsevier BV; 2017. pp. Gessesse A, Hatti-Kaul R, Gashe BA, Mattiasson B. Lisboa, Portugal: Technical University of Lisbon; 2011. Discovery and protein engineering of biocatalysts for organic synthesis. 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 birds nests. These regions can be used for designing probes for identification of new keratinases in unexplored sources (19). Feather-degrading Bacillus species from poultry waste. 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. 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. Accessibility AS-S10-II treatment with EMS resulted in mutants having a higher growth rate and keratinase production (101). Bouacem K, Bouanane-Darenfed A, Zara Jaouadi N, Joseph M, Hacene H, Ollivier B, et al. The environment represents an unlimited source of new keratinolytic strains for the implementation of new industrial processes and improvement of the existing ones (40, 70, 71). Paul T, Das A, Mandal A, Jana A, Maity C, Adak A, et al. Koehn H, Clerens S, Deb-Choudhury S, Morton JD, Dyer JM, Plowman JE. Jaouadi B, Ellouz-Chaabouni S, Ali MB, Messaoud EB, Naili B, Dhouib A, et al. 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). Consequently, final products may be poorly digestible and have a variable nutritional value (8). A current assessment on the production of bacterial keratinases. High-resolution metagenomics targets specific functional types in complex microbial communities. 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). Biochemical characterization of a novel surfactant-stable serine keratinase with no collagenase activity from Brevibacillus parabrevis CGMCC 10798. An unbiased manner to mine natural microbial communities for new keratinases is by screening expression library for the desired enzymatic activity. However, these chemicals are only effective in high concentrations and may have a pungent smell (137). Extracellular serine proteases produced by Thermoactinomyces strains from hot springs and soils of West Anatolia. The rod domains of one acidic and one basic keratin molecule in parallel orientation form heterodimers of keratin molecules, and represent the building blocks of keratin filaments (11). Lv LX, Sim MH, Li YD, Min J, Feng WH, Guan WJ, et al. Despite many advantageous features of Bacillus sp., the use of this host may be limited due to reported plasmid instability (100). 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. Development of some recombinant keratinolytic strains with increased production of enzymes aims to increase and improve keratinous waste decomposition before/during anaerobic digestion (155). Limitation of nutrients reduces GTP concentrations and consequently inactivates CodY (43). 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). Combinatorial and computational challenges for biocatalyst design. Serine peptidases: Classification, structure and function. In addition, some species (for example B. licheniformis) produce significant amounts of indigenous extracellular enzymes (amylases, mannanases, cellulases) which may adversely affect the downstream processes (101). 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). 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. 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). YSA 2130, S. diastaticus SS1, S. corchorusii ST36, S. pactum DSM 40530) and actinomycetes Nocardiopsis and Oerskovia (6365). Hsiao NW, Chen Y, Kuan YC, Lee YC, Lee SK, Chan HH, et al. The primary steps of keratin degradation by fungi involve mechanical attack on the substrate by their mycelium in addition to sulfitolysis (21, 58, 59). It contains engineered protease with increased activity, broader specificity and thermostability, Keratinase product is supposed to regulate the concentration of keratin in pores, therefore helping to eliminate blisters, keratinized skin, it can be used for treatment of dermatophytic and nail diseases, scars and epithelial regeneration, Cleaning pipes and tanks with different enzymes, including keratinases. The role of proteases is to remove an outer layer of coarse fibre, reducing the rough feeling of the wool. Feather meal: A previously unrecognized route for reentry into the food supply of multiple pharmaceuticals and personal care products (PPCPs). National Library of Medicine (34, 35). dehairing enzyme exiguobacterium dg1 extracellular keratinase kera nase 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). In addition, proteomic and transcriptomic tools give us an information on the expression of target genes in specific conditions and explore their mechanisms of action (7781). 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). Zeng YH, Shen FT, Tan CC, Huang CC, Young CC. Novel serine keratinase from Caldicoprobacter algeriensis exhibiting outstanding hide dehairing abilities. The new PMC design is here! So far there have been reports of microorganisms such as Streptomyces sp. 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. The application of keratinases in some areas of industry is already well established, while they remain a promising tool for agriculture and food industry waste management. 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. Zhang et al. Various keratinolytic enzyme preparations for the removal of animal hair without damaging the skin (collagen) are also in use. Lime treatment of keratinous materials for the generation of highly digestible animal feed: 1. Tsiroulnikov K, Rezai H, Bonch-Osmolovskaya E, Nedkov P, Gousterova A, Cueff V, et al. Davidsson . FOIA Search for novel proteolytic enzymes aimed at textile and agro-industrial applications: An overview of current and novel approaches. Subfilamentous protofibril structures in fibrous proteins: Cross-linking evidence for protofibrils in intermediate filaments. The .gov means its official. Nam GW, Lee DW, Lee HS, Lee NJ, Kim BC, Choe EA, et al. This type of screening media used different substrates, including e.g. A major limitation impeding valorization of keratinous substrates is their recalcitrant structure and resistance to hydrolysis by common proteases. According to Zeng et al. 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). (50), Thermoanaerobacter sp. Corneous (hornlike) tissues have a specific role in vertebrates, representing a protective barrier between the organism and its environment. Utilization of keratin-containing biowaste to produce biohydrogen. Behrens GA, Hummel A, Padhi SK, Schtzle S, Bornscheuer UT. Fernndez-Arrojo L, Guazzaroni ME, Lpez-Corts N, Beloqui A, Ferrer M. Metagenomic era for biocatalyst identification. This strategy is based on the ability of bacteria harbouring the target genes to selectively convert substrates to detectable products. official website and that any information you provide is encrypted Kothari D, Rani A, Goyal A. Keratinases: In: Pandey A, Negi S, Soccol CR, editors. Learn more According to the nature of their active site, keratinases belong to serine- and metalloproteases or serine metalloproteases (8). The enzyme practice is a green alternative leading to reduction in environmental pollution and improves the leathers (120). The degradation of keratin is more abundant among Gram-positive than the Gram-negative bacterial genera (8). Increased production can be achieved by two strategies: (i) inserting the plasmid that contains the gene coding for keratinase under a strong promotor, or (ii) using a strain with more keratinase genes integrated in the chromosome.
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