Discovery and protein engineering of biocatalysts for organic synthesis. Protein detection methods in proteomics research. In: Matovic MD, editor. The enzymatic activity is usually tested on solid matrices supplemented with keratinous substrate.

Sivasubramanian S, Murali Manohar B, Puvanakrishnan R. Mechanism of enzymatic dehairing of skins using a bacterial alkaline protease. Keratinases have already found a purpose in pharmaceutical, textile and leather industries. Purification and characterization of an aspartic protease from the Rhizopus oryzae protease extract, peptidase R. New families of carboxyl peptidases: Serine-carboxyl peptidases and glutamic peptidases. New tools for discovering and characterizing microbial diversity. and transmitted securely. 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). Differential extraction of keratin subunits and filaments from normal human epidermis. Keratins are resistant to degradation by conventional proteases (pepsin, trypsin) and insoluble in diluted acids, alkaline reagents, water and organic solvents (7). Paul T, Jana A, Mandal AK, Mandal A, Das Mohpatra PK, Mondal KC.

Despite the fact that E. coli is a preferred expression system for recombinant proteins, the bottleneck of producing the protein in this host is accumulation of inactive inclusion bodies and requirement of in vitro folding of pro-keratinases, which drastically affects final active enzyme yield (8, 19). The sisters -helices of collagen, elastin and keratin recovered from animal by-products: Functionality, bioactivity and trends of application. 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. Enhancement of the thermostability and the catalytic efficiency of Bacillus pumilus CBS protease by site-directed mutagenesis. Reconstruction of Microraptor and the evolution of iridescent plumage. Su C, Gong JS, Zhang RX, Tao LY, Dou WF, Zhang DD, et al. NJ276. Gradiar H, Friedrich J, Kriaj I, Jerala R. Similarities and specificities of fungal keratinolytic proteases: Comparison of keratinases of Paecilomyces marquandii and Doratomyces microsporus to some known proteases. Herrmann H, Wedig T, Porter RM, Lane EB, Aebi U. Lime treatment of keratinous materials for the generation of highly digestible animal feed: 1. The ability of keratin degradation has so far been reported for strains of Bacillus licheniformis (44), B. pumilus, B. cereus, B. subtilis (45) and non-sporogenic bacteria Stenotrophomonas sp.

If the strains themselves are not easy to cultivate, they may represent a source of keratinase genes that can be expressed in another, already established production host.

Fusion construct kerA-stp allowed formation of a recombinant protein keratinase-streptavidin, which was secreted into the medium.

Increase of biogas production at wastewater treatment plants: Addition of urban organic waste and pre-treatment of sludge [PhD Thesis]. Zara Jaouadi N, Rekik H, Ben Elhoul M, Zohra Rahem F, Hila CG, Ben Aicha HS, et al. Serine peptidases: Classification, structure and function. 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.

Nail disorders, mainly associated with fungal infections, are ranging from relatively harmless (pigmentation), to painful conditions such as nail dystrophy (142). Other promising producers are representatives of the genus Streptomyces (S. rectus var. Effective dehairing properties of keratinase from Paenibacillus woosongensis TKB2 obtained under solid state fermentation. Rajendra VB, Baro A, Kumari A, Dhamecha DL, Lahoti SR, Shelke SD. for the keratin degradation. Gegeckas A, Gudiukaite R, Debski J, Citavicius D. Keratinous waste decomposition and peptide production by keratinase from Geobacillus stearothermophilus AD-11. Combining pyrosequencing with SIP allows metagenomics of high resolution that targets specific enzymes (75). Head domain, or N-terminal part of protein, is a globular structure with -turns consisting of variable number of amino acids (50100) with a positive net charge.

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). Engineering strategies for enhanced production of protein and bio-products in Pichia pastoris: A review.

in B. subtilis DB104 and B. licheniformis T399D) (100). Kublanov IV, Tsiroulnikov KB, Kaliberda EN, Rumsh LD, Haertl T, Bonch-Osmolovskaya EA. After enzyme binding and cleavage of disulphide bonds, keratin changes its conformation and exposes multiple sites for hydrolytic action of the enzymes (21). So far there have been reports of microorganisms such as Streptomyces sp. 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). about navigating our updated article layout. Among the latter, proteases are an extremely important group of enzymes due to their broad applicability scope (40, 118).

The primary steps of keratin degradation by fungi involve mechanical attack on the substrate by their mycelium in addition to sulfitolysis (21, 58, 59). Gene shuffling results in different combinations of hybrid genes, originating from different origin cells or even species (106). (38), Fervidobacterium pennavorans (46), F. islandicum (23), Lysobacter sp. The aim of the following review is to approach comprehensively current knowledge on various aspects of these enzymes and their production that may be helpful for future research. 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). Patinvoh RJ, Osadolor OA, Chandolias K, Srvri Horvth I, Taherzadeh MJ.

Structures of protofibrils and microfibres are strengthened by disulphide bonds, hydrogen and hydrophobic interactions, which also play a crucial role in rigidity and recalcitrance of keratin molecules (16). Paul T, Das A, Mandal A, Jana A, Maity C, Adak A, et al. 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. Ismail AMS, Housseiny MM, Abo-Almagd HI, El-Sayed NH, Habib M. Novel keratinase from Trichoderma harzianum MH-20 exhibiting remarkable 24 dehairing capabilities. Limitation of nutrients reduces GTP concentrations and consequently inactivates CodY (43). Serine proteases are functionally rich and diverse group of proteases with nucleophilic serine residues (Ser) located in the enzyme active site. 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). 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). Keratinolytic fungi represent dermatophytes (Microsporum, Epidermophyton and Trichophyton) and related species of the genus Chrysosporium. 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).

Assessment of the anaerobic biodegradability of macropollutants. (140, 141). Wool is composed of structural proteins with high degree of cross-linked disulfide bridges that give fibres mechanical strength and resistance to degradation. Kandasamy S, Duraisamy S, Chinnappan S, Balakrishnan S, Thangasamy S, Muthusamy G, et al.

(50), Thermoanaerobacter sp. 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.

Properties and original source of the enzyme to be expressed affect the choice of the type of heterologous host (which is usually bacterium or yeast strain). The binding of the repressor to the operator prevents transcription of the gene encoding for specific enzyme. The flexibility of UV-inducible mutation in Deinococcus ficus as evidenced by the existence of the imuB-dnaE2 gene cassette and generation of superior feather degrading bacteria. Gloux K, Leclerc M, Iliozer H, LHaridon R, Manichanh C, Corthier G, et al. Wool-associated proteolytic bacteria, isolated from Portuguese Merino breed. Biotechnological and industrial applications of keratinases.

Novel microbial-mediated modifications of wool. Metaproteomics provides functional insight into activated sludge wastewater treatment. 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 glucose and nitrogen starvation response of Bacillus licheniformis.

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. nov., an extremely thermophilic bacterium isolated from a hot spring. Keratinases vis--vis conventional proteases and feather degradation.

The industry has a great interest in microorganisms that are capable of producing large amounts of efficient extracellular keratinases (33, 68, 69). The .gov means its official. (21) have reported a hot spring isolate, belonging to Desulfurococcales (labelled 1507-2), capable of keratin degradation at 70 C and pH=6.

Currently, keratinase expression using heterologous systems would lay the foundation for its genetic manipulation, protein engineering and large scale enzyme preparation (98). This approach has already been used to improve a whole series of enzymes for which the structure-function relationship was not known (115). Dettmer A, Cavalli , Ayub MAZ, Gutterres M. Optimization of the unhairing leather processing with enzymes and the evaluation of inter-fibrillary proteins removal: An environment-friendly alternative. sharing sensitive information, make sure youre on a federal 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. Purification and characterization of a protease produced by Bacillus megaterium RRM2: Application in detergent and dehairing industries.

Feather keratin hydrolysis by a Vibrio sp. This genus has an important role in white biotechnology for several reasons it is very well studied, majority of species are non-pathogenic, generally recognized as safe (for example B. cereus, B. stearothermophilus, B. licheniformis, B. subtilis and so on) and many functional extracellular proteins are secreted directly into the culture medium (60, 62). 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.

Non-dermatophytic keratinolytic fungi are found in two genera, Chrysosporium and Myceliophthora (anamorph) (56). 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). 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). An official website of the United States government. 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.

Expression of the Bacillus licheniformis PWD-1 keratinase gene in B. subtilis. High-resolution metagenomics targets specific functional types in complex microbial communities.

Learn more Structure and functions of keratin proteins in simple, stratified, keratinized and cornified epithelia. Zhang RX, Gong JS, Zhang DD, Su C, Hou YS, Li H, et al. Sharma VK, Kumar N, Prakash T, Taylor TD. Various keratinolytic enzyme preparations for the removal of animal hair without damaging the skin (collagen) are also in use. Keratin is a complex and structurally stable protein found in human and animal hard tissues, such as feathers, wool, hair, hoof and nails. 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). 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. (113) immobilization of keratinases of genus Bacillus sp. Love DC, Halden RU, Davis MF, Nachman KE. Keratinase production and keratin degradation by a mutant strain of Bacillus subtilis. Yamamura S, Morita Y, Hasan Q, Yokoyama K, Tamiya E. Keratin degradation: a cooperative action of two enzymes from Stenotrophomonas sp. A simple method for the detection of protease activity on agar plates using bromocresolgreen dye. Abdullah N, Sulaiman F. The oil palm wastes in Malaysia. Exposing B. subtilis to MNNG, for example, resulted in 2,5-fold higher keratinase activity in mutated strain than in the wild-type (109).

Gopinath SCB, Anbu P, Lakshmipriya T, Tang TH, Chen Y, Hashim U, et al. 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 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. High-throughput screening for biocatalysts. 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).

proteolyticus, S. griseus, S. moderatus NRRL 3150, Streptomyces sp. Zhang RX, Gong JS, Su C, Zhang DD, Tian H, Dou WF, et al. Most of them are monomeric, although there are descriptions of some multimeric enzymes (25). Keratins typically consist of three domains with different secondary structures: head domain, central helical domain and tail domain (6). Chair of Microbiology and Microbial Biotechnology, Department of Animal Science, Biotechnical Faculty, University of Ljubljana, Groblje 3.



applied as host strains for the construction of metagenomic libraries (87). Engineering of enzyme immobilization enables the impro- vement of the efficiency, thermostability and other enzyme characteristics.

Shrinkage has traditionally been controlled using absorbable organic chlorides. 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. Brevibacillus sp. The amino acid replacement N122Y caused 5,6-fold increase in the catalytic efficiency, in comparison to wild-type keratinase (101, 104).

Chaturvedi V, Bhange K, Bhatt R, Verma P. Production of keratinases using chicken feathers as a substrate by a novel multifunctional strain of Pseudomonas stutzeri and its dehairing application. 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). 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). A major limitation impeding valorization of keratinous substrates is their recalcitrant structure and resistance to hydrolysis by common proteases. The sequencing era introduced the need for upgraded classification system based on sequence similarities, also reflecting evolutionary relationships between the proteolytic enzymes.

The production is typically most intensive at the end of the exponential and/or in stationary phase, which is associated with the adaptation to the lack of nutrients (40). Environmentally safe alternatives to the chemicals used in this process are enzyme preparations, typically composed of proteases and lipases.

M49-1 by optimizing the growth conditions using response surface methodology. It is typically used to improve the thermostability, efficiency, activity of keratinases or enzyme yield (promotor mutagenesis) (103). Degradation of keratinous materials by the plant pathogenic fungus Myrothecium verrucaria. 310 amino acids and consists of four right-handed -helical subdomains (1A, 1B, 2A and 2B), which are separated from one another by non-helical -turns called linker regions.

2010. https://doi.org/1010.1016/j.jbiotec.2015.07.007, Successful, safe and effective removal of earwax from the external ear canal, Keratinases present natural alternative to the use of acids for corn and callus removal, Keratoclean Hydra PB, PURE 100 Keratinase, Acne is caused by blockage of sebaceous glands in the presence of large quantities of keratin, therefore keratinases can be used for successful treatment, Keratoclean Sensitive PB, Keratopeel PB, Keratinase product improves the feed ratio and has a positive effect on chicken body mass, Enzyme product reduces cost of cooking and temperature processing of feather, therefore increasing the digestibility and nutritional value of feed, Effective decontamination of medical instruments from prions. 0.1% wool (28), feather meal (25), hair (29, 30), keratin powder, soy flour, etc. Behrens GA, Hummel A, Padhi SK, Schtzle S, Bornscheuer UT. Tail domain or C-terminal part of protein contains globular structure. YSA 2130, S. diastaticus SS1, S. corchorusii ST36, S. pactum DSM 40530) and actinomycetes Nocardiopsis and Oerskovia (6365). Subfilamentous protofibril structures in fibrous proteins: Cross-linking evidence for protofibrils in intermediate filaments. Complete genome sequence of a keratin-degrading bacterium Chryseobacterium gallinarum strain DSM 27622T isolated from chicken. 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). Biochemical characterization of a novel surfactant-stable serine keratinase with no collagenase activity from Brevibacillus parabrevis CGMCC 10798. Lv LX, Sim MH, Li YD, Min J, Feng WH, Guan WJ, et al. 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. Sturmberger L, Wallace PW, Glieder A, Birner-Gruenberger R. Synergism of proteomics and mRNA sequencing for enzyme discovery. Zeng YH, Shen FT, Tan CC, Huang CC, Young CC. 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). 65% of the market (40). A perspective: Metatranscriptomics as a tool for the discovery of novel 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).

Hydrolysis of the amyloid prion protein and nonpathogenic meat and bone meal by anaerobic thermophilic prokaryotes and Streptomyces subspecies. The methane production of poultry slaughtering residues and effects of pre-treatments on the methane production of poultry feather. An unbiased manner to mine natural microbial communities for new keratinases is by screening expression library for the desired enzymatic activity. Davidsson . The main industrial producers of serine proteases are strains belonging to the genus Bacillus (60, 61). Non-ionizing radiation (for example by UV light) results in the excitation of electrons in DNA molecules, which typically results in mutagenic effect. A novel keratinase from Bacillus tequilensis strain Q7 with promising potential for the leather bating process. Brandelli et al. The main waste in poultry industry are feathers, representing 710% of chicken mass. (52) and Chryseobacterium sp. 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. Zhang S, Long L, Yin H, Xiao Z, Li Q, Zhang S, et al. 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). Hard -keratin intermediate filament chains: Substructure of the N-and C-terminal domains and the predicted structure and function of the C-terminal domains of type I and type II chains.

Novel molecular methods for discovery and engineering of biocatalysts from uncultured marine microorganisms. Proteases are classified as hydrolytic enzymes that may either cleave peptide bonds near the ends of polypeptide chain (exoproteases) or inside the chains (endoproteases). Amino acid sequence also affects the secondary structure of keratins, which may be enriched in -helix (typical for -keratins) or -sheet structures (typical for -keratins) (8). The extraction and characterization of bovine epidermal -keratin. Plasmids, bacterial artificial chromosomes (BACs), cosmids and fosmids are commonly in use (82). Microbial production of proteolytic enzymes is a complex and highly regulated process, dependent on the microbial growth stage (39, 40). Wilmes P, Andersson AF, Lefsrud MG, Wexler M, Shah M, Zhang B, et al. Forgcs G, Lundin M, Taherzadeh MJ, Srvri Horvth I. Pretreatment of chicken feather waste for improved biogas production. strain kr2. Lime and sulphide-free dehairing of animal skin using collagenase-free alkaline protease from Vibrio metschnikovii NG155. Reconstruction of biochemical networks in microorganisms.

Due to their protective role, the structure of these proteins is very recalcitrant and resistant to the degradation by widespread enzymes (2). Anaerobic carboxydotrophic bacteria in geothermal springs identified using stable isotope probing. Multistage process of keratin degradation includes two major processes: sulfitolysis or reduction of disulfide bonds and proteolysis (8, 37). Mitola G, Escalona F, Salas R, Garca E, Ledesma A. Morphological characterization of in-vitro human hair keratinolysis, produced by identified wild strains of Chrysosporium species. Another type of target metagenomic enrichment method is stable-isotope probing (SIP) (94), in which microorganisms grow in the presence of isotopically labelled substrates. Descriptions of thermophilic and extremophilic keratinolytic representatives from genera Fervidobacterium sp. Isolation of novel strains producing significant yields of well performing target enzymes is a prerequisite for the development of a viable industrial fermentation process. Jaouadi B, Ellouz-Chaabouni S, Ali MB, Messaoud EB, Naili B, Dhouib A, et al. Despite many advantageous features of Bacillus sp., the use of this host may be limited due to reported plasmid instability (100). Shen J, Rushforth M, Cavaco-Paulo A, Guebitz G, Lenting H. Development and industrialisation of enzymatic shrink-resist process based on modified proteases for wool machine washability. Some of these, like feathers and wool, represent one of the main sources of protein-rich waste with significant potential to be transformed into value-added products such as feed, fertilizers or bioenergy. 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. Novel binding sites for metal ions, active sites, etc. Application of metagenomic techniques in mining enzymes from microbial communities for biofuel synthesis.

Low yield is the result of low accessibility of nutrients due to compact keratin ultrastructure in feathers and can be increased by pretreatment (156). The main bottlenecks limiting wider application of many currently available enzymes are their efficiency on complex natural substrates (such as feather and poor-quality wool), low stability and/or high production costs. 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). This suggests that the production or secretion of proteases may be regulated by nutritional stress, such as the lack of carbon and nitrogen sources. An overview on fermentation, downstream processing and properties of microbial alkaline proteases. Fang Z, Zhang J, Liu B, Jiang L, Du G, Chen J. Cloning, heterologous expression and characterization of two keratinases from Stenotrophomonas maltophilia BBE11-1. Although the mechanism of enzyme adsorption is not yet well understood, it is known that the higher the adsorption capacity, the higher the degree of keratin hydrolysis (22).

Demir T, Gbe , Ycel M, Hame Kocaba EE.

According to the amino acid sequences in the enzyme active sites and associated catalytic mechanisms, proteases can be classified into seven broad groups: serine, cysteine, threonine, aspartic and glutamic proteases, metalloproteases and asparagine peptide lyases (17, 19). Fervidobacterium thailandense sp. Other expression hosts, like Bacillus strains, are also used. In these strains, the significant enzyme yield and higher production have been achieved with the introduction and expression of multiple copies of keratinase genes (e.g.