Keratin Decomposition Research Articles

Current Progress and Biotechnological Applications of Microbial Keratinases

Keratin is a fibrous and recalcitrant protein found in feathers, nails, horns, hooves, and the epidermis of the skin. The presence of the high degree of disulfide bonds, hydrogen bonds, and hydrophobic interactions makes them resistant to mechanical stress and are not degraded by common proteases such as trypsin, pepsin, and papain. Due to the slow degradation of keratinous protein, accumulation of solid wastes from the poultry, slaughterhouse, textile, and leather industries leads to solid waste problems and other environmental and health related problems. In this review, efficient biodegradation of keratinous wastes by microorganisms, as a low-cost, environmentally friendly strategy has been discussed. Keratinases are the microbial proteases and hydrolyze the hard keratin. The decomposition of keratin by keratinases maintains the original structure of the final products, including short peptides, amino acids, and organic nitrogen which are deteriorated when traditional or chemical method is implemented. In this article, the role of keratinases producing bacterial and fungal species and their attributes has been elaborated, along with the biochemical characteristics of keratinases, and further, protein engineering approaches has been discussed, with the prospects to enhance keratinases activity for their biotechnological applications.

The sulfur formation system mediating extracellular cysteine-cystine recycling in Fervidobacterium islandicum AW-1 is associated with keratin degradation.

Most extremophilic anaerobes possess a sulfur formation (Suf) system for Fe-S cluster biogenesis. In addition to its essential role in redox chemistry and stress responses of Fe-S cluster proteins, the Suf system may play an important role in keratin degradation by Fervidobacterium islandicum AW-1. Comparative genomics of the order Thermotogales revealed that the feather-degrading F. islandicum AW-1 has a complete Suf-like machinery (SufCBDSU) that is highly expressed in cells grown on native feathers in the absence of elemental sulfur (S0 ). On the other hand, F. islandicum AW-1 exhibited a significant retardation in the Suf system-mediated keratin degradation in the presence of S0 . Detailed differential expression analysis of sulfur assimilation machineries unveiled the mechanism by which an efficient sulfur delivery from persulfurated SufS to SufU is achieved during keratinolysis under sulfur starvation. Indeed, addition of SufS-SufU to cell extracts containing keratinolytic proteases accelerated keratin decomposition in vitro under reducing conditions. Remarkably, mass spectrometric analysis of extracellular and intracellular levels of amino acids suggested that redox homeostasis within cells coupled to extracellular cysteine and cystine recycling might be a prerequisite for keratinolysis. Taken together, these results suggest that the Suf-like machinery including the SufS-SufU complex may contribute to sulfur availability for an extracellular reducing environment as well as intracellular redox homeostasis through cysteine released from keratin hydrolysate under starvation conditions.

Keratinases from Coriolopsis byrsina as an alternative for feather degradation: applications for cloth cleaning based on commercial detergent compatibility and for the production of collagen hydrolysate.

Keratinases are proteolytic enzymes that emerge as an alternative for dealing with the disposal of chicken feathers. In this study, we aimed to investigate the keratin-degrading enzymes secreted by the fungus Coriolopsis byrsina and their partial biochemical characterization to adapt their use for keratin decomposition, detergent additive applications, and collagen degradation. We observed the secretion of different proteolytic enzymes that possessed caseinolytic activity that peaked at pH 7.0-9.0 and 60-70 °C and at pH 10.5 and 55-60°C, and keratinolytic activity that reached a maximum at pH 7.0-7.5 and 40-55ºC and at pH 9.0 and 55 °C. Keratinolytic activity was maintained at approximately 63% of residual activity for 1h at 50 °C. The caseinolytic activity at pH 10.5 remains stable until 1h at 50 °C, and this is in contrast to the activity at pH 8.5, where the residual activity was 50%. Caseinolytic activity was inhibited only by PMSF, while keratinolytic activity was inhibited by PMSF and EDTA. When investigating the application of C. byrsina peptidases as an additive to commercial detergent, we observed an egg stain removal performance that was similar to that demonstrated by the commercial detergent. Based on their activity and stability at alkaline pH, these enzymes appear to be attractive candidates for use in the detergent industry. Additionally, the collagenolytic activity of these enzymes potentially allows for their use in a wide array of industrial sectors that require collagenolytic enzymes, such as for the production of collagen hydrolysates from residues derived from the meat industry.

In vitro synergistic effects of three enzymes from Bacillus subtilis CH-1 on keratin decomposition.

Extracellular protease Vpr (Vpr), gamma-glutamyltranspeptidase (GGT; EC 2.3.2.2) and glyoxal/methylglyoxal reductase (YvgN; EC 1.1.1.21) are extracellular enzymes involved in feather degradation, which were identified by secretome analyses from an efficient feather-degrading strain Bacillus subtilis CH-1. The encoding sequences corresponding to the three secretory enzymes were cloned into vector pET22b for recombinant expression in Escherichia coli strain BL21 (DE3). Afterward, the proteins containing the C-terminal His-tag were purified using a Ni-IDA column. The optimal temperatures and pH values for protease activity of recombinant Vpr, GGT, and YvgN were identified as 45°C/pH 7.0, 40°C/pH 8.0, and 50°C/pH 6.0 respectively when casein is the substrate. Furthermore, the synergistic effects of the three enzymes were studied using feather powder as substrate. Vpr was the core enzyme to hydrolyze keratin, while GGT and YvgN were coenzymes providing reducing activities for keratin decomposition. The keratinolytic activity was enhanced to about 1.4-folds when YvgN and Vpr applied together in comparison to Vpr alone. And the keratinolytic activity almost reached to 1.5-folds when all the three enzymes were combined to use. The study provides a novel perspective of the mechanism of keratin degradation by microorganisms, and thereby may also be of relevance for the design of an industrial process for enzymatic keratin degradation; however, additional experiments must be done to substantiate this conclusion.

The host range of dermatophytes, it is at all possible? Phenotypic evaluation of the keratinolytic activity of Trichophyton verrucosum clinical isolates.

Dermatophytes are fungi that have an ability to invade keratinised structures. Enzymes secreted by dermatophytes can underlie fungal survival on the host and development of infection. It is possible that the range of activity of keratinases from various dermatophytes is limited to specific species of animals and groups of people. The aim of this study was to carry out phenotypic analysis of the degree of keratinolytic activity of Trichophyton verrucosum strains using hairs of humans and various animal species as substrates. Our results indicated that the activity of keratinases is substrate-induced. The host range of T. verrucosum can be defined as wide. The highest activity of keratinases was recorded in media containing keratin from cow (Bos taurus) and sheep (Ovis aries) hairs in comparison with that from other tested species. The production of keratin-degrading enzymes is a function of time, with the peak of their activity occurring on day 15 of incubation. The role of keratin-degrading enzymes in the pathogenesis of dermatophytosis is becoming increasingly clearer. Given the conceptual understanding that keratin breakdown may require more than just one enzyme, the use of phenotypic methods is an optimal approach to in vitro study of the decomposition of species-specific keratin.

Biodegradation of Feather Waste Keratin by the Keratin-Degrading Strain Bacillus subtilis 8.

Bacillus subtilis 8 is highly efficient at degrading feather keratin. We observed integrated feather degradation over the course of 48 h in basic culture medium while studying the entire process with scanning electron microscopy. Large amounts of ammonia, sulfite, and L-cysteic acid were detected in the fermented liquid. In addition, four enzymes (gamma-glutamyltranspeptidase, peptidase T, serine protease, and cystathionine gamma-synthase) were identified that play an important role in this degradation pathway, all of which were verified with molecular cloning and prokaryotic expression. To the best of our knowledge, this report is the first to demonstrate that cystathionine gamma-synthase secreted by B. subtilis 8 is involved in the decomposition of feather keratin. This study provides new data characterizing the molecular mechanism of feather degradation by bacteria, as well as potential guidance for future industrial utilization of waste keratin.

Microbial decomposition of keratin in nature-a new hypothesis of industrial relevance.

Discovery of keratin-degrading enzymes from fungi and bacteria has primarily focused on finding one protease with efficient keratinase activity. Recently, an investigation was conducted of all keratinases secreted from a fungus known to grow on keratinaceous materials, such as feather, horn, and hooves. The study demonstrated that a minimum of three keratinases is needed to break down keratin, an endo-acting, an exo-acting, and an oligopeptide-acting keratinase. Further, several studies have documented that disruption of sulfur bridges of the keratin structure acts synergistically with the keratinases to loosen the molecular structure, thus giving the enzymes access to their substrate, the protein structure. With such complexity, it is relevant to compare microbial keratin decomposition with the microbial decomposition of well-studied polymers such as cellulose and chitin. Interestingly, it was recently shown that the specialized enzymes, lytic polysaccharide monoxygenases (LPMOs), shown to be important for breaking the recalcitrance of cellulose and chitin, are also found in keratin-degrading fungi. A holistic view of the complex molecular self-assembling structure of keratin and knowledge about enzymatic and boosting factors needed for keratin breakdown have been used to formulate a hypothesis for mode of action of the LPMOs in keratin decomposition and for a model for degradation of keratin in nature. Testing such hypotheses and models still needs to be done. Even now, the hypothesis can serve as an inspiration for designing industrial processes for keratin decomposition for conversion of unexploited waste streams, chicken feather, and pig bristles into bioaccessible animal feed.

Genome and secretome analyses provide insights into keratin decomposition by novel proteases from the non-pathogenic fungus Onygena corvina.

Poultry processing plants and slaughterhouses produce huge quantities of feathers and hair/bristle waste annually. These keratinaceous wastes are highly resistant to degradation. Onygena corvina, a non-pathogenic fungus, grows specifically on feathers, hooves, horn, and hair in nature. Hence, the proteases secreted by O. corvina are interesting in view of their potential relevance for industrial decomposition of keratinaceous wastes. We sequenced and assembled the genome of O. corvina and used a method called peptide pattern recognition to identify 73 different proteases. Comparative genome analysis of proteases in keratin-degrading and non-keratin-degrading fungi indicated that 18 putative secreted proteases from four protease families (M36, M35, M43, and S8) may be responsible for keratin decomposition. Twelve of the 18 predicted protease genes could be amplified from O. corvina grown on keratinaceous materials and were transformed into Pichia pastoris. One of the recombinant proteases belonging to the S8 family showed high keratin-degrading activity. Furthermore, 29 different proteases were identified by mass spectrometry in the culture broth of O. corvina grown on feathers and bristle. The culture broth was fractionated by ion exchange chromatography to isolate active fractions with five novel proteases belonging to three protease families (S8, M28, and M3). Enzyme blends composed of three of these five proteases, one from each family, showed high degree of degradation of keratin in vitro. A blend of novel proteases, such as those we discovered, could possibly find a use for degrading keratinaceous wastes and provide proteins, peptides, and amino acids as valuable ingredients for animal feed.Electronic supplementary materialThe online version of this article (doi:10.1007/s00253-015-6805-9) contains supplementary material, which is available to authorized users.

Keratin decomposition by trogid beetles: evidence from a feeding experiment and stable isotope analysis

The decomposition of vertebrate carcasses is an important ecosystem function. Soft tissues of dead vertebrates are rapidly decomposed by diverse animals. However, decomposition of hard tissues such as hairs and feathers is much slower because only a few animals can digest keratin, a protein that is concentrated in hairs and feathers. Although beetles of the family Trogidae are considered keratin feeders, their ecological function has rarely been explored. Here, we investigated the keratin-decomposition function of trogid beetles in heron-breeding colonies where keratin was frequently supplied as feathers. Three trogid species were collected from the colonies and observed feeding on heron feathers under laboratory conditions. We also measured the nitrogen (δ(15)N) and carbon (δ(13)C) stable isotope ratios of two trogid species that were maintained on a constant diet (feathers from one heron individual) during 70 days under laboratory conditions. We compared the isotopic signatures of the trogids with the feathers to investigate isotopic shifts from the feathers to the consumers for δ(15)N and δ(13)C. We used mixing models (MixSIR and SIAR) to estimate the main diets of individual field-collected trogid beetles. The analysis indicated that heron feathers were more important as food for trogid beetles than were soft tissues under field conditions. Together, the feeding experiment and stable isotope analysis provided strong evidence of keratin decomposition by trogid beetles.

Microsatellite-primed PCR for intra-species genetic relatedness in Trichophyton ajelloi strains isolated in poland from various soil samples.

Trichophyton ajelloi is a geophilic dermatophyte that specializes in the decomposition of native keratin. It exists in soil with a permanent influx of keratin matter. In the present study, two PCR-based methods were used for the identification and intra-species differentiation of T. ajelloi strains isolated from 3 types of soils with different physicochemical properties. The first method, employed for molecular identification, was PCR amplification of the 5.8S rRNA gene and its flanking regions encoding internal transcribed spacers (ITSs), followed by restriction enzyme digestion using endonuclease HinfI. The second method, employed for molecular differentiation, was microsatellite-primed PCR (MSP-PCR) using the repetitive oligonucleotide (GACA)4. All the T. ajelloi strains were also identified using a traditional culture method. Our results showed that molecular identification using the PCR-restriction fragment length polymorphism (PCR-RFLP) method agreed with the identification made using the traditional approach. On the other hand, PCR-RFLP results showed no strain differentiation, while MSP-PCR using the (GACA)4 primer identified different varieties among the T. ajelloi strains. The reasons for the intra-species differentiation of T. ajelloi have been discussed.

Untersuchungen zur Kompostierung von Federabfällen: Studies on the Composting of Feathers

The nitrogen of keratin of feathers is not taken up by plants as a suitable N-source. It becomes available by the keratinolytic activity of microorganisms. The high temperature evolved during the composting process promotes the growth and activity of thermophilic, keratin-decomposing microorganisms, e.g. Thermoactinomyces vulgaris. The increasing in temperature (self-heating) reflects the course of composting. A feather-peat mixture exhibits a considerably high heat production, i.e. a fast decomposition of keratin and other organic matter.

Keratin decomposition by dermatophytes II. Presence of S‐sulfocysteine and cysteic acid in soluble decomposition products

AbstractThe dermatophyte Microsporum gypseum was grown on human hair in a mineral medium. Filtrates of cultures of various age were chromatographed on Sephadex gels and further characterized by chemical analysis and thin‐layer chromatography. The products of keratin decomposition were separated into two fractions. The first fraction formed 10 to 20% of the bulk and contained proteins low in sulfur content. The second, very wide fraction contained oligopeptides and polypeptides with molecular weights from several hundreds up to approximately 13,000 with a peak at 1,000–2,000 daltons. This fraction contained a relatively high amount of cystine, S‐sulfocysteine, and cysteic acid.S‐sulfocysteine was present in the free state and also combined in peptides where its amount exceeded that of cystine. This supports the author's hypothesis on keratin decomposition by dermatophytes. According to this hypothesis, the fungus excretes sulfite which cleaves the disulfide bonds of keratin to cysteine and S‐sulfocysteine. The substrate, denatured by sulfitolysis, is then more easily digestible by fungal proteases.Besides S‐sulfocysteine, comparable amounts of cysteic acid were found in all analyses. This compound most probably originated by air oxidation of sulfur amino acids in the alkaline cultivation fluid.

Keratin decomposition by dermatophytes. II. Presence of s-sulfocysteine and cysteic acid in soluble decomposition products.

The dermatophyte Microsporum gypseum was grown on human hair in a mineral medium. Filtrates of cultures of various age were chromatographed on Sephadex gels and further characterized by chemical analysis and thin-layer chromatography. The products of keratin decomposition were separated into two fractions. The first fraction formed 10 to 20% of the bulk and contained proteins low in sulfur content. The second, very wide fraction contained oligopeptides and polypeptides with molecular weights from several hundreds up to approximately 13,000 with a peak at 1,000-2,000 daltons. This fraction contained a relatively high amount of cystine, S-sulfocysteine, and cysteic acid. S-sulfocysteine was present in the free state and also combined in peptides where its amount exceeded that of cystine. This supports the author's hypothesis on keratin decomposition by dermatophytes. According to this hypothesis, the fungus excretes sulfite which cleaves the disulfide bonds of keratin to cysteine and S-sulfocysteine. The substrate, denatured by sulfitolysis, is then more easily digestible by fungal proteases. Besides S-sulfocysteine, comparable amounts of cysteic acid were found in all analyses. This compound most probably originated by air oxidation of sulfur amino acids in the alkaline cultivation fluid.

Denaturation and Release of Amino Acids in Hairs treated with Cold-waving Lotions, Hair-dyes, Hair-bleaching Solutions and Depilatories

1. Human hair treated with cold-waving lotions, hair bleaching lotions, synthetic organic hair dyes, or depilatories was hydrolyzed with hydrochloric acid, and the hydrolyzed solution was submitted to amino acid autoanalyzer. Determined values of various amino acids were compared with the values of normal hair. A marked depletion of cystine and formation of cysteic acid were observed in the hair treated with cold-waving lotions, hair bleaching solutions, and synthetic organic hair dyes. Formation of cysteic acid was not observed in the hair treated with a depilatories but there was a marked decrease in cystine, with formation of a trace of methionine sulfoxide and methionine sulfone. 2. Amino acids formed by the decomposition of keratin when human hair is treated with cold-waving solutions or hair bleaching solutions were determined, both qualitatively and quantitatively, by amino acid autoanalyzer. Damaging effect of these solutions on the hair was proved by the dissolution of various amino acids.

Keratin decomposition by dermatophytes. I. Sulfite production as a possible way of substrate denaturation.

The dermatophyte Microsporum gypseum was grown on children's hairs in a mineral solution. During the growth of the fungus the substrate digestion, pH of the medium, and concentrations of soluble decomposition products were determined. In aerated and stationary cultures the substrate digestion attained was 56% and 37 to 45%, respectively. The decomposition was accompanied by the alkalinization of the medium and by the release of peptidic substances. The average content of sulfur amino acids in soluble decomposition products was always lower than their content in keratin. Sulfhydryl compounds (cysteine) were found in traces only. The final product of the cystine metabolism was the sulfate excreted in high amounts into the medium. Sulfides, thiosulfates, and free sulfite were not present. However, after the treatment of the culture filtrates with cyanide, arsenite or cysteine well determinable amounts of sulfite were liberated. These reactions are typical of S-sulfocysteine. The author presents the hypothesis that the dermatophytes can produce not only sulfate, but also sulfite in the course of keratinolysis. By the latter compound, disulfide bonds of the substrate could be cleaved to cysteine and S-sulfocysteine. The keratin denatured by “sulfitolysis” would then be easily attacked by the proteases of the fungus.

Keratin decomposition by dermatophytes. I. Sulfite production as a possible way of substrate denaturation

The dermatophyte Microsporum gypseum was grown on children's hairs in a mineral solution. During the growth of the fungus the substrate digestion, pH of the medium, and concentrations of soluble decomposition products were determined. In aerated and stationary cultures the substrate digestion attained was 56% and 37 to 45%, respectively. The decomposition was accompanied by the alkalinization of the medium and by the release of peptidic substances.The average content of sulfur amino acids in soluble decomposition products was always lower than their content in keratin. Sulfhydryl compounds (cysteine) were found in traces only. The final product of the cystine metabolism was the sulfate excreted in high amounts into the medium. Sulfides, thiosulfates, and free sulfite were not present. However, after the treatment of the culture filtrates with cyanide, arsenite or cysteine well determinable amounts of sulfite were liberated. These reactions are typical of S‐sulfocysteine.The author presents the hypothesis that the dermatophytes can produce not only sulfate, but also sulfite in the course of keratinolysis. By the latter compound, disulfide bonds of the substrate could be cleaved to cysteine and S‐sulfocysteine. The keratin denatured by “sulfitolysis” would then be easily attacked by the proteases of the fungus.

Keratin decomposition by dermatophytes: evidence of the sulphitolysis of the protein.

Es wird wahrscheinlich gemacht, dass die Denaturierung des Keratins durch Sulfitausscheidung und Sulfitolyse des Cystins eine wichtige Rolle beim Keratinabbau durch Pilze spielt.

Thiosulphate esters in keratin attacked by dermatophytes in vitro.

A strong basophilia was demonstrated in human hair attacked, in vitro, by Microsporum gypseum. With model substrates e.g. chemically modified keratins, histochemical studies showed that in the regions attacked, S-thiosulphate esters were present apart from the carboxyls originating by proteolysis of peptide chains. Such groups arise from cleavage of cystine bonds by sulphite. Previous personal work showed that sulphite was produced during growth of the fungus on a cystine-containing medium and it is assumed that the denaturation of keratin by sulphitolysis could be a key reaction in keratin decomposition by dermatophytes.

Decomposition of Keratin by Soil Micro-Organisms (With One Text-figure.)

Keratin, prepared from horn meal, was added to moist field and garden soil and allowed to decompose in the laboratory. The keratin was found to undergo a decomposition resulting in a slow, but steady accumulation of ammonia and nitrate. 35–40 per cent, of its nitrogen was transformed into nitrate after 120 days. The addition of keratin produced little or no increase in the number of bacterial colonies on agar platings, but markedly increased the number of actinomycete colonies, especially in garden soil. Two strains of actinomycetes were isolated and found capable of thriving on keratin in pure culture, decomposing the keratin with the formation of ammonia. One of the strains could be recognised asActinomyces citreusKrainsky, as described by Waksman. The other strain could not be named, but corresponded closely to the description of Waksman'sActinomyces145.