Sulfitolytic and keratinolytic potential of Chryseobacterium sp. RBT revealed hydrolysis of melanin containing feathers.
ABSTRACT: In black feathers, melanin is embedded in keratin matrix that makes feather more resistance to the microbial degradation. Chryseobacterium sp. RBT previously isolated from the poultry waste disposable site revealed strong sulfitolytic and keratinolytic activities. Maximum keratinase activity was observed at 48 h (89.12 U ml-1) showed 83 % of native black feather degradation. The concentration of free sulfhydryl groups released during degradation was 0.648 × 10-4 M (12 h), 2.144 × 10-4 M (96 h), and however, declined on prolong incubation to 1.752 × 10-4 M (120 h). Melanin was released in the degradation medium after microbial exploitation of black feather. After purification, melanin was dark brown colored powder insoluble in water, 5 M HCL, ethanol, methanol, benzene, chloroform, and acetone; whereas, soluble in KOH and NaOH. On exposure to oxidizing and reducing reagents feather melanin showed decolorization, while formed a brown precipitate when reacted with FeCl3. The spectroscopic characterization of isolated melanin demonstrated absorption at infra-red region. Similarly, UV-visible scan confirmed that increase in the wavelength progressively declined the absorbance of pigment. The crude keratinase enzyme (2 % v/v) produced during degradation showed complete dehairing of goat skin within 20 h.
Project description:In leather industries and tanneries, large amount of wastes has been disposed; which polluting water, soil, and atmosphere and causing serious human health problems. In particular, chemical dehairing process of leather industries produces fair amount of toxic wastes. It is, thus, urgently needed to use alternative processes free from pollution. As more than 90% of keratin is contained in feather, it is desirable to develop bioremediation process using keratinolytic microorganisms. In the present investigation, therefore, we first identified Bacillus cereus and Pseudomonas sp. to be able to produce keratinase. Then, the optimization was performed to maximize the keratinase activity with respect to cultivation temperature, pH, and incubation time. Moreover, the effects of metal ions and various substrates on keratinase activity were also investigated. The result indicates that keratinase activity became maximum at 50°C for both strains, whereas the optimal pH was 10.0 for B. cereus and 7.0 for Pseudomonas sp. The highest keratinase activity of 74.66?±?1.52?U/mL was attained by B. cereus, whereas 57.66?±?2.52?U/mL was attained by Pseudomonas sp. Enzymatic dehairing efficiency of leathers was also compared with chemical dehairing (Na2S and CaO), where complete dehairing was achieved by treating them with crude keratinase. Partial enzyme purification was performed by acetone precipitation. Batch cultivation of B. cereus using 1?L fermentor indicates a potential candidate for large-scale keratinase production. Thus, keratinase enzyme by degrading poultry wastes (feather) can be an alternative approach to chemical dehairing in leather industries, thus preventing environmental pollution through bioremediation.
Project description:Birds present a stunning diversity of plumage colors that have long fascinated evolutionary ecologists. Although plumage coloration is often linked to sexual selection, it may impact a number of physiological processes, including microbial resistance. At present, the degree to which differences between pigment-based vs. structural plumage coloration may affect the feather microbiota remains unanswered. Using quantitative PCR and DGGE profiling, we investigated feather microbial load, diversity and community structure among two allopatric subspecies of White-shouldered Fairywren, Malurus alboscapulatus that vary in expression of melanin-based vs. structural plumage coloration. We found that microbial load tended to be lower and feather microbial diversity was significantly higher in the plumage of black iridescent males, compared to black matte females and brown individuals. Moreover, black iridescent males had distinct feather microbial communities compared to black matte females and brown individuals. We suggest that distinctive nanostructure properties of iridescent male feathers or different investment in preening influence feather microbiota community composition and load. This study is the first to point to structural plumage coloration as a factor that may significantly regulate feather microbiota. Future work might explore fitness consequences and the role of microorganisms in the evolution of avian sexual dichromatism, with particular reference to iridescence.
Project description:Microbial bioconversion of carbonoclastic materials is an efficient tool for the exploitation and valorization of underutilized agro-industrial wastes. The agro-industrial sector accumulates tones of keratinous wastes biomass which may be valorized into high value products. Consequently, the keratinolytic potentials of some bacteria isolated from terrestrial milieu was evaluated. Soil samples were collected from dumpsites, keratinase producing bacteria were isolated. Bacterial species were identified through 16S rRNA gene sequences. The keratinase activity was assessed in relation to thiol formation, percentage feather degradation and quantitation of keratinase produced. Keratinolytic bacteria were identified as Bacillus spp. (accession numbers: MG214989 - MG214992, MG214997, MG214998, MG215000, MG215002-MG215005) and Arthrobacter sp. (accession numbers; MG215001). The degree of chicken feather degradation ranged from 61.5 ± 0.71 % to 85.0 ± 1.41 %. Similarly, the activity of keratinase, total protein and thiol group ranged from 198.18 ± 15.43-731.83 ± 14.14 U/mL; 0.09 ± 0.01-0.87 ± 0.05 mg/mL; and 0.69 ± 0.12-2.89 ± 0.11 mM respectively. Notably, Bacillus sp. Nnolim-K1 displayed the best keratinolytic potential with extracellular keratinase activity and feather degradation of 731.83 ± 14.14 U/mL and 85.0 ± 1.41 % respectively, and that is an indication of a potential relevance biotechnologically.
Project description:Dehairing is one of the highly polluting operations in the leather industry. The conventional lime-sulfide process used for dehairing produces large amounts of sulfide, which poses serious toxicity and disposal problems. This operation also involves hair destruction, a process that leads to increased chemical oxygen demand (COD), biological oxygen demand (BOD), and total suspended solid (TSS) loads in the effluent. With these concerns in mind, enzyme-assisted dehairing has often been proposed as an alternative method. The main enzyme preparations so far used involved keratinases. The present paper reports on the purification of an extracellular keratinase (KERUS) newly isolated from Brevibacillus brevis strain US575. Matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF/MS) analysis revealed that the purified enzyme was a monomer with a molecular mass of 29121.11 Da. The sequence of the 27 N-terminal residues of KERUS showed high homology with those of Bacillus keratinases. Optimal activity was achieved at pH 8 and 40°C. Its thermoactivity and thermostability were upgraded in the presence of 5 mM Ca(2+). The enzyme was completely inhibited by phenylmethanesulfonyl fluoride (PMSF) and diiodopropyl fluorophosphates (DFP), which suggests that it belongs to the serine protease family. KERUS displayed higher levels of hydrolysis, substrate specificity, and catalytic efficiency than NUE 12 MG and KOROPON® MK EG keratinases. The enzyme also exhibited powerful keratinolytic activity that made it able to accomplish the entire feather-biodegradation process on its own. The kerUS gene encoding KERUS was cloned, sequenced, and expressed in Escherichia coli. The biochemical properties of the extracellular purified recombinant enzyme (rKERUS) were similar to those of native KERUS. Overall, the findings provide strong support for the potential candidacy of this enzyme as an effective and eco-friendly alternative to the conventional chemicals used for the dehairing of rabbit, goat, sheep and bovine hides in the leather processing industry.
Project description:Chicken feathers are predominantly composed of keratin; hence, valorizing the wastes becomes an imperative. In view of this, we isolated keratinase-producing bacteria and identified them through the 16S rDNA sequence. The process condition for keratinase activity was optimized, and electron micrography of the degradation timelines was determined. Keratinolytic bacteria were isolated and identified as Bacillus sp. FPF-1, Chryseobacterium sp. FPF-8, Brevibacillus sp. Nnolim-K2, Brevibacillus sp. FPF-12 and Brevibacillus sp. FSS-1; and their respective nucleotide sequences were deposited in GenBank, with the accession numbers MG214993, MG214994, MG214995, MG214996 and MG214999. The degree of feather degradation and keratinase concentration among the isolates ranged from 62.5 ± 2.12 to 86.0 ± 1.41(%) and 214.55 ± 5.14 to 440.01 ± 20.57 (U/mL), respectively. In the same vein, 0.1% (w/v) xylose, 0.5% (w/v) chicken feather, an initial fermentation pH of 5.0, fermentation temperature of 25 °C and an agitation speed of 150 rpm, respectively, served as the optimal physicochemical conditions for keratinase activity by Bacillus sp. FPF-1. The time course showed that Bacillus sp. FPF-1 yielded a keratinase concentration of 1698.18 ± 53.99(U/mL) at 120 h. The electron microscopic imaging showed completely structural dismemberment of intact chicken feather. Bacillus sp. FPF-1 holds great potential in the valorization of recalcitrant keratinous biomass from the agro sector into useful products.
Project description:The keratinase degrade highly rigid, cross linked structural polypeptides with different efficiency depending on the type of source. Two newly isolated strains of Bacillus subtilis (RSE163 and RSE165; NCBI Accession no JQ887983 and JQ887982) were found to be efficient keratinase producers with unusual catalytic activity result in different morphological changes in degradation pattern of feather, confirmed by their scanned electron micrographs. Maximum keratinolytic activity of both the strains B. subtilis RSE163 and RSE165 were found to be 366 ± 15.79 and 194 ± 7.26 U after 72 h of incubation. While the disulphide reductase activity of RSE163 and RSE165 estimated 0.24 ± 0.05 and 0.15 ± 0.03 U/ml of enzyme after 24 h of incubation. A total of 16 free amino acids of variable concentration were also analyzed in the cell free supernatant of hydrolyzed feather from two strains. Present study demonstrates the action of two different keratinases in feather degradation.
Project description:A new feather-degrading bacterium was isolated from a local feather waste site and identified as Bacillus subtilis based on morphological, physiochemical, and phylogenetic characteristics. Screening for mutants with elevated keratinolytic activity using N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis resulted in a mutant strain KD-N2 producing keratinolytic activity about 2.5 times that of the wild-type strain. The mutant strain produced inducible keratinase in different substrates of feathers, hair, wool and silk under submerged cultivation. Scanning electron microscopy studies showed the degradation of feathers, hair and silk by the keratinase. The optimal conditions for keratinase production include initial pH of 7.5, inoculum size of 2% (v/v), age of inoculum of 16 h, and cultivation at 23 degrees C. The maximum keratinolytic activity of KD-N2 was achieved after 30 h. Essential amino acids like threonine, valine, methionine as well as ammonia were produced when feathers were used as substrates. Strain KD-N2, therefore, shows great promise of finding potential applications in keratin hydrolysis and keratinase production.
Project description:We report the isolation of a keratinolytic-producing Bacillus subtilis strain and the characterization of the exceptional dehairing properties of its subtilisin-like keratinase. This enzyme can be an alternative to sodium sulfide, the major pollutant from tanneries, and may completely replace it. Its unique nonactivity upon collagen enhances its industrial potential.
Project description:Studies of the patterns of diversification of birds on islands have contributed a great deal to the development of evolutionary theory. In white-winged fairy-wrens, Malurus leucopterus, mainland males develop a striking blue nuptial plumage whereas those on nearby islands develop black nuptial plumage. We explore the proximate basis for this divergence by combining microstructural feather analysis with an investigation of genetic variation at the melanocortin-1 receptor locus (MC1R). Fourier analysis revealed that the medullary keratin matrix (spongy layer) of the feather barbs of blue males was ordered at the appropriate nanoscale to produce the observed blue colour by coherent light scattering. Surprisingly, the feather barbs of black males also contained a spongy layer that could produce a similar blue colour. However, black males had more melanin in their barbs than blue males, and this melanin may effectively mask any structural colour produced by the spongy layer. Moreover, the presence of this spongy layer suggests that black island males evolved from a blue-plumaged ancestor. We also document concordant patterns of variation at the MC1R locus, as five amino acid substitutions were perfectly associated with the divergent blue and black plumage phenotypes. Thus, with the possible involvement of a melanocortin receptor locus, increased melanin density may mask the blue-producing microstructure in black island males, resulting in the divergence of plumage coloration between mainland and island white-winged fairy-wrens. Such mechanisms may also be responsible for plumage colour diversity across broader geographical and evolutionary scales.
Project description:Feathers are a major by-product of the poultry industry. They are mainly composed of keratins which have wide applications in different fields. Due to the increasing production of feathers from poultry industries, the untreated feathers could become pollutants because of their resistance to protease degradation. Feathers are rich in amino acids, which makes them a valuable source for fertilizer and animal feeds. Numerous bacteria and fungi exhibited capabilities to degrade chicken feathers by secreting enzymes such as keratinases, and accumulated evidence shows that feather-containing wastes can be converted into value-added products. This review summarizes recent progress in microbial degradation of feathers, structures of keratinases, feather application, and microorganisms that are able to secrete keratinase. In addition, the enzymes critical for keratin degradation and their mechanism of action are discussed. We also proposed the strategy that can be utilized for feather degradation. Based on the accumulated studies, microbial degradation of feathers has great potential to convert them into various products such as biofertilizer and animal feeds.