Keratinase Research Articles

Production of keratinase enzyme from B. subtilis GH2 and its application in feather biodegradation

Production of keratinase enzyme from B. subtilis GH2 and its application in feather biodegradation

KERATINASE ENZYME: VIRULENCE FACTOR IN PATHOGENITY OF DERMATOPHYTES

Pathogenic dermatophytes are prevalent causes of a superficial cutaneous infection, which have the ability to invade keratinized structures such as skin, hairs and nails. Dermatophytes infection in the host involves three main steps: adherence to the host tissue, invading, and the development of a host response. In the first stage of infection, dermatophytes adhere to the surface of the keratinized tissue to reach the epidermis by using some factors that mediate adherence of dermatophytes. Various virulence factors are secreted from dermatophytes during the invading process in order to penetrate the host tissue, to obtain nutrients and survive. The aim of this study was to select keratin degrading fungi isolated from from clinical samples of patients with dermatophytosis. Application of soluble preparation of keratin (KS) of chicken feathers enables a preliminary evaluation of the growth of the fungi and screening of fungal isolates that possesed keratinolytic activity and keratinase enzyme. Five dermatophytes i.e. T.rubrum, T.mentagrophytes, M.gypseum, M.canis and C.tropicum were used in our study for keratinase activity. Out of five dermatophytes species studied, all tested fungal species showed a keratinase activity except one species i.e. M.canis. From our findings, these results provide a scientific validation that dermatophytes have keratinase enzyme activity and responsible for virulence factor in pathogenicity of dermatophytes.

Microbial production of keratinase from Bacillus velezensis strain MAMA: A novel enzyme for eco-friendly degradation of keratin waste

Keratin waste has become an increasingly serious environmental and health hazard. Keratin waste is mainly composed of keratin protein, which is one of the most difficult polymers to break down in nature and is resistant to many physical, chemical, and biological agents. With physical and chemical methods being environment damaging and costly, microbial degradation of keratin using keratinase enzyme is of great significance as it is both environment friendly and cost-effective. The aim of this study was to extract and purify keratinase from bacterial species isolated from the soil. Among the organisms, an isolate of Bacillus velezensis, coded as MAMA could break down chicken feathers within 72 hours (h). The isolated strain produced significant levels of keratinase in mineral salt medium by supplying chicken feathers as the sole source of nitrogen and carbon. Feather deterioration was observed with the naked eye, and enzyme activity was evaluated using a spectrophotometric assay. Sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and zymography results revealed that the keratinase protein produced by Bacillus velezensis had a molecular weight between 40 and 55 kilodalton (kDa).

Decomposed chicken feather: A biostimulant to lettuce (Lactuca sativa) growth

It is crucial to produce plant growth biostimulants from waste and renewable resources. A study was carried during 2020 at the Soil and Water Research Institute, Karaj, Iran to evaluate the effect of decomposed chicken feather as a biostimulant on lettuce (Lactuca sativa L.) growth. A total of 29 soil samples from 15 Iranian regions were used to isolate and identify the bacteria and 31 strains that were able to produce keratinase and capable of feather degrading were isolated. Then, 8 isolates that were able to degrade the feather during 7 days were selected as the superior strains. Based on the results Bacillus siamensis c11, Bacillus methylotrophicus gh1 and Bacillus methylotrophicus a2 were identified as the new strains that can produce keratinase enzyme. Then, the effect of foliar spraying of the solutions produced from feather degradation by the new strains (c11, gh1, and a2) on the growth of lettuce was investigated. The results showed that the solution produced by methylotrophicus gh1 strain significantly increased the fresh and dry weight of the shoot and root (respectively, 25.9, 36.9, 34.1 and 51.9% increase compared to the control). It is concluded that the microbial solution of these three microbes as a consortium could be quite a new addition on one hand, could also reduce the human allergy as a via- medium of waste disposal.

Exploring the potential of proteolytic bacteria for keratinase enzyme synthesis: Study on isolation and screening

Exploring the potential of proteolytic bacteria for keratinase enzyme synthesis: Study on isolation and screening

Production and Characterization of Keratinase Enzyme from Natural Isolate Bacillus Strains

Production and Characterization of Keratinase Enzyme from Natural Isolate Bacillus Strains

Isolation and identification of keratinolytic probiotic Bucillus licheniformis bacteria from the soil below poultry slaughterhouse waste.

Feathers make up 7% of the total weight of adult chickens and keratin protein makes up 85% of the feathers. Today, the keratinase enzymes of some Bacillus strains are used to degrade and process raw keratin waste for animal and poultry feed. According to various studies, the probiotic properties of some spore-shaped Bacillus have also been proven. The study aimed to isolation of the keratinolytic Bacillus bacteria that they have probiotic properties for using in the livestock and poultry feed industry. We were able to isolate 8 strains of Bacillus licheniformis with kreatin degrading properties from the soil of Baharan chicken slaughterhouse (Qom city, Iran) applying heat shock, alcohol- and keratin-rich culture medium, and after microscopic and biochemical analysis, 16S rDNA gene was isolated. The measurement results of keratinase activity showed that the three strains of Bacillus licheniformis pvkr6, pvkr 15, and pvkr41 had the highest activity with 124.08, 101.1, and 100.18 U/ml. The results of probiotic properties evaluation also revealed that among all the isolates, only Bacillus licheniformis pvkr15 and Bacillus licheniformis PTCC 1595 (positive control) were γ-hemolytic strains. The percentage of surface hydrophobicity of the strains was obtained from 3.27 to 30.57. It was also shown that, on average, all the strains had acceptable susceptibility to the tested antibiotics except penicillin G. Bacillus licheniformis pvkr15 with highest keratinase activity (101.1U/ml) was considered an optional probiotics due to its abilities such as (biofilm formation, being safe cause of γ-hemolytic activity, high susceptibility to antibiotics such as streptomycin, gentamicin, cefixime, amoxicillin, tetracycline, vancomycin, erythromycin and having a moderate hydrophilic (hydrophobicity: 19.09%), high survivability in pH 2, 2.5 and 3, strong resistance to bile salts and moderate antagonistic activity against pathogenic bacterium like Proteus mirabilis and the ability to grow under anaerobic conditions). By using this strain, after hydrolysis of keratin protein in the feather structure, to replace part of the protein of livestock and poultry feed, not only is no need to separate bacteria from the feed, but also the strain play role of an useful and effective additive in animal growth.

Bacillus thuringiensis VP4: A potential keratinase producer isolated from poultry waste soils of Himachal Pradesh

Keratinase enzymes are protein hydrolyzing enzymes belonging to the serine hydrolase group and act specifically on keratin proteins to degrade them. The current study was conducted to isolate best keratinase producer bacterial strains. Four Bacillus species (B. cereus PP1- OP748781, B. cereus JP1- OP748782, B. cereus TP1- OP748783 and B. thuringiensis VP4- OP420549), were isolated from the poultry farms of Himachal Pradesh. These strains were evaluated for keratinase production by using chicken feathers in shake flask culture. Among four isolates, Bacillus thuringiensis VP4 showed highest enzymatic activity 343 U/ml. The molecular weight of the keratinase was observed 45 KDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis. The optimization of enzyme production was done with isolate Bacillus thuringiensis VP4 at various parameters such as temperature, pH and effect of metal ions. The maximum keratinase production was observed at 35°C temperature and pH 10.0. The isolated strain Bacillus thuringiensis VP4 might be used to produce poultry compost through degradation of chicken feathers and poultry waste in short time which may also be helpful for farmers and overcoming environmental pollution.

Production and Applications of Keratinases in Industry

Keratin is an insoluble protein with fibrous structure. It is mainly found in hair,feathers,nail,wool and horn of various animals. These animal accessories can be utilized as animals feed, amino-acids and fertilizers. This insoluble protein is very difficult to degrade and has extreme stability because it has disulfide bonds, hydrogen and hydrophobic interaction present in it. But keratin can be easily digested by keratinase enzyme. Keratinase is an extracellular enzyme. It also degrades keratin in prokaryotic organisms. Microorganisms that produce keratinase are species of Bacillus, Actinobacteria and fungi. It can also be obtained from Streptomyces, Aspergillus, Fervidobacterium, Xanthomonas, Chryseobacteriumand Vibrio. Keratinases produced by microbes arevery diverse due to their chemical as well as physical properties. It is a vigorous enzyme that can survive broad pH and temperature. Their optimum pH range is neutral to alkaline and their optimum temperature reported is 40°C to 60°C. And thermophiles have also been reported for stable microbial activity. Microbial keratinases are cheaply available as compared to conventional producers for keratinases production. They have gained great significance in biotechnology by acting on strong, inflexible cross linked polypeptide structure of keratin instead of ordinary proteolytic enzyme papain, trypsin or pepsin. They are mostly extracted by degrading keratin as substrate. Keratin substrates include feather, hair, wool,horn and nails. This degradation process is helpful in converting keratinous wastes in to fertilizer and poultry feed. Other renowned biotechnological implementations include removing hair from leather by using keratinase, development of bio-polymer from keratinous fiber, delivering drug, hydrolysis of prion proteins and detergent industry. They are also used in converting biomass in to biofuel that significantly boosts power preservation and recycling. The main problem for producing enzymes on large scale is their costly production procedures. This hurdle is overcome by utilizing keratin waste eg feathers of chicken as fermentation substrate on industrial scale. The benefit of utilizing this left over is extremely cost effective fermentation process of keratinase and the effluents are environmental friendly. Keratinase enzyme has become popular and provided newly effective and efficient way for managing waste products by using them on industrial scales as substrates which gives rise to environmental friendly industries for nonstop development.

Production and biochemical characterization of keratinase enzyme from Bacillus subtilis ES5 and its potential application in leather dehairing process: a clean leather tanning process

The traditional leather industry releases different chemical wastes into the environment, which calls for the use of alternative, nonpolluting processes. The use of biological catalysts in industrial processing has received a great deal of attention in recent years, mainly for environmental reasons. The main objective of this study was to produce and characterize keratinase enzyme from Bacillus subtilis ES5 and evaluate its potential application for leather dehairing. The gene encoding keratinase was validated with a fragment length of approximately 1100 bp. The keratinase exhibited maximum activity at temperature and pH of 45 °C and 8, respectively. Among the tested metal ions, Ca2+ and Mg2+ improved keratinase activity by 132.56 and 111.78%, respectively, at concentrations of 5 mM. β-mercaptoethanol enhanced keratinolytic activity. However, ethylenediaminetetraacetic acid (EDTA) significantly inhibited it, indicating that the enzyme is a member of the metalloprotease family. It completely dehaired goat/sheep skins within 14 h, resulting in decreased biochemical oxygen demand (BOD5), chemical oxygen demand (COD), total dissolved solids (TDS), total suspended solids (TSS), and overall pollution load by 57.39, 70.53, 93.59, 94.90, and 79.10%, respectively. The results demonstrated the pivotal advantages of this keratinase, addressing environmentally friendly processing and sustainable solid waste management. The improved properties of enzyme-treated dehaired pelts suggest that crude keratinase could be used in ecologically friendly holistic leather manufacturing to avoid the pollution issues associated with the use of chemicals.

Recent Review on the Extraction and Qualitative Assay of Cysteine and Other Amino Acids from Vellore Feather Waste and Molecular Docking Studies of Cysteine for Pharmacological Applications.

Products produced from waste are a relatively recent innovation. Feather substrates are abundant in keratin content and improper disposal can cause ecosystem contamination. However, these pollutants can be transformed into value-added products for industrial application. Physical, chemical and cutting-edge microbiological methods were utilized for decomposing keratin and aid in the identification andestimation of amino acids from poultry feather wastes. These beneficialapproaches are receiving more attention due to their retrieval of harmless and value addedbyproducts. These keratin-based compounds are used widely in pharmaceutical, livestock feed, fertilizer, and a variety of other industrial sectors. Since keratin is primarily consisting of amino acids, it can be utilized to affirm and estimate the amino acids in these feather substrates. This study primarily highlights the various methodologies employed for the qualitative estimation of amino acids in feather waste samples and the inhibitory activity of keratinase enzyme by EDTA and pepstatin in order to accumulate amino acids for drug delivery purpose and their importance in various pharmaceutical industries. In addition to that, molecular docking studies of cysteine with many standard pharmaceutical drugs like acetaminophen, pethidine, methylphenidate, carbamazepine, cillin and amlodipine were performed using autodock to demonstrate how cysteine greatly reduces conventional drug toxicity and its side effects.

Isolation and screening of keratinolytic bacteria from feather dumping soil near in Lucknow and Kanpur city, North region of Indian

Background Keratinases are proteolytic enzymes that have the capability of degrading insoluble keratin substrates. LS4, LS5, and KS3 were newly isolated strains showing ability to hydrolyze keratins and keratin based products. Objective In this study, different bacterial strains were isolated from soil, screened for protease and keratinase production, and the best isolates were identified. Methods Serial dilution and plating method were used for isolation of pure bacterial culture. Isolated, 48 strains were screened, and their feather degrading ability on feather meal agar plates and keratin azure-based agar. The proteolytic activity of the isolates was also screened on casein agar. Results Three strains; LS4, LS5, and KS3 shows keratinolytic activity that reveal the keratinase enzyme on medium; feather meal agar, keratin azure agar, and casein agar. Among all the three strains (LS4, LS5, and KS3), KS3 show largest inhibition zone that show maximum enzymatic activity. For its characterization 16S RNA was performed, sequence KS3 was submitted to GenBank and was identified as Bacillus tropicus KS3. Newly isolated strain B. tropicus KS3 showed the keratinase production and highest keratinase activity (35.06 ± 2.5 U/ml). B. tropicus KS3 also show the highest degradation of feather efficiently showed 84% of whole chicken feather biodegradation at 37 °C within 15 days among all the isolated strains. Conclusion Keratinase enzyme possesses a wide range of potential applications in the bioremediation of feather waste and bio-fertilizer in agricultural land. The industry demands a better source of this enzyme due to its multivariate application and huge market demand. In the present study, a novel keratinase-producing bacteria was isolated and characterized as Bacillus tropicus KS3 from poultry waste contamination soil.

Fusion of Substrate-Binding Domains Enhances the Catalytic Capacity of Keratinases and Promotes Enzymatic Conversion of Feather Waste.

The unique role of keratinases in keratin hydrolysis has garnered huge interest in the recovery of feather waste. However, owing to the high hydrophobicity of feather keratins, the catalytic capacity of keratinases for hydrolyzing feathers is typically low. In this study, we aimed to improve the keratinase feather hydrolysis efficiency by fusing a substrate-binding domain into the enzyme. We screened several carbohydrate-binding modules (CBMs) and linking peptides. We selected the most promising candidates to construct, clone, and express a fusion keratinase enzyme KerZ1/CBM-L8 with a feather hydrolysis efficiency of 7.8 × 10-8 g/U. Compared with those of KerZ1, KerZ1/CBM-L8 has a feather hydrolysis efficiency that is 2.71 times higher, a kcat value that is 179% higher, which translates to higher catalytic efficiency, and Km and binding constant (K) values that are lower, which indicate a higher KerZ1/CBM-L8-keratin binding affinity. Moreover, the number of binding sites to the substrate (N), determined using isothermal titration calorimetry, was 24.1 times higher than that of KerZ1. Thus, the fusion of the substrate-binding domain improved the binding ability of the keratinase enzyme to the hydrophobic substrate, which improved its feather hydrolysis efficiency. Therefore, using the fusion keratinase would significantly improve the recovery of feather waste.

Synthesis and characterization of keratinase laden green synthesized silver nanoparticles for valorization of feather keratin

This study focuses on the efficient and cost-effective synthesis of silver nanoparticles (AgNPs) using plant extracts, which have versatile and non-toxic applications. The research objectives include synthesizing AgNPs from readily available plant extracts, optimizing their production and multi scale characterization, along with exploring their use for enzyme immobilization and mitigation of poultry feather waste. Among the plant extracts tested, the flower extract of Hibiscus rosa-sinensis (HF) showed the most potential for AgNP synthesis. The synthesis of HF-mediated AgNPs was optimized using response surface methodology (RSM) for efficient and environment friendly production. Additionally, the keratinase enzyme obtained from Bacillus sp. NCIM 5802 was covalently linked to AgNPs, forming a keratinase nanocomplex (KNC) whose biochemical properties were evaluated. The KNC demonstrated optimal activity at pH 10.0 and 60 °C and it displayed remarkable stability in the presence of various inhibitors, metal ions, surfactants, and detergents. Spectroscopic techniques such as FTIR, UV–visible, and X-ray diffraction (XRD) analysis were employed to investigate the formation of biogenic HF-AgNPs and KNC, confirming the presence of capping and stabilizing agents. The morphological characteristics of the synthesized AgNPs and KNC were determined using transmission electron microscopy (TEM) and particle size analysis. The study highlighted the antimicrobial, dye scavenging, and antioxidant properties of biogenic AgNPs and KNC, demonstrating their potential for various applications. Overall, this research showcases the effectiveness of plant extract-driven green synthesis of AgNPs and the successful development of keratinase-laden nanocomplexes, opening possibilities for their use in immobilizing industrial and commercial enzymes.

Keratinase of Bacillus cereus Lr3/2 KP015746 from deteriorated leather samples

Keratin is an insoluble fibrous protein found in hair, wool, feather, nail, horns and other epithelial covering. The high resistance of keratin to proteases is due to its densely packed and strongly stabilized by several hydrogen bonds and hydrophobic interactions, in addition to several disulfide bonds. A keratinase enzyme is useful for biotechnological purposes, and also for hydrolysis of de-hairing of bovine hides, poultry feathers, and leather surplus. In the present study, B. cereus KJ72442 was isolated from deteriorated leather samples using feather meal as a substrate and identified by 16SrDNA sequencing and with accession number B. cereus Lr3/2 KP015746). The colony showing the highest zone of keratin hydrolysis on feather meal agar was selected for keratinase production. B. cereus showed efficient keratinase production 41.06±2U/ml at optimum temperature of 37°C and pH 7 after 72h using 1% feather meal as a substrate. B. cereus keratinolytic activity was found to be metalloprotease, as inhibited by 1mM EDTA but not by PMSF.In addition to keratinase, B. cereus KP015746 (Lr3/2) was also proficiently found to produce collagenase, caseinase and gelatinase. On the lab scale, the efficiency of B. cereus Lr3/2 KP015746 keratinase to degrade leather was analyzed and observed excellent results. Hence, the experimental outcome revealed that B.cereus KP015746 (Lr3/2) can be effectively used to treat keratin, collagen rich agro industrial waste ecofriendly.

Isolation and screening of keratinase enzyme producing Bacillus mojavensis par01 from poultry farm

Isolation and screening of keratinase enzyme producing Bacillus mojavensis par01 from poultry farm

Microbial Exploitation of Feather Wastes for Sustainable Production of Keratinase and Collagenase Enzymes by Didymella keratinophila AUMC 15399 in Submerged Fermentation

A distinctive isolate was discovered and visually recognized as a member of the genus Didymella during a routine examination of Coelomycetes isolated from diverse fruit juices. Based on sequencing of the internal transcribed spacer (ITS), the fungus was identified as Didymella keratinophila since it showed a 100% identity to the type strain. The strain thrived and produced keratinase and collagenase enzymes by hydrolyzing native chicken feathers in submerged fermentation (SmF). After 10 days of fermentation at 30 °C, pH 9 using sodium nitrate as a nitrogen supply produced the highest keratinase activity of 8780 ± 620 U/mL/min, while pH 6 and beef extract produced the maximum collagenase activity of 11,230 ± 1290 U/mL/min. The partially-purified keratinase enzyme worked best at pH 7.0 and 45 °C, exhibiting a specific activity of 44,903 ± 1555 U/mg protein. The activity of the partially-purified collagenase enzyme was excellent at pH 6.0 at 35 °C, generating 15,753 ± 110 U/mg enzyme-specific activity. Mn2+ and K+ were the most efficient inhibitors of keratinases and collagenase, respectively. Both EDTA and metal ions significantly decreased the activity of keratinase and collagenase. This report identified a workable supplier of collagenase and keratinase enzymes derived from chicken feathers, offering a reliable way to exploit and manage these wastes for obtaining high-value products.

Degradation of tannery hide raw trimming hairs using keratinolytic bacteria isolated from tannery effluent-contaminated soil.

The disposal of keratinous wastes produced by several leather industries is evolving into a global problem. Around 1 billion tonnes of keratin waste are released into the environment each year. In the breakdown of tannery waste, certain enzymes, such as keratinases produced from microorganisms, might be a better substitute for synthetic enzymes. Keratinase enzymes are able to hydrolyze gelatin, casein, bovine serum albumin and insoluble protein present in wool, feather. Therefore, in this study, bacterial strains from tannery effluent-contaminated soil and bovine tannery hidewere isolated and assessed for their ability to produce the keratinolytic enzyme. Among the six isolates, the strain NS1P showed the highest keratinase activity (298 U/ml) and was identified as Comamonas testosterone through biochemical and molecular characterization. Several bioprocess parameters such as pH, temperature, inoculum size, carbon sources, and nitrogen sources were optimized in order to maximize crude enzyme production. The optimized media were used for inoculum preparation and subsequent biodegradation of hide hairs. The degradation efficacy of the keratinase enzyme produced by Comamonas testosterone was examined by degrading bovine tanneryhide hairs, and it was found to be 73.6% after 30days. The morphology of the deteriorated hair was examined using a field emission scanning electron microscope (FE-SEM), which revealed significant degradation. Thus, our research work has led to the conclusion that Comamonas testosterone may be a promising keratinolytic strain for the biodegradation of tannery bovine hide hair waste and the industrial production of keratinases.

Keratinase mediated fabrication and partial characterization of gold nanoparticles and its antibacterial potential

Keratinase is mainly involved in recycling of keratin waste. Of late, researchers extended its application to nanotechnology. In the present study, we have made an attempt to fabricate and characterize gold nanoparticles using crude keratinase enzyme from Serratia ficaria and also study their biological application, particularly antibacterial activity. The formation of gold nanoparticles (AuNPs) was first verified by UV-Visible Spectroscopy. FTIR spectra confirmed the presence of responsible secondary metabolites for stabilization of nanoparticles. The morphological characteristics and particle size of synthesized nanoparticles were analyzed. The AuNPs showed significant antibacterial activity against Klebsiella pneumonia, Bacillus cereus and Staphylococcus aureus. The highest radical scavenging activity, 60.62% for AuNPs was observed at 500 µg/mL. Results of this study reveals significance of keratinase application in nano-based biological applications.

Optimization of keratinase production by keratinolytic fungus Chrysosporium tropicum and its potentiality in bidegradation of chicken feathers

The current study has been designed to investigate the possible biodegradtion of native keratin by fungi isolated from chicken feathers collected from chicken farms in Egypt. Out of 56 fungal isolates 23 species and 2 species varieties showed different degrees of keratinolytic activity, with Chrysosporium tropicum showing the highest keratinase productivity (23.15 U/ml) as well as the highest value of specific activity for keratinase (197.90 U/mg protein) indicating a great degree of purity for the enzyme. Our present study indicates that the highest production of keratinase by C. tropicum was recorded at pH 8 and a temperature of 35 °C during a period of 20 days. In addition the optimal value of ionic strength for keratinase production was 100 mM NaCl. Monitoring the influence of constituents deprivation from basal salt medium on production of keratinase enzyme showed that K+, Fe2+, Mg2+, Ca2+ and Zn2+ were necessary for keratinase production. Sulfur deprivation has no significant influence on keratinase production. Keratinase activity of C. tropicum showed that, the optimal values of temperature and ionic strength were 35–40 °C and 80 mM NaCl, individually. The optimum pH range was (7.8–9). These promising findings demonstrate a high potentiality of C. tropicum keratinase in keratin proteolysis, suggesting that keratinase producing isolates of C. tropicum may be suitable as a biodegradation agent.