Isolation and characterization of a new keratinolytic bacterium that exhibits significant feather-degrading capability

doi:10.5897/AJB09.545

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Isolation and characterization of a new keratinolytic bacterium that exhibits significant feather-degrading capability

Journal Article•DOI•

Isolation and characterization of a new keratinolytic bacterium that exhibits significant feather-degrading capability

Bo Xu, Qiaofang Zhong, Xianghua Tang, Yunjuan Yang, Zunxi Huang - Show less +1 more

15 Sep 2009-African Journal of Biotechnology (Academic Journals)-Vol. 8, Iss: 18, pp 4590-4596

TL;DR: Potential biotechnological applications of this bacterium that involve hydrolysis of keratin, including the improvement of the nutritional properties of feathers (and other keratins) used as supplementary feedstuffs are suggested.

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Abstract: A novel bacterium, Bacillus licheniformis K-19, which produces a large amount of akeratinase that is extremely thermostable and has a broad resistance to pH, was isolated and characterized. The maximum amount of keratinase activity (about 224 Uml-1) was produced at 37°C when the bacterium was cultured for 72 h in broth containing feather meal with initial pH of 7.5. The keratinase activity was observed over a wide range of temperatures (30 - 90°C) and pH values (pH 6 - 10). It was optimal at 60°C and pH 7.5 - 8 respectively. These results suggest potential biotechnological applications of this bacterium that involve hydrolysis of keratin, including the improvement of the nutritional properties of feathers (and other keratins) used as supplementary feedstuffs. Key words: Bacillus licheniformis, chicken feather, keratin, keratinolytic protease.

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Journal Article•DOI•

Efficacy of crude and immobilizedenzymes from Bacillus licheniformis for production of biodegraded feather meal and their assessment on chickens

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Charles Oluwaseun Adetunji¹, Charles Oluwaseun Adetunji², I. O. Adejumo•Institutions (2)

Jawaharlal Nehru University¹, Landmark University²

01 Aug 2018-Environmental Technology and Innovation

TL;DR: The microbial biodegradation of feather wastes could be a better approach to overcome high feed cost and environmental pollution arising from solid waste disposal.

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Abstract: Keratinase enzymes are a special type of protease that has a bio-degradative potential for degrading keratin-containing substrates by the enzymes they produced during bioprocessing. The study was carried out to investigate the effect of microbial degraded feather meal on broiler chickens. The strain was previously isolated from a feather dumping site. The effects of crude and immobilizedenzyme-degraded feather meal were investigated on growth performance, haematology and intestinal histology of broiler chickens. Maximum activity of the keratinolytic enzyme was at 45 °C, the maximum bio-degradative potential at 48 h of fermentation, while the pH 7 exhibited the maximum keratinolytic activity. The values obtained for feed conversion ratio for birds on feather meal were statistically similar to the value obtained for those on the control diet. The microbial biodegradation of feather wastes could be a better approach to overcome high feed cost and environmental pollution arising from solid waste disposal.

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19 citations

Journal Article•DOI•

Optimization of Keratinase Production for Feather Degradation by Bacillus subtilis

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Somayeh Mousavi¹, Mojtaba Salouti¹, Reza Shapoury¹, Zahra Heidari¹•Institutions (1)

Islamic Azad University¹

01 Oct 2013-Jundishapur Journal of Microbiology

TL;DR: The isolation of feather degrading Bacillus spp.

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Abstract: Background: The feather is an environmental pollutant that can be degraded by bacterial and fungal microorganisms. The keratin sheets constitute 90% of the feather mass. Due to the extremely rigid structure, keratin is insoluble and hard to degrade. Some microorganisms such as Bacillus spp. were reported to be able to degrade keratin by secretion of keratinase. Objectives: The aim of this study was the isolation of feather degrading Bacillus spp. from a poultry waste and the optimization of conditions for the highest enzyme activity and feather degradation. Materials and Methods: The microorganisms were isolated from the waste of a poultry in Miyaneh, Iran, and the Bacillus spp. were identified using morphological, physiological and biochemical tests. The Bacillus spp. cultured in a medium consisted of feather at pH 7.4 and 27 oC for seven days to identify the feather-degrading Bacillus spp. The biochemical tests were performed to determine the strain of the bacterium. The study was repeated under different pH and temperatures to find the optimum conditions for best enzyme activity. Results: The PCR approved the Bacillus genus of the isolates. The strain of Bacillus subtilis was identified using biochemical tests. 40 oC and pH 11 are the optimum condition for maximum keratinase enzyme activity. Conclusions: B. subtilis was found to be able to degrade the feather.

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19 citations

Journal Article•DOI•

Keratinolytic enzyme-mediated biodegradation of recalcitrant poultry feathers waste by newly isolated Bacillus sp. NKSP-7 under submerged fermentation.

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Ikram ul Haq¹, Fatima Akram¹, Zuriat Jabbar¹•Institutions (1)

Government College University¹

16 May 2020-Folia Microbiologica

TL;DR: Keratinolytic enzyme showed stability at 20–65 °C for 4 h over the pH range of 5.5–8.0, and enzyme activity was enhanced by adding β-mercaptoethanol, Na+, Cd2+, and Mn2+.

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Abstract: Microbial and enzymatic degradation of keratin waste is more preferred over various conventional approaches which are costly and not environmentally suitable. Diverse niches are auspicious for the discovery of new microorganisms. To discover novel keratinolytic bacteria, 60 isolates from different poultry dumping sites were initially screened, and among these found a potent keratinolytic isolate (NKSP-7) that displayed higher feather-degrading ability. The selected isolate was identified as Bacillus sp. NKSP-7 based on 16S rDNA sequencing as well as physiochemical and morphological characteristics. The strain NKSP-7 showed complete hydrolysis of native chicken feathers (10 g/L) in nutrient medium after 24 h of incubation at 37 °C under agitation (150 rev/min) and produced thermostable extracellular keratinase. The crude enzyme displayed maximal keratinolytic activity (34.7 U/mL) in phosphate buffer of pH 7.0, and at 60 °C using keratin azure as a substrate. Keratinolytic enzyme showed stability at 20–65 °C for 4 h over the pH range of 5.5–8.0. No obvious inhibitory influence was perceived by cations, organic solvents, EDTA, and detergents. Whereas, enzyme activity was enhanced by adding β-mercaptoethanol, Na+, Cd2+, and Mn2+. All these notable features of keratinase make it a promising candidate for various industrial applications especially for dehairing process in leather industry, bioconversion of poultry waste, and in detergents formulations.

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13 citations

Cites background or result from "Isolation and characterization of a..."

...Hence, the best keratinolytic bacterium with highest chicken feather degradation ability in short period of time and optimal enzyme activity was identified by using various biochemical tests and 16S rRNA gene sequencing after which, preliminary characterization of crude keratinolytic enzyme from potent bacterial isolate (Bacillus sp. NKSP-7) was studied....
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...The results demonstrated that these isolates were identified as the members of genus Bacillus....
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...The 16S rRNA sequencing and gene-based phylogenetic analysis suggested that NKSP-7 and NKSP-9 isolates showed high sequence homology to other species of Bacillus, which confirmed that both strains belong to genus Bacillus....
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...Generally, the activity of keratinases was inhibited with the addition of EDTA (Xu et al. 2009; Vigneshwaran et al. 2010)....
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...3), which is analogous to other keratinolytic enzymes from Bacillus (Xu et al. 2009; Vigneshwaran et al. 2010); and higher than that of other (35–50 °C) (Prasad et al....
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A Potential Strain of Keratinolytic Bacteria VIT RSAS 2 from Katpadi and Its Pharmacological Benefits

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K Revathi, Shaifali Singh, Mohd Azeem Khan, Suneetha

01 Jan 2013

TL;DR: The keratinase activity was observed over a wide range of pH values (pH 3 - 9) and varying substrate concentration and it was optimal at pH 7 – 8 and 0.5 mg substrate /µl enzyme respectively.

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Abstract: Two bacterial strains VIT RSAS1 and VIT RSAS2 obtained from the poultry farms in Vellore were investigated for the keratinase enzyme production. The maximum amount of keratinase activity of strain RSAS2 (about 104 Uml-1) was produced at 37°C when the bacterium was cultured for 432 Hrs. in broth containing feathers with initial pH of 7. The keratinase activity was observed over a wide range of pH values (pH 3 - 9) and varying substrate concentration. It was optimal at pH 7 – 8 and 0.5 mg substrate /µl enzyme respectively. The synthesis of nanoparticles was also monitored. Silver nanoparticles were synthesised using the keratinase enzyme produced by the bacterial strain. Production of silver nanoparticles was monitored by UV- visible spectroscopic analysis. The peak was observed between 400-450 nm indicating their presence. Additionally the antibacterial activity of nanoparticles was checked against E.coli and Staphylococcus aureus which can be used for various research and pharmacological studies.

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12 citations

Cites background from "Isolation and characterization of a..."

...2 Keratinolytic activity has been reported for various bacterial genera, such as Bacillus (8-10), Thermoanaerobacter(11), etc and also by fungal species 12,13 and actinomycetes(14,15) with the enzyme produced both by submerged 18 as well as in solid state fermentation.(19,1,20) The microbial degradation of the poultry waste is an environment friendly biotechnological process, through which one can convert the abundant waste into low-cost, nutrient-rich animal feed (22)....
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Book Chapter•DOI•

Extremophilic Proteases: Developments of Their Special Functions, Potential Resources and Biotechnological Applications

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Aneta Białkowska¹, Ewa Gromek¹, Tomasz Florczak¹, Joanna Krysiak¹, Katarzyna M. Szulczewska¹, Marianna Turkiewicz¹ - Show less +2 more•Institutions (1)

Lodz University of Technology¹

01 Jan 2016

TL;DR: In the chapter below, selected representatives of this most significant, in terms of economy, group of extremophilic proteases are characterized and possible directions for their application in biotechnology are discussed.

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Abstract: Currently, microbiological proteases, along with lipases, are the most significant enzymes for biotechnology. They are produced in great quantity and since they are qualitatively diversified, they can be successfully applied in various branches of industry, including medicine. For several decades, the number as well as the significance of studies on extremophilic proteases have been growing. Extremophilic proteases are isolated from extremophiles which, given their unique kinetic and structural adaptations, can be used at low and high temperatures and in extreme environments (alkaline, acidic, saline). These enzymes have already enriched the range of commercial proteases and the studies on their properties in relation to their structural features stimulated a rational engineering of conventional proteases, aimed at enhancing their ability to adapt to specific conditions. In the chapter below, we characterized selected representatives of this most significant, in terms of economy, group of extremophilic proteases and discussed possible directions for their application in biotechnology.

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11 citations

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Journal Article•DOI•

Purification and Characterization of a Keratinase from a Feather-Degrading Bacillus licheniformis Strain

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Xiang Lin¹, Chung-Ginn Lee¹, Ellen S. Casale¹, Jason C. H. Shih¹•Institutions (1)

North Carolina State University¹

01 Oct 1992-Applied and Environmental Microbiology

TL;DR: The purified keratinase hydrolyzes a broad range of substrates and displays higher proteolytic activity than most proteases and is a useful enzyme for promoting the hydrolysis of feather keratin and improving the digestibility of feather meal.

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Abstract: A keratinase was isolated from the culture medium of feather-degrading Bacillus licheniformis PWD-1 by use of an assay of the hydrolysis of azokeratin. Membrane ultrafiltration and carboxymethyl cellulose ion-exchange and Sephadex G-75 gel chromatographies were used to purify the enzyme. The specific activity of the purified keratinase relative to that in the original medium was approximately 70-fold. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis and Sephadex G-75 chromatography indicated that the purified keratinase is monomeric and has a molecular mass of 33 kDa. The optimum pH and the pI were determined to be 7.5 and 7.25, respectively. Under standard assay conditions, the apparent temperature optimum was 50°C. The enzyme is stable when stored at −20°C. The purified keratinase hydrolyzes a broad range of substrates and displays higher proteolytic activity than most proteases. In practical applications, keratinase is a useful enzyme for promoting the hydrolysis of feather keratin and improving the digestibility of feather meal. Images

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363 citations

"Isolation and characterization of a..." refers background or methods or result in this paper

...Keratinolytic activity was measured using insoluble azokeratin as a substrate (Lin et al., 1992)....
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...…B. licheniformis K-19 can secrete a large amount of a keratinase that is more thermostable and has broader pH resistance than other keratinolytic proteases from Bacillus reported previously (Lin et al., 1992; Cheng et al., 1995; Lin et al., 1999; Suntornsuk et al., 2003; Suntornsuk et al., 2005)....
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...licheniformis K-19 can secrete a large amount of a keratinase that is more thermostable and has broader pH resistance than other keratinolytic proteases from Bacillus reported previously (Lin et al., 1992; Cheng et al., 1995; Lin et al., 1999; Suntornsuk et al., 2003; Suntornsuk et al., 2005)....
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...Many Bacillus species have been reported to produce keratinolytic proteases (Lin et al., 1992; Cheng et al., 1995; Lin et al., 1999; Manczinger et al., 2003; Suntornsuk and Suntornsuk, 2003; Zerdani et al., 2004; Suntornsuk et al., 2005)....
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...The keratinase of B. licheniformis PWD-1, which was isolated from a poultry waste digester, and the gene (kerA) that encodes this keratinase have been isolated and characterized (Williams et al. 1990, Lin et al., 1992, Cheng et al. 1995, Lin et al., 1995)....
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Journal Article•DOI•

Isolation, identification, and characterization of a feather-degrading bacterium.

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C. M. Williams¹, C. S. Richter¹, J. M. MacKenzie¹, Jason C. H. Shih¹•Institutions (1)

North Carolina State University¹

01 Jun 1990-Applied and Environmental Microbiology

TL;DR: A feather-degrading culture was enriched with isolates from a poultry waste digestor and adapted to grow with feathers as its primary source of carbon, sulfur, and energy, indicating a potential biotechnique for degradation and utilization of feather keratin.

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Abstract: A feather-degrading culture was enriched with isolates from a poultry waste digestor and adapted to grow with feathers as its primary source of carbon, sulfur, and energy. Subsequently, a feather-hydrolytic, endospore-forming, motile, rod-shaped bacterium was isolated from the feather-degrading culture. The organism was Gram stain variable and catalase positive and demonstrated facultative growth at thermophilic temperatures. The optimum rate of growth in nutrient broth occurred at 45 to 50°C and at pH 7.5. Electron microscopy of the isolate showed internal crystals. The microorganism was identified as Bacillus licheniformis PWD-1. Growth on hammer-milled-feather medium of various substrate concentrations was determined by plate colony count. Maximum growth (approximately 109 cells per ml) at 50°C occurred 5 days postinoculation on 1% feather substrate. Feather hydrolysis was evidenced as free amino acids produced in the medium. The most efficient conditions for feather fermentation occurred during the incubation of 1 part feathers to 2 parts B. licheniformis PWD-1 culture (107 cells per ml) for 6 days at 50°C. These data indicate a potential biotechnique for degradation and utilization of feather keratin.

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335 citations

Journal Article•

The use of azoalbumin as a substrate in the colorimetric determination or peptic and tryptic activity.

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R M Tomarelli, J Charney, M L Harding

01 Mar 1949-Journal of Laboratory and Clinical Medicine

303 citations

"Isolation and characterization of a..." refers methods in this paper

...Azokeratin was synthesized based on the methodology described for azoalbumin (Tomarelli et al., 1949)....
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Journal Article•DOI•

Characterization of a new keratinolytic bacterium that completely degrades native feather keratin.

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Alessandro Riffel¹, Françoise S. Lucas², Philipp Heeb², Adriano Brandelli¹•Institutions (2)

Universidade Federal do Rio Grande do Sul¹, University of Lausanne²

28 Feb 2003-Archives of Microbiology

TL;DR: A novel feather-degrading microorganism was isolated from poultry waste, producing a high keratinolytic activity when cultured on broth containing native feather, and complete feather degradation was achieved during cultivation.

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Abstract: A novel feather-degrading microorganism was isolated from poultry waste, producing a high keratinolytic activity when cultured on broth containing native feather. Complete feather degradation was achieved during cultivation. The bacterium presents potential use for biotechnological processes involving keratin hydrolysis. Chryseobacterium sp. strain kr6 was identified based on morphological and biochemical tests and 16S rRNA sequencing. The bacterium presented optimum growth at pH 8.0 and 30 degrees C; under these conditions, maximum feather-degrading activity was also achieved. Maximum keratinase production was reached at 25 degrees C, while concentration of soluble protein was similar at both 25 and 30 degrees C. Reduction of disulfide bridges was also observed, increasing with cultivation time. The keratinase of strain kr6 was active on azokeratin and azocasein as substrates, and presented optimum pH and temperature of 7.5 and 55 degrees C, respectively. The keratinase activity was inhibited by 1,10-phenanthroline, EDTA, Hg(2+), and Cu(2+) and stimulated by Ca(2+).

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280 citations

"Isolation and characterization of a..." refers background in this paper

..., 2005), Thermoanaerobacter (Riessen and Antranikian, 2001), Chryseobacterium (Riffel et al., 2003), Flavobacterium (Riffel and Brandelli, 2002; Nam et al....
[...]
...…et al., 2003; Suntornsuk and Suntornsuk, 2003; Zerdani et al., 2004; Suntornsuk et al., 2005), Thermoanaerobacter (Riessen and Antranikian, 2001), Chryseobacterium (Riffel et al., 2003), Flavobacterium (Riffel and Brandelli, 2002; Nam et al., 2002) and Vibrio (Sangali and Brandelli, 2000)....
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...The microbial conversion of feather wastes is a potential technique for the degradation of feathers and their utilization as a feedstuff (Sangali and Brandelli, 2000; Riffel et al., 2003)....
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...Keratinolytic activity has been reported for various bacterial genera, such as Bacillus (Williams et al., 1990; Lin et al., 1999; Manczinger et al., 2003; Suntornsuk and Suntornsuk, 2003; Zerdani et al., 2004; Suntornsuk et al., 2005), Thermoanaerobacter (Riessen and Antranikian, 2001), Chryseobacterium (Riffel et al., 2003), Flavobacterium (Riffel and Brandelli, 2002; Nam et al., 2002) and Vibrio (Sangali and Brandelli, 2000)....
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Journal Article•DOI•

Native-feather degradation by Fervidobacterium islandicum AW-1, a newly isolated keratinase-producing thermophilic anaerobe

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Gae Won Nam¹, Dong Woo Lee¹, Han Seoung Lee¹, Nam Lee¹, Byoung-Chan Kim¹, Eun Ah Choe¹, Jae Kwan Hwang¹, Maggy Thenawidjaja Suhartono², Yu Ryang Pyun¹ - Show less +5 more•Institutions (2)

Yonsei University¹, Bogor Agricultural University²

16 Oct 2002-Archives of Microbiology

TL;DR: The enzyme from F. islandicum AW-1 is a novel, thermostable keratinolytic serine protease that showed higher specific activity for the keratinous substrates than other proteases and catalyzed the cleavage of peptide bonds more rapidly following the reduction of disulfide bridges in feather keratin by 10 mM dithiothreitol.

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Abstract: A native-feather-degrading thermophilic anaerobe was isolated from a geothermal hot stream in Indonesia. Isolate AW-1, identified as a member of the species Fervidobacterium islandicum, was shown to degrade native feathers (0.8%, w/v) completely at 70 °C and pH 7 with a maximum specific growth rate (0.14 h–1) in Thermotoga-Fervidobacterium (TF) medium. After 24 h of culture, feather degradation led to an increase in free amino acids such as histidine, cysteine and lysine. Moreover, nutritionally essential amino acids such as tryptophan and methionine, which are rare in feather keratin, were also produced as microbial metabolites. A homomultimeric membrane-bound keratinolytic protease (>200 kDa; 97 kDa subunits) was purified from a cell extract of F. islandicum AW-1. The enzyme exhibited activity toward casein and soluble keratin optimally at 100 °C and pH 9, and had a half-life of 90 min at 100 °C. The enzyme showed higher specific activity for the keratinous substrates than other proteases and catalyzed the cleavage of peptide bonds more rapidly following the reduction of disulfide bridges in feather keratin by 10 mM dithiothreitol. Therefore, the enzyme from F. islandicum AW-1 is a novel, thermostable keratinolytic serine protease.

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263 citations

"Isolation and characterization of a..." refers background in this paper

...…et al., 2003; Suntornsuk and Suntornsuk, 2003; Zerdani et al., 2004; Suntornsuk et al., 2005), Thermoanaerobacter (Riessen and Antranikian, 2001), Chryseobacterium (Riffel et al., 2003), Flavobacterium (Riffel and Brandelli, 2002; Nam et al., 2002) and Vibrio (Sangali and Brandelli, 2000)....
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..., 2003), Flavobacterium (Riffel and Brandelli, 2002; Nam et al., 2002) and Vibrio (Sangali and Brandelli, 2000)....
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...Keratinolytic activity has been reported for various bacterial genera, such as Bacillus (Williams et al., 1990; Lin et al., 1999; Manczinger et al., 2003; Suntornsuk and Suntornsuk, 2003; Zerdani et al., 2004; Suntornsuk et al., 2005), Thermoanaerobacter (Riessen and Antranikian, 2001), Chryseobacterium (Riffel et al., 2003), Flavobacterium (Riffel and Brandelli, 2002; Nam et al., 2002) and Vibrio (Sangali and Brandelli, 2000)....
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