Isolation and characterization of a new keratinolytic bacterium that exhibits significant feather-degrading capability
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.
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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TL;DR: In this paper, the effect of temperature, keratinolytic activity and the influence of the immobilised and crude enzyme-degraded feather meal on broiler chickens were assessed.
Abstract: The management of solid wastes has been a major concern to many cities of the world due to daily increasing ruralurban migration and globalization. Due to a greater consumption of poultry meat, the disposal of feather wastes has contributed to the daily increasing environmental pollution. Agricultural wastes (such as poultry feathers) are disposed by burning, which consequently constitute environmental pollution and their chemical or mechanical conversion into animal feed normally leads to minimization of amino acids. The application of biotechnology through the utilisation of enzymes is considered an easy and inexpensive means of producing valuable products from poultry feather wastes. Bacillus subtilis was isolated from a dumping site and the plates were incubated on nutrient agar. The treatments containing 200 mL each of crude enzyme, immobilized enzyme and sterilized water were added to the bioreactor for biodegradation of chicken feathers. After hydrolysis, the feathers were dried and the products labelled microbial biodegraded feather meal. The effect of temperature, keratinolytic activity and the influence of the immobilised and crude enzyme-degraded feather meal on broiler chickens were assessed. The optimal activity and biodegradative potential of the keratinolytic enzyme was observed as 45 °C and 48 h after fermentation, respectively. The weight gain of the birds fed immobilised enzyme-degraded feather meal-based diet compared with the control. The enzymedegraded feather meal is safe for inclusion in broilers’ diet and slight feeding manipulations could improve their performance.
6 citations
Cites result from "Isolation and characterization of a..."
...…at which the maximum keratinase activity was observed in the present study is in line with the reports from previous findings, which is an indication that the keratinolytic strain adopted in the present study is a promising candidate for biotechnology (Gupta and Ramnani, 2006; Xu et al., 2009)....
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01 Jan 2015
TL;DR: In this paper, a preliminary study and a detailed solid-state fermentation (SSF) study of 42 keratinolytic fungi were subjected to SSF providing arbitrarily selected conditions of moisture level and incubation period i.e., solid substrate: moistening solution ratio 1:2 and 5 days of incubation.
Abstract: A B S T R A C T The Solid-state fermentation (SSF) is the growth of organism on moist substrates in the absence of free flowing water. It is more techno-economically important compared with the submerged fermentation. Screening of fungi for keratinase production by SSF was done in two steps a preliminary study and the detailed SSF study. In preliminary studies all the 42 keratinolytic fungi were subjected to SSF providing arbitrarily selected conditions of moisture level and incubation period i.e, solid substrate: moistening solution ratio 1:2 and 5 days of incubation. The fungal isolates found to be keratinolytic on keratin agar medium were screened for keratinase production by solid substrate fermentation.Present study showed that the optimum moisture level of 69.92% presented better enzyme production, above which the production started decreasing.The maximum production was obtained at 4 th day of incubation at pH 9 and temperature 55 o C. Carbon and Nitrogen sources were found insignificant in enzyme production. K e y w o r d s Keratinase, Solid state fermentation, Aspergillus flavus, Keratinolytic fungus, Feather keratin, Optimum pH
5 citations
Patent•
08 May 2018
TL;DR: Wang et al. as discussed by the authors revealed that the Bacillus licheniformisZL-1 can be cultured in a liquid culturing medium with pure feathers for three days, the keratinase activity of the BCLZL1 reached 52.14 U/ml, and the degradation rate of feathers reached 90.15%.
Abstract: The invention discloses Bacillus licheniformis ZL-1 and the application thereof. The Bacillus licheniformis ZL-1 is obtained by isolating and screening from a high-temperature stack of feathers combined with edible fungus residues, the Bacillus licheniformis ZL-1 is preserved at Guangdong Provincial Microorganism Culture Collection Center, with a preservation number of GDMCC No. 60284, the preservation address is 5th floor, Building 59, Compound, No. 100 Xianlie Middle Road, Guangzhou, China, and the preservation date is November 24, 2017. Experiments determine that the Bacillus licheniformisZL-1 can be cultured in a liquid culturing medium with pure feathers for three days, the keratinase activity of the Bacillus licheniformis ZL-1 reaches 52.14 U/ml, and the degradation rate of feathersreaches 90.15%. Further more, the Bacillus licheniformis ZL-1 has good tolerance to high temperature and salinity, can grow well at a temperature of 55 DEC G and is resistant to salinity of 14%, so that the Bacillus licheniformis ZL-1 is used for degrading the feathers, preparing compound amino acid solution, conducting high-temperature compost fermentation by using the feathers as raw materialsand preparing compound amino acid fertilizers and keratinase; the Bacillus licheniformis ZL-1 has good application prospects and market value.
5 citations
01 Apr 2015
TL;DR: In this paper, a low cost and energy intensive solid-state fermentation medium was formulated by employing poultry feather for the production of keratinase by Bacillus weihenstephanensis PKD5 following "one variable at a time" (OVAT) and response surface methodology (RSM).
Abstract: A low cost and energy intensive solid-state fermentation medium was formulated by employing poultry feather for the production of keratinase by Bacillus weihenstephanensis PKD5 following ‘one variable at a time’ (OVAT) and response surface methodology (RSM). After OVAT optimization, four most critical factors were identified with significant increase of enzyme production (2.95-fold). Among them, incubation period, incubation temperature, pH and nitrogen source (ammonium chloride) were further optimized statistically by Box-Behnken RSM. The results of analysis of variance and regression of the second-order model of RSM showed that among the parameters, fermentation time (2.85 d), temperature (34.12°C), pH (7.79) and ammonium chloride (0.5%) had the significant influences on keratinase production. Under the optimized conditions, a maximum enzyme production of 164.9 U/g was achieved, which was 3.17-fold higher. During further investigation on milk clotting property, the enzyme had shown the clotting activity of 43.6 SU/mL, suggesting its usefulness as new source of milk-coagulant for cheese making.
4 citations
TL;DR: In this paper , a review of the degradation of keratin waste by biological approaches using keratinase producing microorganisms is presented, focusing on the practical use of Keratinases and the economical importance of bioconverted products of keratinous wastes for different applications.
Abstract: The management of keratinous wastes generated from different industries is becoming a major concern across the world. In each year, more than a billion tons of keratin waste is released into the environment. Despite some trials that have been performed and utilize this waste into valuable products, still a huge amount of keratin waste from different sources is a less explored biomaterial for making valuable products. This indicates that the huge amount of keratin waste is neither disposed properly nor converted into usable products rather thrown away to the environment that causes environmental pollution. Due to the introduction of this waste associated with different pathogenic organisms into soil and water bodies, human beings and other small and large animals are affected by different diseases. Therefore, there is a need for modern and ecofriendly approaches to dispose and convert this waste into usable products. Hence, the objective of this review is to give a concise overview regarding the degradation of keratin waste by biological approaches using keratinase producing microorganisms. The review also focuses on the practical use of keratinases and the economical importance of bioconverted products of keratinous wastes for different applications. Various researches have been studied about the source, disposal mechanisms, techniques of hydrolysis, potential use, and physical and chemical properties of keratin wastes. However, there is negligible information with regard to the use of keratin wastes as media supplements for the growth of keratinolytic microorganisms and silver retrieval from photographic and used X-ray films. Hence, this review differs from other similar reviews in the literature in that it discusses these neglected concerns.
4 citations
References
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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.
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
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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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.
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.
335 citations
Journal Article•
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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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.
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+).
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....
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...…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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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.
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.
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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