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: A newly isolated bacterium identified as Bacillus safensis based on biochemical tests and 16S rRNA analysis and its mutant variant created by exposure to ultraviolet radiation at 254 nm were investigated for keratinolytic activity as mentioned in this paper.
Abstract: A newly isolated bacterium identified as Bacillus safensis based on biochemical tests and 16S rRNA analysis and its mutant variant created by exposure to ultraviolet radiation at 254 nm were investigated for keratinolytic activity. The wild-type strain produced 35.4–50.4 U/mL keratinase over a period of 120 h, while the mutant one yielded 64.4–108.5 U/mL keratinase for the same period of 120 h. The optimal conditions for the enzyme activities were pH 7.5 and 40 °C. The mutant and wild-type strain keratinases retained 59% and 54% of their activity after 12 h pretreatment at 40 °C, and 64% and 60% of their activity after 12 h at pH 7.5, respectively. The keratinases showed high substrate specificity for feathers, but low specificity for human and bovine hairs. The enzymes were activated by Na+, Ca2+, Fe2+ and Mg2+. However, while Mn2+ activated the enzyme from the mutant strain, it inhibited that of the wild type. The mutant and wild-type strain completely degraded whole chicken feathers after 6 and 9 days ...
70 citations
01 Jan 2015
TL;DR: The obtained results showed an improvement in the properties of the mutant strain for use of the micro-organism or its enzyme as biocatalysts and high substrate specificity for feathers, but low specificity for human and bovine hairs.
Abstract: A newly isolated bacterium identified as Bacillus safensis based on biochemical tests and 16S rRNA analysis and its mutant variant created by exposure to ultraviolet radiation at 254 nm were investigated for keratinolytic activity. The wild-type strain produced 35.450.4 U/mL keratinase over a period of 120 h, while the mutant one yielded 64.4108.5 U/mL keratinase for the same period of 120 h. The optimal conditions for the enzyme activities were pH 7.5 and 40 C. The mutant and wild-type strain keratinases retained 59% and 54% of their activity after 12 h pretreatment at 40 C, and 64% and 60% of their activity after 12 h at pH 7.5, respectively. The keratinases showed high substrate specificity for feathers, but low specificity for human and bovine hairs. The enzymes were activated by Na C ,C a 2C ,F e 2C and Mg 2C . However, while Mn 2C activated the enzyme from the mutant strain, it inhibited that of the wild type. The mutant and wild-type strain completely degraded whole chicken feathers after 6 and 9 days at 30 § 2 C, and also completely dehaired goat skin within 12 and 16 h, respectively, without damage to the skin. Similarly, remarkable destaining of blood-stained cloth occurred within 23 h. The obtained results showed an improvement in the properties of the mutant strain for use of the microorganism or its enzyme as biocatalysts.
68 citations
TL;DR: The manuscript first time describes B. weihenstephanensis PKD 5-mediated keratinase production under submerged fermentation and whole chicken feather biodegradation.
Abstract: A new feather-degrading bacterium PKD 5 was isolated from feather dumping soil and identified as Bacillus weihenstephanensis based on morphological and physiochemical characteristics as well as 16S rRNA gene analysis. Extracellular keratinase was produced during submerged aerobic cultivation in a medium containing chicken feather as sole carbon and energy source and supplemented with salt solutions (NaCl 5.0, MgSO4 1.0, K2HPO4 1.0, (NH4)2SO4, 2.0 g/l). The optimal conditions for keratinase production include initial pH of 7.0, inoculum size of 2% (v/v), and cultivation at 40 °C. The maximum keratinase production and keratinolytic activity of PKD 5 was achieved after 7 days of fermentation under shaking condition (120 rpm). The enzyme has found application in developing cost-effective feather by-products for feeds and fertilizers. The manuscript first time describes B. weihenstephanensis PKD 5-mediated keratinase production under submerged fermentation and whole chicken feather biodegradation.
63 citations
TL;DR: Keratin is resistant to degradation by common proteases and chemical catalysts due to high mechanical stability and cross-linked disulphide bonds present in their structure as mentioned in this paper.
Abstract: The environment is under constant threat due to the huge accumulation of the keratinaceous wastes produced by poultry processing industries and poultry farms. Millions of tons of feather wastes are produced each year as a by-product that causes severe environmental pollution. Their degradation is tough due to the presence of insoluble, highly stiff, recalcitrant ‘keratin’ polypeptide. Keratin are resistant to degradation by common proteases and chemical catalysts due to high mechanical stability and cross-linked disulphide bonds present in their structure. Microbial keratinases has emerged as a powerful bio-catalyst that can potentially degrade keratin and transform the keratinaceous wastes into value-added products. Keratinase production is receiving worldwide attention for its application in sustainable development and cleaner production. The enzymatic hydrolysis of keratin have the potential for higher productivity, less energy consumption, less effluent generation and waste management. Keratinases add value to poultry and industrial wastes which brings economic feasibility and environmental sustainability. The present review prioritized the various aspects of keratinolytic proteases in bringing sustainable development to the economy of a nation. It also focuses on the recent advancement in the production of microbial keratinases with emphasis on the biochemical characteristics related to enzyme activity and stability, development of novel recombinant and mutant microbial strains along with different optimization strategies leading to expansive production and yield of keratinases. Furthermore, different properties of microbial keratinases which renders them as a green and sustainable material for industrial applications in waste management, textile, leather and detergents with advantages over conventional treatments have also been discussed.
41 citations
TL;DR: This study revealed that FPH produced by C. sediminis RCM-SSR-7 has the potential to be used as animal feed and organic fertilizer and exhibited radical scavenging activity with an IC50 value of 0.102 mg ml−1.
Abstract: Accumulation of feather waste is becoming a major issue in solid waste management. Towards discovery of keratinolytic bacteria, screening of bacterial strains from feather dumping sites in North East, India was performed and 26 keratinolytic bacterial strains were isolated. Out of these, one isolate RCM-SSR-7 was found to be most promising strain exhibiting feather degradation as well as antioxidant and indole-3-acetic acid production. The strain was identified as Chryseobacterium sediminis RCM-SSR-7. The strain could use chicken feather as sole carbon and nitrogen source for growth. Three parameters (feather concentration, pH and incubation time) were studied to optimize feather protein hydrolysate (FPH) preparation using response surface methodology (RSM). The optimum condition for FPH preparation was achieved at 5% (w/v) feather concentration, pH 7.5, 30 °C and 84 h incubation time upon optimization by RSM. FPH was found to be rich in essential amino acids and trace elements (phosphorous, potassium, calcium, and iron). FPH exhibited radical scavenging activity with an IC50 value of 0.102 mg ml−1. In vitro digestibility showed that FPH is 86% digestible with pepsin and trypsin treatment. This study revealed that FPH produced by C. sediminis RCM-SSR-7 has the potential to be used as animal feed and organic fertilizer.
40 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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