• Journal of Environmental Science International
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• v.19 no.9
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• pp.1077-1082
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• 2010

This study was performed to investigate the nutritional conditions controlling keratinase activity in Bacillus megaterium F7-1. B. megaterium F7-1 produced keratinase using chicken feather as a sole source of carbon, nitrogen and sulfur. Addition of the feather medium with glucose enhanced keratinase production (68.9 U/ml), compared to control without glucose (63.2 U/ml). The synthesis of keratinase was repressed by addition of $NH_4Cl$ in B. megaterium F7-1. The highest keratinase production (70.9 U/ml) was obtained with the feather medium containing glucose and $MgSO_4{\cdot}7H_2O$. Keratinase was produced in the absence of feather (4.9 U/ml), indicating its constitutive synthesis. Feather degradation resulted in free SH group formation. B. megaterium F7-1 effectively degraded chicken feather meal (86%), whereas duck feather, human nail, human hair and sheep wool displayed relatively low degradation rates (8-34%).

• Asian-Australasian Journal of Animal Sciences
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• v.14 no.12
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• pp.1769-1774
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• 2001

Bacillus licheniformis strains L-25 and PWD-1 are two thermophilic feather-degrading bacteria. Despite isolated from different environmental conditions, they were both capable of breaking down chicken feathers and growing in a medium in which feather was the only source of carbon and nitrogen. A 1.46-kb keratinase gene (ker B) was isolated from strain L-25 by a polymerase chain reaction (PCR) using L-25 genomic DNA as templates. Sequencing results reveal that ker B shares great sequence identity with a previously published keratinase gene of B. licheniformis PWD-1 (ker A). Only two amino acids differences were found in the deduced amino acid sequence between the keratinases from L-25 and PWD-1. However several nucleotide changes were found upstream of the putative promoter region. Protease inhibition studies indicated that neutral protease activity accounted for approximate 25 to 30% of total extracellular proteolytic activity produced by strain L-25 in the feather medium. In contrast, no measurable neutral protease activity was produced by strain PWD-1 in the feather medium. When glucose (1%), a common catabolic repressor, was added into the feather medium, L-25 was still able to grow and produce keratinase. Strain PWD-1 produced no neutral protease activity and its growth was severely inhibited in the feather medium containing glucose. L-25 produced an enhanced level of keratinase in the feather medium in comparison with PWD-1.

• Mycobiology
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• v.33 no.2
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• pp.121-123
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• 2005

Extracellular keratinase isolated from Aspergillus flavus K-03 was immobilized on calcium alginate. The properties and reaction activities of free and immobilized keratinase with calcium alginate were characterized. The immobilized keratinase showed proteolytic activity against soluble azo-casein and azo-keratin, and insoluble feather keratin. Heat stability and pH tolerance of keratinase were greatly enhanced by immobilization. It also displayed a higher level of heat stability and an increased tolerance toward alkaline pHs compared with free keratinase. During the durability test at $40^{\circ}C$, 48% of the original enzyme activity of the immobilized keratinase was remained after 7 days of incubation. The immobilized keratinase exhibited better stability, thus increasing its potential for use in industrial application.

• Mycobiology
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• v.35 no.4
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• pp.219-225
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• 2007

A keratinolytic enzyme secreted by Aspergillus flavus K-03 cultured in feather meal basal medium (FMBM) containing 2% (w/v) chicken feather was purified and characterized. Keratinolytic enzyme secretion was the maximal at day 16 of the incubation period at pH 8 and $28^{\circ}C$. No relationship was detected between enzyme yield and increase of fungal biomass. The fraction obtained at 80% ammonium sulfate saturation showed 2.39-fold purification and was further purified by gel filtration in Sephadex G-100 followed by ion exchange chromatography on DEAE-Sephadex A-50, yielding an active protein peak showing 11.53-fold purification. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and zymograms indicated that the purified keratinase is a monomeric enzyme with 31 kDa molecular weight. The extracellular keratinase of A. flavus was active in a board range of pH ($7{\sim}10$) and temperature ($30^{\circ}C{\sim}70^{\circ}C$) profiles with the optimal for keratinase activity at pH 8 and $45^{\circ}C$. The keratinase activity was totally inhibited by protease inhibitors such as phenylmethylsulfonyl fluoride (PMSF), iodoacetic acid, and ethylenediaminetetraacetate (EDTA) while no reduction of activity by the addition of dithiothreitol (DTT) was observed. N-terminal amino acid sequences were up to 80% homologous with the fungal subtilisins produced by Fusarium culmorum. Therefore, on the basis of these characteristics, the keratinase of A. flavus K-03 is determined to be subtilisins-like.

• Journal of Applied Biological Chemistry
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• v.56 no.2
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• pp.119-129
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• 2013

Thirteen different Streptomyces isolates were evaluated for their ability to produce keratinase using chicken feather as a sole carbon and nitrogen sources under solid state fermentation (SSF). Streptomyces sp. NRC 13S produced the highest keratinase activity [1,792 U/g fermented substrate (fs)]. The phenotypic characterization and analysis of 16S rDNA sequencing of the isolate were studied. Optimization of SSF medium for keratinase production by the local isolate, Streptomyces sp. NRC13S, was carried out using the one-variable-at-a-time and the statistical approaches. In the first optimization step, the effect of incubation period, initial moisture content, initial pH value of the fermentation medium, and supplementation of some agro-industrial by-products on keratinase production were evaluated. The strain produced about 2,310 U/gfs when it grew on chicken feather with moisture content of 75% (w/w), feather: fodder yeast ratio of 70:30 (w/w), and initial pH 7 using phosphate buffer after 8 days. Based on these results, the Box-Behnken design and response surface methodology were applied to find out the optimal conditions for the enzyme production. The corresponding maximal production of keratinase was about 2,569.38 U/gfs.

• Journal of Environmental Science International
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• v.19 no.10
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• pp.1307-1314
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• 2010

This study was conducted to isolate and characterize a novel feather-degrading bacterium producing keratinase activity. A strain K9 was isolated from soil at poultry farm and identified as Xanthomonas sp. K9 by phenotypic characters and 16S rRNA gene analysis. The cultural conditions for the keratinase production were 0.3% fructose, 0.1% gelatin, 0.04% $K_2HPO_4$, 0.06% $KH_2PO_4$, 0.05% NaCl and 0.01% $FeSO_4$ with an initial pH 8.0 at $30^{\circ}C$ and 200 rpm. In an optimized medium containing 0.1% chicken feather, production yield of keratinase was approximately 8-fold higher than the yield in basal medium. The strain K9 effectively degraded chicken feather meal (67%) and duck feather (54%), whereas human nail and human hair showed relatively low degradation rates (13-22%). Total free amino acid concentration in the cell-free supernatant was about 25.799 mg/l. Feather hydrolysate produced by the strain K9 stimulated growth of red pepper, indicating Xanthomonas sp. K9 could be not only used to increase the nutritional value of chicken feather but also a potential candidate for the development of natural fertilizer applicable to crop plant soil.

• Journal of Environmental Science International
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• v.23 no.6
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• pp.1095-1100
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• 2014

Keratin wastes are generated in excess of million tons per year worldwide and biodegradation of keratin by microorganisms possessing keratinase activity can be used as an alternative tool to prevent environmental pollution. For practical use of keratinase, its physicochemical properties should be investigated in detail. In this study, we investigated characteristics of keratinase produced by Xanthomonas sp. P5 which is isolated from rhizospheric soil of soybean. The level of keratinase produced by the strain P5 increased with time and reached its maximum (10.6 U/ml) at 3 days. The production of soluble protein had the same tendency as the production of keratinase. Optimal temperature and pH of keratinase were $40^{\circ}C-45^{\circ}C$ and pH 9, respectively. The enzyme showed broad temperature and pH stabilities. Thermostability profile showed that the enzyme retained 94.6%-100% of the original activity after 1 h treatment at $10^{\circ}C-40^{\circ}C$. After treatment for 1 h at pH 6-10, 89.2%-100% of the activity was remained. At pH 11, 71.6% of the original activity was retained after 1 h treatment. Although the strain P5 did not degrade human hair, it degraded duck feather and chicken feather. These results indicate that keratinase from Xanthomonas sp. P5 could be not only used to upgrade the nutritional value of feather hydrolysate but also useful in situ biodegradation of feather.

• Journal of Applied Biological Chemistry
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• v.55 no.3
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• pp.149-156
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• 2012

The effects of solvent type and concentration, solid/liquid ratio, extraction time and repeated extraction on recovery of keratinase from solid-state fermentation (SSF) of chicken feather by a local Streptomyces sp. NRC 13S were investigated in order to establish the experimental conditions for keratinase yield. Among solvents tested, 0.5% (v/v) glycerol was the best. Box-Behnken design was used to investigate the effect of relevant variables on keratinase recovery. The factors investigated were solid/liquid ratio (1:1.66-1:6.66 g/mL), glycerol concentration (0.5-5% v/v) and repeated extraction (1-5 cycle). The results showed that the maximum recovery of keratinase (6933.3 U/gfs) was obtained using 0.5 (v/v) glycerol as extracting solvent, in a solid/liquid ratio of 1:5 and three extraction cycles.

• Journal of Environmental Science International
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• v.21 no.2
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• pp.253-261
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• 2012

We isolated and characterized novel duck feather-degrading bacteria producing keratinase. Twelve strains were isolated from soil and faces at poultry farm, and decayed feathers. They were identified as Bacillus methylotrophicus, Pseudomonas geniculata, Pseudomonas hibiscicola, Exiquobacterium profundum, Bacillus pumilus, Bacillus amyloliquefaciens, Chryseobacterium indologenes, Bacillus thuringiensis, Thermomonas koreensis, respectively, by phenotypic characters and 16S rRNA gene analysis. Generally, the level of keratinase production was not proportional to feather degradation rate. The highest keratinolytic activity was observed in the culture inoculated with Chryseobacterium indologenes D27. Although all strains did not degrade human hair, strains tested effectively degraded chicken feather(53.8-91.4%), wool(40.4-93.0%) and human nail (51.0-82.9%). These results suggest that strains isolated could be not only used to improve the nutritional value of recalcitrant feather waste but also is a potential candidate for biotechnological processes of keratin hydrolysis.

• Journal of Environmental Science International
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• v.22 no.11
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• pp.1457-1464
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• 2013

We investigated the optimal conditions for keratinase production by feather-degrading Pseudomonas geniculata H10 using one variable at a time (OVT) method. The optimal medium composition and cultural condition for keratinase production were determined to be glucose 0.15% (w/v), beef extract 0.08% (w/v), $KH_2PO_4$ 0.12% (w/v), $K_2HPO_4$ 0.02% (w/v), NaCl 0.07% (w/v), $MgSO_4{\cdot}7H_2O$ 0.03%, $MgCl_2{\cdot}6H_2O$ 0.04% along with initial pH 10 at 200 rpm and $25^{\circ}C$, respectively. The production yield of keratinase was 31.6 U/ml in an optimal condition, showing 4.6-fold higher than that in basal medium. The strain H10 also showed plant growth promoting activities. This strain had ammonification activity and produced indoleacetic acid (IAA), siderophore and a variety of hydrolytic enzymes such as protease, lipase and chitinase. Therefore, this study showed that P. geniculata H10 could be not only used to upgrade the nutritional value of feather wastes but also useful in situ biodegradation of feather wastes. Moreover, it is also a potential candidate for the development of biofertilizing agent applicable to crop plant soil.