Biodegradation of feather waste by keratinase produced from newly isolated Bacillus licheniformis ALW1

Keratinase are proteolytic enzymes which have gained much attention to convert keratinous wastes that cause huge environmental pollution problems. Ten microbial isolates were screened for their keratinase production. The most potent isolate produce 25.2?U/ml under static condition and was primarily identified by partial 16s rRNA gene sequence as Bacillus licheniformis ALW1. Optimization studies for the fermentation conditions increased the keratinase biosynthesis to 72.2?U/ml (2.9-fold). The crude extracellular keratinase was optimally active at pH 8.0 and temperature 65?°C with 0.7% soluble keratin as substrate. The produced B. licheniformis ALW1 keratinase exhibited a good stability over pH range from 7 to 9 and over a temperature range 50–60?°C for almost 90?min. The crude enzyme solution was able to degrade native feather up to 63% in redox free system.     

Isolation and characterization of a new <Emphasis Type="Italic">Bacillus</Emphasis> sp. 50-3 with highly alkaline keratinase activity from <Emphasis Type="Italic">Calotes versicolor</Emphasis> faeces

A new native feather-degrading bacterium has been isolated from the faeces of the agamid lizard Calotes versicolor, collected from the Beijing Zoo in China. The isolate, which has been identified as Bacillus sp. 50-3 based on morphological and biochemical and 16S rDNA tests, was shown to degrade native feather completely at 37°C and pH 7.0 within 36 h when using chicken feathers as the sole carbon and nitrogen source. Bacillus sp. 50-3 presented optimum growth at 37°C and pH 7.0 in feather meal medium. Under these conditions, the maximum keratinase activity (680 ± 25 U/ml) was also achieved. The keratinase of Bacillus sp. 50-3 was active over a broad range of pH values and temperatures toward azokeratin, and presented an optimum pH and temperature of 10.0 and 60°C, respectively. Furthermore, it was relatively heat-and alkali-stable. Inhibitor studies showed that it seemed to belong to the serine-metalloprotease type. Therefore, the enzyme from Bacillus sp. 50-3 is a novel, high alkaline keratinase, suggesting its potential use in biotechnological processes.     

Keratinases and sulfide from <Emphasis Type="Italic">Bacillus subtilis</Emphasis> SLC to recycle feather waste

The aim of this study is to investigate the culture conditions of chicken feather degradation and keratinolytic enzyme production by the recently isolated Bacillus subtilis SLC and to evaluate the potential of the SLC strain to recycle feather waste discarded by the poultry industry. The SLC strain was isolated from the agroindustrial waste of a poultry farm in Brazil and was confirmed to belong to Bacillus subtilis by rDNA gene analysis. There was high keratinase production when the medium was at pH 8 (280 U ml⁻¹). Activity was higher using the inoculum propagated for 72 h on 1% whole feathers supplemented with 0.1% yeast extract. In the enzymatic extract, the keratinases were active in the pH range from 2.0 to 12.0 with a maximum activity at pH 10.0 and temperature 60°C. For gelatinase the best pH was 5.0 and the best temperature was 37°C. All keratinases are serine peptidases. The crude enzymatic extract degraded keratin, gelatin, casein, and hemoglobin. Scanning electron microscopy showed Bacillus cells adhered onto feather surfaces after 98 h of culture and degraded feather filaments were observed. MALDI-TOF mass spectrometric analysis showed multiple peaks from 522 to 892 m/z indicating feather degradation. The presence of sulfide was detected on extracellular medium probably participating in the breakdown of sulfide bridges of the feather keratin. External addition of sulfide increased feather degradation.     

Feather degradation by a new keratinolytic <Emphasis Type="Italic">Streptomyces</Emphasis> sp. MS-2

Seventy different actinomycete isolates were evaluated for their ability to produce keratinase using a keratin-salt agar medium containing ball-milled feather as substrate. A novel feather-degrading isolate obtained from marine sediment produced the highest keratinolytic activity when cultured on broth containing whole feather as a primary source of carbon, nitrogen and energy. Based on phenotypic characterization and analysis of 16S rDNA sequencing the isolate was identified as a Streptomyces sp. MS-2. Maximum keratinase activity (11.2 U/mg protein) was achieved when cells were grown on mineral salt liquid medium containing 1% whole chicken feather adjusted to pH 8 and incubated at 35°C for 72 h at 150 rpm. Reduction of disulphide bridges was also detected, increasing with incubation time. Feather degradation led to an increase in free amino acids such as alanine, leucine, valine and isoleucine. Moreover, methionine and phenylalanine were also produced as microbial metabolites.     

Production and characterization of keratinase from<Emphasis Type="Italic">Paracoccus</Emphasis> sp. WJ-98

A bacterial strain WJ-98 found to produce active extracellular keratinase was isolated from the soil of a poultry factory. It was identified asParacoccus sp. based on its 16S rRNA sequence analysis, morphological and physiological characteristics. The optimal culture conditions for the production of keratinase byParacoccus sp. WJ-98 were investigated. The optimal medium composition for keratinase production was determined to be 1.0% keratin, 0.05% urea and NaCl, 0.03% K₂HPO₄, 0.04% KH₂PO₄, and 0.01% MgCl₂·6H₂O. Optimal initial pH and temperature for the production of keratinase were 7.5 and 37°C, respectively. The maximum keratinase production of 90 U/mL was reached after 84 h of cultivation under the optimal culturing conditions. The keratinase fromParacoccus sp. WJ-98 was partially purified from a culture broth by using ammonium sulfate precipitation, ion-exchange chromatography on DEAE-cellulose, followed by gel filtration chromatography on Sephadex G-75. Optimum pH and temperature for the enzyme reaction were pH 6.8 and 50°C, respectively and the enzymes were stable in the pH range from 6.0 to 8.0 and below 50°C. The enzyme activity was significantly inhibited by EDTA, Zn²⁺ and Hg²⁺. Inquiry into the characteristics of keratinase production from these bacteria may yield useful agricultural feed processing applications.     

Isolation and characterization of a feather-degrading bacterium from the poultry processing industry

A Flavobacterium sp. producing a high keratinolytic activity was isolated from a poultry industry after growth on selective feather meal agar. This bacterium grew on feather meal broth, producing keratinase, and was also capable of complete degradation of raw feathers. The proteolytic activity was assessed in the presence of specific protease inhibitors. The crude enzyme showed mainly metalloprotease character. This novel isolate would have potential biotechnological use in processes involving keratin hydrolysis. Received 09 October 2001/ Accepted in revised form 19 July 2002     

Biochemical characterization of an alkaline surfactant-stable keratinase from a new keratinase producer,Bacillus zhangzhouensis

A new keratinase producer, Bacillus sp. BK111, isolated from a poultry feather was identified as Bacillus zhangzhouensis, which is the first report for its keratinolytic activity. The keratinase production was optimized, followed by the enzyme purification and characterization using biochemical assays. A 2.34-fold increase was observed in the enzyme production under optimized conditions. The enzyme was characterized as a serine protease with 42 kDa molecular weight, stable in a wide range of temperature and pH with maximum keratinolytic activity at 60 °C and pH 9.5. The enzyme had a wide range of different substrates with the best performance on the feather meal substrate. Metal ions of Ca²⁺, K⁺, Na⁺ and Mn²⁺ enhanced the enzyme activity. The enzyme showed a great deal of stability in the presence of ethanol, methanol, acetone, 2-propanol, dimethyl sulfoxide, Tween-80 and Triton X-100. Dithiothreitol (DTT), as a reducing agent, caused a twofold increase in keratinolytic activity. The half-life of the enzyme at optimum temperature was calculated to be 125 min and the ratio of keratinolytic:caseinolytic for the enzyme was 0.8. Our results showed the remarkable features of the enzyme that make it suitable for biotechnological usages.

     

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

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+).     

Characterization of a new keratinolytic Trichoderma atroviride strain F6 that completely degrades native chicken feather

Aims:  To isolate novel nonpathogenic fungus that completely degrades native chicken feather and characterize its keratinases. Methods and Results:  Feather‐degrading fungi were isolated from decaying feathers using a novel method based on simulating decaying process in the environment. The isolate F6 with high keratinolytic activity was identified as Trichoderma atroviride based on morphological traits and ITS1‐5·8S‐ITS2 sequence analysis. The purified dominant component of keratinase had a molecular mass of 21 kDa. The purified keratinase belonged to serine protease. Its isoelectric point, molecular weight, optimum pH, optimum temperature, and substrate specificity are different from those of other serine proteases of Trichoderma species. The optimum pH and temperature values of purified keratinase were consistent with those of crude keratinase. However, the differences between crude and purified enzymes such as thermostability, resistance to Ba²⁺, Mn²⁺, Hg²⁺, Zn²⁺, Cu²⁺, 1,10‐phenanthroline, 2,2′‐bipyridyl, and PMSF (phenylmethylsulfonyl fluoride) were observed. Conclusions:  The results suggested the purified keratinase is predominantly extracellular proteins when strain F6 was grown on keratinous substrates. The protease, in combination with other components, is effective in feather degradation. The strain F6 is more suitable for feather degradation than its purified keratinase. Significance and Impact of the Study:  The novel nonpathogenic T. atroviride F6 with high feather‐degrading activity showed potentials in biotechnological process of converting feathers into economically useful feather meal.     

Biodegradation of feather waste by extracellular keratinases and gelatinases from Bacillus spp.

In this study, three feather degrading bacterial strains were isolated from agroindustrial residues from a Brazilian poultry farm. Three Gram-positive, spore-forming, rod-shaped bacteria and were identified as B. subtilis 1271, B. licheniformis 1269 and B. cereus 1268 using biochemical, physiologic and molecular methods. These Bacillus spp. strains grew and produced keratinases and peptidases using chicken feather as the sole source of nitrogen and carbon. B. subtilis 1271 degraded feathers completely after 7 days at room temperature and produced the highest levels of keratinase (446 U ml^?1). Feather hydrolysis resulted in the production of serine, glycine, glutamic acid, valine and leucine as the major amino acids. Enzymography and zymography analyses demonstrated that enzymatic extracts from the Bacillus spp. effectively degraded keratin and gelatin substrates as well as, casein, hemoglobin and bovine serum albumin. Zymography showed that B. subtilis 1271 and B. licheniformis 1269 produced peptidases and keratinases in the 15?C140 kDa range, and B. cereus produced a keratinase of ~200 kDa using feathers as the carbon and nitrogen source in culture medium. All peptidases and keratinases observed were inhibited by the serine specific peptidase inhibitor phenylmethylsulfonyl fluoride (PMSF). The optimum assay conditions of temperature and pH for keratinase activity were 40?C50°C and pH 10.0 for all strains. For gelatinases the best temperature and pH ranges were 50?C70°C and pH 7.0?C11. These isolates have potential for the biodegradation of feather wastes and production of proteolytic enzymes using feather as a cheap and eco-friendly substrate.     

Keratinolytic activity of Bacillus megaterium F7-1, a feather-degrading mesophilic bacterium

The aim of this study was to investigate environmental conditions affecting chicken feather degradation and keratinolytic enzyme production by Bacillus megaterium F7-1, a feather-degrading mesophilic bacterium. B. megaterium F7-1 degraded whole chicken feather completely within 7 days. The bacterium grew with an optimum at pH 7.0–11.0 and 25–40 °C, where maximum keratinolytic activity was also observed. The production of keratinolytic enzyme by B. megaterium F7-1 was inducible with feather. Keratinolytic enzyme production by B. megaterium F7-1 at 0.6% (w/v) skim milk was 468 U/ml, which was about 9.4-fold higher than that without skim milk. The amount of keratinolytic enzyme production depended on feather concentrations. The degradation rate of autoclaved chicken feathers by cell-free culture supernatant was 26% after 24 h of incubation, but the degradation of untreated chicken feathers was unsuccessful. B. megaterium F7-1 effectively degraded feather meal, duck feather and human nail, whereas human hair and sheep wool showed relatively low degradation rates. B. megaterium F7-1 presented high keratinolytic activity and was very effective in feather degradation, providing potential use for biotechnological processes of keratin hydrolysis.     

Multifarious potential applications of keratinase of Bacillus subtilis K-5

Abstract

Bacillus subtilis K-5, an isolate from compost, utilized a wide range of keratinous wastes viz. diverse feather types, nails, hair, scales, etc. for growth and produced a thermostable alkaline protease (keratinase) with broad proteolytic activity. Optimization of cultural and environmental variables using a Plackett–Burman design and response surface methodology resulted in enhanced keratinase production (89%). Keratinase was partially purified (15-fold) by ammonium sulfate precipitation and carboxymethyl cellulose chromatography. The optimum pH and temperature for keratinase activity were 9.0 and 60°C, however, considerable activity and stability was observed over broad pH (5–10) and temperature range (50–90°C). B. subtilis K-5 keratinase exhibited excellent stability toward detergents (cetyl trimethylammonium bromide, Tween 80, and sodium dodecyl sulfate) and organic solvents (benzene, acetonitrile, phenylmethylsulfonyl fluoride); however, metal ions like Mn²⁺, Cu²⁺, Na⁺, Hg²⁺, K⁺, Ca²⁺, and Zn²⁺ inhibited the activity. B. subtilis K-5 protease showed remarkable potential for diverse applications like blood stain removal, gelatin hydrolysis from waste X-ray films and dehairing of animal hide.     

Characterization of a protease of a feather-degrading Microbacterium species

AIMS: To characterize a new feather-degrading bacterium. METHODS AND RESULTS: The strain kr10 producing a high keratinolytic activity when cultured on native feather broth was identified as Microbacterium sp., based on phenotypical characteristics and 16S rDNA sequence. The bacterium presented optimum growth and feather-degrading activity at pH 7.0 and 30 degrees C. Complete feather degradation was achieved during cultivation. The keratinase was partially purified by gel filtration chromatography. It was optimally active at pH 7.0 and 55 degrees C. The enzyme was inhibited by 1,10-phenanthroline, EDTA, p-chloromercuribenzoic acid, 2-mercaptoethanol and metal ions like Hg(2+), Cu(2+) and Zn(2+). SIGNIFICANCE AND IMPACT OF THE STUDY: A new Microbacterium sp. strain was characterized presenting high feather-degrading activity, which appears to be associated to a metalloprotease-type keratinase. This micro-organism has enormous potential for use in biotechnological processes involving keratin hydrolysis.     

Production of keratinolytic proteases through bioconversion of feather meal by the Amazonian bacterium Bacillus sp. P45

Extracellular keratinase production by the feather-degrading Amazonian isolate Bacillus sp. P45 was evaluated with various growth substrates. Higher enzyme production occurred with feather meal (FM) in comparison to casein, gelatin, and cheese whey, suggesting the specificity of this strain for the utilization of keratinous substrates. Supplementation of FM medium with carbohydrates reduced enzyme production, probably due to catabolite repression. Increased keratinase yield was achieved when NH₄Cl was added to FM medium. The effects of FM and NH₄Cl concentrations on enzyme production were investigated using a 2² central composite design. Feather meal was the most significant parameter, while NH₄Cl concentrations resulted in slight differences in enzyme yield. In the range studied, optimal concentrations of FM and NH₄Cl were 43-50 g l⁻¹ and 1.8-8.6 g l⁻¹, respectively, resulting in an effective low-cost medium for the production of keratinolytic protease. Crude keratinase showed maximum activity at 50 °C and pH 7.0, and was strongly inhibited by EDTA, indicating the importance of metal ions for activity/stability. The crude keratinase from mesophilic Bacillus sp. P45 could potentially be used in the bioconversion of recalcitrant keratinous wastes through an environmentally friendly and energy-saving process, producing protein hydrolysates with commercial value for utilization as animal feed and fertilizers.     

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

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.     

Production of alkali-tolerant cellulase-free xylanase by <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. WLUN024 with wheat bran as the main substrate

An alkali-tolerant cellulase-free xylanase producer, WLI-11, was screened from soil samples collected from a pulp and paper mill in China. It was subsequently identified as a Pseudomonas sp. A mutant, WLUN024, was selected by consecutive mutagenesis by u.v. irradiation and NTG treatment using Pseudomonas sp. WLI-11 as parent strain. Pseudomonas sp. WLUN024 produced xylanase when grown on xylosidic materials, such as hemicellulose, xylan, xylose, and wheat bran. Effects of various nutritional factors on xylanase production by Pseudomonas sp. WLUN024 with wheat bran as the main substrate were investigated. A batch culture of Pseudomonas sp. WLUN024 was conducted under suitable fermentation conditions, where the maximum activity of xylanase reached 1245 U ml⁻¹ after incubating at 37 °C for 24 h. Xylanase produced by Pseudomonas sp. WLUN024 was purified and the molecular weight was estimated as 25.4 kDa. Primary studies on the characteristics of the purified xylanase revealed that this xylanase was alkali-tolerant (optimum pH 7.2–8.0) and cellulase-free. In addition, the xylanase was also capable of producing high quality xylo-oligosaccharides, which indicated its application potential in not only pulp bio-bleaching processes but also in the nutraceutical industry.     

Feather keratin hydrolysis by a Vibrio sp. strain kr2

The aim of the study was to characterize feather-degrading bacteria isolated from poultry industry waste. A Vibrio sp. strain kr2 producing a high keratinolytic activity when cultured on native feather-containing broth was isolated. The bacterium grew with an optimum at pH 6.0 and 30 degrees C, where maximum featherdegrading activity was also observed. Keratinase production was similar at both 25 and 30 degrees C, while the maximum concentration of soluble protein was reached at 30 degrees C. Reduction of disulphide bridges was also observed, increasing with cultivation time. The keratinase of strain kr2 was active on azokeratin, azocasein, benzoyl-arginine-p-nitroanilide and Ala-Ala-p-nitroanilide as substrates. The amino acid composition of the feather hydrolysate was determined, presenting similarities with that reported for feather lysate, feather meal and raw feathers. A novel feather-degrading bacterium was isolated and characterized, showing high keratinolytic activity. Complete feather degradation was achieved during cultivation. Strain kr2 shows potential for use for biotechnological processes involving keratin hydrolysis.     

Characterization of a keratinolytic protease produced by the feather-degrading Amazonian bacterium Bacillus sp. P45

Abstract

An extracellular keratinolytic protease produced by Bacillus sp. P45 was purified and characterized. The keratinase had a molecular weight of approximately 26 kDa and was active over wide pH and temperature ranges, with optimal activity at 55°C and pH 8.0. However, this enzyme displayed low thermostability, being completely inactivated after 10 min at 50°C. Keratinase activity increased with Ca²⁺, Mg²⁺, Triton X-100, ethanol and DMSO, was stable in the presence of the reducing agent 2-mercaptoethanol, and was inactivated by SDS. PMSF (phenylmethylsulfonyl fluoride) completely inactivated and EDTA strongly inhibited the enzyme, indicating that the keratinase is a serine protease depending on metal ions for optimal activity and/or stability. Accordingly, analysis of tryptic peptides revealed sequence homologies which characterize the keratinase as a subtilisin-like serine protease. The purified enzyme was able to hydrolyze azokeratin and keratin azure. Casein was hydrolyzed at higher rates than keratinous substrates, and 2-mercaptoethanol tended to enhance keratin hydrolysis. With synthetic substrates, the keratinase showed a preference for aromatic and hydrophobic residues at the P1 position of tetrapeptides; the enzyme was not active, or the activity was drastically diminished, towards shorter peptides. Keratinase from Bacillus sp. P45 might potentially be employed in the production of protein hydrolysates at moderate temperatures, being suitable for the bioconversion of protein-rich wastes through an environmentally friendly process requiring low energy inputs.     

Isolation and characterization of a feather-degrading enzyme from Bacillus pseudofirmus FA30-01

We isolated the feather-degrading Bacillus pseudofirmus FA30-01 from the soil sample of poultry farm. The isolate completely degraded feather pieces after liquid culture at 30°C (pH 10.5) for 3 days. Strain FA30-01 is a Gram-positive, spore-forming, rod-shaped bacterium and was identified with B. pseudofirmus based on 16S rDNA analysis. The keratinase enzyme produced by strain FA30-01 was refined using ammonium sulfate precipitation, negative-ion DEAE Toyopearl exchange chromatography, and hydroxyapatite chromatography. The refinement level was 14.5-fold. The molecular weight of this enzyme was 27.5 kDa and it had an isoelectric point of 5.9. The enzyme exhibited activity at pH 5.1–11.5 and 30–80°C with azokeratin as a substrate, although the optimum pH and temperature for keratinase activity were pH 8.8–10.3 and 60°C, respectively. This enzyme is one of the serine-type proteases. Subtilisin ALP I and this enzyme had 90% homology in the N-terminal amino acid sequence. Since this enzyme differed from ALP I in molecular weight, heat resistance and isoelectric point, they are suggested to be different enzymes.     

Fermentation production of keratinase from Bacillus licheniformisPWD-1 and a recombinant B. subtilis FDB-29

Bacillus licheniformis PWD-1, the parent strain, and B. subtilis FDB-29, a recombinant strain. In both strains, keratinase was induced by proteinaceous media, and repressed by carbohydrates. A seed culture of B. licheniformis PWD-1 at early age, 6–10 h, is crucial to keratinase production during fermentation, but B. subtilis FDB-29 is insensitive to the seed culture age. During the batch fermentation by both strains, the pH changed from 7.0 to 8.5 while the keratinase activity and productivity stayed at high levels. Control of pH, therefore, is not necessary. The temperature for maximum keratinase production is 37°C for both strains, though B. licheniformis is thermophilic and grows best at 50°C. Optimal levels of dissolved oxygen are 10% and 20% for B. licheniformis and B. subtilis respectively. A scale-up procedure using constant temperature at 37°C was adopted for B. subtilis. On the other hand, a temperature-shift procedure by which an 8-h fermentation at 50°C for growth followed by a shift to 37°C for enzyme production was used for B. licheniformis to shorten the fermentation time and increase enzyme productivity. Production of keratinase by B. licheniformis increased by ten-fold following this new procedure. After respective optimization of fermentation conditions, keratinase production by B. licheniformis PWD-1 is approximately 40% higher than that by B. subtilis FDB-29. Received 16 July 1998/ Accepted in revised form 07 March 1999