Nutritional regulation of keratinolytic activity in Bacillus pumilis

Bacillus pumilis F3-4 utilized feather as a sole source of carbon, nitrogen and sulfur. Supplementation of the feather medium with glucose or MgSO₄ · 7H₂O increased keratinolytic protease production (14.6–16.7 U/mg). The synthesis of keratinolytic protease was repressed by an exogenous nitrogen source. Keratinolytic protease was produced in the absence of feather (9.4 U/mg). Feather degradation resulted in sulfhydryl group formation (0.8–2.6 μM). B. pumilis F3-4 effectively degraded chicken feather (75%), duck feather (81%) and feather meal (97%), whereas human nails, human hair and sheep wool under went less degradation (9–15%). An erratum to this article can be found at     

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.     

Keratinolytic potential of a novel Bacillus sp. P45 isolated from the Amazon basin fish Piaractus mesopotamicus

Bacillus sp. P45, isolated from the intestine of the Amazon basin fish Piaractus mesopotamicus, showed proteolytic activity when grown on skimmed milk and feather meal agar plates. The keratinolytic potential of this strain was evaluated on whole feather broth and human hair broth. Bacillus sp. P45 degraded almost 90% of chicken feathers after 72 h of submerged cultivation on whole feather broth, and the production of extracellular proteases was observed. The formation of thiol groups was also detected during growth, indicating the contribution of sulphitolysis to the efficient hydrolysis of feather keratin. Nevertheless, Bacillus sp. P45 was unable to degrade hair keratin, possibly due to the conformational diversity of this substrate in comparison to feather keratin. Additionally, preliminary results demonstrated that this strain might be utilized in the degradation of recalcitrant collagen-containing wastes. The keratinolytic character of Bacillus sp. P45 might be utilized in environmental-friendly processes such as bioconversion of waste feathers, representing an alternative way of waste management that could lead to the production of value-added products such as microbial biomass, protein hydrolysates and proteolytic enzymes.     

Hide depilation and feather disintegration studies with keratinolytic serine protease from a novel Bacillus subtilis isolate

Keratinases play an important role in biotechnological applications such as improvement of feather meal, enzymatic dehairing and production of amino acids or peptides from high molecular weight substrates. Bacillus subtilis P13, isolated from Vajreshwari hot spring (45–50°C) near Mumbai, India, produces a neutral serine protease and has an optimum temperature of 65°C. This enzyme preparation was keratinolytic in nature and could disintegrate whole chicken feathers, except for the remnants of shafts. The enzyme preparation also exhibited depilation of goat hides with the recovery of intact animal hair. The enzyme preparation could release peptides from ground feathers and bring about their weight reduction; however, similar action on hair was relatively weak. A single major PMSF-sensitive protease band could be detected upon zymogram analysis, indicating that a single enzyme may be responsible for feather degradation and hide depilation. The importance of these findings in the biotechnological application for feather and leather industries is discussed.     

Chicken feathers: a complex substrate for the co-production of α-amylase and proteases by <Emphasis Type="Italic">B. licheniformis</Emphasis> NH1

This study is concerned with the co-production of alkaline proteases and thermostable α-amylase by some feather-degrading Bacillus strains: B. mojavensis A21, B. licheniformis NH1, B. subtilis A26, B. amyloliquefaciens An6 and B. pumilus A1. All strains produced both enzymes, except B. pumilus A1, which did not exhibit amylolytic activity. The best enzyme co-production was obtained by the NH1 strain when chicken feathers were used as nitrogen and carbon sources in the fermentation medium. The higher co-production of both enzymes by B. licheniformis NH1 strain was achieved in the presence of 7.5 g/l chicken feathers and 1 g/l yeast extract. Strong catabolic repression on protease and α-amylase production was observed with glucose. Addition of 0.5% glucose to the feather medium suppressed enzyme production by B. licheniformis NH1. The growth of B. licheniformis NH1 using chicken feathers as nitrogen and carbon sources resulted in its complete degradation after 24 h of incubation at 37°C. However, maximum protease and amylase activities were attained after 30 h and 48 h, respectively. Proteolytic activity profiles of NH1 enzymatic preparation grown on chicken feather or casein-based medium are different. As far as we know, this is the first contribution towards the co-production of α-amylase and proteases using keratinous waste. Strain NH1 shows potential use for biotechnological processes involving keratin hydrolysis and industrial α-amylase and proteases co-production. Thus, the utilization of chicken feathers may result in a cost-effective process suitable for large-scale production.     

Enhanced stability of immobilized keratinolytic protease on electrospun nanofibers

Abstract

Feathers from poultry industries are considered a major pollutant and its degradation is a challenging problem due to its recalcitrant nature. The high cost of energy and loss of essential amino acids by conventional methods have paved a way for an environmentally benign approach using microbial keratinolytic proteases. The widespread application of keratinolytic proteases is limited due to autolysis and denaturation of the enzyme upon storage. Immobilization overcomes these disadvantages by adsorbing the enzyme onto a solid support. Recently, electrospun nanofibers have been used due to their increased surface area and porous structure. The biocompatible and hydrophilic polyvinyl alcohol (PVA) has been blended with biodegradable chitosan for immobilization in electrospinning. The present study focuses on feather degradation by immobilized keratinolytic proteases on electrospun nanofibers. The keratinolytic protease production was enhanced by using a media containing hydrolyzed feather under optimized conditions. The immobilized keratinolytic protease on electrospun PVA chitosan (PVA-Ch) nanofibers (100–150?nm diameter) degraded the chicken feathers with 88% efficiency at the end of 72?hr.     

Identification of a <Emphasis Type="Italic">Candida parapsilosis</Emphasis> Strain Producing Extracellular Serine Peptidase with Keratinolytic Activity

A yeast strain isolated from feather waste from a chicken processing plant was identified as Candida parapsilosis by biochemical tests and morphological studies. The yeast was able to grow in phosphate-buffered saline supplemented with 1% native feather as the sole carbon and nitrogen source. A keratin substrate was obtained from the feathers by dimethylsulphoxide extraction. A 20-fold concentrated culture supernatant from Candida parapsilosis grown on feathers was analysed by SDS–PAGE electrophoresis containing either 1% gelatin or 1% keratin as copolymerised substrates. The presence of a single band with an approximate molecular mass of 60 kDa with gelatinolytic and keratinolytic activities was observed. This proteolytic activity was fully inhibited by phenylmethylsulphonyl fluoride. These results suggest that the extracellular enzyme belongs to the serine peptidase class. This is the first report of an extracellular serine peptidase produced by C. parapsilosis with keratinolytic activity. The role of this enzyme in yeast–host interactions is discussed.     

Characterization of a multifunctional feather-degrading <Emphasis Type="Italic">Bacillus subtilis</Emphasis> isolated from forest soil

In this study, we isolated and characterized a novel feather-degrading bacterium that shows keratinolytic, antifungal and plant growth-promoting activities. A bacterium S8 was isolated from forest soil and confirmed to belong to Bacillus subtilis by BIOLOG system and 16S rRNA gene analysis. The improved culture conditions for the production of keratinolytic protease were 0.1% (w/v) sorbitol, 0.3% (w/v) KNO₃, 0.1% (w/v) K₂HPO₄, 0.06% (w/v) KH₂PO₄ and 0.04% (w/v) MgCl₂·6H₂O (pH 8.0 and 30°C), respectively. In the improved medium containing 0.1% (w/v) feather, keratinolytic protease production was around 53.3 ± 0.3 U/ml at 4 day; this value was 10-fold higher than the yield in the basal feather medium (5.3 ± 0.1 U/ml). After cultivation for 5 days in the improved medium, intact feather was completely degraded. Feather degradation resulted in free –SH group, soluble protein and amino acids production. The concentration of free –SH group in the culture medium was 15.5 ± 0.2 μM at 4 days. Nineteen amino acids including all essential amino acids were produced in the culture medium; the concentration of total amino acid produced was 3360.4 μM. Proline (2809.9 μM), histidine (371.3 μM) and phenylalanine (172.0 μM) were the major amino acids released in the culture medium. B. subtilis S8 showed the properties related to plant growth promotion: hydrolytic enzymes, ammonification, indoleacetic acid (IAA), phosphate solubilization, and broad-spectrum antimicrobial activity. Interestingly, the strain S8 grown in the improved medium produced IAA and antifungal activity, indicating simultaneous production of keratinolytic and antifungal activities and IAA by B. subtilis S8. These results suggest that B. subtilis S8 could be not only used to improve the nutritional value of feather wastes but also is useful in situ biodegradation of feather wastes. Furthermore, it could also be a potential biofertilizer or biocontrol agent applicable to crop plant soil.     

Production of keratinolytic enzyme by a newly isolated feather-degrading Stenotrophomonas maltophilia that produces plant growth-promoting activity

A novel feather-degrading Stenotrophomonas maltophilia R13 was isolated from rhizospheric soil of reed. The strain R13 produces keratinolytic enzyme using chicken feather as the sole carbon and nitrogen source. Addition of 0.1% glucose and 0.12% polypeptone to the feather medium increased the enzyme production. The optimum temperature and initial pH for the enzyme production were 30 °C and 7.0. The maximum yield of the enzyme was 82.3 ± 1.0 U/ml in the optimal feather medium; this value was about 5.5-fold higher than the yield in the basal feather medium. S. maltophilia R13 possessed disulfide reductase activity along with keratinolytic activity. As a result of feather degradation, 18 free amino acids were produced in the culture; the concentration of total amino acid was 2298.8 μM. The strain R13 produced IAA in the optimal feather medium without l-tryptophan supplementation, indicating simultaneous production of keratinolytic activity and IAA by S. maltophilia R13. The strain R13 grown in the optimal feather medium also inhibited mycelial growth of some phytopathogenic fungi. This result suggests that antifungal activity of the strain R13 could be produced in the same conditions observed for keratinolytic activity. Thus, S. maltophilia R13 could be not only used to enhance the nutritional value of feather meal but is also a potential bioinoculant in agricultural environments.     

Application of statistical experimental design for optimization of keratinases production by Bacillus pumilus A1 grown on chicken feather and some biochemical properties

A new keratinolytic enzyme-producing bacterium was isolated from slaughter house polluted water and identified as Bacillus pumilus A1. Medium composition and culture conditions for the keratinases production by B. pumilus A1 were optimized using two statistical methods: Plackett–Burman design applied to find the key ingredients and conditions for the best yield of enzyme production and central composite design used to optimize the concentration of the five significant variables: feathers meal, soy peptone, NaCl, KCl, and KH₂PO₄. The medium optimization resulted in a 3.4-fold increase in keratinase production (87.73 U/ml) compared to that of the initial medium (25.9 U/ml). The zymography analysis shows the presence of at least five keratinolytic enzymes. The keratinolytic activity of the extracellular proteinases was examined by incubation with non-autoclaved chicken feathers. Complete solubilisation of whole feathers was observed after a 6-h incubation at temperatures ranging from 45 °C to 60 °C. The crude enzyme exhibited maximal activity at 60 °C and pH 8.5 or 55 °C and pH 9.0 using casein or keratin as substrates, respectively.     

Production of feather protein hydrolysate by keratinolytic bacterium Vibrio sp. kr2

A feather protein hydrolysate was produced using the keratinolytic bacterium Vibrio sp. strain kr2. Complete feather degradation was observed in medium containing up to 60 g L(-1) raw feathers. Cultivation on 40, 60 or 80 g L(-1) feathers for five days resulted in similar amounts of soluble protein, reaching maximum values around 2.5 g L(-1). Maximum yields of soluble protein were achieved at 30 degrees C and initial pH ranging from 6.0 to 8.0. Strain kr2 was effective in producing keratin hydrolysate from chicken feathers. Bacterial feather hydrolysate has the potential for utilization as an ingredient in animal feed or as organic fertilizer, thereby reducing the environmental impact of feather waste from the poultry industry.     

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     

Isolation and characterization of feather degrading enzymes from Bacillus megaterium SN1 isolated from Ghazipur poultry waste site

The SN1 strain of Bacillus megaterium, isolated from soil of Ghazipur poultry waste site (India) produced extracellular caseinolytic and keratinolytic enzymes in basal media at 30°C, 160 rpm in the presence of 10% feather. Feathers were completely degraded after 72 h of incubation. The caesinolytic enzyme was separated from the basal media following ammonium sulphate precipitation and ion exchange chromatography. We report 29.3-fold purification of protease after Q Sepharose chromatography. The molecular weight of this enzyme was estimated to be 30 kDa as shown by SDS-PAGE and zymography studies. Protease activity increased by 2-fold in presence of 10 mM Mn²⁺ whereas Ba²⁺ and Hg²⁺ inhibited it. Ratio of milk clotting activity to caseinolytic activity was found to be 520.8 for the 30–60% ammonium sulphate fraction in presence of Mn²⁺ ion suggesting potential application in dairy industry. Keratinase was purified to 655.64 fold with specific activity of 544.7 U/mg protein and 12.4% recovery. We adopted the strategy of isolating the keratinolytic and caesinolytic producing microorganism by its selective growing in enriched media and found that feather protein can be metabolized for production of animal feed protein concentrates.     

Fermentation characteristics and secretion of proteases of a new keratinolytic strain of Bacillus licheniformis

A keratin-degrading strain of Bacillus licheniformis (K-508) was isolated from partially-degraded feathers and characterised. It had high chicken feather-degrading activity when cultured in feather-containing broth, with a growth optimum at pH 7 and 47 °C. Broth filtrates were active towards N-Bz-Phe-Val-Arg-p-nitroanilide and N-Suc-Ala-Ala-Pro-Phe-p-nitroanilide, as chromogenic protease substrates at pH 8. Strain K-508 displays keratinolytic activity against native feather keratin (without any pretreatment) in the presence of SH-reducing compounds. It constitutively secreted both trypsin-like and chymotrypsin-like proteases.     

Response surface methodology based optimization of keratinase from Bacillus velezensis strain ZBE1 and nanoparticle synthesis,biological and molecular characterization

The increasing demands of keratinases for biodegradation of recalcitrant keratinaceous waste like chicken feathers has lead to research on newer potential bacterial keratinases to produce high-value products with biological activities. The present study reports a novel keratinolytic bacterium Bacillus velezensis strain ZBE1 isolated from deep forest soil of Western Ghats of Karnataka, which possessed efficient feather keratin degradation capability and induced keratinase production. Production kinetics depicts maximum keratinase production (11.65 U/mL) on 4th day with protein concentration of 0.61 mg/mL. Effect of various physico-chemical factors such as, inoculum size, metal ions, carbon and nitrogen sources, pH and temperature influencing keratinase production were optimized and 3.74 folds enhancement was evidenced through response surface methodology. Silver (AgNP) and zinc oxide (ZnONP) nanoparticles with keratin hydrolysate produced from chicken feathers by the action of keratinase were synthesized and verified with UV–Visible spectroscopy that revealed biological activities like, antibacterial action against Bacillus cereus and Escherichia coli. AgNP and ZnONP also showed potential antioxidant activities through radical scavenging activities by ABTS and DPPH. AgNP and ZnONP revealed cytotoxic effect against MCF-7 breast cancer cell lines with IC₅₀ of 5.47 µg/ml and 62.26 µg/ml respectively. Characterizations of nanoparticles were carried out by Fourier transform infrared spectroscopy, scanning electron microscopy with energy dispersive X-ray, X-ray diffraction, thermogravimetric analysis and atomic force microscopy analysis to elucidate the thermostability, structure and surface attributes. The study suggests the prospective applications of keratinase to trigger the production of bioactive value-added products and significant application in nanotechnology in biomedicine.     

Substrate specificity characterization of a thermostable keratinase from <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> KS-1

A feather-degrading strain of Pseudomonas aeruginosa KS-1 was used in the present study. Its crude cell-free fermentation broth completely degraded chicken feather within 12 h, in the absence of disulphide reductase activity. Keratinase from its extracellular broth was purified and characterized, assuming that it would be a potential β-keratin-degrading enzyme with prospective applications in degradation of β-plaques of prions. The keratinase was purified by using Q-Sepharose anion exchange chromatography and its molecular weight, as determined by SDS–PAGE analysis, was 45 kDa. It was an alkaline, serine protease with pH and temperature optima of 9 and 60°C, respectively. The enzyme was highly thermostable with a t _1/2 > 2 h at 80°C and had a very high K to C (keratinolytic to caseinolytic) ratio of 2.5. Besides feather keratin, it also hydrolyzed a variety of other complex substrates including fibrin, gelatin and meat protein. Its activity on synthetic substrates revealed that it efficiently cleaves them in the order phenylalanine > lysine > alanine > leucine p-nitroanilides. It also cleaved insulin B chain between Val¹²-Glu¹³, Ala¹⁴-Leu¹⁵, Gly²⁰-Glu²¹ and Arg²²-Gly²³ residues.     

Degradation of chicken feathers by Chrysosporium georgiae

Using a baiting technique, Chrysosporium georgiae was isolated from chicken feathers. Twenty-eight different fungal isolates were evaluated for their ability to produce keratinase enzymes using a keratin–salt agar medium containing either white chicken feathers or a prepared feather keratin suspension (KS). The Chrysosporium species were able to use keratin and grow at different rates. Chrysosporium georgiae completely degraded the added keratin after 9 days of incubation. Degradation of feathers by C. georgiae was affected by several cultural factors. Highest keratinolytic activity occurred after 3 weeks of incubation at 6 and 8~pH at 30 °C. Chrysosporium georgiae was able to degrade white chicken feathers, whereas bovine and human hair and sheep wool were not degraded and did not support fungal growth. Addition of 1% glucose to the medium containing keratin improved fungal growth and increased enzyme production. Higher keratin degradation resulted in high SH accumulation and the utilization of the carbohydrate carbon in the medium resulted in high keto-acid accumulation but decreased ammonia accumulation. Supplementation of the keratin–salt medium with minerals such as NH4Cl and MgSO4 slightly increased mycelial growth, but decreased production of extracelluar keratinase. Keratinase enzymes were very poorly produced in the absence of keratin, indicating its inducible nature. Analysis of endocellular keratinases in the mycelial homogenate indicated higher activity of intracellular keratinase as compared to the extracellular enzyme in culture filtrates. Chrysosporium georgiae was the most superior for keratinase production among the Chrysosporium species tested in the presence or absence of glucose. It produced more of the intracellular enzymes than the exocellular ones. This revised version was published online in June 2006 with corrections to the Cover Date.     

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.     

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.     

Feather degradation by Kocuria rosea in submerged culture

A strain of Kocuria rosea with keratinolytic activity was studied. In batch culture, the optimum temperature for feather degradation, bacterial growth and protease secretion was at 40 °C. A specific growth rate of 0.17 h⁻¹ was attained in basal medium with feathers as fermentation substrate. Under these conditions, after 36 h of incubation, biomass and caseinolytic activity reached 3.2 g/l and 0.15 U/ml, respectively. Extracellular protease secretion was associated with the exponential growth phase. In batch fermentation, feather degradation up to 51% in 72 h was obtained with a conversion yield in biomass of 0.32 g/g. No organic acids were detected in the fermentation broth in significant amount. This revised version was published online in July 2006 with corrections to the Cover Date.