Secretion of keratinolytic enzymes and keratinolysis by Scopulariopsis brevicaulis and Trichophyton mentagrophytes: regression analysis

A survey on keratinophilic fungi from poultry-farm soils at Namakkal and from feather dumping soils at Chennai, India, revealed the existence of 34 species of fungi. Most of the fungi exhibited variable efficiency in producing extracellular keratinase when grown in plates with chicken feathers as the sole carbon and nitrogen source. The fungi Aspergillus flavus, Aspergillus niger, Aspergillus versicolor, Chrysosporium state of Arthroderma tuberculatum, Paecilomyces carneus, Scopulariopsis brevicaulis, Trichoderma viride, and Trichophyton mentagrophytes were efficient candidates to degrade the feathers. However, when cultivating the strains in submerged conditions in a medium containing chicken feathers as the sole nutrients source, Aspergillus glaucus, Chrysosporium keratinophilum, Curvularia lunata, Fusarium solani, and Penicillium citrinum also proved to be potent. Among all species, S. brevicaulis and Trichophyton mentagrophytes produced higher amounts of keratinase in both methods. Conditions for keratinase production were optimized by statistical design and surface plots. The highest keratinase activity was estimated by S. brevicaulis (3.2 KU/mL) and Trichophyton mentagrophytes (2.7 KU/mL) in the culture medium with chicken feathers and shows (79% and 72.2% of degrading ability, respectively).     

Influence of the carbon and nitrogen sources on keratinase production by <Emphasis Type="Italic">Myrothecium verrucaria</Emphasis> in submerged and solid state cultures

Myrothecium verrucaria is a nondermatophytic filamentous fungus able to grow and to produce keratinase in submerged (93.0 ± 19 U/ml) and solid state (98.8 ± 7.9 U/ml) cultures in which poultry feather powder (PFP) is the only substrate. The purpose of the present work was to verify how different carbon and nitrogen sources can influence the production of keratinase by this fungus. Addition of carbohydrates, such as glucose and sucrose, caused only slight improvements in keratinase production, but the addition of starch caused a significant improvement (135.0 ± 25 U/ml). The highest levels of keratinase activity, however, were obtained by supplementing the PFP cultures with cassava bagasse, 168.0 ± 28 U/ml and 189.0 ± 26 U/ml in submerged and solid state cultures, respectively. Contrarily, the supplementation of PFP medium with organic or inorganic nitrogen sources, such as casein, soy bean protein, gelatin, ammonium nitrate and alanine, decreased the production of keratinase in both types of cultures (around 20 U/ml), showing that the production of keratinase by M. verrucaria is repressed by nitrogen sources. The results obtained in this work suggest that the association of the two residues PFP plus cassava bagasse could be an excellent option as a cheap culture medium for the production of keratinase in submerged and solid state cultures.     

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     

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+).     

Keratinolytic potential of Bacillus licheniformis RG1: structural and biochemical mechanism of feather degradation

Keratinolytic Bacillus licheniformis RG1 was used to study the mechanism of keratinolysis. Scanning electron microscopy studies revealed that bacterial cells grew closely adhered to the barbules of feathers, completely degrading them within 24 h. Biochemical studies indicated that the Bacillus strain produced an extracellular protease, which had keratinolytic potential. The extracellular keratinolytic activity (425 U) was synergistically enhanced by the addition of intracellular disulfide reductases (1712 U). However, these enzymes alone (keratinase and disulfide reductase), without live bacterial cells, failed to degrade the feather. Complete feather degradation was obtained only when living bacterial cells were present, emphasizing that bacterial adhesion plays a key role during the degradation process. The bacterial cells probably provide a continuous supply of reductant to break disulfide bridges. In addition, sulfite detected in the extracellular broth during feather degradation indicated that sulfitolysis may also play a role in feather degradation by the bacterium.     

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.

     

产角蛋白酶耐热金孢菌的初筛研究

用鸡羽毛粉或人头发粉作为唯一碳、氮源,对9个耐热的金孢属(Chrysosporium spp.)真菌菌株进行了产角蛋白酶筛选。研究结果表明:菌株H-49-2对鸡羽毛的利用能力最强,而菌株H-143-1对人头发的利用能力最强。对H-49-2菌株进行液体发酵产酶试验的结果表明,培养6d时酶活性最大,为9.6U/mL。     

Optimizing the fermentation conditions and enhanced production of keratinase from Bacillus cereus isolated from halophilic environment

Keratinase degrading Bacillus cereus was isolated from the halophilic environment in Tamilnadu, India and keratinase production was optimized using wheat bran substrate. Of the screened bacterial isolates, four were found to have the ability to produce keratinolytic enzyme. The process parameters were optimized using one-variable-at-a-time approach and response surface methodology. Supplementation of 1% lactose supported more keratinase production (120?U/g). Among the selected nitrogen sources, addition of casein significantly enhanced maximum keratinase production (132.5?U/g). Among the ions, manganese chloride significantly enhanced keratinsase production (102.6?U/g), however addition of zinc sulphate and copper sulphate decreased keratinase production. The maximum keratinase production was obtained in the wheat bran medium containing 1% lactose, 0.5% manganese with 80% moisture (292?U/g). Statistics based contour plots were generated to explore the variations in the response surface and to find the relationship between the keratinase yield and the bioprocess conditions.     

Degradation of keratinous materials by the plant pathogenic fungus <Emphasis Type="Italic">Myrothecium verrucaria</Emphasis>

In this paper it is described for the first time the capability of Myrothecium verrucaria to grow in submerged and solid state cultures using poultry feathers as the only substrate. The fungus produced a protease with an unusual keratinolytic activity among plant pathogenic fungi. Its crude protease hydrolyzed keratinous substrates at pH 9.0 and 40 °C in the following order: poultry feather keratin > sheep wool keratin > human nail keratin > human hair keratin. Protease activity was highly sensitive to phenylmethyl sulphonyl fluoride (PMSF) indicating that the enzyme belonged to the serine protease family.     

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.     

Production, purification and characterization of an extracellular keratinase from Lysobacter NCIMB 9497

AIMS: The aim of this study was to determine the keratinolytic ability of a range of bacteria and subsequently, to characterize the keratinase showing the greatest biotechnological potential. METHODS AND RESULTS: Nine bacteria, reported to produce extracellular proteases, were screened for production of keratinases. Of these, Lysobacter NCIMB 9497 exhibited the highest keratinolytic activity. The keratinase from this strain (Mr 148 kDa) was purified and characterized. Optimum activity occurred at 50 degrees C; no inhibition was demonstrated by phenylmethylsulphonyl fluoride (PMSF), but inhibition by EDTA was demonstrated. CONCLUSIONS: This study indicates that keratinase is a metalloprotease with a high degree of keratinolytic activity and stability. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first detailed report of a metalloprotease with keratinolytic activity. The novel reaction mechanism, degree of keratinolytic activity and stability indicate considerable biotechnological potential in the leather industry, and in the processing of poultry waste.     

Thermophilic protease production by Streptomyces sp. 594 in submerged and solid-state fermentations using feather meal

AIMS: Protease production by Streptomyces sp. 594 in submerged (SF) and solid-state fermentation (SSF) using feather meal, an industrial poultry residue, and partial characterization of the crude enzyme. METHODS AND RESULTS: Streptomyces sp. 594 produced proteases in SF (7.2 +/- 0.2 U ml(-1)) and SSF (15.5 +/- 0.41 U g(-1)), with pH increase in both media. Considering protease activity, values obtained in the liquid extract after SSF (6.3 +/- 0.17 U ml(-1)) were lower than those from SF. The proteases, which belong to serine and metalloproteinase classes, were active over a wide range of pH (5.0-10.0) and high temperatures (55-80 degrees C). Strain 594 was also able to degrade feather in agar and liquid media. Keratinase activity (80 U l(-1)) also confirmed the keratin degrading capacity of this streptomycete. CONCLUSIONS: Proteases produced using residues from poultry industry have shown interesting properties for industrial purposes. SIGNIFICANCE AND IMPACT OF THE STUDY: As far as we are concerned, this is the first contribution towards the production of thermophilic protease by a streptomycete in SSF using a keratinous waste.     

Similarities and specificities of fungal keratinolytic proteases: comparison of keratinases of Paecilomyces marquandii and Doratomyces microsporus to some known proteases

Based on previous screening for keratinolytic nonpathogenic fungi, Paecilomyces marquandii and Doratomyces microsporus were selected for production of potent keratinases. The enzymes were purified and their main biochemical characteristics were determined (molecular masses, optimal temperature and pH for keratinolytic activity, N-terminal amino acid sequences). Studies of substrate specificity revealed that skin constituents, such as the stratum corneum, and appendages such as nail but not hair, feather, and wool were efficiently hydrolyzed by the P. marquandii keratinase and about 40% less by the D. microsporus keratinase. Hydrolysis of keratin could be increased by the presence of reducing agents. The catalytic properties of the keratinases were studied and compared to those of some known commercial proteases. The profile of the oxidized insulin B-chain digestion revealed that both keratinases, like proteinase K but not subtilisin, trypsin, or elastase, possess broad cleavage specificity with a preference for aromatic and nonpolar amino acid residues at the P-1 position. Kinetic studies were performed on a synthetic substrate, succinyl-Ala-Ala-Pro-Phe-p-nitroanilide. The keratinase of P. marquandii exhibited the lowest Km among microbial keratinases reported in the literature, and its catalytic efficiency was high in comparison to that of D. microsporus keratinase and proteinase K. All three keratinolytic enzymes, the keratinases of P. marquandii and D. microsporus as well as proteinase K, were significantly more active on keratin than subtilisin, trypsin, elastase, chymotrypsin, or collagenase.     

降解鸡毛真菌板蜡蚧的鉴定及产酶培养优化

鉴定降解鸡毛真菌并通过单因素和正交实验优化其产角蛋白酶发酵培养条件.从加入鸡毛粉钓饵的医院花坛土中分离筛选获得3株角蛋白高效降解真菌,利用形态学和分子系统学鉴定均为板蜡蚧(Lecani-cillium testudineum).单因素实验表明,对优选菌株1Y2-12产酶能力具促进作用的碳源为乳糖,氮源为酵母膏,无机离子...     

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.     

Isolation of keratinase from bacterial isolates of poultry soil for waste degradation

Isolation of two keratinolytic bacterial strains from poultry soil as well as purification and properties of keratinase were investigated. Isolates were designated as KI8101 and KI8102 (KI, keratin isolates) and were identified as Bacillus subtilis and B. licheniformis respectively. The purified enzyme from KI8102 exhibited a high specific activity of 500 U/mg with 71‐fold purification and 41% yield. SDS‐PAGE analysis indicated that the purified keratinase had a molecular mass of 32 kDa. The optimum temperature and pH were 50°C and 7.5, respectively. Its K_m was 83.3 μM and V_max was 71.4 μmol/mL min. The bacterium could potentially degrade keratin waste such as human hair, nails, bovine hair and wool. Therefore, the enzyme could improve the nutritional value of meat and poultry‐processing waste containing keratin and could be a potential candidate for biotechnological processing involving keratin hydrolysis.     

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.     

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.     

Screening fungi for synthesis of keratinolytic enzymes

Almost 300 common fungi were screened for synthesis of extracellular keratinases. About 54% of the fungi grew on agar plates with soluble keratin and excreted the enzymes. Some representatives of Fusarium , Acremonium and Geotrichum were the most active. However, when cultivating the promising strains in submerged conditions in a medium with porcine nail as the sole source of carbon and nitrogen, other fungi proved to be potent. Aspergillus flavus was the most powerful producer of extracellular keratinases, followed by Alternaria radicina , Trichurus spiralis and Stachybotrys atra.