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.     

Protein quality of mixtures of animal protein meals

The protein quality of two-component mixtures of meat-and-bone meal, bone meal, blood meal and feather meal was characterized by rat experiments (net protein utilisation (NPU)) and by chemical indices calculated from amino acid composition (limiting essential amino acid, sum of essential amino acids, chemical score (CS), essential amino acid index (EAA-index) and predicted value (PV)).Substitution of meat-and-bone meal for bone meal on a nitrogen basis reduced the NPU value. This was also indicated by amino acid indices.Substitution of meat-and-bone meal for blood meal on a nitrogen basis also reduced the NPU value; however, this was not indicated by the chemical indices.In the mixtures examined, feather meal complemented meat-and-bone meal as indicated by both the NPU value and chemical indices. A complementary effect was also found with the mixtures of feather meal and blood meal.A significant correlation was found between NPU value and either predicted value (r=0.89), chemical score (0.83) or EAA-index (0.62), as calculated on cumulated data (n=20).     

Keratinolytic Activity of Aspergillus fumigatus Fresenius

Aspergillus fumigatus can utilize chicken feather keratin as its sole carbon and nitrogen source. Because enzymatic conversion of native keratin into readily usable products is of economic interest, this fungus was studied for its capacity to produce and secrete keratin-hydrolyzing proteinases. Substantial keratin-azure hydrolyzing activity was present in the culture fluid of keratin-containing media. Considerably lower activity was present in cultures containing glucose and nitrate as the carbon and nitrogen sources, or keratin plus glucose and nitrate. Secretion of keratin-hydrolyzing activity in A. fumigatus was induced by keratin but repressed by low-molecular-weight carbon and nitrogen sources. The amount of keratinolytic enzyme present in the culture fluid was dependent on the initial pH of the culture medium. The crude enzyme also hydrolyzed native keratin and casein in vitro. Hydrolysis was optimal at pH 9 and 45°C. The crude enzyme was remarkably thermostable. At 70°C, it retained about 90% of its original activity for 1.5 h. The obtained results indicated that the A. fumigatus keratinolytic enzyme may be suitable for enzymatic improvement of feather meal. Received: 25 April 1996 / Accepted: 18 June 1996     

一株高效羽毛降解菌株的分离与鉴定

采用以羽毛粉为唯一碳源和氮源的培养基,从自然界中分离到一株能够高效降解羽毛角蛋白的细菌,经形态学观察,生理生化实验和16SrRNA基因鉴定,初步确定该菌株为短小芽孢杆菌(Bacillus pumilus),且命名为短小芽孢杆菌WHK4。发酵48 h时,羽毛粉降解率达到85.76%。本研究为微生物降解羽毛角蛋白提供了优良的菌株,在蛋白饲料生产中具有潜在的广泛的应用前景。     

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     

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.     

Two-dimensional Fourier analysis of the spongy medullary keratin of structurally coloured feather barbs

We conducted two-dimensional (2D) discrete Fourier analyses of the spatial variation in refractive index of the spongy medullary keratin from four different colours of structurally coloured feather barbs from three species of bird: the rose-faced lovebird, Agapornis roseicollis (Psittacidae), the budgerigar, Melopsittacus undulatus (Psittacidae), and the Gouldian finch, Poephila guttata (Estrildidae). These results indicate that the spongy medullary keratin is a nanostructured tissue that functions as an array of coherent scatterers. The nanostructure of the medullary keratin is nearly uniform in all directions. The largest Fourier components of spatial variation in refractive index in the tissue are of the appropriate size to produce the observed colours by constructive interference alone. The peaks of the predicted reflectance spectra calculated from the 2D Fourier power spectra are congruent with the reflectance spectra measured by using microspectrophotometry. The alternative physical models for the production of these colours, the Rayleigh and Mie theories, hypothesize that medullary keratin is an incoherent array and that scattered waves are independent in phase. This assumption is falsified by the ring-like Fourier power spectra of these feathers, and the spacing of the scattering air vacuoles in the medullary keratin. Structural colours of avian feather barbs are produced by constructive interference of coherently scattered light waves from the optically heterogeneous matrix of keratin and air in the spongy medullary layer.     

雪被斑块对川西亚高山两个森林群落冬季土壤氮转化的影响

为了解气候变暖情景下雪况变化对高寒森林冬季土壤氮转化的影响,测定了川西亚高山冷杉(Abies faxoniana)+红桦(Betula albo-sinensis)混交林(MF)和冷杉次生林(SF)三类雪被斑块(浅雪被、中厚度雪被和厚雪被)内冬季土壤氮矿化特征。结果表明:经过一个冬季(2011-2012),两个森林群落土壤净氮氨化量都为负值,净氮硝化量都为正值,且净氮硝化量显著高于净氮氨化量;冬季土壤氮氨化、硝化、矿化和固持量都是中度雪被厚度最高,但各雪被斑块之间都未达到显著水平。各雪被斑块下,冷杉次生林土壤氮矿化参数都显著高于针阔混交林,但雪被斑块和林型交互作用对冬季土壤氮矿化无显著影响。这表明,该区冬季土壤氮矿化以硝化过程为主,硝化和氨化过程可能受不同微生物群落调控;短时期内,未来气候变化所导致的雪被减少对该区森林冬季土壤氮转化影响可能不明显。     

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.     

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     

尕海湿地草甸土退化过程土壤氮矿化演变特征

氮矿化是生态系统循环的重要环节之一,影响着生态系统功能和氮素生物地球化学循环,因此研究高寒湿地退化过程中土壤氮矿化演变特征,对揭示气候变化和人为活动干扰背景下的湿地土壤氮素循环过程具有重要意义。以尕海湿地4种不同退化梯度(未退化、轻度退化、中度退化、重度退化)土壤为研究对象,采用野外树脂芯原位培养方法,通过对植物生长季不同生长阶段(生长初期、生长盛期、枯萎期)土壤氮素矿化作用研究,分析湿地退化演替过程中土壤氮矿化时空变化特征及其与土壤环境因子和酶活性之间的关系。结果表明：尕海湿地退化对土壤氮矿化过程有显著抑制作用,与未退化(0.143 mg kg^-1 d^-1)相比,轻度退化、中度退化、重度退化的土壤净氮矿化速率分别减小了0.018 mg kg^-1 d^-1、0.025 mg kg^-1 d^-1、0.020 mg kg^-1 d^-1;随着退化程度加剧,土壤净氨化速率逐渐减小或者不变,而净硝化速率却增大。随时间推移,各退化...     

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.     

A keratin-decomposing streptomycete

A proteolytic actinomycete was isolated from an Indian soil sample. It degraded hair, silk, wool and feather. Protease activity was reported for growth of the organism on these keratin substrates. The organism was taxonomically studied and designated as Streptomyces sp. S₇.     

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.     

蚂蚁筑巢对西双版纳热带森林土壤有机氮矿化的影响

蚂蚁作为生态系统工程师能够调节土壤微生物及理化环境,进而对热带森林土壤有机氮矿化速率及其时间动态产生显著影响。以西双版纳白背桐热带森林群落为研究对象,采用室内需氧培养法测定土壤有机氮矿化速率,比较蚁巢和非蚁巢土壤有机氮矿化速率的时间动态,揭示蚂蚁筑巢活动引起土壤无机氮库、微生物生物量碳及化学性质改变对有机氮矿化速率时间动态的影响。结果表明：（1）蚂蚁筑巢显著影响土壤有机氮矿化速率（P<0.01）,相较于非蚁巢,蚁巢土壤有机氮矿化速率提高了261%;（2）土壤有机氮矿化速率随月份推移呈明显的单峰型变化趋势,即6月最大（蚁巢1.22 mg kg^-1 d^-1、非蚁巢0.41 mg kg^-1 d^-1）,12月最小（蚁巢0.82 mg kg^-1 d^-1、非蚁巢0.18 mg kg^-1 d^-1）;（3）两因素方差分析表明,不同月份及不同处理对土壤有机氮矿化速率、NH₄-N及NO₃-N产生显著影响（P<0.05）,但对NO₃-N的交互作用不显著;（4）蚂蚁筑巢显著提高了无机氮库（NH₄-N与NO₃-N）、微生物生物量碳、有机质、水解氮、全氮及易氧化有机碳等土壤养分含量,而降低了土壤pH值;（5）回归分析表明,铵态氮和硝态氮对土壤有机氮矿化速率产生显著影响,分别解释87.89%、61.84%的有机氮矿化速率变化;（6）主成份分析表明NH₄-N、微生物生物量碳及有机质是影响有机氮矿化速率时间动态的主要因素,而全氮、NO₃-N、易氧化有机碳、水解氮及pH对土壤有机氮矿化速率的影响次之,且pH与土壤有机氮矿化速率呈显著负相关。总之,蚂蚁筑巢活动主要通过影响土壤NH₄-N、微生物生物量碳及有机质的状况,进而调控西双版纳热带森林土壤有机氮矿化速率的时间动态。研究结果将有助于进一步提高对土壤氮矿化生物调控机制的认识。     

Purification and characterization of an alkaline serine endopeptidase from a feather-degrading Xanthomonas maltophilia strain

A keratinolytic Xanthomonas maltophilia strain (POA-1), cultured on feather meal broth, using keratin as its sole source of carbon and nitrogen, secretes several extracellular peptidases. The major serine peptidase was purified to homogeneity by a five-step procedure. Its purity was evaluated by capillary zone electrophoresis. This enzyme has a molecular mass of 36 kDa, an optimum pH of 9.0, and an optimum temperature of 60 degrees C. The inhibitory profile using protease inhibitors shows that this enzyme is a serine endopeptidase. Besides keratin, the enzyme is active upon the substrates azokeratin, azocasein, and the following fluorogenic peptide substrates: Abz-Leu-Gly-Met-Ile-Ser-Leu-Met-Lys-Arg-Pro-Gln-EDDnp, Abz-Lys-Leu-Cys(SBzl)-Gly-Pro-Lys-Gln-EDDnp, and Abz-Lys-Pro-Cys(SBzl)-Phe-Ser-Lys-Gln-EDDnp.     

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.     

Identification and characterization of a novel antioxidant peptide from feather keratin hydrolysate

Objectives

To improve the potential value of feather, which is a valuable protein resource, we have separated and identified antioxidant peptide(s) from feather hydrolysate.

Results

Feather hydrolysate was prepared by fermentation with Bacillus subtilis S1–4. Antioxidative peptides were separated by sequential acid precipitation, cation exchange, and reversed-phase fast performance liquid chromatography. Finally, a peptide with antioxidative activity was identified as Ser-Asn-Leu-Cys-Arg-Pro-Cys-Gly by MALDI time-of-flight (TOF)/TOF analysis, and determined to represent a portion of feather keratin near its N-terminal. A synthesized peptide with the same sequence was used to characterize its antioxidative properties, including scavenging free radicals, reducing power, and Fe²⁺ chelation. In terms of the peptide’s amino acid composition, the antioxidative activity might be mainly attributed to Cys and other amino acid residues.

Conclusion

Feather keratin is a good source for the quantitative preparation of antioxidative peptides.

     

How are feathers digested by raptors?

In order to determine the cause of the evident degradation of feathers from ingested prey in pellets regurgitated by raptors, in vitro digestions of whole feather barbs by pellet extracts, pepsin or trypsin were carried out. The material was analysed by using biochemical and electron microscopic methods. The results show that the changes in the feathers which occur in the stomach of the Falconidae do not arise from digestion of keratin but from hydrolysis of protein acting as a cement matter in the feather.