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.     

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.     

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+).     

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.     

一株羽毛降解菌的分离与鉴定

【目的】从土壤中分离并鉴定羽毛降解菌,测定其生长最适温度及起始pH,并观察酶活动态。【方法】采用系列稀释法和选择培养基法筛选目的菌株,基于16S rRNA基因序列及Biolog方法鉴定其分类地位,利用全自动生长曲线分析仪监测菌株的最适生长条件,并通过测定蛋白水解活性观察其酶活动态。【结果】从混合羽毛的土壤样品中筛选到一株羽毛降解菌,命名为菌株GIMN1.015,初步判定该菌株属于芽孢八叠球菌属(Sporosarcina)。最适生长pH为9.0,温度为30°C。蛋白水解活性最高值出现在培养后96 h。【结论】菌株GIMN1.015在利用羽毛角蛋白资源中具有潜在的应用价值。这是芽孢八叠球菌在羽毛降解方面的首次报道。     

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.     

Chemical warfare? Effects of uropygial oil on feather‐degrading bacteria

Anti-microbial activity is a commonly suggested but rarely tested property of avian uropygial oil. Birds may defend themselves against feather-degrading and other potentially harmful bacteria using this oil. We preliminarily identified 13 bacterial isolates taken from the plumage of wild house finches Carpodacus mexicanus , measured bacterial production of the enzyme keratinase as an index of feather-degrading activity, and used the disc-diffusion method to test bacterial response to uropygial oil of house finches. For comparison, we performed the same tests on a type strain of the known feather-degrading bacterium Bacillus licheniformis . Uropygial oil inhibited the growth of three strongly feather-degrading isolates (including Bacillus licheniformis ), as well as one weakly feather-degrading isolate and one non-feather-degrading isolate. Uropygial oil appeared to enhance the growth of one weakly feather-degrading isolate. Growth of the remaining isolates was unaffected by uropygial oil. These results suggest that birds may defend themselves against some feather-degrading bacteria using uropygial oil.     

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.     

Purification and Characterisation of Keratinase from a Thermotolerant Feather-degrading Bacterium

Summary Isolation and identification of a thermotolerant feather-degrading bacterial strain from Thai soil as well as purification and properties of its keratinase were investigated. The thermotolerant bacterium was identified as Bacillus licheniformis. The keratinase was purified to homogeneity by three-step chromatography. The purified enzyme exhibited a high specific activity (218 U mg⁻¹) with 86-fold purification and 25% yield. The enzyme was monomeric and had a molecular mass of 35 kDa. The optimum pH and temperature for the enzyme were 8.5 and 60 °C, respectively. The enzyme activity was significantly inhibited by PMSF and partly inhibited by EDTA and iodoacetamide, but was stimulated by metal ions. It hydrolysed soluble proteins with a relative activity of 4–100% and insoluble proteins, including keratins, with a relative activity of 3–35%. Therefore, the enzyme could improve the nutritional value of meat- and poultry-processing wastes containing keratins, collagen and gelatin.     

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.     

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.     

Production, characterization and application of keratinase from Streptomyces gulbargensis

A Streptomyces gulbargensis newly isolated, thermotolerant feather-degrading bacterial strain was investigated for its ability to produce keratinase enzyme. Maximum keratinolytic activity was observed at 45 degrees C and pH 9.0 at 120 h of incubation. Activity was completely stable (100%) between 30 and 45 degrees C and pH 7.0-9.0, respectively. Addition of starch to the growth medium affects the activity by means of increase in keratinase secretion. After seven days of cultivation, 10-fold increase (14.3 U ml(-1)) in keratinase activity was observed in the presence of 3g starch (per liter) of the medium. The enzyme was monomeric and had a molecular mass of 46 kDa. The enzyme activity was significantly inhibited by CaCl(2) and partly inhibited by EDTA, whereas, Na(2)SO(3) enhance the enzyme activity by 2.9 times more. In addition, native chicken feather was completely degraded at 96 h of incubation. The results obtained showed that newly isolated strain S. gulbargensis could be a useful in biotechnology in terms of valorization of keratin-containing wastes or in the leather industry.     

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.     

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.     

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.     

Isolation,identification and some cultural conditionsof a protease-producing thermophilic Streptomyces straingrown on chicken feather as a substrate

Six strains of thermophilic actinomycetes were isolated from soil using an enrichmenttechnique with feathers as the sole carbon and nitrogen source. They showed clear proteolyticactivity on casein agar medium. The most active strain was tentatively identified as Streptomycesthermonitrificans. This isolate was grown in a basal medium with feathers and:or other carbon andnitrogen sources. Supernatant from centrifuged cultures was examined for protease activity andtemperature and pH optima were determined for enzyme activity. Optimum proteolytic activity onbasal liquid medium containing 1% chicken feather pieces was obtained at 50°C, in a mediumadjusted at pH8 and incubated for 72 h at 150 rpm. Proteolytic activity was further increased by1.5% feather pieces and the time required for maximal activity was 96 h. The keratinolytic activityof S. thermonitrificans was examined by incubation with native chicken feather pieces and it wasfound that it is significantly active. The degradation of whole intact feathers by S.thermonitrificans was obtained after 48 h of incubation at 50°C. The pH and temperature optimafor proteolytic activity were 9.0 and 50°C, respectively. The proteolytic activity was stable at40°C for 1 h. The proteolytic activity was inhibited by DFP but not by EDTA or pCMB. Theseresults inidicated that the enzyme(s) can be classified as an alkaline protease. 1999 ElsevierScience Ltd. All rights reserved.     

Expression of the Bacillus licheniformis PWD-1 keratinase gene in B. subtilis

The kerA gene which encodes the enzyme keratinase was isolated from the feather-degrading bacterium Bacillus licheniformis PWD-1. The entire gene, including pre-, pro- and mature protein regions, was cloned with P_ker, its own promoter, P43, the vegetative growth promoter, or the combination of P43-P_ker into plasmid pUB18. Transformation of the protease-deficient strain B. subtilis DB104 with these plasmids generated transformant strains FDB-3, FDB-108 and FDB-29 respectively. All transformants expressed active keratinase in both feather and LB media, in contrast to PWD-1, in which kerA was repressed when grown in LB medium. With P43-P_ker upstream of kerA, FDB-29 displayed the highest activity in feather medium. Production of keratinase in PWD-1 and transformants was further characterized when glucose or casamino acids were supplemented into the feather medium. These studies help understand the regulation of kerA expression and, in the long run, can help strain development and medium conditioning for the production of this industrially important keratinase. Received 31 December 1996/ Accepted in revised form 23 June 1997     

Purification and characterization of a keratinolytic metalloprotease from Chryseobacterium sp. kr6

The Chryseobacterium sp. kr6 strain has been described as a highly keratinolytic bacterium showing effective feather-degrading and de-hairing activities. A keratinase Q1 enzyme was purified from Chryseobacterium sp. kr6 culture by Phenyl Sepharose and Superose 12HR chromatography. This enzyme showed a specific activity of 967U/mg for keratin azure. Electrophoresis under denaturing conditions showed a monomeric protein with approximately 64kDa. The enzyme showed pH and temperature optima of 8.5 and 50 degrees C, respectively. The inhibitory effect of EDTA, EGTA and 1,10-phenanthroline characterized Q1 enzyme as a Zn-metalloprotease. Its activity was increased by three-fold in the presence of Ca(2+). ESI-MS/MS analysis of peptides generated from a tryptic digestion revealed sequence homology which may characterize the Q1 keratinase as a member of the M14 metalloprotease family, with a consensus glycosylation region similar to proteins from Chryseobacerium meningosepticum.     

Purification and characterization of four key enzymes from a feather-degrading Bacillus subtilis from the gut of tarantula Chilobrachys guangxiensis

A feather-degrading bacterium was isolated from the gut of the tarantula Chilobrachys guangxiensis, and was classified as Bacillus subtilis (named Bacillus subtilis CH-1) according to both the phenotypic characteristics and 16S rRNA profile. The improved culture conditions for feather-degrading were 10.0 g l⁻¹ mannitol, 10.0 g l⁻¹ tryptone, 0.1 g l⁻¹ MgCl₂, 0.4 g l⁻¹ KH₂PO₄, 0.3 g l⁻¹ K₂HPO₄, 0.5 g l⁻¹ NaCl, and 2.0 g l⁻¹ intact feather, with pH 8.5 and 37 °C. In the optimized medium, the intact black feather was completely degraded by Bacillus subtilis CH-1 in 24 h. Furthermore, four kinds of enzymes which include extracellular protease Vpr, peptidase T, γ-glutamyl transpeptidase and glyoxalmethylglyoxal reductase were identified as having principal roles. Simultaneously, the relationship between the disulfide bond reducing activity (DRT) and the keratinase activity (KT) in B. subtilis CH-1 fermentation system was discussed. This is the first report for a feather-degrading enteric bacterium from tarantula. The identification of the enzymes shines a light on further understanding the molecular mechanism of feather-degrading by microbes.