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.     

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.     

Keratinolytic activity from new recombinant fusant AYA2000, derived from endophytic Micromonospora strains

Two different endophytic strains, ESRAA1997 and ALAA2000, were isolated from the Egyptian herbal plant Anastatica hierochuntica. The 2 strains produced alkaline serine protease and were identified based on their phenotypic and chemotypic characteristics as different strains of Micromonospora spp. Both strains grew and produced keratinase, using different keratinous waste substances as the sole source of carbon and nitrogen. In our study, the activity and properties of keratinase enzymes of the wild strains ESRAA1997 and ALAA2000 were altered by genetic recombination through protoplast fusion between them, leading to a potent keratinolytic fusant Micromonospora strain AYA2000 with improved properties (activity, stability, specificity, and tolerance to inhibitors). Using a mixture of yeast extract, peptone, and malt extract as a supplement to the bovine hair medium increased keratinase production by 48%, and addition of 1% glucose suppressed enzyme production by Micromonospora strain AYA2000. The enzyme was purified by ammonium sulphate precipitation and DEAE-cellulose chromatography followed by gel filtration. The molecular weight, estimated using SDS-PAGE, was 39?kDa. The enzyme exhibited remarkable activity towards all keratinous wastes used and could also adapt to a broad range of pH and temperatures, with optima at pH?11 and 60?°C. The enzyme was not influenced by chelating reagents, metal ions, or alcohols. These properties make AYA2000 keratinase an ideal candidate for biotechnological application.     

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.     

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     

Hydrolysis of native proteins by keratinolytic protease of Doratomyces microsporus

A keratinolytic protease from the fungus Doratomyces microsporus was investigated for its ability to hydrolyse different native proteins. The purified enzyme was incubated for up to 24 h with keratinous substrates as well as with non-keratinous proteins. The results showed that the enzyme was broad specific since it hydrolysed various globular and fibrillar proteins. The hydrolysis of keratinous substrates decreased in the following order: skin keratins > nail keratins > hair keratins. With non-keratinous substrates, the order was: casein > BSA > elastin. Feather keratin and collagen could not be hydrolysed. Comparison of the enzyme with some known proteolytic enzymes showed that on keratin from stratum corneum the activity of the keratinase was comparable to that of proteinase K, other enzymes were less active. Hydrolysis of porcine skin with the keratinase revealed the degradation of the epidermis while dermis was not damaged.     

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

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

     

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

Abstract

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

Purification and characterization of a keratinolytic serine protease from Purpureocillium lilacinum LPS # 876

A keratinolytic serine protease secreted by Purpureocillium lilacinum (formerly Paecilomyces lilacinus) upon culture in a basal medium containing 1% (w/v) hair waste as carbon and nitrogen source was purified and characterized. After purification the keratinase was resolved by SDS-PAGE as a homogeneus protein band of molecular mass 37.0 kDa. The extracellular keratinase of P. lilacinum was characterized by its appreciable stability over a broad pH range (from 4.0 to 9.0), and up to 65 °C, along with its strong inhibition by phenylmethylsulphonyl fluoride among the protease inhibitors tested (98.2% of inhibition), thus suggesting its nature as a serine protease. The enzyme was active and stable in the presence of organic solvents such as dimethylsulfoxide, methanol, and isopropanol; certain surfactants such as Triton X-100, sodium dodecylsulfate, and Tween 85; and bleaching agents such as hydrogen peroxide. These biochemical characteristics suggest the potential use of this enzyme in numerous industrial applications.     

Isolation and partial characterisation of extracellular keratinase from a wool degrading thermophilic actinomycete strain Thermoactinomyces candidus.

The keratinase production by the thermophilic actinomycete strain Thermoactinomyces candidus was induced by sheep wool as the sole source of carbon and nitrogen in the cultivation medium. For complete digestion of wool by the above strain, both keratinolytic serine proteinase and cellular reduction of disulfide bonds were involved. Evidence was presented that substrate induction was a major regulatory mechanism and the keratinase biosynthesis was not completely repressed by addition of other carbon (glucose) and nitrogen (NH4C1) sources. The enzyme was purified 62-fold by diethylaminoethyl-anion exchange and Sephadex G-75 gel permeation chromatographies. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the purified keratinase is a monomeric enzyme with a molecular mass of 30 kDa. The pH and temperature optima were determined to be 8.6 and 70 degrees C, respectively. The purified thermophilic keratinase catalyses the hydrolysis of a broad range of substrates and displays higher proteolytic activity against native keratins than other proteinases. Ca2+ was found to have a stabilizing effect on the enzyme activity at elevated temperatures.     

Keratinolytic activity of purified alkaline keratinase produced by Scopulariopsis brevicaulis (Sacc.) and its amino acids profile

Sodium dodecyl sulfate–polyacrlyamide gel electrophoresis (SDS–PAGE) was used to assess the purity and molecular weight of the previously purified alkaline keratinase enzyme of Scopulariopsis brevicaulis. The enzyme was homogenous, as seen by a single band of protein, and had an apparent molecular weight of 28.5 kDa. Amino acid profile of the purified keratinase revealed that it was composed of 14 different amino acids with high proportions of glutamic acid (20.86%), alanine (14.52%), glycine (14.21%), leucine (8.59%) and serine (7.81%). The enzyme contained moderate amounts of valine (6.01%), threonine (5.58%) and phenyl alanine (5.22%). The purified enzyme of S. brevicaulis exerted a potent keratinolytic activity and was capable to hydrolyze different keratinaceous materials with highest activity on chicken feathers followed by human nails and human hair.     

Keratinolytic activity of Bacillus subtilis AMR using human hair

Aims:  To determine the ability of a novel Bacillus subtilis AMR isolated from poultry waste to hydrolyse human hair producing peptidases including keratinases and hair keratin peptides.
Methods and Results:  The Bacillus subtilis AMR was identified using biochemical tests and by analysis of 16S rDNA sequence. The isolate was grown in medium containing human hair as the sole source of carbon and nitrogen. The supplementation of hair medium (HM) with 0·01% yeast extract increased the keratinolytic activity 4·2-fold. B. subtilis AMR presented high keratinase production on the 8th day of fermentation in hair medium (HM) supplemented with 0·01% yeast extract (HMY) at pH 8·0. Keratinase yield was not correlated with increase in biomass. Zymography showed keratin-degrading peptidases migrating at c. 54, 80 and 100 kDa and gelatin-degrading bands at c. 80, 70 63, 54 32 and 15 kDa. Keratinases were optimally active at 50°C and pH 9·0 and was fully inhibited by the serine proteinase inhibitor (PMSF). Scanning electron microscopy showed complete degradation of the hair cuticle after exposure to B. subtilis AMR grown in HMY. MALDI-TOF analysis of culture supernatant containing peptides produced during enzymatic hydrolysis of hair by B. subtilis AMR revealed fragments in a range of 800–2600 Da.
Conclusions:  This study showed that B. subtilis AMR was able to hydrolyse human hair producing serine peptidases with keratinase and gelatinase activity as well as hair keratin peptides.
Significance and Impact of the Study:  This is the first report describing the production and partial characterization of keratinases by a B. subtilis strain grown in a medium containing human hair . These data suggest that peptides obtained from enzymatic hair hydrolysis may be useful for future applications on pharmaceutical and cosmetic formulations.     

Keratin degradation: a cooperative action of two enzymes from Stenotrophomonas sp

A novel keratin-degrading bacterium Stenotrophomonas sp. strain D-1, isolated from deer fur, produced two types of extracellular proteins: proteolytic and disulfide bond-reducing. The results on the biochemical properties suggest that this protease belongs to the serine protease, and the disulfide bond-reducing protein could be the disulfide reductase type. None of these enzymes showed keratinolytic activity independently. However, after mixing of the two enzymes, the keratinolytic activity was increased tremendously (more than 50-fold) over that of the protease only. This keratinolytic activity was more than 2-fold higher than that of the combination with proteinase K (also known for its high keratinolytic activity). Since the two enzymes discovered in this study acted cooperatively and resulted in higher keratinolytic activity, a new mechanism of keratin degradation has been revealed. To our knowledge, this is the first report on the cooperative action of two enzymes resulting in the effective degradation of keratin.     

Purification and characterization of a novel exocellular keratinase from Kocuria rosea

A keratinolytic protease activity secreted by Kocuria rosea when cultured in bioreactors using feathers as unique carbon and nitrogen source was purified and characterized. This novel keratinase activity was purified from the bioreaction broth growing media to apparent homogeneity after single step, (24-fold purification with a high yield of 54%) using DEAE column chromatography. The native molecular mass of the enzyme determined by gel filtration chromatography was 240 kDa. K. rosea extracellular keratinase was stable in a broad range of pH (8–11) and temperature (10–60 °C) profile with optimums at pH 10 and 40 °C. Crystalline soybean trypsin inhibitor (type I-S), 4-(2-aminoethyl) benzenesulfonyl floride (AEBSF) and chymostatin, strongly inhibited the keratinolytic activity indicating that the keratinase belongs to the serine protease family. The K_m for the soluble keratin degradation from feathers was 242 μM. The enzyme was resistant to denaturing or reducing agents such as dithiotreitol and 2-mercaptoethanol. All of the biochemical characteristics, raising the potential use of this enzyme in numerous industrial applications.     

New Feather-Degrading Filamentous Fungi

Among 106 filamentous fungi isolated from poultry farm waste, 13 species belonging to seven genera (Aspergillus, Acremonium, Alternaria, Beauvaria, Curvularia, Paecilomyces, and Penicillium) were able to grow and produce keratinase in stationary cultures using poultry feather powder as the only substrate. The four most efficient keratinase producers were selected for a comparative study of keratinase production in submerged and stationary conditions. The highest keratinolytic activities were produced after 4-6 days of cultivation in submerged conditions: 53.8 +/- 6.1 U/mL (Alternaria tenuissima), 51.2 +/- 5.4 U/mL (Acremonium hyalinulum), 55.4 +/- 5.2 U/mL (Curvularia brachyspora), and 62.8 +/- 4.8 U/mL (Beauveria bassiana). These novel nondermatophytic keratinolytic fungi have potential use in biotechnological processes involving keratin hydrolysis. The results of this work contribute to show that keratinolytic activity is relatively widespread among common filamentous fungi and may have an important rule in feather decomposition in natural settings.     

Purification and properties of a keratinolytic metalloprotease from Microbacterium sp

AIMS: This study was developed to purify and to characterize a keratinolytic protease from the bacterium Microbacterium sp. strain kr10. METHODS AND RESULTS: Enzyme purification was carried out by sequential liquid chromatography on Sephadex G-100 and Q-Sepharose columns. The purification was about 255-fold, with a yield of 34%, as determined with azocasein as substrate. The molecular weight of the enzyme was estimated as 42,000 Da by SDS-PAGE. The enzyme had pH and temperature optima of 7.5 and 50 degrees C respectively. This keratinase was inhibited by EDTA and 1,10-phenanthroline, and analysis of metal content indicates that Zn(2+) and Mg(2+) are present. A 2(2) factorial design was developed to investigate the effect of keratinase and mercaptoacetate concentration on feather keratinolysis. Statistical analysis showed that both variables have a significant effect on hydrolysis of keratin. CONCLUSIONS: A new keratinase produced by Microbacterium sp. was purified and characterized. SIGNIFICANCE AND IMPACT OF THE STUDY: This keratinolytic enzyme offers an interesting potential for the hydrolysis of keratin wastes to be used as feed supplement or bioconversion to added-value products.     

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.     

Keratinase of Doratomyces microsporus

 The fungus Doratomyces microsporus produced an extracellular keratinase during submerged aerobic cultivation in a medium containing a protein inducer for enzyme synthesis. The keratinase was purified to homogeneity using hydrophobic interaction chromatography followed by gel chromatography. The molecular weight was estimated to be 33 kDa (from SDS-PAGE analysis) or 30 kDa (by gel chromatography), suggesting a monomeric structure. The isoelectric point of the enzyme was determined to be around 9. The optimal pH and temperature for keratinolytic activity were pH 8–9 and 50 °C, respectively. The serine protease inhibitor PMSF totally inhibited the keratinase. The enzyme was not glycosylated. It was capable of hydrolysing different keratinous materials as well as some non-keratinous proteins. Hydrolysis of some synthetic substrates, specific for known proteinases, suggested that the keratinase of D. microsporus is close to proteinase K. Received: 9 July 1999 / Received revision: 13 September 1999 / Accepted: 17 September 1999