Feather-degrading Activity Research Articles

Genomic characterization and production of antimicrobial lipopeptides by Bacillus velezensis P45 growing on feather by-products.

To investigate the potential of novel Bacillus velezensis P45 as an eco-friendly alternative for bioprocessing poultry by-products into valuable antimicrobial products. The complete genome of B. velezensis P45 was sequenced using the Illumina MiSeq platform, showing 4455 protein and 98 RNA coding sequences according to the annotation on the RAST server. Moreover, the genome contains eight gene clusters for the production of antimicrobial secondary metabolites and 25 putative protease-related genes, which can be related to feather-degrading activity. Then, in vitro tests were performed to determine the production of antimicrobial compounds using feather, feather meal and brain-heart infusion (BHI) cultures. Antimicrobial activity was observed in feather meal and BHI media, reaching 800 and 3200AUml-1 against Listeria monocytogenes respectively. Mass spectrometry analysis indicates the production of antimicrobial lipopeptides surfactin, fengycin and iturin. The biotechnological potential of B. velezensis P45 was deciphered through genome analysis and in vitro studies. This strain produced antimicrobial lipopeptides growing on feather meal, a low-cost substrate. The production of antimicrobial peptides by this keratinolytic strain may represent a sustainable alternative for recycling by-products from poultry industry. Furthermore, whole B. velezensis P45genome sequence was obtained and deposited.

Isolation of a feather-degrading strain of bacterium from spider gut and the purification and identification of its three key enzymes.

A novel feather-degrading bacterium named CA-1 was isolated from the gut of the spider Chilobrachys guangxiensis, which degrades native whole chicken feathers within 20h. The CA-1 was confirmed to belong to Stenotrophomonas maltophilia based on morphologic and molecular analysis. Maximum feather degradation activity of the bacterium was observed at 37°C in basal feather medium (NaCl 0.5g/L, KH2PO4 0.3g/L, K2HPO4 0.4g/L, feather powder 10.0g/L, pH 8.0), which was inhibited when glucose and ammonium nitrate were added in the medium. Furthermore, the purified enzymes under the optimal and suppressive conditions were analyzed respectively by SDS-PAGE and LC-MS/MS. Three enzymes, namely alkaline serine protease (29.1kDa), ABC transporter permease (27.5kDa), and alkaline phosphatase (40.8kDa), were isolated and identified from the supernatant of the optimal culture and were considered to play principal roles. On the other hand, the potential synergic effects of the three proteins in S. maltophilia CA-1 feather degradation system were analyzed theoretically. CA-1 may product outer-membrane vesicles comprised of membranes and periplasmic proteins in the feather medium. The newly identified CA-1 and its synergic enzymes provide a new insight into further understanding the molecular mechanism of feather degradation by microbes. They also have potential application in cost-effectively degrading feathers into feeds and fertilizers through careful optimization and engineering of the three newly identified enzymes.

Feather degrading, phytostimulating, and biocontrol potential of native actinobacteria from North Eastern Indian Himalayan Region.

Twelve actinobacterial strains were isolated from tomato rhizospheric soil from Manipur, a state in North East Indian Himalayan Region and screened for keratinolytic and plant growth promoting traits. Nine promising isolates were identified as Streptomyces species using partial 16S rRNA gene sequencing. Among the seven isolates showing chicken feather degradation activity, three keratinolytic strains RCM-SSR-2, -6, and -12 were found to be the most efficient feather degrading strains achieving 90% feather weight loss within 48 h of incubation. They also showed maximum keratinase and soluble peptide production. Strain RCM-SSR-2, -5, -6, -8, and -11 showed positive results for all plant growth promoting traits tested. Maximum indole-3-acetic acid production was exhibited by RCM-SSR-6. Strain RCM-SSR-1, -2, -5, -6, -9, and -11 showed antagonistic activity against three important plant pathogens. Feather hydrolysate of RCM-SSR-6 was also evaluated for in vitro seed germination test using garden pea seeds. Higher concentration of feather protein hydrolysate (3 mg ml-1 ) inhibited shoot and root length of the germinating embryo. However, lower concentration (0.01 mg ml-1 ) of feather protein hydrolysate promoted seed germination. Among the 12 strains, four isolates namely RCM-SSR-1, -2, -5, and -6 were found to be promising as multi-traits plant growth promoting rhizobacteria for development of organic fertilizer, phytostimulator, and biocontrol agents.

Synergistic effect of Chryseobacterium gleum sp. SUK with ACC deaminase activity in alleviation of salt stress and plant growth promotion in Triticum aestivum L.

Soil salinity is major abiotic stresses affecting morphological, biochemical and physiological processes of plant growth. Chryseobacterium gleum sp. SUK isolated from salt-stressed soil exhibited ACC (1-aminocyclopropane-1-carboxylate) deaminase activity with IAA (indole acetic acid), siderophore, ammonia, hydrogen cyanide production, 2% salt tolerance and fungal cell wall degrading enzyme production (cellulase, protease). The isolate also showed a poultry feather degrading activity which is the main waste material of poultry industry and opulent source of proteins, amino acids, nitrogen, phosphorous, calcium, potassium, manganese, zinc and copper. Application of feather-degraded lysate with the degrading isolate, C. gleum sp. SUK denotes triple role of bioformulation to surmount salinity stress, management of poultry waste disposal and utilization of feathers degraded products as a biostimulant for better growth of plants as well as strain SUK having multifarious plant growth promoting traits. Wheat crops exposed to salt stressor were inoculated with studied bioformulation. Results of plant analysis showed improvement in root and shoot length, fresh and dry weight, chlorophyll, proteins, amino acids, phenolics, flavonoids content and decreased level of proline. In addition, Na+ uptake was decreased and K+ uptake was increased. Therefore, application of novel bioformulation could increase the yield of crops by ameliorating growth of plants and alleviating the salinity stress.

Isolation and Selection of Feather-Degrading Aerobic Bacteria From Poultry Processing Plants in Mekong Delta of Vietnam

Studies on keratinolytic microorganism have been mainly related to biotechnological applications based on their capability for decomposition of keratin-containing wastes to produce animal feed and bio-fertilizer. However, these organisms have ecological relevance to recycling keratinous residues in nature. This study aimed to select and identify new local potential feather - degrading bacteria for applications in biotechnological processes in Mekong Delta. A total of 21 aerobic, rod-shaped isolates from Mekong Delta poultry processing plants showed capability to growth on feather meal agar medium. They all expressed clear zones around the colonies on milk agar medium indicating proteolytic activity. They also hydrolyzed 24.43% to 58.13% of chicken feather dry weight after 7 days incubation at 37 o C and partially disintegrated the whole feather after 10 days. The isolates K15 and K14 showed the most prominent feather-degrading activity were further identified according to their morphology and physiological characteristics. Molecular identification based on their 16S rDNA gene sequence also carried out and revealed that K14 as Chryseobacterium indologenes ; whereas K15 was highly related to Bacillus subtilis.

Effect of pH and temperature on stability and kinetics of novel extracellular serine alkaline protease (70 kDa)

A novel extracellular serine protease (70kDa by SDS-PAGE) was purified and characterized. This enzyme retained more than 93% of its initial activity after preincubation for 30min at 37°C in the presence of 25% (v/v) tested organic solvents and showed feather degradation activity. The purified enzyme was deactivated at various combinations of pH and temperature to examine the interactive effect of them on enzyme activity. The deactivation process was modeled as first-order kinetics and the deactivation rate constant (kd) was found to be minimum at pH 9 and 37°C. The kinetic analysis of enzyme over a range of pH values indicated two pK values at 6.21 and at 10.92. The lower pK value was likely due to the catalytic histidine in the free enzyme and higher pK value likely reflected deprotonation of the proline moiety of the substrate but ionization of the active site serine is another possibility. Inhibition kinetic showed that enzyme is serine protease because enzyme was competitively inhibited by antipain and aprotinin as these compounds are known to be competitive inhibitors of serine protease. The organic solvent, thermal and pH tolerances of enzyme suggested that it may have potential for use as a biocatalyst in industry.

Iron containing keratinolytic metallo-protease produced by Chryseobacterium gleum

Chryseobacterium gleum exhibited complete dissolution of whole chicken-feathers (10gl−1, pH 8) after 72h at 30°C through synthesis of keratinolytic protease when inoculated at 1% (v/v). This enzyme was purified to 67-fold with yield of 2.25% having a specific activity of 1670Umg−1 and ∼36kDa Mw. MALDI-TOF MS of this keratinase showed some similarity with the keratinase peptides of Bacillus subtilis (BOFXJ2). The keratinase action was inhibited by EDTA, iodoacetamide and metal ions like mercury, copper and zinc (1mM each), while it was enhanced by iron and calcium. Keratinase showed presence of 3mM of FeM−1 as tested by atomic absorption spectroscopy and addition of Fe in its apoenzyme retained about 79% of original residual feather degradation activity which portrayed it to be metalloprotease. Purified keratinase revealed significant degradation (85%) of feather concentrate (20gl−1) to 3.9μMml−1 of free amino groups in 24h at an initial pH of 8.0, 30°C and 120rpm shaking. This keratinase activity can be controlled precisely by presence of chemical or metal ions which could be of use in biotechnology industry while the culture can be used in poultry waste management.

Alkaline proteases produced by Bacillus licheniformis RP1 grown on shrimp wastes: Application in chitin extraction, chicken feather-degradation and as a dehairing agent

The current increase in the amount of shrimp wastes produced by the shrimp industry has led to the need in finding new methods for shrimp wastes disposal. In this study, Bacillus licheniformis RP1 was shown to produce proteases when grown in media containing shrimp wastes powder as a sole carbon and nitrogen source, indicating that this bacteria could obtain its carbon and nitrogen requirements directly from shrimp wastes. The maximum protease production was obtained when the strain was grown in a medium containing (g/L): shrimp wastes powder 30, KCl 1.5, K2HPO4 0.5, and KH2PO4 0.5. Using casein zymography, the crude protease preparation was found to produce at least seven proteases. The proteases of B. licheniformis RP1 were tested for shrimp waste deproteinization in the preparation of chitin. The percent of protein removal after 3 h hydrolysis at 60°C and at an enzyme/substrate (E/S) ratio of 0.5 and 5 (Unit of enzyme/mg of protein) were about 68 and 81%, respectively. Additionally, B. licheniformis RP1 showed important feather degrading activity. Complete solubilisation of whole feathers was observed after 24 h of incubation at 50°C. More interestingly, the RP1 proteolytic preparation demonstrated powerful dehairing capabilities for hair removal from skin. Collagen, which is the major leather-forming protein, was not significantly degraded. Considering its promising properties, B. licheniformis RP1 enzymatic preparation may be considered a potential candidate for future use in several biotechnological processes.

Statistical optimization of production and tannery applications of a keratinolytic serine protease from Bacillus subtilis P13

Bacillus subtilis P13, an isolate from Vajreshwari hot springs near Mumbai, India, produces an extracellular serine protease that catalyses dehairing of hides as well as feather degradation. Present work reports the optimization of production medium at shake flask level, resulting in a 3.2 fold increase in enzyme activity with a concomitant reduction in submerged culturing time by 24h. Soaking, dehairing and bating of hides were efficiently carried using enzyme from optimized medium. The growth pattern in the optimized medium was different from that in the unoptimized medium, showing entry into stationary phase 4h earlier. Zymogram profiling of the extracellular proteases initially showed the appearance of a major ∼31kDa protease, but at later growth stages additional proteases of ∼66, 43 and 20kDa were prominent. By correlating the caseinolytic activity, feather degrading activity and zymogram patterns in different media combinations used for the statistical optimization, the 31kDa protease could be identified to be responsible for feather disintegration. The purified 31kDa protease is a PMSF-sensitive serine protease with keratinase and sulfitolysis activity and is proficient at feather degradation and hide depilation. Its N-terminal amino acid sequence is similar to that of subtilisins of other B. subtilis strains.

Characterization of detergent stable and feather degrading serine proteases from Bacillus mojavensis A21

The present study describes the characterization of crude protease from Bacillus mojavensis A21 and its evaluation in detergent and chicken feathers hydrolysis. The strain was found to produce at least six major extracellular proteases as shown by casein-zymography. The optimum pH and temperature for proteolytic activity were 8.0–11.0 and 60 °C, respectively. The crude protease showed extreme stability towards non-ionic (5% Tween 80 and 5% Triton X-100) and anionic (1% SDS) surfactants, and relative stability towards oxidizing agents. Additionally, it showed excellent stability and compatibility with various solid (7 mg/ml) and liquid (1%; v/v) detergents at temperatures from 30 to 50 °C. In the presence of solid detergents, the enzyme preparation retained 100% of its initial activity after pre-incubation for 1 h at 40 °C with Axion and Ariel, followed by Nadhif (87%), Dixan (85%) and New Det (82%). With liquid detergents, the enzyme preparation retained 100% of its original activity after pre-incubation for 1 h at 40 °C with Dixan and Nadhif. Wash performance analysis revealed that A21 crude protease could effectively remove blood-stains. In addition, B. mojavensis A21 proteolytic preparation showed important feather degrading activity. Considering its promising properties, B. mojavensis A21 enzymatic preparation may be considered a potential candidate for future use in biotechnological processes, particularly in detergent and in the processing of poultry waste.

Decomposition of Intact Chicken Feathers by a Thermophile in Combination with an Acidulocomposting Garbage-Treatment Process

In order to develop a practical method for the decomposition of intact chicken feathers, a moderate thermophile strain, Meiothermus ruber H328, having strong keratinolytic activity, was used in a bio-type garbage-treatment machine working with an acidulocomposting process. The addition of strain H328 cells (15 g) combined with acidulocomposting in the garbage machine resulted in 70% degradation of intact chicken feathers (30 g) within 14 d. This degradation efficiency is comparable to a previous result employing the strain as a single bacterium in flask culture, and it indicates that strain H328 can promote intact feather degradation activity in a garbage machine currently on the market.

Characterization of a novel Stenotrophomonas isolate with high keratinase activity and purification of the enzyme

A feather-degrading bacterium was isolated from poultry decomposition feathers in China. The strain, named L1, showed significant feather-degrading activity because it grew and reproduced quickly on basal medium containing 10 g/L of native feather as the source of energy, carbon, and nitrogen. According to the phenotypic characteristics and 16S rRNA profile, the isolate belongs to Stenotrophomonas maltophilia. Keratinase activity of the isolate was determined during cultivation on raw feathers at different temperatures and initial pH. Maximum growth and feather-degrading activity of the bacterium were observed at 40 degrees C and initial pH ranging from 7.5 to 8.0. The crude enzyme was purified by ammonium sulphate precipitation, Sephadex G-100 chromatographic and ceramic hydroxyapatite (CHT) chromatographic. Its molecular mass estimated as 35.2 kDa in SDS-PAGE. The enzyme had an optimum activity at the pH was 7.8 and the temperature was 40 degrees C. The keratinase was wholly inhibited by a serine protease inhibitor, PMSF. Its activity was activated or inhibited by different metal ions. The keratinase activity of enzyme from strain L1 functioned on different keratins, such as feather, hair, wool, horn, and so on.

Biochemical and molecular characterization of a detergent-stable serine alkaline protease from Bacillus pumilus CBS with high catalytic efficiency

We have described previously the potential use of an alkaline protease from Bacillus pumilus CBS as an effective additive in laundry detergent formulations [B. Jaouadi, S. Ellouz-Chaabouni, M. Ben Ali, E. Ben Messaoud, B. Naili, A. Dhouib, S. Bejar, A novel alkaline protease from Bacillus pumilus CBS having a high compatibility with laundry detergent and a high feather-degrading activity, Process Biochem, submitted for publication]. Here, we purified this enzyme (named SAPB) and we cloned, sequenced and over-expressed the corresponding gene. The enzyme was purified to homogeneity using salt precipitation and gel filtration HPLC. The pure protease was found to be monomeric protein with a molecular mass of 34598.19 Da as determined by MALDI-TOF mass spectrometry. The NH 2-terminal sequence of first 21 amino acids (aa) of the purified SAPB was AQTVPYGIPQIKAPAVHAQGY and was completely identical to proteases from other Bacillus pumilus species. This protease is strongly inhibited by PMSF and DFP, showing that it belongs to the serine proteases superfamily. Interestingly, the optimum pH is 10.6 while the optimum temperature was determined to be 65 °C. The enzyme was completely stable within a wide range of pH (7.0–10.6) and temperature (30–55 °C). One of the distinguishing properties is its catalytic efficiency ( k cat/ K m) calculated to be 45,265 min −1 mM −1 and 147,000 min −1 mM −1 using casein and AAPF as substrates, respectively, which is higher than that of Subtilisin Carlsberg, Subtilisin BPN′ and Subtilisin 309 determined under the same conditions. In addition, SAPB showed remarkable stability, for 24 h at 40 °C, in the presence of 5% Tween-80, 1% SDS, 15% urea and 10% H 2O 2, which comprise the common bleach-based detergent formulation. The sapB gene encoding SAPB was cloned, sequenced and over-expressed in Escherichia coli. The purified recombinant enzyme (rSAPB) has the same physicochemical and kinetic properties as the native one. SapB gene had an ORF of 1149 bp encoding a protein of 383 aa organized into a signal peptide (29 aa), a pro-protein (79 aa) and a mature enzyme (275 aa). The deduced amino acid sequence inspection displays an important homology with other bacterial proteases. The highest homology of 98.1% was found with BPP-A protease from Bacillus pumilus MS-1, with only 8 aa of difference.

Characterization of a new keratinolytic Trichoderma atroviride strain F6 that completely degrades native chicken feather

To isolate novel nonpathogenic fungus that completely degrades native chicken feather and characterize its keratinases. 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 Ba(2+), Mn(2+), Hg(2+), Zn(2+), Cu(2+), 1,10-phenanthroline, 2,2'-bipyridyl, and PMSF (phenylmethylsulfonyl fluoride) were observed. 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. The novel nonpathogenic T. atroviride F6 with high feather-degrading activity showed potentials in biotechnological process of converting feathers into economically useful feather meal.

Characterization of a Novel Feather-Degrading <I>Bacillus</I> sp. Strain

Feather waste is generated in large amounts as a byproduct of commercial poultry processing. This residue is almost pure keratin, which is not easily degradable by common proteolytic enzymes. A feather-degrading bacterium was isolated from poultry feathers in decomposition. The strain identified as kr16 showed important feather-degrading activity when grown on basal medium containing 10 g/L of native feather as the source of energy, carbon, and nitrogen. The isolate was characterized according to the phenotypical characteristics and biochemical profiling that belong to the Bacillus genus. Keratinolytic activity of this isolate was monitored during cultivation of the bacterium on raw feathers at different temperatures. Maximum growth and feather-degrading activity were observed at 30-37 degrees C. The keratinolytic enzyme had a pH optimum ranging from 8.0 to 11.0 and a temperature optimum of 45-65 degrees C. The keratinase was strongly inhibited by EDTA and the metal ions Hg2+ and Sn2+.

Characterization of a protease of a feather-degrading Microbacterium species.

To characterize a new feather-degrading bacterium. 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+). 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+).

Isolation and characterization of a new keratinolytic Bacillus licheniformis strain

A keratin-degrading bacterium strain (K-508) was isolated from partially degraded feathers and characterized. This isolate exhibited a high chicken feather-degrading activity when cultured in feather-containing broth with a growth optimum of pH 7.0 and 47 °C. On the basis of its phenotypic characteristics (quickly moving, Gram-positive rods), the results of metabolic tests and rDNA sequence analysis, it was identified as Bacillus licheniformis. Its fermentation broth showed activity on N-Bz-l-Phe-l-Val-l-Arg-p-nitroanilide, N-Suc-l-Ala-l-Ala-l-Pro-l-Phe-p-nitroanilide, N-CBZ-Gly-Gly-l-Leu-p-nitroanilide and N-CBZ-l-Ala-l-Ala-l-Leu-p-nitroanilide as chromogenic protease substrates at near neutral pH. Both trypsin-like and chymotrypsin-like proteases were constitutively secreted by this strain.

Secretion of a trypsin-like thiol protease by a new keratinolytic strain of Bacillus licheniformis

When cultured in feather-containing broth with a growth optimum of pH 7.0 and 47°C, a Bacillus licheniformis strain exhibited a high chicken feather-degrading activity. A trypsin-like protease was isolated from its ferment broth and was partially characterized. The enzyme was constitutively secreted and was highly active towards N-benzoyl-Phe-Val-Arg- p-nitroanilide as chromogenic substrate. Its pH optimum was 8.5 and it exhibited the highest activity at 52°C. Fractionation on Sephadex G-100 column revealed that its molecular mass was about 42 kDa. The enzyme, which is new for the genus Bacillus, is a thiol protease, as tosyl- L-phenylalanine chloromethyl ketone, tosyl- L-lysine chloromethyl ketone, phenylmethylsulfonyl fluoride and ethylenediamine tetraacetate did not inhibit it, while HgCl 2 and para-chloromercuribenzoate lowered its activity.

Secretion of a trypsin-like thiol protease by a new keratinolytic strain of Bacillus licheniformis

When cultured in feather-containing broth with a growth optimum of pH 7.0 and 47 degrees C, a Bacillus licheniformis strain exhibited a high chicken feather-degrading activity. A trypsin-like protease was isolated from its ferment broth and was partially characterized. The enzyme was constitutively secreted and was highly active towards N-benzoyl-Phe-Val-Arg-p-nitroanilide as chromogenic substrate. Its pH optimum was 8.5 and it exhibited the highest activity at 52 degrees C. Fractionation on Sephadex G-100 column revealed that its molecular mass was about 42 kDa. The enzyme, which is new for the genus Bacillus, is a thiol protease, as tosyl-L-phenylalanine chloromethyl ketone, tosyl-L-lysine chloromethyl ketone, phenylmethylsulfonyl fluoride and ethylenediamine tetraacetate did not inhibit it, while HgCl2 and para-chloromercuribenzoate lowered its activity.