Keratinolytic activity of Streptomyces sp. S.K1–02: a new isolated strain

A Streptomyces sp. producing a high keratinolytic activity when cultured on feather meal medium was isolated from a naturally degraded feather. Maximal keratin degradation using supernatant fluid obtained from batch culture of this organism was observed at 70 °C and pH 10. Keratinolytic activity was only partially inhibited by EDTA or PMSF, suggesting that the overall keratinolytic activity was supported by different proteases. Comparisons between proteolytic activities derived from this new strain ( S .K_1–02) and commercial proteases indicated that S .K_1–02 could be a useful biotechnological tool in valorization of keratin-containing wastes, or in the depilation process in the leather industry.     

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.     

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.     

Evaluation of environmental conditions for production of bacteriocin-like substance by <Emphasis Type="Italic">Bacillus</Emphasis> sp. strain P34

The influence of temperature, pH and media on bacteriocin production by Bacillus sp. P34 was investigated. The effect of temperature and initial pH was evaluated by factorial design and response surface methodology (RSM). Statistical analysis of results showed that, in the range studied, the two variables have a significant effect on bacteriocin production. Response-surface data showed maximum antimicrobial activity production at initial pH values between 6.0 and 8.0 and temperatures between 25 and 37 °C. No relationship between bacterial growth and bacteriocin production was observed. RSM proved to be a powerful tool in optimizing the production of antimicrobial activity by Bacillus sp. P34. When different media were tested, maximum bacteriocin production was observed in soybean protein-based medium, but antimicrobial activity was not achieved by cultivation on fish meal, feather meal, whey and grape waste.     

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).     

降解鸡毛真菌板蜡蚧的鉴定及产酶培养优化

鉴定降解鸡毛真菌并通过单因素和正交实验优化其产角蛋白酶发酵培养条件。从加入鸡毛粉钓饵的医院花坛土中分离筛选获得3株角蛋白高效降解真菌,利用形态学和分子系统学鉴定均为板蜡蚧（Lecanicillium testudineum）。单因素实验表明,对优选菌株1Y2-12产酶能力具促进作用的碳源为乳糖,氮源为酵母膏,无机离子Zn²⁺。正交实验结果表明,对菌株1Y2-12产酶活力的影响大小排序依次为氮源>无机离子>碳源;促酶活力最佳组合为氮源添加量为0.15%,碳源添加量为2%,无机离子添加量为0.01%。在此优化条件下,菌株1Y2-12的酶活力达22.03 U/mL,是未优化前的2.1倍。研究结果首次发现蜡蚧属真菌能较好降解鸡毛角蛋白,鉴定的3个菌株均为板蜡蚧,是中国新记录种。     

Production and properties of keratinolytic proteases from three novel Gram-negative feather-degrading bacteria isolated from Brazilian soils

The keratinolytic potential and protease properties of three novel Gram-negative feather-degrading bacteria isolated from Brazilian soils was described. Aeromonas hydrophila K12, Chryseobacterium indologenes A22 and Serratia marcescens P3 were able to degrade feather meal, producing high amounts of soluble proteins and forming thiol groups. The proteases of strains K12, A22 and P3 had optimal pH of 8.0, 7.5 and 6.0, respectively; this last is an uncommon feature for bacterial keratinases. The optimal temperature was in the range 45–55°C. All three proteases were active towards azokeratin and were inhibited by EDTA, suggesting that they are keratinolytic metalloproteases. The proteolytic activity of K12 was stimulated by organic solvents and the detergent SDS, suggesting its potential application for detergent formulations and peptide synthesis. Strains A22, K12 and P3 have great potential for use in biotechnological processes involving hydrolysis of keratinous byproducts.     

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     

Effect of feather meal as proteic feeder on combi-CLEAs preparation for grape juice clarification

This work describes the preparation of combi-CLEAs of pectinases using feather meal as novel proteic feeder. Initially, four solvents were tested as precipitant agents, and ethanol was chosen as the best solvent. Subsequently, a 2³ factorial design was carried out to define the optimal conditions for combi-CLEAs preparation, which were set as 110 mM of glutaraldehyde, 0.66 mg protein mL⁻¹ and 5 h of reaction time. Then, the effect of BSA or feather meal (FM) as proteic feeders was evaluated. Combi-CLEAs, combi-CLEAs-BSA and combi-CLEAs-FM were characterized and evaluated by their activity recovery, optimal pH and temperature, operational and thermal stabilities and e effectiveness on juice clarification. The addition of feather meal increased two-fold the activity recovery compared to BSA. The thermal stability was similar for all combi-CLEAs, providing stabilization factors of 1.12, 1.48 and 1.53, respectively, for the combi-CLEA, combi-CLEA-FM and combi-CLEA-BSA, compared to free enzyme. CLEAs presented turbidity reduction near to 50%, 2.5-fold than soluble enzyme. Feather meal was used for the first time as proteic feeder for CLEA preparations and appears to be a good, and low price, alternative.     

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.     

A biotechnological process for treatment and recycling poultry feathers as a feed ingredient

A strain of Kocuria rosea with keratinolytic capacity was cultured aerobically on submerged feathers to obtain a fermented feather meal (FFM). This FFM enriched with cells of K. rosea mainly contains crude protein (71%). The pepsin digestibility of the fermented product (88%) was similar to the value of the commercial feather meal and more than 70% greater that untreated feathers. The bacterial biomass improved the content of amino acids lysine (3.46%), histidine (0.94%) and methionine (0.69%). Additionally, the amino acid availability tested by in vivo assay was greater than commercial feather meal. The microbial cells also supplied carotenoid pigments to FFM (68 ppm). These results suggest that feather meal enriched with K. rosea may be useful in animal feeding as protein and pigment source.     

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     

Optimization of metallo-keratinase production by Pseudomonas sp. LM19 as a potential enzyme for feather waste conversion

Locally isolated bacterium Pseudomonas sp. LM19, a metallo-keratinase producer was used to hydrolyze the highly rigid keratin recalcitrant in this study. The production of crude keratinase by Pseudomonas sp. LM19 is influenced by both physical and nutritional parameters. The highest keratinase activity of 127?U/ml (2.15-fold) was observed in feather meal medium supplemented with fructose and peptone at a C/N ratio of 40. The optimum pH and temperature for keratinase production were found to be pH 8 and 30?°C, using 1% (w/v) feather as substrate. The degradation rate of the feathers was increased 2.4-fold at optimized physical and nutritional conditions. Feather degradation by Pseudomonas sp. LM19 led to the production of free amino acids such as arginine, glycine, leucine, and serine. The information on the production of keratinase by Pseudomonas sp. LM19 obtained from this study warrants further research for possible commercial application.     

Hide depilation and feather disintegration studies with keratinolytic serine protease from a novel Bacillus subtilis isolate

Keratinases play an important role in biotechnological applications such as improvement of feather meal, enzymatic dehairing and production of amino acids or peptides from high molecular weight substrates. Bacillus subtilis P13, isolated from Vajreshwari hot spring (45–50°C) near Mumbai, India, produces a neutral serine protease and has an optimum temperature of 65°C. This enzyme preparation was keratinolytic in nature and could disintegrate whole chicken feathers, except for the remnants of shafts. The enzyme preparation also exhibited depilation of goat hides with the recovery of intact animal hair. The enzyme preparation could release peptides from ground feathers and bring about their weight reduction; however, similar action on hair was relatively weak. A single major PMSF-sensitive protease band could be detected upon zymogram analysis, indicating that a single enzyme may be responsible for feather degradation and hide depilation. The importance of these findings in the biotechnological application for feather and leather industries is discussed.     

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.     

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.

     

Optimal protease production condition for Prevotella ruminicola 23 and characterization of its extracellular crude protease

In this study, Prevotella ruminicola 23 (ATCC 19189), a ruminal proteolytic bacterium, was used as protease producer to examine the optimal condition for protease production. The best carbon and nitrogen sources for the maximum growth were glucose with peptone. Both sucrose and glucose could stimulate high protease production. Casein and peptone are better nitrogen sources for protease production than other choice in this study. The best enzyme production condition was 18-20 h incubation which was at late log phase in the broth of 5% glucose or sucrose as carbon source with 0.1% ammonium chloride and 0.2% peptone as nitrogen sources. Most of the protease activity was secreted into broth (65%) and on cell surface (18%). The optimal temperature and pH for protease reaction were 40 degrees C and pH 6.8, respectively. After incubation for 6h, the crude extract maintained 50% of original protease activity at 30 and 50 degrees C, and protease activity was stable between pH 6 and 8. The protease inhibitor test showed that serine, aspartic acid and metallo-protease inhibitors could cause inhibition of proteolysis. Protein feedstuff degradation experiments suggested that protease in crude extract had higher degradation ability on fish meal, whey, and feather meal (2.39, 2.60 and 1.76 micromol aminoacid/mg enzyme/h) in comparison to soybean meal and blood meal (1.11 and 1.09 micromol aminoacid/mg enzyme/h). The protease in the crude extract should have application potential in term of improving utilization of fish meal and feather meal for monogastric animals.     

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.     

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.     

Fertilizer potential of some commercially available forms of keratin and microbial biomass

The mineralization of nitrogen from citric acid waste mycelium (Aspergillus niger) and Pruteen (Methylophilus methylotrophus), and nitrogen and sulphur mineralization from keratin (feather meal) have been studied in a fertile loam soil. Ammonium nitrogen was rapidly released from the organic nitrogen sources and was subsequently nitrified. Sulphate release from feather meal was also demonstrated. Both the advantages and disadvantages of using these commercially available forms of microbial biomass and keratin as fertilizers are discussed.