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。     

Chicken feathers: a complex substrate for the co-production of α-amylase and proteases by <Emphasis Type="Italic">B. licheniformis</Emphasis> NH1

This study is concerned with the co-production of alkaline proteases and thermostable α-amylase by some feather-degrading Bacillus strains: B. mojavensis A21, B. licheniformis NH1, B. subtilis A26, B. amyloliquefaciens An6 and B. pumilus A1. All strains produced both enzymes, except B. pumilus A1, which did not exhibit amylolytic activity. The best enzyme co-production was obtained by the NH1 strain when chicken feathers were used as nitrogen and carbon sources in the fermentation medium. The higher co-production of both enzymes by B. licheniformis NH1 strain was achieved in the presence of 7.5 g/l chicken feathers and 1 g/l yeast extract. Strong catabolic repression on protease and α-amylase production was observed with glucose. Addition of 0.5% glucose to the feather medium suppressed enzyme production by B. licheniformis NH1. The growth of B. licheniformis NH1 using chicken feathers as nitrogen and carbon sources resulted in its complete degradation after 24 h of incubation at 37°C. However, maximum protease and amylase activities were attained after 30 h and 48 h, respectively. Proteolytic activity profiles of NH1 enzymatic preparation grown on chicken feather or casein-based medium are different. As far as we know, this is the first contribution towards the co-production of α-amylase and proteases using keratinous waste. Strain NH1 shows potential use for biotechnological processes involving keratin hydrolysis and industrial α-amylase and proteases co-production. Thus, the utilization of chicken feathers may result in a cost-effective process suitable for large-scale production.     

Characterization of a novel <Emphasis Type="Italic">Stenotrophomonas</Emphasis> 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°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°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.     

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.     

Application of protease from Nocardiopsis sp. as a laundry detergent additive

The application of protease as a laundry detergent additive from a newly isolated Nocardiopsis sp., isolated from a soil sample collected in Northeast Brazil is reported. The optimal pH and temperature for protease activity were pH 10.5 and 50 °C, respectively. The enzyme was stable in a long-term incubation, showed 73.5% of initial activity at pH 10.5 and 61.7% at pH 12.0 for 120 min. Approximately 60% of initial activity remained after 120 min at 50 °C or after 30 min at 80 °C. Almost 87% of enzyme activity was retained in the presence of 10% (v/v) of peroxide at 40 °C, after 1 h. The protease also was stable in the presence of oxidants and surfactants such as SDS, saponin, Tween 20 and Tween 80 after 30 min. In the presence of Omo^®, the enzyme retained 64% of its activity at 40 °C for 1 h. An increase in the proteolytic activity (6–17%) was observed with K⁺, Na⁺, and Mg⁺⁺ ions. At pH 8.0, the protease hydrolysed casein maximally (50 U/mg).     

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.     

Degradation of chicken feathers by Chrysosporium georgiae

Using a baiting technique, Chrysosporium georgiae was isolated from chicken feathers. Twenty-eight different fungal isolates were evaluated for their ability to produce keratinase enzymes using a keratin–salt agar medium containing either white chicken feathers or a prepared feather keratin suspension (KS). The Chrysosporium species were able to use keratin and grow at different rates. Chrysosporium georgiae completely degraded the added keratin after 9 days of incubation. Degradation of feathers by C. georgiae was affected by several cultural factors. Highest keratinolytic activity occurred after 3 weeks of incubation at 6 and 8~pH at 30 °C. Chrysosporium georgiae was able to degrade white chicken feathers, whereas bovine and human hair and sheep wool were not degraded and did not support fungal growth. Addition of 1% glucose to the medium containing keratin improved fungal growth and increased enzyme production. Higher keratin degradation resulted in high SH accumulation and the utilization of the carbohydrate carbon in the medium resulted in high keto-acid accumulation but decreased ammonia accumulation. Supplementation of the keratin–salt medium with minerals such as NH4Cl and MgSO4 slightly increased mycelial growth, but decreased production of extracelluar keratinase. Keratinase enzymes were very poorly produced in the absence of keratin, indicating its inducible nature. Analysis of endocellular keratinases in the mycelial homogenate indicated higher activity of intracellular keratinase as compared to the extracellular enzyme in culture filtrates. Chrysosporium georgiae was the most superior for keratinase production among the Chrysosporium species tested in the presence or absence of glucose. It produced more of the intracellular enzymes than the exocellular ones. This revised version was published online in June 2006 with corrections to the Cover Date.     

一株羽毛降解菌产酶特性的初步研究

通过富集培养从土壤中分离到一株能降解羽毛角蛋白的芽孢八叠球菌（编号为GIMN1.015）。以天然羽毛为底物,初步研究了温度、起始pH、辅助碳源以及羽毛底物含量对该菌株的蛋白酶水解活性的影响。结果表明,在羽毛发酵培养基中,菌株GIMN1.015在初始pH 11.0、温度30℃时,蛋白酶活力最强;与培养基中只含有羽毛的发酵过程相比,添加葡萄糖有利于提高蛋白酶的活性;底物浓度为1.5%时蛋白酶活性最高。本试验结果为进一步利用角蛋白降解微生物实现羽毛角蛋白的资源化利用奠定了基础。     

Production of feather hydrolysate by <Emphasis Type="Italic">Elizabethkingia meningoseptica</Emphasis> KB042 (MTCC 8360) in submerged fermentation

A keratinolytic bacterium Elizabethkingia meningoseptica KB042 was isolated from dropped off feathers. The bacterium showed 82.50 ± 0.3% feather degradation when grown on medium containing 10 g/l chicken feathers with initial pH 7.0 at 37°C, 150 rpm in 6 days. The pH of the medium was increased up to 10.02 ± 0.10 during 6 days of incubation. Soluble protein and amino acids concentration in the culture fluid was also found increased until the end of incubation. During the cultivation of strain KB042 on feather as sole source of carbon and nitrogen, the maximum cysteine release was noted on the 3rd day. Varying feather concentration 1.0–2.0% in basal medium resulted in soluble protein release between 1814.42 and 1954.61 μg/ml. The amino acid concentration was found to be maximum, i.e. 937.85 ± 11.9 μg/ml in the cultures grown with 2% feather. The hydrolysate was also found rich in essential amino acids valine, tryptophan, threonine, leucine and cysteine and contains minor amount of methionine and arginine. These data indicate a potential biotechnology for biotransformation and utilization of feather keratin as a source of protein which can be used as animal feed after successful animal trials.     

Alkaline proteases produced by <Emphasis Type="Italic">Bacillus licheniformis</Emphasis> 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.     

Biodegradation of feather waste by extracellular keratinases and gelatinases from Bacillus spp.

In this study, three feather degrading bacterial strains were isolated from agroindustrial residues from a Brazilian poultry farm. Three Gram-positive, spore-forming, rod-shaped bacteria and were identified as B. subtilis 1271, B. licheniformis 1269 and B. cereus 1268 using biochemical, physiologic and molecular methods. These Bacillus spp. strains grew and produced keratinases and peptidases using chicken feather as the sole source of nitrogen and carbon. B. subtilis 1271 degraded feathers completely after 7 days at room temperature and produced the highest levels of keratinase (446 U ml^?1). Feather hydrolysis resulted in the production of serine, glycine, glutamic acid, valine and leucine as the major amino acids. Enzymography and zymography analyses demonstrated that enzymatic extracts from the Bacillus spp. effectively degraded keratin and gelatin substrates as well as, casein, hemoglobin and bovine serum albumin. Zymography showed that B. subtilis 1271 and B. licheniformis 1269 produced peptidases and keratinases in the 15?C140 kDa range, and B. cereus produced a keratinase of ~200 kDa using feathers as the carbon and nitrogen source in culture medium. All peptidases and keratinases observed were inhibited by the serine specific peptidase inhibitor phenylmethylsulfonyl fluoride (PMSF). The optimum assay conditions of temperature and pH for keratinase activity were 40?C50°C and pH 10.0 for all strains. For gelatinases the best temperature and pH ranges were 50?C70°C and pH 7.0?C11. These isolates have potential for the biodegradation of feather wastes and production of proteolytic enzymes using feather as a cheap and eco-friendly substrate.     

Fungal Proteolytic Enzymes

Two kinds of proteolytic enzyme, tentatively named acid protease A and B which showed a single peak on electrophoresis individually, were isolated from the crude enzyme powder obtained from the broth filtrate cultured with Asper gillus niger var. macrosporus. Acid protease B is similar too the fungal acid protease previously reported, bccause the enzyme exhibits optimum activity on milk casein at about pH 2.6 and 55°C when the incubation was done at pH 2.6. Acid protease A is a new proteolytic enzyme, because the enzyme exhibits optimum activity on milk casein at about 2.0 and 70°C or 60°C when the incubation was done at pH 2.6 or 1.5 respectively.     

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.     

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.     

Characterization of a keratinolytic serine proteinase from Streptomyces pactum DSM 40530.

A serine protease from the keratin-degrading Streptomyces pactum DSM 40530 was purified by casein agarose affinity chromatography. The enzyme had a molecular weight of 30,000 and an isoelectric point of 8.5. The proteinase was optimally active in the pH range from 7 to 10 and at temperatures from 40 to 75 degrees C. The enzyme was specific for arginine and lysine at the P1 site and for phenylalanine and arginine at the P1' site. It showed a high stereoselectivity and secondary specificity with different synthetic substrates. The keratinolytic activity of the purified proteinase was examined by incubation with the insoluble substrates keratin azure, feather meal, and native and autoclaved chicken feather downs. The S. pactum proteinase was significantly more active than the various commercially available proteinases. After incubation with the purified proteinase, a rapid disintegration of whole feathers was observed. But even after several days of incubation with repeated addition of enzymes, less than 10% of the native keratin substrate was solubilized. In the presence of dithiothreitol, degradation was more than 70%.     

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     

Production of Extracellular Acid Proteases by Aspergillus Clavatus

The production of extracellular acid proteases from Aspergillus clavatus was evaluated in a culture filtrate medium, with different carbon and nitrogen sources. The fungus was cultivated at three different temperatures during 10 days. The proteolytic activity was determined on haemoglobin pH 5.0 at 37 °C. The highest acid proteolytic activity (80 U/ml) was observed in culture medium containing glucose and gelatin at 1%(w/v) at 30 °C at the third day of incubation. Cultures developed in Vogel medium with glucose at 2%(w/v) showed at about 45% of proteolytic activity when compared to the cultures with 1% of the same sugar. The optimum pH of enzymatic activity was 2.0 and the enzyme was stable at pH values ranging from 2.0 to 4.0. The optimum temperature was 40 °C and the half-lives at 40, 45 and 50 °C were 30, 10 and 5 min, respectively. This revised version was published online in July 2006 with corrections to the Cover Date.     

Feather degradation by Kocuria rosea in submerged culture

A strain of Kocuria rosea with keratinolytic activity was studied. In batch culture, the optimum temperature for feather degradation, bacterial growth and protease secretion was at 40 °C. A specific growth rate of 0.17 h⁻¹ was attained in basal medium with feathers as fermentation substrate. Under these conditions, after 36 h of incubation, biomass and caseinolytic activity reached 3.2 g/l and 0.15 U/ml, respectively. Extracellular protease secretion was associated with the exponential growth phase. In batch fermentation, feather degradation up to 51% in 72 h was obtained with a conversion yield in biomass of 0.32 g/g. No organic acids were detected in the fermentation broth in significant amount. This revised version was published online in July 2006 with corrections to the Cover Date.     

Production of alkaline protease by an alkaliphilic bacteria isolated from an alkaline soda lake

A new alkaliphilic strain of Microbacterium producing an alkaline protease was isolated from an alkaline soda lake in Ethiopia. High level of protease activity was produced in the presence of glucose and sucrose as carbon sources. The optimum temperature and pH for activity were 65°C and 9.5-11.5 respectively. Above 50°C, Ca²⁺ was required for enzyme activity and stability. At 55 and 60°C it retained 100 and 85% of its original activity respectively after 1 h incubation. The enzyme was stable over the pH range of 5-12. This revised version was published online in November 2006 with corrections to the Cover Date.