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Keratinolytic potential of Bacillus licheniformis RG1: Structural and biochemical mechanism of feather degradation
Abstract
Keratinolytic Bacillus licheniformis RG1 was used to study the mechanism of keratinolysis. Scanning electron microscopy studies revealed that bacterial cells grew closely adhered to the barbules of feathers, completely degrading them within 24 h. Biochemical studies indicated that the Bacillus strain produced an extracellular protease, which had keratinolytic potential. The extracellular keratinolytic activity (425 U) was synergistically enhanced by the addition of intracellular disulfide reductases (1712 U). However, these enzymes alone (keratinase and disulfide reductase), without live bacterial cells, failed to degrade the feather. Complete feather degradation was obtained only when living bacterial cells were present, emphasizing that bacterial adhesion plays a key role during the degradation process. The bacterial cells probably provide a continuous supply of reductant to break disulfide bridges. In addition, sulfite detected in the extracellular broth during feather degradation indicated that sulfitolysis may also play a role in feather degradation by the bacterium.
Supplementary resource (1)
Nucleotide Sequence
March 2004
... Feather keratin has a dense structure that acts as a hydrophobicity resistance barrier, and the breaking of disulfide bonds is the rate-limiting step for its degradation [24]. It has been reported that the degradation of keratins in some Bacillus species relies on extracellular proteases and sulphitolytic systems [17,37]. To verify whether sulfite is the reducing force for strain CN2 to break disulfide bonds, the effect of reducing agents on promoting the hydrolysis of feather keratin by keratinase in vitro was studied. ...
... Once T3 γ-glutamyltransferase is involved, the feather keratin is rapidly degraded in a short time (Fig. 8). This result indicates that only the combination of T3 γ-glutamyltransferase and endopeptidase can effectively degrade keratin, which is different from the keratin degradation mode of other Bacillus, which use disulfide reductase to utilize keratin [37]. ...
Background
Keratin, the main component of chicken feather, is the third most abundant material after cellulose and chitin. Keratin can be converted into high-value compounds and is considered a potential high-quality protein supplement; However, its recalcitrance makes its breakdown a challenge, and the mechanisms of action of keratinolytic proteases-mediated keratinous substrates degradation are not yet fully elucidated. Bacillus sp. CN2, having many protease-coding genes, is a dominant species in keratin-rich materials environments. To explore the degradation patterns of feather keratin, in this study, we investigated the characteristics of feather degradation by strain CN2 based on the functional-degradomics technology.
Results
Bacillus sp. CN2 showed strong feather keratin degradation activities, which could degrade native feathers efficiently resulting in 86.70% weight loss in 24 h, along with the production of 195.05 ± 6.65 U/mL keratinases at 48 h, and the release of 0.40 mg/mL soluble proteins at 60 h. The extracellular protease consortium had wide substrate specificity and exhibited excellent biodegradability toward soluble and insoluble proteins. Importantly, analysis of the extracellular proteome revealed the presence of a highly-efficient keratin degradation system. Firstly, T3 γ-glutamyltransferase provides a reductive force to break the dense disulfide bond structure of keratin. Then S8B serine endopeptidases first hydrolyze keratin to expose more cleavage sites. Finally, keratin is degraded into small peptides under the synergistic action of proteases such as M4, S8C, and S8A. Consistent with this, high-performance liquid chromatography (HPLC) and amino acid analysis showed that the feather keratin hydrolysate contained a large number of soluble peptides and essential amino acids.
Conclusions
The specific expression of γ-glutamyltransferase and co-secretion of endopeptidase and exopeptidase by the Bacillus sp. CN2 play an important role in feather keratin degradation. This insight increases our understanding of the keratinous substrate degradation and may inspire the design of the optimal enzyme cocktails for more efficient exploration of protein resources in industrial applications.
... Based on the fact, that A. oryzae hydrolysis did not provide detectable sulfite but high level of FM degradation, it can be hypothesized that A. oryzae secretes true keratinase as it is able to degrade keratin without generation of reducing chemicals like sulfite (Qiu et al., 2020), while B. licheniformis relies on sulfite assisted sulfitolysis to help its keratinases action. However, we couldn't determine also other factors that contributes to disulfide bonds reduction, like intracellular disulfide reductases and the redox systems from living and lysate cells, which are also a possibility (Alam et al., 2018) and it was previously reported for B. licheniformis (Ramnani et al., 2005). The concentration of macrominerals (Ca, Na, Mg, K, P) and macronutrients (C, N and S) in FM, control FMB without FM, and FM hydrolysates from B. licheniformis and A. oryzae after 12 days of cultivation are presented in Table 1. ...
... For example, Bockle found that fresh culture filtrate and homogenate during feather degradation by Streptomyces pactum had little or no reducing power, while washed cells had great reducing power for disulfide bonds (Bockle et al. 1997). Ramnani also found that neither keratinase nor intracellular soluble substances with disulfide-reducing activity can completely degrade feathers, and the presence of living cells is vital for complete degradation (Ramnani et al. 2005). The biological membrane potential theory relies on the activity of living cells to generate an unlimited reduction capacity to break disulfide bonds, but it is unclear how this is done. ...
Keratin is regarded as the main component of feathers and is difficult to be degraded by conventional proteases, leading to substantial abandonment. Keratinase is the only enzyme with the most formidable potential for degrading feathers. Although there have been in-depth studies in recent years, the large-scale application of keratinase is still associated with many problems. It is relatively challenging to find keratinase not only with high activity but could also meet the industrial application environment, so it is urgent to exploit keratinase with high acid and temperature resistance, strong activity, and low price. Therefore, researchers have been keen to explore the degradation mechanism of keratinases and the modification of existing keratinases for decades. This review critically introduces the basic properties and mechanism of keratinase, and focuses on the current situation of keratinase modification and the direction and strategy of its future application and modification.
Key points
•The research status and mechanism of keratinase were reviewed.
•The new direction of keratinase application and modification is discussed.
•The existing modification methods and future modification strategies of keratinases are reviewed.
Microbial keratinases have prominent potential in biotransformation of recalcitrant keratin substrates to value-added products which has made keratinases a research focus in the past decades. In this study, an efficient feather-degrading bacterium was isolated and identified as a novel species in Ectobacillus genus and designated as Ectobacillus sp. JY-23. The degradation characteristics analysis revealed that Ectobacillus sp. JY-23 could utilize chicken feathers (0.4% w/v) as the sole nutrient source and degraded 92.95% of feathers in 72 h. A significant increase in sulfite and free sulfydryl group content detected in the feather hydrolysate (culture supernatant) indicated efficient reduction of disulfide bonds, which inferred that the degradation mechanism of isolated strain was a synergetic action of sulfitolysis and proteolysis. Moreover, abundant amino acids were also detected, among which proline and glycine were the predominant free amino acids. Then, the keratinase of Ectobacillus sp. JY-23 was mined and Y1_15990 was identified as the keratinase encoding gene of Ectobacillus sp. JY-23 and designated as kerJY-23. Escherichia coli strain overexpressing kerJY-23 degraded chicken feathers in 48 h. Finally, bioinformatics prediction of KerJY-23 demonstrated that it belonged to the M4 metalloprotease family, which was a third keratinase member in this family. KerJY-23 showed low sequence identity to the other two keratinase members, indicating the novelty of KerJY-23. Overall, this study presents a novel feather-degrading bacterium and a new keratinase in the M4 metalloprotease family with remarkable potential in feather keratin valorization.
Fungal enzymes are vital for various biotechnological processes and their impact is going to be felt much more in coming years. Fungal enzymes such as proteases, amylases, lipases, pectinases and tannases are used in meat, sugar and other food industries. Fungal keratinases are the proteases, which hydrolyse hard-to-degrade keratin, with a lot of potentials whose demand is gradually increasing due to their broad specificity towards variety of insoluble keratin substrates. Biotechnological methods employing microbial keratinases play a key role in processing keratin waste. Amongst microbial diversiform, fungal sources have become more perpetual choice for keratinase production, due to their low cost, easy recovery and high enzyme activity. These are inducible enzymes, which are produced extracellularly or intracellularly primarily from dermatophytic and broad saprotrophic non-specialized ascomycetous fungi in the presence of keratinous substrates. Fungal keratinases are a great resource pool for industrial sector and sustainable environmental management, and a source of value-added products. These enzymes can cause solubilization of keratinous waste through proteolysis and sulfitolysis as well as fascinate researchers for applications in skin/hide dehairing, textile processing, detergent formulation, pharmaceutical and cosmetic industries and bioactive peptide production. Microbial degradation of keratinous waste is preferred over chemical methods since it is more specific, saves energy and produces value-added hydrolysate that can find application in food, feed and agriculture. Fungal keratinases can easily penetrate into keratinous substrate due to filamentous growth of the producer that produces high enzyme titres. This chapter focuses on the potential applications of fungal keratinases in different industries. The biotechnological conversion of keratinous waste into soluble products and subsequent application in food, feed and agroindustry would be discussed.KeywordsKeratinous wasteManagementBioremediationKeratinolytic fungiNutritional enhancement
Keratin-rich wastes generated from different anthropogenic activities especially during animal products processing, accumulate and cause serious environmental and human health problems. Different chemical and physiological treatment methods to remove these wastes generate other pollutants causing further degradation of the environment. Bio-valorisation of the wastes by some microorganisms using keratinolytic enzymes, keratinases, have shown to be safe, economic and eco-friendly method of converting keratin-rich wastes into value added products with numerous biotechnological applications. The fields of tissue medicine, pharmaceuticals, cosmetics and food industries have immensely profited from the products of keratinolysis. Recombinant technology and protein engineering further aided in keratin production and purification for future applications. Combination of valorised keratin with other components improves their quality and usage for different purposes. This article reviews the different procedures involved in bulk reduction and utilization of keratin-rich wastes for their effective management and applications.
Keratinase plays an important role in the leather tanning industry. The industry requires enzymes that are stable in a high salt environment process. One source of those enzymes is microbes that live in salt ponds. Previously, 8 halophilic isolates were isolated from Pasuruan salt pond. TG-3 and TG-6 have been characterized and tested for their ability to produce keratinase using chicken feathers with the highest activity of 5.893 U/mL and 9.685 U/mL. In this study, TG-5 isolate was characterized and tested for its ability to produce keratinase. The results exhibited the TG-5 isolate was Gram- negative with a rode shape and was not a new type of microbe because the sensitivity to the Salinivibrio proteolyticus strain DV was above 97%, which was 99.20%, and it was called Salinivibrio proteolyticus TG5. The optimum activity of TG-5 keratinase was achieved at pH 8, temperature 43 °C, 5% NaCl, and the presence of 1 mM Mg²⁺ ions. Optimization of keratinase production by SSF method showed day 3 of incubation, at pH 7, and humidity ratio of 1:4 (chicken feather: salt solution) with enzyme activity value of 6.094 U/mL. The results indicate that salinivibrio proteolyticcus TG-5 has potential in tanning leather.
Enormous amounts of keratinaceous waste make a significant and unexploited protein reserve that can be utilized through bioconversion into high-value products using microbial keratinases. This study was intended to assess the keratinase production from a newly isolated B. velezensis NCIM 5802 that can proficiently hydrolyze chicken feathers. Incubation parameters used to produce keratinase enzyme were optimized through the Response Surface Methodology (RSM) with chicken feathers as substrate. Optimization elevated the keratinase production and feather degradation by 4.92-folds (109.7 U/mL) and 2.5 folds (95.8%), respectively. Time-course profile revealed a direct correlation among bacterial growth, feather degradation, keratinase production and amino acid generation. Biochemical properties of the keratinase were evaluated, where it showed optimal activity at 60 °C and pH 10.0. The keratinase was inhibited by EDTA and PMSF, indicating it to be a serine–metalloprotease. Zymography revealed the presence of four distinct keratinases (Mr ~ 100, 62.5, 36.5 and 25 kDa) indicating its multiple forms. NMR and mass spectroscopic studies confirmed the presence of 18 free amino acids in the feather hydrolysates. Changes in feather keratin brought about by the keratinase action were studied by X-ray diffraction (XRD) and spectroscopic (FTIR, Raman) analyses, which showed a decrease in the total crystallinity index (TCI) (1.00–0.63) and confirmed the degradation of its crystalline domain. Scanning electron microscopy (SEM) revealed the sequential structural changes occurring in the feather keratin during degradation. Present study explored the use of keratinolytic potential of the newly isolated B. velezensis NCIM 5802 in chicken feather degradation and also, unraveled the underlying keratin hydrolysis mechanism through various analyses.
Feathers represent a natural source of proteins packed in a highly stable structure. Because of their recalcitrance, feathers can take considerable time to naturally decompose. To help avoid their accumulation in the environment, feather waste needs to be sustainably handled, e.g., by converting them into more valuable products through microbial processes. Here, we screened bacteria from the rhizosphere of Mimosa pudica for their ability to degrade feathers and use them as their substrate. Three candidates produced proteases that were active in a wide range of temperature (30–70 °C) and pH (5–11). One of the isolates, identified as Chryseobacterium sp. A9.9, effectively degraded feathers, yielding 45% mass shrinkage in 24 h. Laboratory adaptation of Chryseobacterium sp. A9.9 using feathers as its sole carbon and nitrogen source for 30 passages improved its ability to degrade feathers and to produce proteases with higher activity, yielding higher total protein content. In addition, because Chryseobacterium sp. A9.9 produces flexirubin, a yellow pigment with potential biomedical applications, the system may add value to the bioconversion process. The use of microorganisms from local resources and simple methods to improve their performance to degrade recalcitrant wastes such as feathers provides an attractive approach to biomass valorization.
Keratinolytic microorganisms are highly valuable for decomposition of poultry waste. this study aimed to isolate keratin-decomposing actinobacteria from poultry farm soils and examine their capacity to decompose feathers. Soil samples were placed in a basal medium with feather meal, which is a deposit of carbon and nitrogen. Nine actinobacterial strains were isolated. Actinobacteria were cultured in the media to show clear feather-decomposing potential. Actinobacterial strains were identified using 16S ribosomal RNA (rRNA) sequencing as being related to Streptomyces rochei AM8. thus, the supernatant of S. rochei AM8 exhibited keratinolytic enzyme activity. increased biodecomposition of feathers was recorded in a keratinase assay (0.782 U/ml) for separated cultures. the ability of the selected microorganisms to decompose feathers may be an effective biotechnological solution for managing feather waste from poultry.
A strain of Kocuria rosea able to secrete keratin-hydrolysing proteinases (keratinases) in submerged batch cultures with finely milled feathers as carbon and nitrogen sources was studied. The highest production of keratinases was obtained when feathers were used as the only fermentation substrate (17 U/mg). Considerably lower activity was present in cultures containing glucose and others nutrient supplements. The optimum temperature and pH for keratinolytic activity was 40 C and 10, respectively. Gelatin-sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) electrophoresis analysis showed that Kocuria rosea grown on feathers secreted at least two alkaline extracellular proteases with apparent molecular weights of 90.2 and >200 kDa, respectively. These proteolytic activities appear sequentially during microbial growth. Keratinolytic activity was strongly inhibited by phenylmethanesulphonyl fluoride (PMSF), chymostatin and crystalline soybean trypsin inhibitor, indicating the presence of serine proteases. Proteolytic enzymes derived from the biodegradation of feathers by this microorganism could be a useful biotechnological tool in the leather, food and cosmetic industries.
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 degrees 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.
Thermostable alkaline proteinase was produced by a strain of Chrysosporium keratinophilum when cultured in lactose/mineral salt medium incorporating keratin solubilized with DMSO. The proteinase, partially purified by cold-acetone precipitation followed by gel-filtration on Sephadex G-75, was optimally active at pH 9 and stable from pH 7 to 10 with over 90% relative residual activity after incubation at 25°C for 24 h. The optimum temperature for enzyme activity was 90°C at which the activity half-life was 30 min. Enzyme activity was stimulated by Fe2+ and inhibited by 1,10 o-phenanthroline. Gel-filtration indicated an M
r of 69 kDa.
Advances in microbial enzyme technology, keratinolytic proteases in this case, offer considerable opportunities for a low-energy consuming technology for bioconversion of poultry feathers from a potent pollutant to a nutritionally upgraded protein-rich feedstuff for livestock. A compendium of recent information on microbial keratinolysis in nature and infection (dermatophytoses) has been provided as underscoring feasible harnessing of the biotechnology for nutritional improvement of feathers, and as an alternative to conventional hydrothermal processing. Supporting evidence of a nutritional (amino acid) upgrading sequel to diverse microbial treatments of feathers, and positive results obtained from growth studies in rats and chicks have been presented. The paper concludes with suggestions for avenues of application of biotechnology for nutritional improvement of feather (and other keratins) as feedstuffs for livestock
In cultures of Streptomyces fradiae on wool as the only source of nutrition inorganic thiosulfate (in amounts up to 0.5 mg of Na2S2O35 H2O/ml) was formed as the final product of metabolization of sulfur from cystine of keratin proteins. The presence of thiosulfate was proved by qualitative tests and thin-layer chromatography and estimated quantitatively by spectrophotometry, titrimetry, and capillary isotachophoresis. Metabolization of organic sulfur to thiosulfate excreted into the medium is a process not yet described in microorganisms.
Two alkaline protease producing alkaliphilic bacterial strains, designated as AL-20 and AL-89, were isolated from a naturally occurring alkaline habitat. The two strains were identified as Nesternkonia sp. and Bacillus pseudofirmus, respectively. Both strains grew and produced alkaline protease using feather as the sole source of carbon and nitrogen. Addition of 0.5% glucose to the feather medium increased protease production by B. pseudofirmus AL-89 and suppressed enzyme production by Nesternkonia sp. AL-20. The enzymes from both organisms were purified to electrophoretic homogeneity following ammonium sulphate precipitation, ion exchange, hydrophobic interaction, and gel filtration chromatography. The molecular weight, determined using SDS–PAGE, was 23 kDa for protease AL-20 and 24 kDa for protease AL-89. Protease AL-20 was active in a broad pH range displaying over 90% of its maximum activity between pH 7.5 and 11.5 with a peak at pH 10. The enzyme is unique in that unlike all other microbial serine proteases known so far, it did not require Ca2+ for activity and thermal stability. Its optimum temperature for activity was at 70 °C and was stable after 1 h incubation at 65 °C both in the presence and absence of Ca2+. These properties make protease AL-20 an ideal candidate for detergent application. Protease AL-89 on the other hand require Ca2+ for activity and stability at temperature values above 50 °C. Its optimum activity was at 60 and 70 °C in the absence and presence of Ca2+, respectively. It displayed a pH optimum of 11 and retained about 70% or more of its original activity between pH 6.5 and 11. B. pseudofirmus AL-89, and the protease it produce offers an interesting potential for the enzymatic and/or microbiological hydrolysis of feather to be used as animal feed supplement.
Feather-degrading bacteria were isolated from poultry waste. Among those isolates, three strains identified as Bacillus subtilis, Bacillus pumilis and Bacillus cereus degraded feathers effectively and produced 142, 96 and 109 units of keratinolytic activities, respectively. The production of keratinolytic protease by B. pumilis and B. cereus was inducible with feathers, but B. subtilis produced the enzyme constitutively in the presence of various proteins such as casein, feather and BSA. The optimal conditions for the enzyme production by B. subtilis were 40 °C and pH 5–9, for B. pumilis 40 °C and pH 5–6 and for B. cereus 30 °C and pH 7.0. The maximum keratinolytic activities of B. subtilis and B. pumilis were 161 and 149 units/ml after 84 and 72 h of cultivation, respectively. With B. cereus, the maximum enzyme activity was 117 units/ml after 60 h of cultivation. The strains showed maximum enzyme activities in the late logarithmic growth phase or the beginning of stationary phase, and the production of soluble protein showed the same tendency as that of keratinolytic protease.
An extracellular, thiol-dependent and oxidation-stable alkaline serine protease (molecular mass 30 kDa on sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE)) from Bacillus mojavensis was purified to homogeneity with 17-fold purification as unbound fractions using a single step anion exchange chromatography on fast flow Q-sepharose column pre-equilibrated with buffer of pH 10.5. The N-terminal sequence of first 15 amino acids of purified protease showed 91% similarity with other subtilisins and alkaline proteases. The enzyme exhibited pH and temperature optima of 10.5 and 60 °C, respectively, and was stable between a wide pH range of 7.0 and 11.5 for 48 h. The half-lives of protease at 60, 65, and 70 °C were 150, 15 and 7 min, respectively. Various stabilizers and additives stabilized protease for more than 4 h at 60 °C, and 45 min at 65 °C. Specific protease inhibitors, such as phenylmethyl sulfonyl fluoride (PMSF), bestatin, chymostatin, iodoacetic acid, and N-bromosuccinimide completely inhibited the enzyme activity, whereas, the enzyme activity was increased up to more than two-fold in presence of 2-mercaptoethanol, glutathione, and dithiothreitol, suggesting it to be a thiol-dependent serine protease. Among metal ions, Cu2+ and Mn2+ ions increased enzyme activity up to 36%. The enzyme was also stable towards several commercially available laboratory bleaches (H2O2, sodium perborate), surfactants (tweens, Triton X-100, sodium choleate), and other commercial detergents used in laundries. The protease hydrolyzed several native proteinacious substrates, such as gelatin, elastin, albumin, haemoglobin, and skim milk, thus making it suitable for its use as an effective detergent additive. Wash performance analysis of enzyme revealed that it could effectively remove a variety of stains, such as blood, beetle and grass most effectively at 60 °C.