RESUMEN

Keratin is one of the major components of solid waste, and the degradation products have extensive applications in various commercial industries. Due to the complexity of the structure of keratin, especially the disulfide bonds between keratin polypeptides, keratinolytic activity is efficient with a mixture of proteins with proteases, peptidases, and oxidoreductase activity. The present work aimed to create an engineered chimeric protein with a disulfide reductase domain and a protease domain connected with a flexible linker. The structure, stability, and substrate interaction were analyzed using the protein modeling tools and codon-optimized synthetic gene cloned, expressed, and purified using Ni2+-NTA chromatography. The keratinolytic activity of the protein was at its maximum at 70 °C. The suitable pH for the enzyme activity was pH 8. While Ni2+, Mg2+, and Na+ inhibited the keratinolytic activity, Cu2+, Ca2+, and Mn2+ enhanced it significantly. Biochemical characterization of the protease domain indicated significant keratinolytic activity at 70 °C at pH 10.0 but was less efficient than the chimeric protein. Experiments using feathers as the substrate showed a clear degradation pattern in the SEM analysis. The samples collected from the degradation experiments indicated the release of proteins (2-fold) and amino acids (8.4-fold) in a time-dependent manner. Thus, the protease with an added disulfide reductase domain showed excellent keratin degradation activity and has the potential to be utilized in the commercial industries.

Asunto(s)

Péptido Hidrolasas , Proteínas Recombinantes de Fusión , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Concentración de Iones de Hidrógeno , Péptido Hidrolasas/química , Péptido Hidrolasas/metabolismo , Péptido Hidrolasas/genética , Queratinas/química , Queratinas/metabolismo , Estabilidad de Enzimas , Animales , Ingeniería de Proteínas/métodos , Temperatura , Dominios Proteicos , Modelos Moleculares , Plumas/química , Especificidad por Sustrato

RESUMEN

Microbial degradation of feathers offers potential for bioremediation, yet the microbial response mechanisms warrant additional investigation. In prior work, Pseudomonas aeruginosa Gxun-7, which demonstrated robust degradation of feathers at elevated concentrations, was isolated. However, the molecular mechanism of this degradation remains only partially understood. To investigate this, we used RNA sequencing (RNA-seq) to examine the genes that were expressed differentially in P. aeruginosa Gxun-7 when exposed to 25 g/L of feather substrate. The RNA-seq analysis identified 5571 differentially expressed genes; of these, 795 were upregulated and 603 were downregulated. Upregulated genes primarily participated in proteolysis, amino acid, and pyruvate metabolism. Genes encoding proteases, as well as those involved in sulfur metabolism, phenazine synthesis, and type VI secretion systems, were notably elevated, highlighting their crucial function in feather decomposition. Integration of Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) taxonomies, combined with a review of the literature, led us to propose that metabolic feather degradation involves environmental activation, reducing agent secretion, protease release, peptide/amino acid uptake, and metabolic processes. Sulfite has emerged as a critical activator of keratinase catalysis, while cysteine serves as a regulatory mediator. qRT-PCR assay results for 11 selected gene subset corroborated the RNA-seq findings. This study enhances our understanding of the transcriptomic responses of P. aeruginosa Gxun-7 to feather degradation and offers insights into potential degradation mechanisms, thereby aiding in the formulation of effective feather waste management strategies in poultry farming.

RESUMEN

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.

Asunto(s)

Pollos , Plumas , Animales , Plumas/metabolismo , Plumas/microbiología , Péptido Hidrolasas/metabolismo , Metaloproteasas/metabolismo , Queratinas/metabolismo , Aminoácidos/metabolismo , Concentración de Iones de Hidrógeno

RESUMEN

Feather keratin has a complex structure, hard texture and must be treated to improve its bioavailability. In this paper, according to the designability of DES, some deep eutectic solvents (DESs) were prepared to degrade feathers and extract keratin. Calculations by quantum chemical methods showed that DESs were considered molecular scissors with the ability to break initial hydrogen bonds and form new bonds only when the Gibbs free energy change for the degradation process was ΔG < 0, i.e., hydrogen binding energy ΔE < -0.3038 kcal/mol. Then, the degradation mechanism was predicted to provide guidance for the molecular design of DES. Finally, experimental results showed that the same ratio of choline chloride-based DESs had higher catalytic performance, in which [ChCl][P][ZnCl2] 1:5:2 was used with a high yield of keratin of 85.46 %. DES had a high catalytic performance after multiple recycling cycles and this method has no H2S gas generation, which improves the atomic utilization.

Asunto(s)

Disolventes Eutécticos Profundos , Plumas , Animales , Solventes/química , Queratinas , Colina/química

RESUMEN

Chicken feather (CF) has been deemed as one of the main poultry byproducts with a large amount produced globally. However, the robust chemical nature of chicken feathers has been limiting in its wide-scale utilization and valorization. The study proposed a strategy of keratin regeneration from chicken feather combining ultrasound and Cysteine (Cys)-reduction for keratin regeneration. First, the ultrasonic effect on feather degradation and keratin properties was systematically explored based on Cys-reduction. Results showed that the feather dissolution was significantly improved by increasing both ultrasonic time and power, and the former had a greater impact on keratin yield. However, the treatment time over 4 h led to a decrease of keratin yield, producing more soluble peptides, > 9.7 % of which were < 0.5 kDa. Meanwhile, prolonging time decreased the thermal stability with weight loss at a lower temperature and amino acids content (e.g., Ser, Pro and Gly) of keratin. Conversely, no remarkable damage in chemical structure and thermal stability of regenerated keratin was observed by only increasing ultrasonic power, while the keratin solubility was notably promoted and reached 745.72 mg·g-1 in NaOH (0.1 M) solution (400 W, 4 h). The regenerated keratin under optimal conditions (130 W, 2.7 h, and 15 % of Cys) possessed better solubility while without obvious damage in chemical structure, thermal stability, and amino acids composition. The study illustrated that ultrasound physically improved CF degradation and keratin solubility without nature damage and provided an alternative for keratin regeneration involving no toxic reagent, probably holding promise in the utilization and valorization of feather waste.

Asunto(s)

Plumas , Queratinas , Animales , Plumas/química , Queratinas/química , Pollos , Péptidos , Aminoácidos/análisis

RESUMEN

The aim of this present work was to explore the potential feather-degrading bacterial isolates were isolated from poultry farm soil. Isolation and screening of keratinase-producing bacterial isolates were performed in keratin agar medium. The potential keratinase-producing bacterial isolates were identified using morphological, biochemical and molecular characterization. Degradation of chicken feather was optimized using different nutrient or physical factors in feather meal broth medium. Soluble peptide, amino acid and free thiol group liberation during feather degradation were estimated too. The isolated bacterial isolates were found significantly degrading the chicken feathers with keratinase enzyme production. The present study revealed a significantly novel feather-degrading Geobacillus thermodenitrificans PS41 bacterial isolate, isolated from poultry farm soil.

Asunto(s)

Plumas , Aves de Corral , Animales , Pollos , Medios de Cultivo/metabolismo , Granjas , Plumas/química , Plumas/metabolismo , Plumas/microbiología , Geobacillus , Concentración de Iones de Hidrógeno , Péptido Hidrolasas/metabolismo , Aves de Corral/microbiología , Suelo

RESUMEN

Keratin-containing wastes become pollution to the environment if they are not treated properly. On the other hand, these wastes can be converted into value-added products applicable to many fields. Organic fertilizers and biofertilizers are important for sustainable agriculture by providing nutrients to enhance the growth speed of the plant and production. Keratin-containing wastes, therefore, will be an important resource to produce organic fertilizers. Many microorganisms exhibit capabilities to degrade keratins making them attractive to convert keratin-containing wastes into valuable products. In this review, the progress in microbial degradation of keratins is summarized. In addition, perspectives in converting keratin into bio- and organic fertilizers for agriculture are described. With proper treatment, feather wastes which are rich in keratin can be converted into high-value fertilizers to serve as nutrients for plants, reduce environmental pressure and improve the quality of the soil for sustainable agriculture.

RESUMEN

Bacteria play an important role in the biodegradation of feather waste. The exploration of the related microbial community structure and diversity is essential to improve the performance of feather waste treatment processes. In the present work, an in-situ soil sampled from a poultry farm was directly used to simulate and accelerate the natural degradation processes of feather waste under laboratory conditions, in which the dynamics of the microbial communities was further analyzed by Illumina HiSeq high-throughput 16S rRNA gene sequencing. Biochemical factors, including pH, feather degradation rate and soluble protein content were also determined in this study. The biochemical results showed that the in-situ soil exhibited an effective degradability on chicken feathers, and the degradation rate of feathers reached 57.95 ± 3.09% at 120 h of cultivation. Meanwhile, soluble protein content and pH reached 33.62 ± 1.45 mg/mL 8.99 ± 0.08, respectively. The results of bacterial diversity analysis showed that bacterial community structure and composition significantly varied in each phase of degradation. Additionally, the bacteria system with feather degradability might consist of Bacillus, Chryseobacterium, Lysobacter, Brevibacillus, and Stenotrophomonas genera. This system may include the following key pathways: carbohydrate metabolism, amino acid metabolism, nucleotide metabolism, membrane transport, replication and repair, translation, signal transduction and energy metabolism. Moreover, the bacterial communities may occur community succession during the degradation processes of chicken feathers. In summary, the present work provided valuable insights into the understanding of microbial community and metabolic functions for feather degradation, although the in-situ biodegradation process was conducted under laboratory conditions. Supplementary Information: The online version contains supplementary material available at 10.1007/s12088-021-00996-6.

RESUMEN

BACKGROUND: Feathers are the most abundant agricultural waste produced by poultry farms. The accumulation of a large number of feathers not only seriously pollutes the environment but also causes the waste of protein resources. The degradation of feather waste by keratinase-producing strains is currently a promising method. Therefore, screening high-producing keratinase strains from marine environment and studying the fermentation conditions, enzymatic properties and feather degradation mechanism are crucial for efficient degradation of feathers. RESULTS: A novel efficient feather-degrading bacteria, Gxun-17, isolated from the soil sample of a marine duck farm of Beibu Gulf in Guangxi, China, was identified as Bacillus tropicus. The optimum fermentation conditions were obtained by single factor and orthogonal tests as follows: feather concentration of 15 g/L, maltose concentration of 10.0 g/L, MgSO4 concentration of 0.1 g/L, initial pH of 7.0 and temperature of 32.5 °C. The strain completely degraded the feathers within 48 h, and the highest keratinase activity was 112.57 U/mL, which was 3.18-fold that obtained with the basic medium (35.37 U/mL). Detecting the keratinase activity and the content of sulphur-containing compounds in the fermentation products showed that the degradation of feathers by the strain might be a synergistic effect of the enzyme and sulphite. The keratinase showed optimal enzyme activity at pH 7.0 and temperature of 60 °C. The keratinase had the best performance on the casein substrate. When casein was used as the substrate, the Km and Vmax values were 15.24 mg/mL and 0.01 mg/(mL·min), respectively. Mg2+, Ca2+, K+, Co2+, Al3+, phenylmethylsulphonyl fluoride and isopropanol inhibited keratinase activity, which indicated that it was a serine keratinase. Conversely, the keratinase activity strongly increased with the addition of Mn2+ and ß-mercaptoethanol. CONCLUSIONS: A novel feather-degrading B. tropicus Gxun-17 was obtained from marine environment. The strain adapted the extreme conditions such as low temperature, high salt and high pressure. Thus, the keratinase had high activity, wide range of temperature and pH, salt tolerance and other characteristics, which had potential application value.

Asunto(s)

Caseínas , Plumas , Animales , Bacillus , Caseínas/metabolismo , Pollos/metabolismo , China , Plumas/química , Concentración de Iones de Hidrógeno , Queratinas/análisis , Queratinas/química , Queratinas/metabolismo , Péptido Hidrolasas/metabolismo , Temperatura

RESUMEN

Chryseobacterium carnipullorum 9_R23581T, isolated from raw chicken meat, was evaluated for its potential to degrade keratin found in feathers. The focus of this study was to heterologously express and characterise a keratinolytic enzyme produced by C. carnipullorum. Chryseobacterium carnipullorum secretes proteolytic enzymes that have feather degrading capabilities during its exponential growth phase. This study concluded that the most likely main component of the keratinolytic enzymes of C. carnipullorum was peptidase M64, a serine-endopeptidase with a molecular weight in crude form of 49.46 kDa. Primers were designed on the selected gene of interest, which was amplified from the genome of C. carnipullorum (accession number NZ-FRCD01000002.1). The gene coding for peptidase M64 was further cloned, propagated and expressed in E. coli BL21 [DE3] cells. Purification was by Immobilised Metal Affinity Chromatography (IMAC). The molecular weight of the keratinase was about 50 kDa after purification while its optimum temperature and pH were 50 °C and 8.5, respectively. The activity of this keratinase was inhibited by phenylmethylsulfonyl fluoride (PMSF) and it was enhanced by the presence of divalent metal ions such as Mg2+ and Ca2+. Enzyme activity was further assayed by application to chicken feathers and observed degradation was an indication of keratinolytic potential.

Asunto(s)

Proteínas Bacterianas , Chryseobacterium , Péptido Hidrolasas , Proteínas Recombinantes , Animales , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Pollos/microbiología , Chryseobacterium/enzimología , Chryseobacterium/genética , Estabilidad de Enzimas , Escherichia coli/genética , Plumas/metabolismo , Péptido Hidrolasas/química , Péptido Hidrolasas/genética , Péptido Hidrolasas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Temperatura

RESUMEN

The keratin-degrading bacterium Actinomadura viridilutea DZ50 secretes a keratinase (KERDZ) with potential industrial interest. Here, the kerDZ gene was extracellularly expressed in Escherichia coli BL21(DE3)pLysS using pTrc99A vector. The recombinant enzyme (rKERDZ) was purified and biochemically characterized. Results showed that the native and recombinant keratinases have similar biochemical characteristics. The conventional dehairing with lime and sodium sulfide degrades the hair to the extent that it cannot be recovered. Thus, these chemical processes become a major contributor to wastewater problem and create a lot of environmental concern. The complete dehairing was achieved with 2000 U/mL rKERDZ for 10 h at 40 °C. In fact, keratinase assisted dehairing entirely degraded chicken feather (45 mg) and removed wool/hair from rabbit, sheep, goat, or bovine' hides (1.6 kg) while preserving the collagen structure. The enzymatic process is the eco-friendly option that reduces biological (BOD) (50%) and chemical (COD) oxygen demands (60%) in leather processing. Consequently, the enzymatic hair removal process could solve the problem of post-treatments encountering the traditional leather processing. The enzymatic (rKERDZ) dehaired leather was analyzed by scanning electron microscopic (SEM) studies, which revealed similar fiber orientation and compactness compared with control sample. Those properties support that the rKERDZ enzyme-mediated process is greener to some extent than the traditional one.

Asunto(s)

Actinomycetales , Plumas , Actinomadura , Animales , Bovinos , Péptido Hidrolasas , Conejos , Ovinos

RESUMEN

Keratinases are valuable enzymes, given their application in keratin-rich waste recycling. Considering that keratinases usually require reducing agents to efficiently degrade keratin, improving the stability of keratinases under reducing conditions is highly desirable for practical applications. Here, we show that the introduction of several tyrosine derivatives containing para-substituted long-chain haloalkanes into the keratinase KerBL, which enabled proximity-triggered covalent crosslinking by rational design, could improve both the thermostability and autolytic resistance of the enzyme. After screening a series of noncanonical amino acid (ncAA)-based variants generated by rational design, two variants, N159C/Y260BprY and N159C/Y260BbtY, with enhanced keratinolytic activity were obtained. Both variants increased the Tm of the enzyme by approximately 10 °C. The potential mechanism underlying these improvements was investigated by molecular dynamics (MD) analysis. The results indicated that BprY-Cys and BbtY-Cys covalent bonds in the N159C/Y260TAG variant could significantly decrease the flexibility and fluctuations of the long loop (residues 151-162).

Asunto(s)

Bacillus licheniformis/enzimología , Queratinas/metabolismo , Péptido Hidrolasas/química , Péptido Hidrolasas/metabolismo , Bacillus licheniformis/genética , Secuencia de Bases , Clonación Molecular , Activación Enzimática , Péptido Hidrolasas/genética , Plásmidos/genética , Conformación Proteica , Proteolisis , Análisis de Secuencia , Relación Estructura-Actividad

RESUMEN

The increasing amount of recalcitrant keratinous wastes generated from the poultry industry poses a serious threat to the environment. Keratinase have gained much attention to convert these wastes into valuable products. Ever since primitive feathers first appeared on dinosaurs, microorganisms have evolved to degrade this most recalcitrant keratin. In this study, we identified a promising keratinolytic bacterial strain for bioconversion of poultry solid wastes. A true keratinolytic bacterium was isolated from the slaughterhouse soil and was identified and designated as Bacillus pumilus AR57 by 16S rRNA sequencing. For enhanced keratinase production and rapid keratin degradation, the media components and substrate concentration were optimized through shake flask culture. White chicken feather (1% w/v) was found to be the good substrate concentration for high keratinase production when supplemented with simple medium ingredients. The biochemical characterization reveals astounding results which makes the B. pumilus AR57 keratinase as a novel and unique protease. Optimum activity of the crude enzyme was exhibited at pH 9 and 45 °C. The crude extracellular keratinase was characterized as thermo-and-solvent (DMSO) stable serine keratinase. Bacillus pumilus AR57 showed complete degradation (100%) of white chicken feather (1% w/v) within 18 h when incubated in modified minimal medium supplemented with DMSO (1% v/v) at 150 rpm at 37 °C. Keratinase from modified minimal medium supplemented with DMSO exhibits a half-life of 4 days. Whereas, keratinase from the modified minimal medium fortified with white chicken feather (1% w/v) was stable for 3 h only. Feather meal produced by B. pumilus AR57 was found to be rich in essential amino acids. Hence, we proposed B. pumilus AR57 as a potential candidate for the future application in eco-friendly bioconversion of poultry waste and the keratinase could play a pivotal role in the detergent industry. While feather meal may serve as an alternative to produce animal feed and biofertilizers.

Asunto(s)

Bacillus pumilus/enzimología , Bacillus pumilus/genética , Péptido Hidrolasas/biosíntesis , Péptido Hidrolasas/química , Serina Proteasas/biosíntesis , Serina Proteasas/química , Álcalis/química , Aminoácidos/análisis , Animales , Bacillus pumilus/clasificación , Bacillus pumilus/crecimiento & desarrollo , Fenómenos Bioquímicos , Medios de Cultivo/química , Plumas/química , Plumas/metabolismo , Concentración de Iones de Hidrógeno , Iones/química , Queratinas/química , Queratinas/metabolismo , Péptido Hidrolasas/efectos de los fármacos , Péptido Hidrolasas/aislamiento & purificación , Aves de Corral , Inhibidores de Proteasas/farmacología , ARN Ribosómico 16S , Serina Proteasas/efectos de los fármacos , Serina Proteasas/aislamiento & purificación , Residuos Sólidos , Solventes/química , Tensoactivos/química , Temperatura , Administración de Residuos/métodos

RESUMEN

Feathers, rich in keratin, are usually the unused by-products of poultry industries. In addition, the cast-off X-ray films serve as secondary sources of silver, and the traditional method of silver extraction from this source is costly and not eco-friendly. Therefore, the current study focuses on protease production using a freshwater bacterium Chryseobacterium cucumeris SARJS-2, aiming to convert these wastes into useful products. The protease production was optimized by one-factor-at-a-time (OFAT), followed by Plackett Burman design (PBD) and response surface methodology (RSM). The protease production got enhanced by more than two folds after the statistical optimisation. Upon partial purification, the enzyme activity increased by approximately three folds. The protease was active in the range of temperatures from 25 to 75 °C, but the optimum temperature was recorded as 35 °C. The protease exhibited detergent compatibility and organic solvent stability. The detergent compatibility suggests the protease could be a detergent additive. It was also found that the presence of Fe+2 enhanced protease activity. The protease was tested for stain removal, feather degradation and silver recovery applications. It was found that the protease could efficiently remove stains of blood and tomato sauce. In addition, the protease was found to be a successful candidate for feather degradation, thereby feather-hydrolysate production which has prominent roles as nature-friendly fertilizer and animal feed ingredient. The protease also degraded gelatin from the X-ray films to release the silver-halides for silver recovery. The results recommend that the SARJS-2 protease is a potential candidate for use in eco-friendly applications in various industrial sectors.

RESUMEN

Extracellular protease Vpr (Vpr), gamma-glutamyltranspeptidase (GGT; EC 2.3.2.2) and glyoxal/methylglyoxal reductase (YvgN; EC 1.1.1.21) are extracellular enzymes involved in feather degradation, which were identified by secretome analyses from an efficient feather-degrading strain Bacillus subtilis CH-1. The encoding sequences corresponding to the three secretory enzymes were cloned into vector pET22b for recombinant expression in Escherichia coli strain BL21 (DE3). Afterward, the proteins containing the C-terminal His-tag were purified using a Ni-IDA column. The optimal temperatures and pH values for protease activity of recombinant Vpr, GGT, and YvgN were identified as 45 °C/pH 7.0, 40 °C/pH 8.0, and 50 °C/pH 6.0 respectively when casein is the substrate. Furthermore, the synergistic effects of the three enzymes were studied using feather powder as substrate. Vpr was the core enzyme to hydrolyze keratin, while GGT and YvgN were coenzymes providing reducing activities for keratin decomposition. The keratinolytic activity was enhanced to about 1.4-folds when YvgN and Vpr applied together in comparison to Vpr alone. And the keratinolytic activity almost reached to 1.5-folds when all the three enzymes were combined to use. The study provides a novel perspective of the mechanism of keratin degradation by microorganisms, and thereby may also be of relevance for the design of an industrial process for enzymatic keratin degradation; however, additional experiments must be done to substantiate this conclusion.

RESUMEN

Bacillus subtilis S1-4, isolated from chicken feather could efficiently degrade feathers by secreting several extracellular proteases. In order to get insight into the individual protease involved in keratin hydrolysis, a keratinase designed as BsKER71 was cloned and expressed in Bacillus subtilis WB600. In silico analysis revealed that BsKER71 protein contained a mature protein of 36.1 kDa. Further, purified BsKER71 could hydrolyze a variety of natural proteins, such as fibrous protein, collagen protein, casein, keratin and bovine serum albumin. In addition, this keratinase exhibited high enzyme activity in a wide range of pH and optimal pH of 10.0 and 9.0 in the hydrolysis of casein and keratin, respectively. Similarly, the optimal temperature was 55 °C and 50 °C for the hydrolysis of above two substrates, respectively. The hydrolytic activity was significantly inhibited by phenylmethanesulfonyl fluoride (PMSF), indicating the presence of serine residue in the active site. Moreover, ethylenediaminetetraacetic acid (EDTA) and phenanthroline moderately inhibited the hydrolytic activity. The catalytic activity was stimulated by Mg2+ and Ca2+, but greatly inhibited by Cu2+. Furthermore, several chemicals exhibited different effects on the hydrolysis of casein and keratin by BsKER71. These results provided a better understanding of BsKER71 from feather degrading bacterium B. subtilis S1-4.

RESUMEN

Proteinase K (PROK) from Parengyodontium album hydrolyzes keratin, a major protein component of poultry feathers, which are an inexpensive and renewable protein resource. Based on structural studies for analysis of amino acid flexibility near the catalytic center, identification of highly conserved residues, and experimental screening, we obtained a mutant R218S with residual activity 1.6-fold higher than that of PROK after incubation at 60 °C for 1 h. Molecular dynamics simulation indicated that substitution of Arg218 with Ser leads to three hydrogen bonds being introduced into the structure, stabilizing the ß-sheet in which Ser218 is located, and thus improvement of thermostability. Additionally, the mutant R218S had a 15% increase in specific activity compared to PROK and improvement in the rate and thoroughness of feather degradation compared with PROK. We confirmed the positive effects of enhancing catalytic center rigidity on enzyme thermostability, a finding which may have broad applications.

Asunto(s)

Biocatálisis , Endopeptidasa K/metabolismo , Plumas/metabolismo , Hypocreales/enzimología , Animales , Endopeptidasa K/química , Endopeptidasa K/genética , Simulación de Dinámica Molecular , Mutación , Conformación Proteica

RESUMEN

Black chicken feathers generated in large amount from poultry and slaughter houses are highly recalcitrant to microbial degradation due to their tough structural nature. A novel keratinolytic bacterium that possessed high affinity for black feather was isolated from chicken manure and identified as Pseudochrobactrum sp. IY-BUK1. Keratinase and feather soluble protein were effectively produced by the free living cells of the bacterium in media containing only black feathers and a mixture of equal amount of black-, brown- and white-coloured feathers. Complete degradation of 5 g/L of black feathers was completed in 3 days following optimisation of physico-chemical conditions. However, the bacterium selectively completed the degradation of black feather in a medium containing mixture of feathers in 144 h leaving behind approximately 33% and 45% of brown and white feathers in the medium respectively. Gellan gum-immobilised cells of strain IY-BUK1 enhanced the keratinase production by about 150% and were used repeatedly for ten cycles to degrade 5 g/L of black feather in a semi continuous fermentation of 18 h per cycle with enhanced and stable production of soluble protein. The study demonstrated the potential use of Pseudochrobactrum sp. IY-BUK1 not only in biodegradation of highly recalcitrant black feathers, but also in producing keratinase enzymes and valuable soluble proteins for possible industrial usage.

Asunto(s)

Biodegradación Ambiental , Brucellaceae/aislamiento & purificación , Plumas/metabolismo , Hidrolisados de Proteína/biosíntesis , Animales , Brucellaceae/metabolismo , Pollos , Heces/microbiología , Péptido Hidrolasas/biosíntesis , Polisacáridos Bacterianos/metabolismo

RESUMEN

Feathers from poultry industries are considered a major pollutant and its degradation is a challenging problem due to its recalcitrant nature. The high cost of energy and loss of essential amino acids by conventional methods have paved a way for an environmentally benign approach using microbial keratinolytic proteases. The widespread application of keratinolytic proteases is limited due to autolysis and denaturation of the enzyme upon storage. Immobilization overcomes these disadvantages by adsorbing the enzyme onto a solid support. Recently, electrospun nanofibers have been used due to their increased surface area and porous structure. The biocompatible and hydrophilic polyvinyl alcohol (PVA) has been blended with biodegradable chitosan for immobilization in electrospinning. The present study focuses on feather degradation by immobilized keratinolytic proteases on electrospun nanofibers. The keratinolytic protease production was enhanced by using a media containing hydrolyzed feather under optimized conditions. The immobilized keratinolytic protease on electrospun PVA chitosan (PVA-Ch) nanofibers (100-150 nm diameter) degraded the chicken feathers with 88% efficiency at the end of 72 hr.

Asunto(s)

Bacillus amyloliquefaciens/enzimología , Quitosano/química , Enzimas Inmovilizadas/química , Queratinas/química , Nanofibras/química , Péptido Hidrolasas/química , Alcohol Polivinílico/química , Animales , Pollos , Estabilidad de Enzimas , Plumas/química , Hidrólisis , Proteolisis

RESUMEN

The ability of gellan gum-immobilised cells of the heavy metal-tolerant bacterium Alcaligenes sp. AQ05-001 to utilise both heavy metal-free and heavy metal-polluted feathers (HMPFs) as substrates to produce keratinase enzyme was studied. Optimisation of the media pH, incubation temperature and immobilisation parameters (bead size, bead number, gellan gum concentration) was determined for the best possible production of keratinase using the one-factor-at-a-time technique. The results showed that the immobilised cells could tolerate a broader range of heavy metal concentrations and produced higher keratinase activity at a gellan gum concentration of 0.8% (w/v), a bead size of 3 mm, bead number of 250, pH of 8 and temperature of 30 °C. The entrapped bacterium was used repeatedly for ten cycles to produce keratinase using feathers polluted with 25 ppm of Co, Cu and Ag as substrates without the need for desorption. However, its inability to tolerate/utilise feathers polluted with Hg, Pb, and Zn above 5 ppm, and Ag and Cd above 10 ppm resulted in a considerable decrease in keratinase production. Furthermore, the immobilised cells could retain approximately 95% of their keratinase production capacity when 5 ppm of Co, Cu, and Ag, and 10 ppm of As and Cd were used to pollute feathers. When the feathers containing a mixture of Ag, Co, and Cu at 25 ppm each and Hg, Ni, Pb, and Zn at 5 ppm each were used as substrates, the immobilised cells maintained their operational stability and biological activity (keratinase production) at the end of 3rd and 4th cycles, respectively. The study indicates that HMPF can be effectively utilised as a substrate by the immobilised-cell system of Alcaligenes sp. AQ05-001 for the semi-continuous production of keratinase enzyme.