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1.
Braz. j. biol ; 84: e257473, 2024. tab, graf, ilus
Artigo em Inglês | VETINDEX | ID: biblio-1374639

RESUMO

Feathers make up 7% of the total weight of adult chickens and keratin protein makes up 85% of the feathers. Today, the keratinase enzymes of some Bacillus strains are used to degrade and process raw keratin waste for animal and poultry feed. According to various studies, the probiotic properties of some spore-shaped Bacillus have also been proven. The study aimed to isolation of the keratinolytic Bacillus bacteria that they have probiotic properties for using in the livestock and poultry feed industry. We were able to isolate 8 strains of Bacillus licheniformis with kreatin degrading properties from the soil of Baharan chicken slaughterhouse (Qom city, Iran) applying heat shock, alcohol- and keratin-rich culture medium, and after microscopic and biochemical analysis, 16S rDNA gene was isolated. The measurement results of keratinase activity showed that the three strains of Bacillus licheniformis pvkr6, pvkr 15, and pvkr41 had the highest activity with 124.08, 101.1, and 100.18 U/ml. The results of probiotic properties evaluation also revealed that among all the isolates, only Bacillus licheniformis pvkr15 and Bacillus licheniformis PTCC 1595 (positive control) were γ-hemolytic strains. The percentage of surface hydrophobicity of the strains was obtained from 3.27 to 30.57. It was also shown that, on average, all the strains had acceptable susceptibility to the tested antibiotics except penicillin G. Bacillus licheniformis pvkr15 with highest keratinase activity (101.1U/ml) was considered an optional probiotics due to its abilities such as (biofilm formation, being safe cause of γ-hemolytic activity, high susceptibility to antibiotics such as streptomycin, gentamicin, cefixime, amoxicillin, tetracycline, vancomycin, erythromycin and having a moderate hydrophilic (hydrophobicity: 19.09%), high survivability in pH 2, 2.5 and 3, strong resistance to bile salts and moderate antagonistic activity against pathogenic bacterium like Proteus mirabilis and the ability to grow under anaerobic conditions). By using this strain, after hydrolysis of keratin protein in the feather structure, to replace part of the protein of livestock and poultry feed, not only is no need to separate bacteria from the feed, but also the strain play role of an useful and effective additive in animal growth.


As penas representam 7% do peso total das galinhas adultas e a proteína de queratina compõe 85% das penas. Hoje, as enzimas queratinase de algumas cepas de Bacillus são usadas para degradar e processar resíduos de queratina brutos para alimentação de animais e aves. De acordo com vários estudos, as propriedades probióticas de alguns Bacillus em forma de esporos também foram comprovadas. O estudo teve como objetivo o isolamento das bactérias queratinolíticas Bacillus que possuem propriedades probióticas para uso na indústria de ração animal e avícola. Conseguimos isolar 8 cepas de Bacillus licheniformis com propriedades degradantes de creatina do solo do abatedouro de frangos de Baharan (cidade de Qom, Irã) aplicando choque térmico, meio de cultura rico em álcool e queratina e, após análise microscópica e bioquímica, o gene 16S rDNA foi isolado. Os resultados da medição da atividade da queratinase mostraram que as três cepas de Bacillus licheniformis pvkr6, pvkr15 e pvkr41 tiveram a maior atividade com 124,08, 101,1 e 100,18 U/ml. Os resultados da avaliação das propriedades probióticas também revelaram que dentre todos os isolados apenas Bacillus licheniformis pvkr15 e Bacillus licheniformis PTCC 1595 (controle positivo) eram cepas γ-hemolíticas. A porcentagem de hidrofobicidade superficial das cepas foi obtida de 3,27 a 30,57. Também foi demonstrado que, em média, todas as cepas apresentaram suscetibilidade aceitável aos antibióticos testados, exceto penicilina G. Bacillus licheniformis pvkr15 com maior atividade de queratinase (101,1U/ml) foi considerado um probiótico opcional devido às suas habilidades como formação de biofilme, sendo causa segura de atividade γ-hemolítica, alta suscetibilidade a antibióticos como estreptomicina, gentamicina, cefixima, amoxicilina, tetraciclina, vancomicina, eritromicina e ter uma hidrofílica moderada (hidrofobicidade: 19,09%), alta capacidade de sobrevivência em pH 2, 2,5 e 3, forte resistência aos sais biliares e atividade antagonista moderada contra bactérias patogênicas como Proteus mirabilis e a capacidade de crescer em condições anaeróbicas. Ao utilizar esta cepa, após a hidrólise da proteína queratina na estrutura da pena, para substituir parte da proteína da ração de gado e aves, não só não há necessidade de separar as bactérias da ração, mas também a cepa desempenha um papel útil e eficaz aditivo no crescimento animal.


Assuntos
Animais , Solo , Resíduos , Probióticos , Bacillus licheniformis , Queratinas , Ração Animal
2.
Braz. J. Biol. ; 83: 1-8, 2023. graf
Artigo em Inglês | VETINDEX | ID: vti-765419

RESUMO

Poultry industry is expanding rapidly and producing million tons of feather waste annually. Massive production of keratinaceous byproducts in the form of industrial wastes throughout the world necessitates its justified utilization. Chemical treatment of keratin waste is proclaimed as an eco-destructive approach by various researchers since it generates secondary pollutants. Keratinase released by a variety of microbes (bacteria and fungi) can be used for the effective treatment of keratin waste. Microbial degradation of keratin waste is an emerging and eco-friendly approach and offers dual benefits, i.e., treatment of recalcitrant pollutant (keratin) and procurement of a commercially important enzyme (keratinase). This study involves the isolation, characterization, and potential utility of fungal species for the degradation of chicken-feather waste through submerged and solid-state fermentation. The isolated fungus was identified and characterized as Aspergillus (A.) flavus. In a trial of 30 days, it was appeared that 74 and 8% feather weight was reduced through sub-merged and solid-state fermentation, respectively by A. flavus. The pH of the growth media in submerged fermentation was changed from 4.8 to 8.35. The exploited application of keratinolytic microbes is, therefore, recommended for the treatment of keratinaceous wastes to achieve dual benefits of remediation.(AU)


A indústria avícola está se expandindo rapidamente e produzindo milhões de toneladas de resíduos de penas anualmente. A produção massiva de subprodutos queratinosos na forma de resíduos agrícolas e industriais em todo o mundo exige sua utilização justificada. O tratamento químico de resíduos de queratina é proclamado como uma abordagem ecodestrutiva por vários pesquisadores, uma vez que gera poluentes secundários. A queratinase liberada por uma variedade de micróbios (bactérias e fungos) pode ser usada para o tratamento eficaz de resíduos de queratina. A degradação microbiana de resíduos de queratina é uma abordagem emergente e ecológica e oferece benefícios duplos, ou seja, tratamento de poluente recalcitrante (queratina) e obtenção de uma enzima comercialmente importante (queratinase). Este estudo envolve o isolamento, caracterização e utilidade potencial de espécies de fungos para a degradação de resíduos de penas de frango por meio da fermentação submersa e em estado sólido. O fungo isolado foi identificado e caracterizado como Aspergillus (A.) flavus. Em um ensaio de 30 dias, constatou-se que 74% e 8% do peso das penas foram reduzidos por A. flavus, respectivamente, por meio da fermentação submersa e em estado sólido. O pH do meio de crescimento em fermentação submersa foi alterado de 4,8 para 8,35. A aplicação explorada de micróbios queratinolíticos é, portanto, recomendada para o tratamento de resíduos ceratinosos para obter benefícios duplos de remediação.(AU)


Assuntos
Aspergillus flavus/isolamento & purificação , Queratinas/análise , Queratinas/toxicidade , Biotransformação
3.
Braz. j. biol ; 83: e246389, 2023. graf
Artigo em Inglês | LILACS, VETINDEX | ID: biblio-1285638

RESUMO

Abstract Poultry industry is expanding rapidly and producing million tons of feather waste annually. Massive production of keratinaceous byproducts in the form of industrial wastes throughout the world necessitates its justified utilization. Chemical treatment of keratin waste is proclaimed as an eco-destructive approach by various researchers since it generates secondary pollutants. Keratinase released by a variety of microbes (bacteria and fungi) can be used for the effective treatment of keratin waste. Microbial degradation of keratin waste is an emerging and eco-friendly approach and offers dual benefits, i.e., treatment of recalcitrant pollutant (keratin) and procurement of a commercially important enzyme (keratinase). This study involves the isolation, characterization, and potential utility of fungal species for the degradation of chicken-feather waste through submerged and solid-state fermentation. The isolated fungus was identified and characterized as Aspergillus (A.) flavus. In a trial of 30 days, it was appeared that 74 and 8% feather weight was reduced through sub-merged and solid-state fermentation, respectively by A. flavus. The pH of the growth media in submerged fermentation was changed from 4.8 to 8.35. The exploited application of keratinolytic microbes is, therefore, recommended for the treatment of keratinaceous wastes to achieve dual benefits of remediation.


Resumo A indústria avícola está se expandindo rapidamente e produzindo milhões de toneladas de resíduos de penas anualmente. A produção massiva de subprodutos queratinosos na forma de resíduos agrícolas e industriais em todo o mundo exige sua utilização justificada. O tratamento químico de resíduos de queratina é proclamado como uma abordagem ecodestrutiva por vários pesquisadores, uma vez que gera poluentes secundários. A queratinase liberada por uma variedade de micróbios (bactérias e fungos) pode ser usada para o tratamento eficaz de resíduos de queratina. A degradação microbiana de resíduos de queratina é uma abordagem emergente e ecológica e oferece benefícios duplos, ou seja, tratamento de poluente recalcitrante (queratina) e obtenção de uma enzima comercialmente importante (queratinase). Este estudo envolve o isolamento, caracterização e utilidade potencial de espécies de fungos para a degradação de resíduos de penas de frango por meio da fermentação submersa e em estado sólido. O fungo isolado foi identificado e caracterizado como Aspergillus (A.) flavus. Em um ensaio de 30 dias, constatou-se que 74% e 8% do peso das penas foram reduzidos por A. flavus, respectivamente, por meio da fermentação submersa e em estado sólido. O pH do meio de crescimento em fermentação submersa foi alterado de 4,8 para 8,35. A aplicação explorada de micróbios queratinolíticos é, portanto, recomendada para o tratamento de resíduos ceratinosos para obter benefícios duplos de remediação.


Assuntos
Animais , Galinhas , Plumas , Fermentação , Fungos , Resíduos Industriais , Queratinas/metabolismo
4.
Braz. j. biol ; 83: e248026, 2023. tab, graf
Artigo em Inglês | LILACS-Express | VETINDEX | ID: biblio-1374638

RESUMO

Abstract Poultry industry is amongst highly developed industries of Pakistan, fulfilling the protein demand of rapidly increasing population. On the other hand, the untreated poultry waste is causing several health and environmental problems. The current study was designed to check the potential of keratinolytic fungal species for the conversion of chicken-feather waste into biofortified compost. For the purpose, three fungal species were isolated from soil samples. These strains were pure cultured and then characterized phenotypically and genotypically. BLAST searches of 18S rDNA nucleotide sequence of the fungal isolates revealed that the two fungal isolates belonged to genus Aspergillus and one belonged to genus Chrysosporium. Optimum temperature for Aspergillus flavus, Aspergillus niger and Chrysosporium queenslandicum was 29, 26 and 25 oC, respectively. A. flavus showed maximum (53%) feather degradation, A. niger degraded feather waste up to 37%, while C. queenslandicum showed 21% keratinolytic activity on chicken feathers at their respective temperature optima. The degradation potential of these fungal species showed their ability to form compost that has agro-industrial importance.


Resumo A indústria avícola está entre as indústrias altamente desenvolvidas do Paquistão, atendendo a demanda de proteína da população em rápido crescimento. Por outro lado, os resíduos de aves não tratados estão causando diversos problemas de saúde e ambientais. O presente estudo foi desenhado para verificar o potencial de espécies de fungos queratinolíticos para a conversão de resíduos de penas de frango em composto biofortificado. Para tanto, três espécies de fungos foram isoladas de amostras de solo. Essas cepas foram cultivadas puramente e, em seguida, caracterizadas fenotipicamente e genotipicamente. As pesquisas do BLAST da sequência de nucleotídeos do rDNA 18S dos isolados de fungos revelaram que os dois isolados de fungos pertenciam ao gênero Aspergillus e um pertencia ao gênero Chrysosporium. A temperatura ótima para Aspergillus flavus, Aspergillus niger e Chrysosporium queenslandicum foi de 29, 26 e 25 oC, respectivamente. A. flavus apresentou degradação máxima de penas (53%), A. niger degradou resíduos de penas em até 37%, enquanto C. queenslandicum apresentou 21% de atividade queratinolítica em penas de frango em suas respectivas temperaturas ótimas. O potencial de degradação dessas espécies de fungos mostrou sua capacidade de formar composto de importância agroindustrial.

5.
BMC Biotechnol ; 22(1): 26, 2022 09 08.
Artigo em Inglês | MEDLINE | ID: mdl-36076195

RESUMO

BACKGROUND: With the growing concern for the environment, there are trends that bio-utilization of keratinous waste by keratinases could ease the heavy burden of keratinous waste from the poultry processing and leather industry. Especially surfactant-stable keratinases are beneficial for the detergent industry. Therefore, the production of keratinase by Bacillus cereus YQ15 was improved; the characterization and use of keratinase in detergent were also studied. RESULTS: A novel alkaline keratinase-producing bacterium YQ15 was isolated from feather keratin-rich soil and was identified as Bacillus cereus. Based on the improvement of medium components and culture conditions, the maximum keratinase activity (925 U/mL) was obtained after 36 h of cultivation under conditions of 35 °C and 160 rpm. Moreover, it was observed that the optimal reacting temperature and pH of the keratinase are 60 °C and 10.0, respectively; the activity was severely inhibited by PMSF and EDTA. On the contrary, the keratinase showed remarkable stability in the existence of the various surfactants, including SDS, Tween 20, Tween 60, Tween 80, and Triton X-100. Especially, 5% of Tween 20 and Tween 60 increased the activity by 100% and 60%, respectively. Furtherly, the keratinase revealed high efficiency in removing blood stains. CONCLUSION: The excellent compatibility with commercial detergents and the high washing efficiency of removing blood stains suggested its suitability for potential application as a bio-detergent additive.


Assuntos
Bacillus cereus , Detergentes , Animais , Bacillus cereus/metabolismo , Detergentes/química , Estabilidade Enzimática , Plumas/metabolismo , Concentração de Íons de Hidrogênio , Queratinas/metabolismo , Peptídeo Hidrolases/metabolismo , Polissorbatos , Tensoativos , Temperatura
6.
Arch Microbiol ; 204(9): 565, 2022 Aug 18.
Artigo em Inglês | MEDLINE | ID: mdl-35982264

RESUMO

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.


Assuntos
Plumas , Aves Domésticas , Animais , Galinhas , Meios de Cultura/metabolismo , Fazendas , Plumas/química , Plumas/metabolismo , Plumas/microbiologia , Geobacillus , Concentração de Íons de Hidrogênio , Peptídeo Hidrolases/metabolismo , Aves Domésticas/microbiologia , Solo
7.
Front Microbiol ; 13: 918262, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35794912

RESUMO

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.

8.
Sci Total Environ ; 845: 157161, 2022 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-35817113

RESUMO

Keratinase-catalyzed degradation of keratin waste has been shown to be a promising recycling method. Although the recombinant KerZ1 derived from Bacillus subtilis has shown the highest activity among the keratinases reported so far, the low thermal stability caused by the unstable flexible loops limited its keratin-degrading ability. To this end, the flexible loops of KerZ1 were engineered to be more hydrophobic and rigid through B-factor calculations, molecular dynamics simulations, and ß-turn redesign. We developed several highly thermostable keratinase variants and showed enhanced keratin degradation activity. In particular, the loop regions of the variants KerZ1A128D/L240N, KerZ1T77E/L240N and KerZ1T77C/A128D were designed to be more stable, with Tm values increased by 8 °C, 6 °C and 5 °C, and corresponding t1/2 increased by 2.3, 3.3 and 5.0 times. The keratin degradation activity of the variant KerZ1T77C/A128D at 60 °C was enhanced by 46 % compared with KerZ1WT. The strategy of this research and the obtained keratinase variants will be a significant improvement in the complete degradation of keratin.


Assuntos
Queratinas , Peptídeo Hidrolases , Bacillus subtilis/metabolismo , Concentração de Íons de Hidrogênio , Peptídeo Hidrolases/química , Peptídeo Hidrolases/metabolismo
9.
J Genet Eng Biotechnol ; 20(1): 81, 2022 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-35612674

RESUMO

BACKGROUND: Antarctica is one of the harshest environments in the world. Despite this fact, it has been colonized by microorganisms, which had to develop different adaptations in order to survive. By studying their enzymes, we can harness these adaptations in order to use them in various industrial processes. Keratinases (E.C. 3.4.99.11) are characterized by their robustness in withstanding extreme conditions and, along with other enzymes, are commonly added to laundry detergents, which makes their study of industrial interest. RESULTS: In this work, a novel keratinase producer, Pedobacter sp. 3.14.7 (MF 347939.1), isolated from Antarctic birds' nests, was identified. This psychrotolerant isolate displays a typical psychrotolerant growth pattern, with an optimal temperature of 20 °C (µmax=0.23 h-1). After 238 h, maximum proteolytic (22.00 ± 1.17 U ml-1) and keratinolytic (33.04 ± 1.09 U ml-1) activities were achieved with a feather sample conversion of approximately 85%. The keratinase present in crude extract was characterized as a metalloprotease with a molecular weight of 25 kDa, stable in a wide range of pH, with an optimum pH of 7.5. Optimum temperature was 55 °C. Wash performance at 20 °C using this crude extract could remove completely blood stain from cotton cloth. CONCLUSION: We report a new keratinolytic bacteria from maritime Antarctica. Among its biochemical characteristics, its stability in the presence of different detergents and bleaching agents and its wash performance showed promising results regarding its potential use as a laundry detergent additive.

10.
Poult Sci ; 101(6): 101913, 2022 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-35525153

RESUMO

The objective of this study was to assess the effects of dietary supplementation of keratinase on the production of broilers fed a diet containing feather meal. A total of 162 1-d-old Cobb 500 male broiler (n = 9 cages/diet with 6 chicks/cage) were randomly allocated to 3 dietary treatments. The broilers were fed a corn-soybean-feather meal based diet (BD), or BD supplemented with keratinase at 100,000 or 200,000 U/kg for 6 weeks. Compared to the control, dietary supplementation with 200,000 U/kg keratinase increased (P < 0.05) body weight gain (3.6-4.3%) and reduced feed conversion ratio (2.4-5.6%) during the various experimental periods, and also improved (P < 0.05) apparent total tract digestibility of ash and calcium by 45.0% and 8.8%, respectively. Meanwhile, dietary supplementation of keratinase at 100,000 U/kg reduced (P < 0.05) the drip loss (29.2%), while 200,000 U/kg keratinase supplementation increased (P < 0.05) the pH value (1.6%) at 45 min and decreased (P < 0.05) the lightness (L* value; 13.6%) and drip loss (22.1%) of pectoral muscle. Moreover, dietary supplementation of keratinase at both levels of 100,000 and 200,000 U/kg increased (P < 0.05) Glutathione peroxidase activity (82.5-87.5%) and decreased the Malondialdehyde concentration (14.5-18.3%) in the pectoral muscle. In conclusion, dietary supplementation of keratinase at 200,000 U/kg can improve the performance, meat quality, apparent total tract digestibility of nutrients, and redox status of broiler chickens fed a diet containing feather meal.


Assuntos
Fenômenos Fisiológicos da Nutrição Animal , Galinhas , Ração Animal/análise , Animais , Galinhas/fisiologia , Dieta/veterinária , Suplementos Nutricionais , Plumas , Masculino , Carne/análise , Oxirredução , Peptídeo Hidrolases
11.
Front Microbiol ; 13: 794738, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35359724

RESUMO

Wool keratin is difficult to degrade as comparing to feathers because of its tough secondary structure. In order to develop an approach for high-value utilization of wool fiber waste by keratinolytic microorganisms, which is produced from shearing, weaving, and industrial processing of wool, screening of wool-degrading bacterium with high degradation efficiency were performed in this study. To this end, Lysobacter brunescens YQ20 was identified and characterized. The optimized conditions for wool degradation were pH 9.0 and 37°C with 20% liquid volume of Erlenmeyer flask. After fermentation, 15 essential amino acids were detected when wool fiber waste was fermented. The total amino acids produced from 1% wool per hour were 13.7 mg/L. The concentration was 8.6-fold higher than that produced by the strain Stenotrophomonas maltophilia BBE11-1, which had previously been reported to have the highest wool-degrading capacity. Our study reports the first Lysobacter strain that exhibits efficient wool degradation and yields higher concentrations of amino acids than previously reported strains. Whole-genome sequencing indicated that there were 18 keratinase-like genes in the genome of YQ20, which exhibited a long evolutionary distance from those of Bacillus. Therefore, L. brunescens YQ20 may have applications in the environmentally friendly management of wool waste as fertilizer in agriculture.

12.
BMC Biotechnol ; 22(1): 11, 2022 03 20.
Artigo em Inglês | MEDLINE | ID: mdl-35307009

RESUMO

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.


Assuntos
Caseínas , Plumas , Animais , Bacillus , Caseínas/metabolismo , Galinhas/metabolismo , China , Plumas/química , Concentração de Íons de Hidrogênio , Queratinas/análise , Queratinas/química , Queratinas/metabolismo , Peptídeo Hidrolases/metabolismo , Temperatura
13.
3 Biotech ; 12(4): 90, 2022 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-35330961

RESUMO

The poultry industry produces millions of tons of feathers waste that can be transformed into valuable products through bioprocess. The study describes the enhanced keratinase and feather hydrolysate production by Bacillus subtilis AMR. The metabolism of each microorganism is unique, so optimization tools are essential to determine the best fermentation parameters to obtain the best process performance. The evaluation of different propagation media indicated the constitutive production of two keratinases of approximately 80 kDa. The combination of Mn2+, Ca2+, and Mg2+ at 0.5 mM improved the keratinolytic activity and feather degradation 1.5-fold, while Cu2+ inhibited the enzymatic activity completely. Replace yeast extract for sucrose increased the feather hydrolysate production three times. The best feather concentration for hydrolysate production was 1.5% with an inoculum of 108 CFU/mL and incubation at 30 °C. None of the inorganic additional nitrogen sources tested increased hydrolysate production, although (NH4)2SO4 and KNO3 improved enzymatic activity. The optimization process improved keratinolytic activity from 205.4 to 418.7 U/mL, the protein concentration reached 10.1 mg/mL from an initial concentration of 3.9 mg/mL, and the feather degradation improved from 70 to 96%. This study characterized keratinase and feather hydrolysate production conditions offering valuable information for exploring and utilizing AMR keratinolytic strain for feather valorization. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-022-03153-y.

14.
Biology (Basel) ; 11(2)2022 Feb 04.
Artigo em Inglês | MEDLINE | ID: mdl-35205110

RESUMO

Environmental safety and economic factors necessitate a search for new ways of processing poultry farm feathers, which are 90% ß-keratin and can be used as a cheap source of amino acids and peptones. In this study, feather-decomposing bacteria were isolated from a site of accumulation of rotten feathers and identified as Bacillus. Among them, the Bacillus sp. A5.3 isolate showed the best keratinolytic properties. Scanning electron microscopy indicated that Bacillus sp. A5.3 cells closely adhere to the feather surface while degrading the feather. It was found that Bacillus sp. A5.3 secretes thermostable alkaline proteolytic and keratinolytic enzymes. Zymographic analysis of the enzymatic extract toward bovine serum albumin, casein, gelatin, and ß-keratin revealed the presence of proteases and keratinases with molecular weights 20-250 kDa. The proteolytic and keratinolytic enzymes predominantly belong to the serine protease family. Proteome analysis of the secreted proteins by nano-HPLC coupled with Q-TOF mass spectrometry identified 154 proteins, 13 of which are proteases and peptidases. Thus, strain Bacillus sp. A5.3 holds great promise for use in feather-processing technologies and as a source of proteases and keratinases.

15.
N Biotechnol ; 68: 19-27, 2022 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-35032710

RESUMO

Keratinases are proteases that can catalyze the degradation of insoluble keratinous biomass. Keratinases in protease family M36 (MEROPS database) are endo-acting proteases. In total, 687 proteases are classified in family M36. In the present study, new keratinolytic enzymes were identified in protease family M36 using the bioinformatics tool Conserved Unique Peptide Patterns (CUPP). Via CUPP, M36 family members were classified into 11 groups, with CUPP group 1 containing the three currently known and sequenced family M36 keratinases (derived from the fungi Fusarium oxysporum, Microsporum canis and Onygena corvina) as well as an additional 71 uncharacterized M36 proteases. In order to assess the relevance of CUPP group 1 categorization to keratinolytic function, four uncharacterized M36 proteases and the known keratinase from F. oxysporum (in CUPP group 1) were selected for recombinant expression and keratinolytic activity assessment. The four hitherto unknown M36 proteases were from Phaeosphaeria nodorum, Aspergillus clavatus, Pseudogymnoascus pannorum and Nectria haematococca, and represent four different fungal taxonomical classes. The genes encoding the selected M36 proteases were individually expressed in Pichia pastoris and all proteases displayed keratinase activity on keratin azure. Additionally, the activity on different keratinase substrates, optimal reaction conditions and thermal stability were determined for the two most active new keratinases. The results validate the applicability of CUPP for function-based discovery of non-characterized keratinases and present new robust keratinases for potential use in keratin upgrading.


Assuntos
Biologia Computacional , Fungos/enzimologia , Peptídeo Hidrolases , Endopeptidases , Queratinas , Peptídeo Hidrolases/metabolismo
16.
Microorganisms ; 10(1)2022 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-35056542

RESUMO

In this study, five keratinolytic bacteria were isolated from poultry farm waste of Eastern Province, Saudi Arabia. The highest keratinase activity was obtained at 40-45 °C, pH 8-9, feather concentration 0.5-1%, and using white chicken feather as keratin substrate for 72 h. Enhancement of keratinase activity through physical mutagen UV radiation and/or chemical mutagen ethyl methanesulfonate (EMS) resulted in five mutants with 1.51-3.73-fold increased activity over the wild type. When compared with the wild type, scanning electron microscopy validated the mutants' effectiveness in feather degradation. Bacterial isolates are classified as members of the S8 family peptidase Bacillus cereus group based on sequence analysis of the 16S rRNA and keratinase genes. Interestingly, keratinase KerS gene shared 95.5-100% identity to keratinase, thermitase alkaline serine protease, and thermophilic serine protease of the B. cereus group. D137N substitution was observed in the keratinase KerS gene of the mutant strain S13 (KerS13uv+ems), and also seven substitution variations in KerS26 and KerS26uv of strain S26 and its mutant S26uv. Functional analysis revealed that the subtilisin-like serine protease domain containing the Asp/His/Ser catalytic triad of KerS gene was not affected by the predicted substitutions. Prediction of physicochemical properties of KerS gene showed instability index between 17.5-19.3 and aliphatic index between 74.7-75.7, which imply keratinase stability and significant thermostability. The docking studies revealed the impact of substitutions on the superimposed structure and an increase in binding of mutant D137N of KerS13uv+ems (affinity: -7.17; S score: -6.54 kcal/mol) and seven mutants of KerS26uv (affinity: -7.43; S score: -7.17 kcal/mol) compared to the wild predicted structure (affinity: -6.57; S score: -6.68 kcal/mol). Together, the keratinolytic activity, similarity to thermostable keratinases, and binding affinity suggest that keratinases KerS13uv+ems and KerS26uv could be used for feather processing in the industry.

17.
Bioresour Technol ; 346: 126513, 2022 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-34890818

RESUMO

Advances in microbial enzyme technology offer a significant opportunity for developing low-energy bioconversion solutions for industrial wastes as inexpensive feedstocks for useful products. In this short communication, two agro-food industrial wastes, chicken feather powder (CFP) and okara, were converted into peptides by a Bacillus licheniformis mutant using solid-state fermentation (SSF). The optimum SSF conditions for okara to CFP ratio, inoculum size, and time were 0.7 (7:10), 15%, and 90 h, respectively, which produced 185.99 mg/g peptides, with 910.12 U/g keratinase activity and 85.03% antioxidant scavenging activity. Compared to okara, CFP with mutant strain showed 11.28% higher keratinase activity and produced higher amounts of peptides (5.51%).


Assuntos
Bacillus licheniformis , Bacillus , Bacillus/genética , Bacillus licheniformis/genética , Fermentação , Resíduos Industriais , Peptídeos
18.
Sci Total Environ ; 818: 151824, 2022 Apr 20.
Artigo em Inglês | MEDLINE | ID: mdl-34808176

RESUMO

Keratinase has shown great significance and application potentials in the biodegradation and recycle of keratin waste due to its unique and efficient hydrolysis ability. However, the inherent instability of the enzyme limits its practical utilization. Herein, we obtained a thermostability-enhanced keratinase based on a combination of bioinformatics analysis and rational design strategies for the efficient biodegradation of feathers. A systematical in silico analysis combined with filtering of virtual libraries derived a smart library for experimental validation. Synergistic mutations around the highly flexible loop, the calcium binding site and the non-consensus amino acids generated a dominant mutant which increased the optimal temperature of keratinase from 40 °C to 60 °C, and the half-life at 60 °C was increased from 17.3 min to 66.1 min. The mutant could achieve more than 66% biodegradation of 50 g/L feathers to high-valued keratin product with a major molecular weight of 36 kDa. Collectively, this work provided a promising keratinase variant with enhanced thermostability for efficient conversion of keratin wastes to valuable products. It also generated a general strategy to facilitate enzyme thermostability design which is more targeted and predictable.


Assuntos
Biologia Computacional , Plumas , Animais , Plumas/química , Queratinas/química , Peptídeo Hidrolases/química , Peptídeo Hidrolases/genética , Peptídeo Hidrolases/metabolismo , Temperatura
19.
Appl Biochem Biotechnol ; 194(4): 1546-1565, 2022 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-34806139

RESUMO

Keratinase is one of the important proteases, which is widely used for converting keratin of the keratinaceous materials into various value-added products. In this study, a popular keratinase producer, Bacillus licheniformis PWD-1, was exposed to ultraviolet (UV) and He-Ne laser irradiations to develop high keratinase-producing mutants. Laser irradiation showed a higher lethality of cells (94%) than UV treatment (92%), whereas laser treatment required a longer time (75 min) than UV treatment (20 min). A total of 58 mutants were selected from 176 isolates to study protein and keratinase production capability of the mutants. The highest keratin-to-casein (K:C) ratio (1.43) was exhibited by LU11 mutant, which was obtained from the combined laser and UV irradiations. The purified keratinase (65 kDa) of LU11 showed 40% yield 1.7-fold purity, while the respective value for wild enzyme was 29% and 1.3-fold. Both enzymes showed optimal activity at 55 ℃ and pH 8, with a Z value of 15.78 ℃ for LU11 and 19.72 ℃ for wild strain. The Vmax and specific constant (Kcat/Km) of the mutant enzyme were 357.17 U/ml and 33.11 min-1 mM-1, respectively, which were significantly higher than the respective values of wild enzyme (102.04 U/ml and 28.36 min-1 mM-1).


Assuntos
Bacillus licheniformis , Bacillus licheniformis/genética , Bacillus licheniformis/metabolismo , Concentração de Íons de Hidrogênio , Queratinas , Mutagênese , Peptídeo Hidrolases/metabolismo
20.
Front Microbiol ; 12: 731262, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34745034

RESUMO

The current study reported a new keratinolytic bacterium, which was characterized as Bacillus paramycoides and identified by 16S rRNA, and the sequence was then deposited in the GenBank (MW876249). The bacterium was able to degrade the insoluble chicken feather keratin (CFK) into amino acids (AA) through the keratinase system. The statistical optimization of the biodegradation process into AA was performed based on the Plackett-Burman design and rotatable central composite design (RCCD) on a simple solid-state fermentation medium. The optimum conditions were temperature, 37°C, 0.547 mg KH2PO4, 1.438 mg NH4Cl, and 11.61 days of incubation. Innovatively, the degradation of the CFK process was modeled using the artificial neural network (ANN), which was better than RCCD in modeling the biodegradation process. Differentiation of the AA by high-performance liquid chromatography (HPLC) revealed the presence of 14 AA including essential and non-essential ones; proline and aspartic acids were the most dominant. The toxicity test of AA on the HepG2 cell line did not show any negative effect either on the cell line or on the morphological alteration. B. paramycoides ZW-5 is a new eco-friendly tool for CFK degradation that could be optimized by ANN. However, additional nutritional trials are encouraged on animal models.

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