Isolation and identification of keratinolytic probiotic Bucillus licheniformis bacteria from the soil below poultry slaughterhouse waste / Isolamento e identificação de bactérias probióticas queratinolíticas Bacillus licheniformis do solo sob resíduos de abatedouro de aves

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.

Fungal decomposition of chicken-feather waste in submerged and solid-state fermentation

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.

Fungal decomposition of chicken-feather waste in submerged and solid-state fermentation / Decomposição fúngica de resíduos de pena de frango em fermentação submersa e de estado sólido

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.

Fungal decomposition of chicken-feather waste in submerged and solid-state fermentation / Decomposição fúngica de resíduos de pena de frango em fermentação submersa e de estado sólido

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.

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.

Fungal decomposition of chicken-feather waste in submerged and solid-state fermentation / Decomposição fúngica de resíduos de pena de frango em fermentação submersa e de estado sólido

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)

Fungal conversion of chicken-feather waste into biofortified compost / Conversão fungosa de resíduos de pena de frango em composto biofortificado

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.

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.

Production and characterization of a new detergent-stable keratinase expressed by Pedobacter sp. 3.14.7, a novel Antarctic psychrotolerant keratin-degrading bacterium.

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.

The extremophile Anoxybacillus sp. PC2 isolated from Brazilian semiarid region (Caatinga) produces a thermostable keratinase.

The aim of this study was to select and identify thermophilic bacteria from Caatinga biome (Brazil) able to produce thermoactive keratinases and characterize the keratinase produced by the selected isolate. After enrichment in keratin culture media, an Anoxybacillus caldiproteolyticus PC2 was isolated. This thermotolerant isolate presents a remarkable feature producing a thermostable keratinase at 60°C. The partially purified keratinase, identified as a thermolysin-like peptidase, was active at a pH range of 5.0-10.0 with maximal activity at a temperature range of 50-80°C. The optimal activity was observed at pH 7.0 and 50-60°C. These characteristics are potentially useful for biotechnological purposes such as processing and bioconversion of keratin.

Citrobacter diversus-derived keratinases and their potential application as detergent-compatible cloth-cleaning agents.

Currently, poultry farming is one of the sectors that have a significant impact on the global economy. In recent years, there has been an increase in the production of broilers, inflicting this segment of the industry to generate tons of keratin due to huge disposal of chicken feathers. This points to the need to degrade these chicken feathers, as they have emerged as a major threat to the environment. Thus, in this study we aimed to identify keratinases that are produced by the bacterium Citrobacter diversus and further investigate the biochemical characteristics of these keratin-degrading enzymes. In a submerged medium, the bacterium was capable of degrading chicken feathers almost completely after 36 h of fermentation. We found a maximum caseinolytic activity at pH 9-10.5 and 50-55 °C, and keratinolytic activity at pH 8.5-9.5 and 50 °C. Thus, given its stability at higher temperatures, upon incubation of this enzyme extract for 1 h at 50 °C, it showed approximately 50% of the keratinolytic and 100% of the caseinolytic activity. Further, under pH stability for 48 h at 4 °C, the enzyme extract maintained greater residual activity in the pH range 6-8. Caseinolytic activity was inhibited by EDTA and PMSF, whereas the keratinolytic activity was inhibited only by EDTA. Additionally, due to its alkaline activity and detergent compatibility, this enzyme extract could improve washing performance when added to a commercial detergent formulation. Using application tests, we could demonstrate a potential use of this bacterial enzyme extract as an additive in detergents to remove egg stains from cloth.

Fusarium oxysporum and Aspergillus sp. as Keratinase Producers Using Swine Hair From Agroindustrial Residues.

Technological processes mediated by microorganisms and enzymes are promising alternatives for treatment of recalcitrant residues. Keratinases hydrolyze keratin, the primary component of some wastes generated in many industrial activities. The present study was designed to evaluate strategies for obtaining keratinases produced by fungi using submerged fermentation and two residues as substrates, chicken feathers and swine hair. Two fungi isolated from feather residues showed potential for keratinase production, Fusarium oxysporum and Aspergillus sp. These were subjected to submerged fermentation using chicken feathers and swine hair prepared in three conditions (microbial concentration reduction, sterilization and hydrogen peroxide). The residual mass was quantified and tested for keratinase production. The most potent enzymatic extract was used in the precipitation technique with salts and organic solvents. The best results of enzymatic activity were obtained using F. oxysporum, on the 6thday of fermentation, obtaining 243.25 U mL-1 using sterilized swine hair as the substrate. Aspergillus sp. showed the highest keratinolytic activity on the 9thday, 113.50 U mL-1 using feathers as the substrate. The highest degradation percentage was 59.20% (w/w) in swine hair and the precipitation technique, with relative activities close to 50%. The results are promising for the application of residues and microorganisms in biotechnological processes of economic and environmental interest.

Simultaneous production of proteases and antioxidant compounds from agro-industrial by-products.

The use of processes for simultaneous production of bioproducts as enzymes and bioactive compounds is an interesting alternative to reduce environmental impacts. Thus, the aim of this study was to produce simultaneously, using the biorefinery concept, both proteases and bioactive compounds with antioxidant activity from Bacillus sp. P45 cultivation by using different by-products. The integrated process developed in this study enabled to obtain enzymes with proteolytic and keratinolytic properties in a process with alternate substrates from agro-industrial by-products (feather meal, residual feather meal and biomass), thus, creating an interesting alternative to managing them. The residual biomass provided the highest protease activity (1306.6U/mL) and the reused feather meal reached the highest keratinolytic activity (89U/mL), both at 32h of cultivation. Moreover, hydrolysates produced in cultivation using feather meal and residual biomass had high antioxidant activity, they have great potential as natural antioxidants.

Keratinolytic abilities of Micrococcus luteus from poultry waste.

Keratinolytic microorganisms have become the subject of scientific interest due to their ability to biosynthesize specific keratinases and their prospective application in keratinic waste management. Among several bacterial classes, actinobacteria remain one of the most important sources of keratin-degrading strains, however members of the Micrococcaceae family are rarely scrutinized in regard to their applicatory keratinolytic potential. The tested Micrococcus sp. B1pz isolate from poultry feather waste was identified as M. luteus. The strain, grown in the medium with 1-2% chicken feathers and a yeast extract supplement, produced keratinases of 32 KU and lower level of proteases, 6 PU. It was capable to effectively decompose feathers or "soft" keratin of stratum corneum, in contrast to other "hard" hair-type keratins. The produced keratinolytic enzymes were mainly a combination of alkaline serine or thiol proteases, active at the optimum pH 9.4, 55 °C. Four main protease fractions of 62, 185, 139 and 229 kDa were identified in the crude culture fluid. The research on the auxiliary role of reducing factors revealed that reducing sulfur compounds could be applied in keratinolysis enhancement during enzymatic digestion of keratin, rather than in culture conditions. The presented M. luteus isolate exhibits a significant keratinolytic potential, which determines its feasible applicatory capacity towards biodegradation of poultry by-products or formulation of keratin-based feed components.

Keratinolytic abilities of Micrococcus luteus from poultry waste

Keratinolytic microorganisms have become the subject of scientific interest due to their ability to biosynthesize specific keratinases and their prospective application in keratinic waste management. Among several bacterial classes, actinobacteria remain one of the most important sources of keratin-degrading strains, however members of the Micrococcaceae family are rarely scrutinized in regard to their applicatory keratinolytic potential. The tested Micrococcus sp. B1pz isolate from poultry feather waste was identified as M. luteus. The strain, grown in the medium with 1–2% chicken feathers and a yeast extract supplement, produced keratinases of 32 KU and lower level of proteases, 6 PU. It was capable to effectively decompose feathers or “soft” keratin of stratum corneum, in contrast to other “hard” hair-type keratins. The produced keratinolytic enzymes were mainly a combination of alkaline serine or thiol proteases, active at the optimum pH 9.4, 55 °C. Four main protease fractions of 62, 185, 139 and 229 kDa were identified in the crude culture fluid. The research on the auxiliary role of reducing factors revealed that reducing sulfur compounds could be applied in keratinolysis enhancement during enzymatic digestion of keratin, rather than in culture conditions. The presented M. luteus isolate exhibits a significant keratinolytic potential, which determines its feasible applicatory capacity towards biodegradation of poultry by-products or formulation of keratin-based feed components.

Keratinolytic abilities of Micrococcus luteus from poultry waste

Keratinolytic microorganisms have become the subject of scientific interest due to their ability to biosynthesize specific keratinases and their prospective application in keratinic waste management. Among several bacterial classes, actinobacteria remain one of the most important sources of keratin-degrading strains, however members of the Micrococcaceae family are rarely scrutinized in regard to their applicatory keratinolytic potential. The tested Micrococcus sp. B1pz isolate from poultry feather waste was identified as M. luteus. The strain, grown in the medium with 1–2% chicken feathers and a yeast extract supplement, produced keratinases of 32 KU and lower level of proteases, 6 PU. It was capable to effectively decompose feathers or “soft” keratin of stratum corneum, in contrast to other “hard” hair-type keratins. The produced keratinolytic enzymes were mainly a combination of alkaline serine or thiol proteases, active at the optimum pH 9.4, 55 °C. Four main protease fractions of 62, 185, 139 and 229 kDa were identified in the crude culture fluid. The research on the auxiliary role of reducing factors revealed that reducing sulfur compounds could be applied in keratinolysis enhancement during enzymatic digestion of keratin, rather than in culture conditions. The presented M. luteus isolate exhibits a significant keratinolytic potential, which determines its feasible applicatory capacity towards biodegradation of poultry by-products or formulation of keratin-based feed components..(AU)

Peptidase with Keratinolytic Activity Secreted by Aspergillus terreus During Solid-State Fermentation

The aim of this study was to evaluate peptidase production by Aspergillus terreus in solid-state bioprocess and evaluate its parameters. The best conditions were 5.0 g of wheat bran as substrate, incubation temperature 30°C, inoculum 2.0x105 spores/g and 75% saline volume, with production reaching 677 U/mL (5400 U/g culture medium) after 72 h of fermentation. Biochemical characterization of the crude enzymatic extract showed the optimum pH and temperature of 6.5 and 55°C, respectively. The stability at different temperatures and pH values showed that the extract could endure different pH. The evaluation of the ions influence and inhibitors proved that the enzyme required an ion for better activity, which was corroborated with the inhibition of EDTA and PMSF, characterizing serine and/or metallo peptidase. The extract was also tested for specific activities and showed promising results for keratinolytic and collagenolytic activities (0.252 and 0.165 OD/mL, respectively).

Studies on PVA pectin cryogels containing crosslinked enzyme aggregates of keratinase.

Polyvinyl alcohol-pectin (PVA-P) films containing enrofloxacin and keratinase were developed to treat wounds and scars produced by burns and skin injuries. However, in order to prevent enzyme inactivation at the interface between the patch and the scars, crosslinked enzyme aggregates (CLEAs) from a crude extract of keratinase produced by Paecilomyces lilacinus (LPSC#876) were synthesized by precipitation with acetone and crosslinking with glutaraldehyde. Soluble vs. CLEA keratinase (K-CLEA) activities were tested in 59% (v/v) hydrophobic (isobutanol and n-hexane) and hydrophilic (acetone and dimethylsulfoxide) solvents mixtures. K-CLEA activity was 1.4, 1.7 and 6.6 times higher in acetone, n-hexane and isobutanol than the soluble enzyme at 37 °C after 1 h of incubation, respectively. K-CLEA showed at least 45% of enzyme residual activity in the 40-65 °C range, meanwhile the soluble biocatalyst was fully inactivated at 65 °C after 1h incubation. Also, the soluble enzyme was completely inactivated after 12 h at pH 7.4 and 45 °C, even though K-CLEA retained full activity. The soluble keratinase was completely inactivated at 37 °C after storage in buffer solution (pH 7.4) for 2 months, meanwhile K-CLEAs kept 51% of their activity. K-CLEA loaded into polyvinyl alcohol (PVA) and PVA-P cryogels showed six times lower release rate compared to the soluble keratinase at skin pH (5.5). Small angle X-ray scattering (SAXS) analysis showed that K-CLEA bound to pectin rather than to PVA in the PVA-P matrix.

Immobilized keratinase and enrofloxacin loaded on pectin PVA cryogel patches for antimicrobial treatment.

A keratinase isolated from Paecilomyces lilacinus (LPS #876) was tested against proteins present in the skin but the high enzyme activity was detected on collagen. Keratinase was physically immobilized onto PVA-pectin cryogels and enzyme release was 20.8±2.1%, 63.8±0.2%, 41.5±3.5% and 26.0±3.5% in cryogels containing pectins with esterification degrees (DE) 33.0%, 55.0%, 62.7% and 71.7% respectively at 37°C after 3h incubation. In presence of 0.75 M NaCl, the percentage of enzyme release changed to: 57.5±1.5, 65.8±3.8, 57.3±0.2 and 34.0±4.0 for the four pectins respectively. In-vitro studies of enrofloxacin release from PVA-pectin cryogels at pH close to the human skin (pH=5.5) showed 15.0% free antibiotic following first order kinetic at 37°C after 5h incubation. However, in the presence of keratinase only 6.9% of enrofloxacin was released under the same experimental conditions.

Isolation of Microsporum gypseum in soil samples from different geographical regions of Brazil, evaluation of the extracellular proteolytic enzymes activities (keratinase and elastase) and molecular sequencing of selected strains

A survey of Microsporum gypseum was conducted in soil samples in different geographical regions of Brazil. The isolation of dermatophyte from soil samples was performed by hair baiting technique and the species were identified by morphology studies. We analyzed 692 soil samples and the recuperating rate was 19.2%. The activities of keratinase and elastase were quantitatively performed in 138 samples. The sequencing of the ITS region of rDNA was performed in representatives samples. M. gypseum isolates showed significant quantitative differences in the expression of both keratinase and elastase, but no significant correlation was observed between these enzymes. The sequencing of the representative samples revealed the presence of two teleomorphic species of M. gypseum (Arthroderma gypseum and A. incurvatum). The enzymatic activities may play an important role in the pathogenicity and a probable adaptation of this fungus to the animal parasitism. Using the phenotypical and molecular analysis, the Microsporum identification and their teleomorphic states will provide a useful and reliable identification system.

Isolation of Microsporum gypseum in soil samples from different geographical regions of brazil, evaluation of the extracellular proteolytic enzymes activities (keratinase and elastase) and molecular sequencing of selected strains.

A survey of Microsporum gypseum was conducted in soil samples in different geographical regions of Brazil. The isolation of dermatophyte from soil samples was performed by hair baiting technique and the species were identified by morphology studies. We analyzed 692 soil samples and the recuperating rate was 19.2%. The activities of keratinase and elastase were quantitatively performed in 138 samples. The sequencing of the ITS region of rDNA was performed in representatives samples. M. gypseum isolates showed significant quantitative differences in the expression of both keratinase and elastase, but no significant correlation was observed between these enzymes. The sequencing of the representative samples revealed the presence of two teleomorphic species of M. gypseum (Arthroderma gypseum and A. incurvatum). The enzymatic activities may play an important role in the pathogenicity and a probable adaptation of this fungus to the animal parasitism. Using the phenotypical and molecular analysis, the Microsporum identification and their teleomorphic states will provide a useful and reliable identification system.

Isolation of Microsporum gypseum in soil samples from different geographical regions of Brazil, evaluation of the extracellular proteolytic enzymes activities (keratinase and elastase) and molecular sequencing of selected strains

A survey of Microsporum gypseum was conducted in soil samples in different geographical regions of Brazil. The isolation of dermatophyte from soil samples was performed by hair baiting technique and the species were identified by morphology studies. We analyzed 692 soil samples and the recuperating rate was 19.2%. The activities of keratinase and elastase were quantitatively performed in 138 samples. The sequencing of the ITS region of rDNA was performed in representatives samples. M. gypseum isolates showed significant quantitative differences in the expression of both keratinase and elastase, but no significant correlation was observed between these enzymes. The sequencing of the representative samples revealed the presence of two teleomorphic species of M. gypseum (Arthroderma gypseum and A. incurvatum). The enzymatic activities may play an important role in the pathogenicity and a probable adaptation of this fungus to the animal parasitism. Using the phenotypical and molecular analysis, the Microsporum identification and their teleomorphic states will provide a useful and reliable identification system.