RESUMO
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.
Assuntos
Queratinas/metabolismo , Micrococcus luteus/enzimologia , Micrococcus luteus/metabolismo , Peptídeo Hidrolases/metabolismo , Animais , Biodegradação Ambiental , Galinhas/microbiologia , Plumas/microbiologia , Micrococcus luteus/isolamento & purificação , NADH NADPH Oxirredutases/metabolismo , Oxirredução , Aves Domésticas/microbiologia , Compostos de Enxofre/metabolismo , Gerenciamento de ResíduosRESUMO
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
Assuntos
Animais , Queratinas/metabolismo , Micrococcus luteus/enzimologia , Micrococcus luteus/metabolismo , Peptídeo Hidrolases/metabolismo , Biodegradação Ambiental , Galinhas/microbiologia , Plumas/microbiologia , Micrococcus luteus/isolamento & purificação , NADH NADPH Oxirredutases/metabolismo , Oxirredução , Aves Domésticas/microbiologia , Compostos de Enxofre/metabolismo , Gerenciamento de ResíduosRESUMO
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
Assuntos
Animais , Queratinas/metabolismo , Micrococcus luteus/enzimologia , Micrococcus luteus/metabolismo , Peptídeo Hidrolases/metabolismo , Biodegradação Ambiental , Galinhas/microbiologia , Plumas/microbiologia , Micrococcus luteus/isolamento & purificação , ética , Oxirredução , Aves Domésticas/microbiologia , Compostos de Enxofre/metabolismo , Gerenciamento de ResíduosRESUMO
BACKGROUND: UV radiation can produce mutations in skin cells and correlates strongly with the onset of actinic keratoses and basal and squamous cell carcinomas. Xeroderma pigmentosum (XP) is a heritable disease characterized by an extreme sensitivity of skin to UV radiation. Recently, studies in cultured cells as well as in XP patients have demonstrated that the recombinant T4 endonuclease V UV-specific endonuclease could enhance repair of UV-induced photoproducts. OBJECTIVE: We aimed to obtain a stable UV-specific DNA recombinant endonuclease, pharmacologically active in mammalian cells so as to be used in treatment and prophylaxis of sun damage. METHODS: The UV-specific DNA endonuclease gene obtained from Micrococcus luteus, was fused to a leader peptide and expressed (alphaUveA), refolded and purified. A construction under the control of an eukaryotic promoter was used to transfect XP fibroblasts deficient in DNA damage repair. Transformed cells were UV irradiated and cell survival was assessed. RESULTS: alphaUveA was obtained as a highly active UV-specific repair enzyme stable for at least 2 years. XP fibroblasts transfected with alphaUveA gene increased the resistance to UV radiation and, in consequence, cell survival. CONCLUSION: alphaUveA is stable and pharmacologically active in human cells. The topical administration of this long-term stable new active principle could help diminish the risks of skin cancer after sun exposure.
Assuntos
Reparo do DNA , Linhagem Celular , Sobrevivência Celular , DNA/metabolismo , Dano ao DNA , DNA Ligase Dependente de ATP , DNA Ligases/metabolismo , Desoxirribonuclease (Dímero de Pirimidina)/metabolismo , Relação Dose-Resposta à Radiação , Eletroforese em Gel de Ágar , Fibroblastos/metabolismo , Vetores Genéticos , Humanos , Luz , Micrococcus luteus/enzimologia , Neoplasias/metabolismo , Peptídeos/química , Regiões Promotoras Genéticas , Biossíntese de Proteínas , Proteínas Recombinantes de Fusão/química , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Queimadura Solar , Temperatura , Fatores de Tempo , Transfecção , Raios Ultravioleta , Proteínas Virais/metabolismo , Xeroderma Pigmentoso/metabolismoRESUMO
We present herein an improved assay for detecting the presence of extracellular proteases from microorganisms on agar plates. Using different substrates (gelatin, BSA, hemoglobin) incorporated into the agar and varying the culture medium composition, we were able to detect proteolytic activities from Pseudomonas aeruginosa, Micrococcus luteus and Serratia marcescens as well as the influence that these components displayed in the expression of these enzymes. For all microorganisms tested we found that in agar-BHI or yeast extract medium containing gelatin the sensitivity of proteinase detection was considerably greater than in BSA-agar or hemoglobin-agar. However, when BSA or hemoglobin were added to the culture medium, there was an increase in growth along with a marked reduction in the amount of proteinase production. In the case of M. luteus the incorporation of glycerol in BHI or yeast extract gelatin-agar induced protease liberation. Our results indicate that the technique described here is of value for detecting extracellular proteases directly in the culture medium, by means of a qualitative assay, simple, inexpensive, straight forward method to assess the presence of the proteolytic activity of a given microorganism colony with great freedom in substrate selection.
Assuntos
Ágar , Bactérias/enzimologia , Peptídeo Hidrolases/isolamento & purificação , Micrococcus luteus/enzimologia , Pseudomonas aeruginosa/enzimologia , Serratia marcescens/enzimologiaRESUMO
Photoaffinity labeling is a special type of chemical modification, where the label is activated by the action of light. This article presents the general principles and limitations of this technique, its application to the study of Micrococcus luteus ATPase and the use of photoaffinity crosslinking to probe the structure of this enzyme.
Assuntos
Difosfato de Adenosina/química , Trifosfato de Adenosina/química , Marcadores de Afinidade/química , Ativação Enzimática/fisiologia , Radicais Livres/química , Ligantes , Micrococcus luteus/enzimologia , Peptídeos/química , Conformação Proteica , ATPases Translocadoras de Prótons/química , ATPases Translocadoras de Prótons/ultraestruturaRESUMO
Photoaffinity labeling is a special type of chemical modification, where the label is activated by the action of light. This article presents the general principles and limitations of this technique, its application to the study of Micrococcus luteus ATPase and the use of photoaffinity crosslinking to probe the structure of this enzyme