Pseudogymnoascus da Antártica: taxonomia e prospecção de enzimas queratinolíticas
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Data
2022-03-04
Autores
Turunday, Morgana Ferreira
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Editor
Universidade Estadual Paulista (Unesp)
Resumo
A Antártica é considerada o continente dos extremos devido as suas condições climáticas. No entanto alguns micro-organismos desenvolveram adaptações que os permitem colonizar este habitat, como é o caso dos fungos dos gêneros Geomyces e Pseudogymnoascus. Representantes desses gêneros são capazes de secretar uma grande variedade de enzimas, incluindo as queratinases que são proteases capazes de degradar a queratina. No presente trabalho foram examinados 86 fungos filamentosos do acervo de pesquisa da Central de Recursos Microbianos da UNESP (CRM-UNESP) obtidos a partir de diferentes amostras coletadas na Baía do Almirantado, Ilha Rei George (Antártica) e previamente identificados como pertencentes a esses dois gêneros, com o objetivo de autenticar a caracterização taxonômica dos mesmos e investigar a capacidade de produção de enzimas queratinolíticas. Os fungos foram submetidos à checagem de purificação através de culturas monospóricas, análises macromorfológicas baseadas na observação da área da placa de Petri e análises filogenéticas, as quais foram realizadas com o marcador ITS e MCM7 através de Inferência Bayesiana no software MrBayes v. 3.1.1. Um representante de cada um dos 9 morfotipos foi submetido as análises enzimáticas. Para a análise enzimática qualitativa os isolados foram cultivados por 15 dias a 15 ºC em placas de Petri contendo meio mínimo sólido com adição de penas de frango como fonte de nutrientes. Todos os isolados apresentaram crescimento em meio sólido com penas de frango e foram submetidos a quantificação da atividade enzimática. Para tanto, os fungos foram cultivados em meio caldo de pena por 7 dias a 15°C e 120 rpm. Os 9 isolados apresentaram atividade de queratinase com destaque para o fungo LAMAI 539 e LAMAI 1777 que produziram 4,11 U ml-1 e 2,8 U ml-1 da enzima, respectivamente. As análises filogenéticas revelaram que todos os isolados são pertencentes ao gênero Pseudogymnoascus, sendo possível chegar no nível de espécie (Pseudogymnoascus sinensis, P. roseus e P. verrucosus) com um valor de probabilidade significativo para 27 dos 52 isolados analisados. Os dados de filogenia demostraram que os morfotipos não necessariamente agruparam em clados diferentes. Em relação a distribuição do gênero, os resultados revelaram que estes são capazes de colonizar substratos distintos além de regiões geográficas diferentes e distantes. Além disso, diferentes espécies podem ser encontradas em um mesmo substrato de um mesmo local.
Antarctica is considered the continent of extremes due to its climatic conditions. However, some microorganisms have developed adaptations that allow them to colonize this habitat, such as the fungi of the genera Geomyces and Pseudogymnoascus. Representatives of these genera are capable of secreting a wide variety of enzymes, including keratinases, which are proteases capable of degrading keratin. In this work, 86 filamentous fungi from the research collection of the UNESP Microbial Resources Center (CRM-UNESP) obtained from different samples collected in Admiralty Bay, King George Island (Antarctica) and previously identified as belonging to these two genera were examined. The aim of this work was to authenticate the taxonomic characterization of these genera and investigate the production of keratinolytic enzymes. For that, these fungi were submitted to a purification check through monosporic cultures, macromorphological analysis based on the observation of the Petri dish area, and phylogenetic analysis, which were performed with the ITS and MCM7 marker through Bayesian Inference in software MrBayes v. 3.1.1. One representative of each 9 morphotypes was submitted to enzymatic analysis. For qualitative enzymatic analysis, the isolates were cultured for 15 days at 15 ºC in Petri dishes containing minimal solid medium with the addition of chicken feathers as a source of nutrients. All isolates showed growth on solid medium with chicken feathers and were submitted to quantification of enzymatic activity. For this, the fungi were cultivated in feather broth medium for 7 days at 15°C and 120 rpm. The 9 isolates showed keratinase activity, especially the fungus LAMAI 539 and LAMAI 1777, which produced 4.11 U ml-1 and 2.8 U ml-1 of the enzyme, respectively. Phylogenetic analyzes revealed that all isolates belong to the genus Pseudogymnoascus, and it was possible to reach the species level with a significant probability value (Pseudogymnoascus sinensis, P. roseus and P. verrucosus) for 27 of the 52 isolates analyzed. The phylogeny data showed that the morphotypes did not necessarily group into different clades. Regarding the distribution of this genus, our study showed that they are able to colonize very different substrates in addition to different and distant geographic regions. Besides, different species can be found in the same substrate in the same place.
Antarctica is considered the continent of extremes due to its climatic conditions. However, some microorganisms have developed adaptations that allow them to colonize this habitat, such as the fungi of the genera Geomyces and Pseudogymnoascus. Representatives of these genera are capable of secreting a wide variety of enzymes, including keratinases, which are proteases capable of degrading keratin. In this work, 86 filamentous fungi from the research collection of the UNESP Microbial Resources Center (CRM-UNESP) obtained from different samples collected in Admiralty Bay, King George Island (Antarctica) and previously identified as belonging to these two genera were examined. The aim of this work was to authenticate the taxonomic characterization of these genera and investigate the production of keratinolytic enzymes. For that, these fungi were submitted to a purification check through monosporic cultures, macromorphological analysis based on the observation of the Petri dish area, and phylogenetic analysis, which were performed with the ITS and MCM7 marker through Bayesian Inference in software MrBayes v. 3.1.1. One representative of each 9 morphotypes was submitted to enzymatic analysis. For qualitative enzymatic analysis, the isolates were cultured for 15 days at 15 ºC in Petri dishes containing minimal solid medium with the addition of chicken feathers as a source of nutrients. All isolates showed growth on solid medium with chicken feathers and were submitted to quantification of enzymatic activity. For this, the fungi were cultivated in feather broth medium for 7 days at 15°C and 120 rpm. The 9 isolates showed keratinase activity, especially the fungus LAMAI 539 and LAMAI 1777, which produced 4.11 U ml-1 and 2.8 U ml-1 of the enzyme, respectively. Phylogenetic analyzes revealed that all isolates belong to the genus Pseudogymnoascus, and it was possible to reach the species level with a significant probability value (Pseudogymnoascus sinensis, P. roseus and P. verrucosus) for 27 of the 52 isolates analyzed. The phylogeny data showed that the morphotypes did not necessarily group into different clades. Regarding the distribution of this genus, our study showed that they are able to colonize very different substrates in addition to different and distant geographic regions. Besides, different species can be found in the same substrate in the same place.
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Palavras-chave
Microbiologia, Filogenia, Ambientes extremos, Queratinase, Microbiology, Keratinase, Phylogeny, Extremophilic environments, Enzimas queratinolíticas, Autenticação taxonômica, Keratinolytic enzymes, Taxonomic authentication