Caracterização estrutural e funcional de uma alfa-N-arabinofuranosidase recombinante expressa em Escherichia coli.
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Data
2019-07-01
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Universidade Estadual Paulista (Unesp)
Resumo
As abordagens metagenômicas têm sido amplamente utilizadas para isolar novos biocatalisadores de amostras ambientais, uma vez que as comunidades microbianas podem apresentar grande diversidade e, na grande maioria dos casos, não podem ser cultivadas em laboratório. Dessa maneira, estudos direcionados apenas para o isolamento e cultivo não podem fornecer informações suficientes do papel biológico de uma grande quantidade de microrganismos membros de uma comunidade, sendo estas informações melhor entendidas por abordagem metagenômica. Inicialmente investigamos as características das ORFs presentes no genoma de bactéria do gênero Chitinophaga com o intuito de avaliar o potencial biotecnológico da comunidade microbiana, e a partir disso a sequência CB10.2_116 foi selecionada. A clonagem e expressão da α-L-arabinofuranosidase codificada pela sequência CB10.2_116 foi realizada, sendo essa selecionada de um banco de dados (CB10) (http://200.145.102.111/cb10/) construído a partir do sequenciamento de um consórcio microbiano. Após ser prospectada por similaridade e analisada in silico, a proteína codificada apresenta 780 resíduos de aminoácidos, sendo identificada como uma α-L-arabinofuranosidase (EC 3.2.1.55) da família de glicosil hidrolases 127, com 80,3% de similaridade com uma proteína não caracterizada da bactéria Chitinophaga jiangningensis. A clonagem se deu em vetor pET28-a. Para expressar a proteína de interesse, a qual apresenta uma massa molecular estimada de 87,99 kDa em célula competente de E. coli, uma colônia transformada foi isolada e inoculada em meio Lúria Bertani (LB) contendo canamicina a 50 μg/mL, e deixada sob agitação constante a 37oC até atingir a fase logarítmica de crescimento, com DO600nm entre 0,6 e 0,8. A cultura foi induzida com isopropil-β-D- tiogalactopiranosídeo (IPTG). A confirmação da expressão da proteína foi realizada através da eletroforese desnaturante em gel de poliacrilamida 12% - SDS-PAGE. Neste estudo não foi possível alcançar a expressão ideal da enzima de interesse, acredita-se que em virtude do sistema de expressão escolhido.
Metagenomic approaches have been widely used to isolate new biocatalysts from environmental samples, since microbial communities can exhibit great diversity and, in the vast majority of cases, can not be grown in the laboratory. Thus, studies directed only at isolation and cultivation can not provide sufficient information on the biological role of a large number of microorganisms members of a community, and this information is better understood by metagenomic approach. We initially investigated the characteristics of the ORFs present in the genome of Chitinophaga bacteria in order to evaluate the biotechnological potential of the microbial community, and from that the sequence CB10.2_116 was selected. The cloning and expression of the α-L-arabinofuranosidase encoded by the sequence CB10.2_116 was performed, being selected from a database (CB10) (http://200.145.102.111/cb10/) constructed from the sequencing of a consortium microbial. After being prospected for similarity and analyzed in silico, the encoded protein shows 780 amino acid residues, being identified as an α-Larabinofuranosidase (EC 3.2.1.55) from the family of glycosyl hydrolases 127, with 80.3% similarity to a uncharacterized protein from the bacterium Chitinophaga jiangningensis. Cloning took place in vector pET28-a. To express the protein of interest, which has an estimated molecular mass of 87.99 kDa in a competent E. coli cell, a transformed colony was isolated and inoculated into Bertani lutein medium (LB) containing 50 μg / ml kanamycin, and left under constant stirring at 37 ° C until reaching the logarithmic growth stage, with DO600nm between 0.6 and 0.8. The culture was induced with isopropyl-β-D-thiogalactopyranoside (IPTG). Confirmation of protein expression was performed by denaturing electrophoresis on 12% polyacrylamide gel - SDS-PAGE. In this study it was not possible to achieve the ideal expression of the enzyme of interest, it is believed that by virtue of the chosen expression system.
Metagenomic approaches have been widely used to isolate new biocatalysts from environmental samples, since microbial communities can exhibit great diversity and, in the vast majority of cases, can not be grown in the laboratory. Thus, studies directed only at isolation and cultivation can not provide sufficient information on the biological role of a large number of microorganisms members of a community, and this information is better understood by metagenomic approach. We initially investigated the characteristics of the ORFs present in the genome of Chitinophaga bacteria in order to evaluate the biotechnological potential of the microbial community, and from that the sequence CB10.2_116 was selected. The cloning and expression of the α-L-arabinofuranosidase encoded by the sequence CB10.2_116 was performed, being selected from a database (CB10) (http://200.145.102.111/cb10/) constructed from the sequencing of a consortium microbial. After being prospected for similarity and analyzed in silico, the encoded protein shows 780 amino acid residues, being identified as an α-Larabinofuranosidase (EC 3.2.1.55) from the family of glycosyl hydrolases 127, with 80.3% similarity to a uncharacterized protein from the bacterium Chitinophaga jiangningensis. Cloning took place in vector pET28-a. To express the protein of interest, which has an estimated molecular mass of 87.99 kDa in a competent E. coli cell, a transformed colony was isolated and inoculated into Bertani lutein medium (LB) containing 50 μg / ml kanamycin, and left under constant stirring at 37 ° C until reaching the logarithmic growth stage, with DO600nm between 0.6 and 0.8. The culture was induced with isopropyl-β-D-thiogalactopyranoside (IPTG). Confirmation of protein expression was performed by denaturing electrophoresis on 12% polyacrylamide gel - SDS-PAGE. In this study it was not possible to achieve the ideal expression of the enzyme of interest, it is believed that by virtue of the chosen expression system.
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Palavras-chave
α-L-arabinofuranosidase, Enzima recombinante, Metagenômica, Recombinant enzyme, Metagenomics