Busca de biomoléculas com potencial biotecnológico em database metagenômico por sequence-driven
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Data
2017-07-24
Autores
Lima, Natália Sarmanho Monteiro [UNESP]
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Universidade Estadual Paulista (Unesp)
Resumo
O mercado industrial está se tornando cada vez mais dependente do uso de enzimas como catalisadores. Dentre as enzimas utilizadas industrialmente estão as de origem microbiana, por serem ativas nas mais diversas condições. As lacases são enzimas com alto potencial, pois atuam sobre diversos compostos, o que possibilita uma vasta diversidade de possíveis aplicações. Entre as formas de prospecção de novas enzimas microbianas, encontra-se a metagenômica. Diante disto, foi realizada a busca por potenciais lacases seguindo uma estratégia “sequence-driven” em uma base de dados metagenomicos composta por amostras de diversos ambientes, pertencente ao Laboratório de Bioquímica de Microrganismos e Plantas (LBMP). As ORFs previamente anotadas por similaridade de sequências e domínios conservados como possíveis “Multicopper oxidases” (grupo no qual se encontram as lacases) foram prospectadas pela região conservada para lacases L3 (HLHGH). Esta estratégia retornou três ORFs, a ORF50MF; a ORF51ME e a ORF13SE (lacmeta), as quais foram clonadas e submetidas as análises de super-expressão e purificação. LacMeta, similar em 82% com a sequência de proteína hipotética de Streptomyces rubidus [WP_069465126.1], foi a proteína que apresentou o melhor resultado de expressão e purificação, sendo então submetida a caracterização cinética. A proteína fusionada a uma cauda de hexa-histidina foi purificada na forma homo-trimérica, com 107 kDa, e apresentou atividade ótima em pH ácido para o substrato ABTS, para o qual obteve os parâmetros catalíticos kcat, Km e Vmax iguais a 1,85 (±0,07) min-1; 4,04 mM (±0,37) e 24,43 (±0,94) μM min-1, respectivamente. Quanto à estabilidade de estocagem, a enzima apresentou considerável estabilidade temporal, permanecendo com 50% da atividade após 150 dias a 4°C na presença de glicerol 5% como estabilizador estrutural. A enzima LacMeta apresentou atividade em uma ampla faixa de temperatura, atuando entre 10-60 °C com atividade acima de 50%. Sob o aspecto de aplicação biotecnológica, a enzima demonstrou elevado potencial para o tratamento de água residual de indústria têxtil, uma vez que foi capaz de degradar sete corantes de cinco classes distintas sem a presença de um mediador redox.
The industrial market is becoming increasingly dependent on the use of enzymes as biocatalysts. Among the enzymes used industrially are those of microbial origin, because they are active in the most diverse conditions. Laccases are enzymes with high potential, since they act on several compounds, which allows a wide diversity of possible applications. Among the search for new forms of microbial enzymes, is the metagenomic. In this way, the search for potential laccases was carried out following a sequence-driven strategy in a metagenomic database composed of samples from different environments, belonging to the Laboratory of Biochemistry of Microorganisms and Plants (LBMP). The ORFs previously annotated by similarity of sequences and conserved domains as possible "Multicopper oxidases" (group in which the laccases are) were prospected by the region conserved for laccases L3 (HLHGH). This strategy returned three ORFs, the ORF50MF; the ORF51ME and the ORF13SE (lacmeta), which were cloned and subjected to super-expression and purification analyzes. LacMeta, similar in 82% to the hypothetical protein sequence of Streptomyces rubidus [WP_069465126.1], was the protein that presented the best expression and purification result and was then subjected to kinetic characterization. The protein fused to a hexa-histidine tail was purified in the homo-trimeric form with 128.22 kDa and presented optimum acidic pH activity for the ABTS substrate, for which obtained the catalytic parameters kcat, Km and Vmax equal to 1.85(± 0.07) min -1 , 4.04 (± 0.37) mM and 24.43 (± 0.94) μM min -1 , respectively. Regarding the storage stability, the enzyme showed considerable time stability, with 50% remaining activity after 150 days at 4 ° C in the presence of 5% glycerol as a structural stabilizer. The enzyme presented a wide temperature range, acting between 10-60 ° C with activity above 50%. Under the aspect of biotechnological application, the enzyme demonstrated a high potential for the treatment of waste water from the textile industry, since it was able to degrade seven dyes of five distinct classes without the presence of a redox mediator.
The industrial market is becoming increasingly dependent on the use of enzymes as biocatalysts. Among the enzymes used industrially are those of microbial origin, because they are active in the most diverse conditions. Laccases are enzymes with high potential, since they act on several compounds, which allows a wide diversity of possible applications. Among the search for new forms of microbial enzymes, is the metagenomic. In this way, the search for potential laccases was carried out following a sequence-driven strategy in a metagenomic database composed of samples from different environments, belonging to the Laboratory of Biochemistry of Microorganisms and Plants (LBMP). The ORFs previously annotated by similarity of sequences and conserved domains as possible "Multicopper oxidases" (group in which the laccases are) were prospected by the region conserved for laccases L3 (HLHGH). This strategy returned three ORFs, the ORF50MF; the ORF51ME and the ORF13SE (lacmeta), which were cloned and subjected to super-expression and purification analyzes. LacMeta, similar in 82% to the hypothetical protein sequence of Streptomyces rubidus [WP_069465126.1], was the protein that presented the best expression and purification result and was then subjected to kinetic characterization. The protein fused to a hexa-histidine tail was purified in the homo-trimeric form with 128.22 kDa and presented optimum acidic pH activity for the ABTS substrate, for which obtained the catalytic parameters kcat, Km and Vmax equal to 1.85(± 0.07) min -1 , 4.04 (± 0.37) mM and 24.43 (± 0.94) μM min -1 , respectively. Regarding the storage stability, the enzyme showed considerable time stability, with 50% remaining activity after 150 days at 4 ° C in the presence of 5% glycerol as a structural stabilizer. The enzyme presented a wide temperature range, acting between 10-60 ° C with activity above 50%. Under the aspect of biotechnological application, the enzyme demonstrated a high potential for the treatment of waste water from the textile industry, since it was able to degrade seven dyes of five distinct classes without the presence of a redox mediator.
Descrição
Palavras-chave
Slac, Lacase, Metagenômica, Corantes, Laccase, Metagenomics, Dyes