Análise in silico, expressão e purificação de uma nova lacase bacteriana de Chitinophaga sp. CB10
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Data
2019-02-28
Autores
Buzzo, Bárbara Bonfá
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Universidade Estadual Paulista (Unesp)
Resumo
As enzimas são catalisadores biológicos que cada vez mais despertam o interesse da indústria em aplicá-las em seus processos. As lacases, uma classe de enzimas pertencente à família das “multicopper oxidases”, possuem alto potencial industrial pois atuam em diversos substratos, tendo assim, uma diversidade de aplicações. Dentre as lacases, as bacterianas são as de maior interesse para os processos industriais em comparação com as de fungos, uma vez que bactérias apresentam maior tolerância a ambientes extremos e apresentam um tempo de reprodução mais rápido. Diante disto, foi realizada a prospecção de uma lacase bacteriana no genoma de Chitinophaga sp. CB10 a partir do banco de dados genômico, pertencente ao Laboratório de Bioquímica de Microrganismos e Plantas (LBMP), com base em genes ortólogos e busca por HMM (“Hidden Markov Model”) seguida pela busca de lacases utilizando como sequência isca o padrão conversado L3 (HLHGH), que é uma assinatura específica de lacases. Esta prospecção revelou uma ORF CB10_180.4889 a qual foi clonada e submetida às análises de expressão, extração e purificação. A proteína se mostrou retida em corpúsculos de inclusão e só foi possível extraí-la em sua fração solúvel utilizando agentes desnaturantes, como ureia e guanidina. Após a extração, a proteína desnaturada foi submetida a uma purificação concomitantemente a renaturação, que se mostrou eficiente de acordo com os testes de visualização de degradação de corante. A partir das análises “in silico”, observou-se que a enzima CB10_180.4889 possui três domínios de cobre (cupredoxina) e pertence à classe das CopAs, classe em que se encontram somente proteínas bacterianas com maior resistência ao cobre. Considerando a possível aplicação biotecnológica, a enzima apresentou potencial para a degradação de corantes.
Enzymes are biological catalysts that increasingly arouse industry interest in applying them in their processes. Among all the enzymes, laccases are a class of enzymes belonging to the family of Multicopper oxidases, with high industrial potential because they act on several substrates, thus having a diversity of applications. Between all laccases, bacterial are greater interest for industrial processes than fungi, once bacteria have a greater tolerance to extreme environments than these, besides presenting a time of reproduction and cloning faster. Therefore, a bacterial laccase was prospected in the genome of Chitinophaga sp. CB10 from the genomic database belonging to the Laboratory of Biochemistry of Microorganisms and Plants (LBMP), based on orthologous genes and search for HMM ("Hidden Markov Model") followed by the search for laccases using as motif the standard L3 (HLHGH which is a specific signature of laccases. This prospection revealed a ORF CB10_180.4889 which was cloned and subjected to expression, extraction and purification analyzes. The protein was included in inclusion bodies and it was only possible to extract it in its soluble fraction using denaturing agents, such as urea and guanidine. After extraction, the denatured protein was subjected to purification with concomitantly renaturation, which proved to be efficient according to the dye degradation screening tests. From the in silico analyzes, it was observed that the enzyme CB10_180.4889 has three domains of copper (cupredoxin) and belongs to the class of CopAs, characteristic only of bacterial laccases with greater resistance to copper. Considering the possible biotechnological application, the enzyme presents potential for the degradation of dye.
Enzymes are biological catalysts that increasingly arouse industry interest in applying them in their processes. Among all the enzymes, laccases are a class of enzymes belonging to the family of Multicopper oxidases, with high industrial potential because they act on several substrates, thus having a diversity of applications. Between all laccases, bacterial are greater interest for industrial processes than fungi, once bacteria have a greater tolerance to extreme environments than these, besides presenting a time of reproduction and cloning faster. Therefore, a bacterial laccase was prospected in the genome of Chitinophaga sp. CB10 from the genomic database belonging to the Laboratory of Biochemistry of Microorganisms and Plants (LBMP), based on orthologous genes and search for HMM ("Hidden Markov Model") followed by the search for laccases using as motif the standard L3 (HLHGH which is a specific signature of laccases. This prospection revealed a ORF CB10_180.4889 which was cloned and subjected to expression, extraction and purification analyzes. The protein was included in inclusion bodies and it was only possible to extract it in its soluble fraction using denaturing agents, such as urea and guanidine. After extraction, the denatured protein was subjected to purification with concomitantly renaturation, which proved to be efficient according to the dye degradation screening tests. From the in silico analyzes, it was observed that the enzyme CB10_180.4889 has three domains of copper (cupredoxin) and belongs to the class of CopAs, characteristic only of bacterial laccases with greater resistance to copper. Considering the possible biotechnological application, the enzyme presents potential for the degradation of dye.
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Palavras-chave
Multicopper oxidase, Corpúsculos de inclusão, Expressão