Expressão heteróloga de uma protease coagulante produzida por Thermomucor indicae-seudaticae N31 em Escherichia coli e Pichia pastoris
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Data
2019-04-26
Autores
Pereira, Waldir Eduardo Simioni
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Universidade Estadual Paulista (Unesp)
Resumo
Proteases são enzimas cuja função é a hidrólise de proteínas e peptídeos, gerando produtos de variados tamanhos. São produzidas pelos mais diversos organismos vivos e possuem um amplo espectro de aplicações. O objetivo desse trabalho foi promover a expressão heteróloga de uma protease coagulante produzida por Thermomucor indicae-seudaticae em Escherichia coli e Pichia pastoris, através da identificação do gene codificador da enzima. Para isso, foi realizada a comparação entre o genoma de T. indicae-seudaticae e Rhizomucor pusillus utilizado como referência. Os vetores pET 28ª (+) e pPICZαA foram sintetizados contendo o gene de interesse para a transformação em E. coli e P. pastoris respectivamente. Células competentes de cinco diferentes linhagens de E. coli foram transformadas e submetidas a teste de expressão em 20 °C e 37 °C por 24 horas, analisadas tanto a fração solúvel, quanto insolúvel, onde apresentaram, aparentemente, a expressão com maior quantidade em fração insolúvel. Células de P. pastoris foram transformadas e submetidas a expressão por 72 horas. O extrato enzimático bruto do produto gerado da expressão foi caracterizado bioquimicamente. A enzima recombinante produzida por E. coli apresentou máxima atividade de 250,89 U mL-1 utilizando caseína como substrato nas condições de pH 5,0 e temperatura de 50 °C, porém com estabilidade após 24 horas apenas nos pH 6,0 e 6,5, demonstrando assim ser sensível em pH alcalino. Enquanto que a enzima recombinante produzida por P. pastoris apresentou máxima atividade de 57,82 U mL-1 nas condições de pH 5,0 e temperatura de 60 °C. Ainda a enzima produzida por P. pastoris apresentou melhor termoestabilidade quando comparada com a produzida por E. coli e ambas as enzimas tiveram forte inibição da atividade por pepstatina A, confirmando pertencer a classe das proteases aspárticas e também pelo íon mercúrio o que confirma a presença de pontes dissulfeto na estrutura da enzima.
Proteases are enzymes whose function is the hydrolysis of proteins and peptides, generating products of various sizes. They are produced by the most diverse living organisms and have a wide spectrum of applications. The objective of this work was to promote the heterologous expression of a coagulant protease produced by Thermomucor indicae-seudaticae in Escherichia coli and Pichia pastoris through the identification of the gene encoding the enzyme. For this, a comparison was made between the genome of T. indicae-seudaticae and Rhizomucor pusillus used as reference. The vectors pET 28a (+) and pPICZαA were synthesized containing the gene of interest for transformation in E. coli and P. pastoris respectively. Competent cells from five different strains of E. coli were transformed and submitted to the expression test at 20 ° C and 37 ° C for 24 hours, analyzed both the soluble and the insoluble fraction, where they apparently presented the expression with the highest amount in insoluble fraction. P. pastoris cells were transformed and submitted to expression for 72 hours. The crude enzyme extract of the expression product generated was biochemically characterized. The recombinant enzyme produced by E. coli presented maximum activity of 250.89 U mL-1 using caseine as substrate under conditions of pH 5.0 and temperature of 50 ° C, but with stability after 24 hours only at pH 6.0 and 6.5, thus demonstrating to be sensitive at alkaline pH. While the recombinant enzyme produced by P. pastoris presented maximum activity of 57.82 U mL-1 under conditions of pH 5.0 and temperature of 60 ° C. In addition, the enzyme produced by P. pastoris presented better thermostability when compared to that produced by E. coli and both enzymes had strong inhibition of the activity by pepstatin A, confirming that it belongs to the class of aspartic proteases and also by the mercury ion, which confirms the presence of disulfide bonds in the structure of the enzyme.
Proteases are enzymes whose function is the hydrolysis of proteins and peptides, generating products of various sizes. They are produced by the most diverse living organisms and have a wide spectrum of applications. The objective of this work was to promote the heterologous expression of a coagulant protease produced by Thermomucor indicae-seudaticae in Escherichia coli and Pichia pastoris through the identification of the gene encoding the enzyme. For this, a comparison was made between the genome of T. indicae-seudaticae and Rhizomucor pusillus used as reference. The vectors pET 28a (+) and pPICZαA were synthesized containing the gene of interest for transformation in E. coli and P. pastoris respectively. Competent cells from five different strains of E. coli were transformed and submitted to the expression test at 20 ° C and 37 ° C for 24 hours, analyzed both the soluble and the insoluble fraction, where they apparently presented the expression with the highest amount in insoluble fraction. P. pastoris cells were transformed and submitted to expression for 72 hours. The crude enzyme extract of the expression product generated was biochemically characterized. The recombinant enzyme produced by E. coli presented maximum activity of 250.89 U mL-1 using caseine as substrate under conditions of pH 5.0 and temperature of 50 ° C, but with stability after 24 hours only at pH 6.0 and 6.5, thus demonstrating to be sensitive at alkaline pH. While the recombinant enzyme produced by P. pastoris presented maximum activity of 57.82 U mL-1 under conditions of pH 5.0 and temperature of 60 ° C. In addition, the enzyme produced by P. pastoris presented better thermostability when compared to that produced by E. coli and both enzymes had strong inhibition of the activity by pepstatin A, confirming that it belongs to the class of aspartic proteases and also by the mercury ion, which confirms the presence of disulfide bonds in the structure of the enzyme.
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Palavras-chave
Clonagem molecular, Enzima proteolítica, DNA recombinante, Caracterização bioquímica, Molecular cloning, Proteolitic enzyme, Recombinant DNA, Biochemical characterization