Sistemas de Duas Fases Aquosas: uma ferramenta biocompatível para extração de lipase de interesse biotecnológico
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Data
2019-04-24
Autores
Nascimento, Paloma Andrade Martins [UNESP]
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Editor
Universidade Estadual Paulista (Unesp)
Resumo
As lipases são biocatalisadores que têm como função natural a hidrólise de triglicerídeos na interface lipídeo-água, e apresentam também a capacidade de síntese de ésteres. Quando estas enzimas são produzidas de microrganismos, dependendo da sua aplicação final, existe a necessidade de extrair e purificar a enzima do meio fermentado. Desta maneira, o objetivo deste trabalho foi estudar a estabilidade da lipase comercial de Aspergillus niger frente aos agentes formadores de Sistemas de Duas Fases Aquosas (SDFA) e a capacidade de diferentes SDFA como plataforma de extração da lipase comercial de Aspergillus niger e posterior da lipase de Aspergillus sp. produzida em meio fermentado. Primeiramente foi realizado o estudo de atividade e estabilidade da enzima comercial frente aos agentes formadores dos SDFA, empregando tensoativo, polímeros, Líquidos Iônicos (LIs) e carboidratos. Os resultados mostraram que a lipase comercial é estável em solução aquosa de Triton X- 114, PEG 600 e PPG 400, enquanto, que com PPG 425 a atividade foi mantida em tampão Mcllvaine pH 5,5. Os polímeros PPG 725 e NaPA 8.000 inibiram a atividade enzimática tanto em solução aquosa quanto em tampão pH 5,5. Na presença dos LIs, o aumento da cadeia alquílica catiônica, para os cloretos de imidazólios ([Cnmim]Cl), e aniônica, para a família das colinas ([Ch]X), acarretaram em perda de atividade comprometendo o comportamento catalítico da enzima. Na presença dos carboidratos a lipase manteve sua estabilidade. Na última etapa do trabalho, foi avaliada a capacidade de extração das lipases utilizando os Sistemas Micelares de Duas Fases Aquosas com LIs como adjuvantes (SMDFA-LIs) e Sistemas Poliméricos de Duas Fases Aquosas (SPDFA) com carboidrato. Os SMDFA-LIs apresentaram pequena influência no particionamento das lipases, i.e., a adição de LIs como adjuvantes não aumentou significativamente os coeficientes de partição da enzima. Para a lipase de Aspergillus sp., o melhor resultado foi com 11% (m/m) de Triton + 0,05 M [Ch][Prop], o qual apresentou uma eficiência de extração (EE) de 64% e fator de purificação de 8,33. Os SPDFA com carboidrato foram mais promissores, apresentando uma EE de 100%. Embora os estudos de estabilidade tenham sido realizados com a lipase comercial, o trabalho permitiu elucidar as interações entre a lipase e os diferentes agentes formadores do SDFA. Os SDFA empregados neste trabalho propõem diferentes aplicações, uma vez que os SMDFA-LIs são propícios para purificação de lipases, enquanto os SPDFA com carboidrato se mostram efetivos para extração em uma única etapa do processo downstream (i.e. menos tempo e energia), o que os tornam viáveis economicamente, além de serem ambientalmente mais benignos e biocompatíveis com lipases.
Lipases are natural biocatalysts for the hydrolysis of triglycerides at the lipid-water interface and with the ability to synthesize esters. When these enzymes are produced by microorganisms, depending on its final application, it is needed to extract and purify them. Therefore, the aim of this work was to study the stability of the Aspergillus niger commercial lipase in the presence of different aqueous two-phase systems (ATPS) forming agents, and to evaluate the ability of different ATPS as extractive platforms of the Aspergillus niger commercial lipase and after Aspergillus sp. lipase produced in fermented medium. Initially, the activity and stability of the commercial lipase in the presence of ATPS forming agents were studied, namely: surfactant, polypropylene glycol, ionic liquids (ILs) and carbohydrates. The results showed that commercial lipase is stable in the presence of Triton X-114, PEG 600 and PPG 400 aqueous solutions, whereas with PPG 425 aqueous solutions the enzyme maintained its activity at Mcllvaine buffer pH 5.5. Both PPG 725 and NaPA 8,000 aqueous solutions (water and buffered at pH 5.5) inhibited the catalytic activity. In the presence of ILs, the increase of the cationic alkyl chain, using of the imidazolium-based chlorides family ([Cnmim]Cl), and anionic, for the cholinium family ([Ch]X), resulted in a loss of enzymatic activity, compromising the catalytic behavior of the lipase. On the other hand, lipase remained stable in the presence of all carbohydrates aqueous solutions. In the last part, the lipases’ extraction capability of Aqueous Two-Phase Micellar Systems using ILs as adjuvants (ATPMS-ILs) and Aqueous Two-Phase Polymeric Systems (ATPPS) with carbohydrates was evaluated. ATPMS-ILs did not influence the partitioning of lipases, i.e. the addition of ILs as adjuvants did not increase the partition coefficients of the enzyme. For the Aspergillus sp., the best result was obtained with the system composed of 11% (w/w) of Triton and 0.05 M of [Ch][Prop], which showed an extraction efficiency (EE %) of 64% and a purification factor of 8.33. The ATPPS with carbohydrates were more promising, showing in general EE of 100%. Although stability studies have been performed with commercial lipase, the work has elucidated the interactions between lipase and different ATPS forming agents. The ATPS used in this work propose different applications, since the ATPMS-ILs are suitable for lipase purification, whereas the ATPPS with carbohydrates are effective for extraction in a single step of the downstream process (i.e. less time and energy), make them economically viable, as well as being environmentally bening and biocompatible with lipases.
Lipases are natural biocatalysts for the hydrolysis of triglycerides at the lipid-water interface and with the ability to synthesize esters. When these enzymes are produced by microorganisms, depending on its final application, it is needed to extract and purify them. Therefore, the aim of this work was to study the stability of the Aspergillus niger commercial lipase in the presence of different aqueous two-phase systems (ATPS) forming agents, and to evaluate the ability of different ATPS as extractive platforms of the Aspergillus niger commercial lipase and after Aspergillus sp. lipase produced in fermented medium. Initially, the activity and stability of the commercial lipase in the presence of ATPS forming agents were studied, namely: surfactant, polypropylene glycol, ionic liquids (ILs) and carbohydrates. The results showed that commercial lipase is stable in the presence of Triton X-114, PEG 600 and PPG 400 aqueous solutions, whereas with PPG 425 aqueous solutions the enzyme maintained its activity at Mcllvaine buffer pH 5.5. Both PPG 725 and NaPA 8,000 aqueous solutions (water and buffered at pH 5.5) inhibited the catalytic activity. In the presence of ILs, the increase of the cationic alkyl chain, using of the imidazolium-based chlorides family ([Cnmim]Cl), and anionic, for the cholinium family ([Ch]X), resulted in a loss of enzymatic activity, compromising the catalytic behavior of the lipase. On the other hand, lipase remained stable in the presence of all carbohydrates aqueous solutions. In the last part, the lipases’ extraction capability of Aqueous Two-Phase Micellar Systems using ILs as adjuvants (ATPMS-ILs) and Aqueous Two-Phase Polymeric Systems (ATPPS) with carbohydrates was evaluated. ATPMS-ILs did not influence the partitioning of lipases, i.e. the addition of ILs as adjuvants did not increase the partition coefficients of the enzyme. For the Aspergillus sp., the best result was obtained with the system composed of 11% (w/w) of Triton and 0.05 M of [Ch][Prop], which showed an extraction efficiency (EE %) of 64% and a purification factor of 8.33. The ATPPS with carbohydrates were more promising, showing in general EE of 100%. Although stability studies have been performed with commercial lipase, the work has elucidated the interactions between lipase and different ATPS forming agents. The ATPS used in this work propose different applications, since the ATPMS-ILs are suitable for lipase purification, whereas the ATPPS with carbohydrates are effective for extraction in a single step of the downstream process (i.e. less time and energy), make them economically viable, as well as being environmentally bening and biocompatible with lipases.
Descrição
Palavras-chave
Lipase, Líquidos Iônicos, Estabilidade, Sistema de Duas Fases Aquosas, Extração, Aqueous Two-Phase System, Ionic liquids