Lipase livre e imobilizada em quitosana : caracterização e potencial de aplicação em reações de hidrólise e síntese

Detalhes bibliográficos
Ano de defesa: 1999
Autor(a) principal: Pereira, Ernandes Benedito
Orientador(a): Não Informado pela instituição
Banca de defesa: Não Informado pela instituição
Tipo de documento: Dissertação
Tipo de acesso: Acesso aberto
Idioma: por
Instituição de defesa: Universidade Estadual de Maringá
Brasil
Departamento de Engenharia Química
Programa de Pós-Graduação em Engenharia Química
UEM
Maringá, PR
Centro de Tecnologia
Programa de Pós-Graduação: Não Informado pela instituição
Departamento: Não Informado pela instituição
País: Não Informado pela instituição
Palavras-chave em Português:
Link de acesso: http://repositorio.uem.br:8080/jspui/handle/1/3779
Resumo: The objective of this work was the production of immobilized lipase derivatives on chitosan (cheap support), aiming their application on hydrolytic and synthetic reactions. The derivatives were obtained by physical adsorption and covalent binding using hexane as dispersion medium. Two types of chitosan (analytical and pharmaceutical grade) were used for immobilizing lipase from microbial source (Candida rugosa) and animal cell (porcine pancreas). The best results with respect to the recovery of total activity afier immobilization (protein retention of 80% and immobilization efficiency of 15%) were obtained for microbial lipase and pharmaceutical grade chitosan. This set was selected for further immobilization studies, including fiilly characterization of the immobilized derivative under aqueous and non-aqueous media. Under aqueous medium, a comparative study between free and immobilized lipase was provided in terms of p11, temperature, thermal stability and operational stability. Slightly lower value for optimum p11 (6.0) was found for the immobilized form in comparison with that attained for the free lipase (7.0). The optimal reaction temperature shifted from 37°C for the free lipase to 50°C for the chitosan lipase. The patterns of heat stability indicated that the immobilization process tends to stabilize the enzyme. The half life of the free lipase at 55°C was equal to 0.41 h (K1= 1.7 h1), whereas for the immobilized lipase was found to be 0.86 h (kd = 0.81 h-1). The operation stability of the immobilized lipase was tested by repeated assays (olive oil hydrolysis for 10 mm! 3 7°C) and a half-life of 5 hours was observed. Under organic medium, the potential application of the immobilized derivative was verified by testing its performance on the esterification reaction using several aliphatic alcohols and fatty acids. The system butanol (ButOH)I butyric acid (Abut) was selected for a detailed study employing statistical experimental design. A full 2 factorial design at two levels was used to determine the influence of three factors: temperature (37 and 50°C), amount of lipase (0.25 and 0.5g) and molar ratio between Abut: ButOH (1 and 2) on the variable response (yield of ester). Three runs were carried out at the center point level for experiment error estimation. The results were analyzed by Analysis of Variance (ANOVA) and the most significant main effect on the formation of butyl butyrate was the initial lipase amount. For the experiment range studied, there was no evidence on the interaction of the lipase amount with the other two factors. The immobilized lipase was used repeatedly (seven times) in batch esterification reactions of butanol with butyric acid and the activity loss in long-term was about 28%, however there was a significant decrease on the esterification yields (83%). This suggested that no desorption of the enzyme from the support occurred. It is therefore likely that reactants and product bind to the solid enzyme phase resulting in drastic changes in the enzyme synthetic activity for the next cycle. In this sense, techniques allowing the removal of these potential inhibitors from the enzyme solid phase, are expected to improve the operational stability of this immobilized derivative.
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spelling Lipase livre e imobilizada em quitosana : caracterização e potencial de aplicação em reações de hidrólise e sínteseFree and immobilized lipase on chitosan: characterization and potential application on hydrolysis and synthesis reactionsDesenvolvimento de processosQuitosanaBrasil.EngenhariasEngenharia QuímicaThe objective of this work was the production of immobilized lipase derivatives on chitosan (cheap support), aiming their application on hydrolytic and synthetic reactions. The derivatives were obtained by physical adsorption and covalent binding using hexane as dispersion medium. Two types of chitosan (analytical and pharmaceutical grade) were used for immobilizing lipase from microbial source (Candida rugosa) and animal cell (porcine pancreas). The best results with respect to the recovery of total activity afier immobilization (protein retention of 80% and immobilization efficiency of 15%) were obtained for microbial lipase and pharmaceutical grade chitosan. This set was selected for further immobilization studies, including fiilly characterization of the immobilized derivative under aqueous and non-aqueous media. Under aqueous medium, a comparative study between free and immobilized lipase was provided in terms of p11, temperature, thermal stability and operational stability. Slightly lower value for optimum p11 (6.0) was found for the immobilized form in comparison with that attained for the free lipase (7.0). The optimal reaction temperature shifted from 37°C for the free lipase to 50°C for the chitosan lipase. The patterns of heat stability indicated that the immobilization process tends to stabilize the enzyme. The half life of the free lipase at 55°C was equal to 0.41 h (K1= 1.7 h1), whereas for the immobilized lipase was found to be 0.86 h (kd = 0.81 h-1). The operation stability of the immobilized lipase was tested by repeated assays (olive oil hydrolysis for 10 mm! 3 7°C) and a half-life of 5 hours was observed. Under organic medium, the potential application of the immobilized derivative was verified by testing its performance on the esterification reaction using several aliphatic alcohols and fatty acids. The system butanol (ButOH)I butyric acid (Abut) was selected for a detailed study employing statistical experimental design. A full 2 factorial design at two levels was used to determine the influence of three factors: temperature (37 and 50°C), amount of lipase (0.25 and 0.5g) and molar ratio between Abut: ButOH (1 and 2) on the variable response (yield of ester). Three runs were carried out at the center point level for experiment error estimation. The results were analyzed by Analysis of Variance (ANOVA) and the most significant main effect on the formation of butyl butyrate was the initial lipase amount. For the experiment range studied, there was no evidence on the interaction of the lipase amount with the other two factors. The immobilized lipase was used repeatedly (seven times) in batch esterification reactions of butanol with butyric acid and the activity loss in long-term was about 28%, however there was a significant decrease on the esterification yields (83%). This suggested that no desorption of the enzyme from the support occurred. It is therefore likely that reactants and product bind to the solid enzyme phase resulting in drastic changes in the enzyme synthetic activity for the next cycle. In this sense, techniques allowing the removal of these potential inhibitors from the enzyme solid phase, are expected to improve the operational stability of this immobilized derivative.Objetivou-se, neste trabalho, a produção de amostras de lipase imobilizada em quitosana (suporte de baixo custo), visando aplicação em reações de hidrólise e síntese. A lipase imobilizada foi obtida por adsorção fisica ou ligação covalente usando hexano como meio de dipersão. Dois tipos de quitosana (grau analítico e farmacêutico) foram usados para imobilizar a lipase de fonte microbiana (Candida rugosa) e de células animais (pâncreas de porco). Os melhores resultados com relação à retenção de proteína (8 0%) e eficiência de imobilização (15%) foram obtidos para a lipase microbiana imobilizada em quitosana de grau farmacêutico. Este par foi selecionado para estudos de imobilização adicionais, incluindo a caracterização do derivado imobilizado em meio aquoso e não-aquoso. Em meio aquoso, um estudo comparativo entre lipase livre e imobilizada foi efetuado em termos de pH, temperatura, estabilidade térmica e estabilidade operacional. Mediante o procedimento de imobilização, ocorreram modificações para pH mais ácido (6,0) e para maior valor de temperatura ótima (45°C) quando comparado com os valores originais da lipase na sua forma livre (pH ótimo 7,0 e temperatura de 37°C). Os perfis das curvas de estabilidade térmica sugerem que o procedimento de imobilização tende a aumentar a estabilidade térmica da enzima lipase. O tempo de meia-vida da lipase livre a 55°C foi da ordem de 0,41 h (Kd = 1.7 h-1enquanto para a lipase imobilizada foi encontrado um valor 0,86 h (kd = 0,81 h). A estabilidade operacional da lipase imobilizada em meio não-aquoso, verificada em bateladas cíclicas de hidrólise do azeite de oliva (3 7°C/10 minutos), revelou um tempo de meia-vida de 5 horas a 37°C. Na caracterização em meio orgânico, a potencialidade da lipase imobilizada foi inicialmente verificada pela quantificação do seu desempenho na esterificação direta de diversos álcoois alifáticos e ácidos graxos, sendo selecionado para um estudo mais detalhado o sistema butanol (ButOH)/ ácido butírico (Abut), empregando a metodologia de planejamento estatístico. Um planejamento fatorial completo 2 em dois níveis foi usado para determinar a influência dos fatores: temperatura (37 e 50°C), massa de lipase (0,25 e 0,5 gramas) e razão molar entre Abut: ButOH (1 e 2) na variável resposta (rendimento de éster). Para cálculo do erro experimental, foram realizados três experimentos no nível médio. Os resultados foram analisados pela técnica da Análise de Variância (ANOVA), sendo verificado como fator mais significativo na formação de butirato de butila a massa de enzima. Dentro da região experimental avaliada, não houve evidência de interação da massa de lipase com os outros dois fatores. A lipase imobilizada foi usada sucessivamente (24 h/ 3 7°C) em reações de esterificação do butanol com ácido butírico e ao final do sétimo reciclo foi constatada uma redução significativa no rendimento do éster (83%) apesar de uma perda da atividade hidrolítica de apenas 28%. Isto sugere que não ocorre desligamento da enzima do suporte mas uma alteração das condições de hidratação da preparação imobilizada, afetando diretamente a atividade sintética no novo ciclo. Nesse sentido, técnicas que permitam a remoção de reagentes não consumidos e produto formado da fase sólida da enzima podem melhorar a estabilidade operacional da lipase imobilizada. Por meio da técnica adotada foi constatado que, além da simplicidade do próprio método, a conformação da enzima e do seu sítio ativo foram preservados por não haver o envolvimento de nenhuma espécie reativa, o que propiciou a obtenção de um sistema imobilizado com atividade média de 50 U!mg de suporte seco, cujas propriedades catalíticas foram consideradas adequadas tanto para conduzir reações de hidrólise (hidrólise de azeite de oliva) como de esterificação (síntese do butirato de butila ou ésteres similares).xviii, 127 f. : il., tabs. (algumas color.)Universidade Estadual de MaringáBrasilDepartamento de Engenharia QuímicaPrograma de Pós-Graduação em Engenharia QuímicaUEMMaringá, PRCentro de TecnologiaGisella Maria ZaninMaria Helena Andrade SantanaFlávio Faria de Moraes - UEMPereira, Ernandes Benedito2018-04-17T17:43:54Z2018-04-17T17:43:54Z1999info:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/masterThesishttp://repositorio.uem.br:8080/jspui/handle/1/3779porinfo:eu-repo/semantics/openAccessreponame:Repositório Institucional da Universidade Estadual de Maringá (RI-UEM)instname:Universidade Estadual de Maringá (UEM)instacron:UEM2024-03-19T21:14:34Zoai:localhost:1/3779Repositório InstitucionalPUBhttp://repositorio.uem.br:8080/oai/requestopendoar:2024-04-23T14:56:56.020236Repositório Institucional da Universidade Estadual de Maringá (RI-UEM) - Universidade Estadual de Maringá (UEM)false
dc.title.none.fl_str_mv Lipase livre e imobilizada em quitosana : caracterização e potencial de aplicação em reações de hidrólise e síntese
Free and immobilized lipase on chitosan: characterization and potential application on hydrolysis and synthesis reactions
title Lipase livre e imobilizada em quitosana : caracterização e potencial de aplicação em reações de hidrólise e síntese
spellingShingle Lipase livre e imobilizada em quitosana : caracterização e potencial de aplicação em reações de hidrólise e síntese
Pereira, Ernandes Benedito
Desenvolvimento de processos
Quitosana
Brasil.
Engenharias
Engenharia Química
title_short Lipase livre e imobilizada em quitosana : caracterização e potencial de aplicação em reações de hidrólise e síntese
title_full Lipase livre e imobilizada em quitosana : caracterização e potencial de aplicação em reações de hidrólise e síntese
title_fullStr Lipase livre e imobilizada em quitosana : caracterização e potencial de aplicação em reações de hidrólise e síntese
title_full_unstemmed Lipase livre e imobilizada em quitosana : caracterização e potencial de aplicação em reações de hidrólise e síntese
title_sort Lipase livre e imobilizada em quitosana : caracterização e potencial de aplicação em reações de hidrólise e síntese
author Pereira, Ernandes Benedito
author_facet Pereira, Ernandes Benedito
author_role author
dc.contributor.none.fl_str_mv Gisella Maria Zanin
Maria Helena Andrade Santana
Flávio Faria de Moraes - UEM
dc.contributor.author.fl_str_mv Pereira, Ernandes Benedito
dc.subject.por.fl_str_mv Desenvolvimento de processos
Quitosana
Brasil.
Engenharias
Engenharia Química
topic Desenvolvimento de processos
Quitosana
Brasil.
Engenharias
Engenharia Química
description The objective of this work was the production of immobilized lipase derivatives on chitosan (cheap support), aiming their application on hydrolytic and synthetic reactions. The derivatives were obtained by physical adsorption and covalent binding using hexane as dispersion medium. Two types of chitosan (analytical and pharmaceutical grade) were used for immobilizing lipase from microbial source (Candida rugosa) and animal cell (porcine pancreas). The best results with respect to the recovery of total activity afier immobilization (protein retention of 80% and immobilization efficiency of 15%) were obtained for microbial lipase and pharmaceutical grade chitosan. This set was selected for further immobilization studies, including fiilly characterization of the immobilized derivative under aqueous and non-aqueous media. Under aqueous medium, a comparative study between free and immobilized lipase was provided in terms of p11, temperature, thermal stability and operational stability. Slightly lower value for optimum p11 (6.0) was found for the immobilized form in comparison with that attained for the free lipase (7.0). The optimal reaction temperature shifted from 37°C for the free lipase to 50°C for the chitosan lipase. The patterns of heat stability indicated that the immobilization process tends to stabilize the enzyme. The half life of the free lipase at 55°C was equal to 0.41 h (K1= 1.7 h1), whereas for the immobilized lipase was found to be 0.86 h (kd = 0.81 h-1). The operation stability of the immobilized lipase was tested by repeated assays (olive oil hydrolysis for 10 mm! 3 7°C) and a half-life of 5 hours was observed. Under organic medium, the potential application of the immobilized derivative was verified by testing its performance on the esterification reaction using several aliphatic alcohols and fatty acids. The system butanol (ButOH)I butyric acid (Abut) was selected for a detailed study employing statistical experimental design. A full 2 factorial design at two levels was used to determine the influence of three factors: temperature (37 and 50°C), amount of lipase (0.25 and 0.5g) and molar ratio between Abut: ButOH (1 and 2) on the variable response (yield of ester). Three runs were carried out at the center point level for experiment error estimation. The results were analyzed by Analysis of Variance (ANOVA) and the most significant main effect on the formation of butyl butyrate was the initial lipase amount. For the experiment range studied, there was no evidence on the interaction of the lipase amount with the other two factors. The immobilized lipase was used repeatedly (seven times) in batch esterification reactions of butanol with butyric acid and the activity loss in long-term was about 28%, however there was a significant decrease on the esterification yields (83%). This suggested that no desorption of the enzyme from the support occurred. It is therefore likely that reactants and product bind to the solid enzyme phase resulting in drastic changes in the enzyme synthetic activity for the next cycle. In this sense, techniques allowing the removal of these potential inhibitors from the enzyme solid phase, are expected to improve the operational stability of this immobilized derivative.
publishDate 1999
dc.date.none.fl_str_mv 1999
2018-04-17T17:43:54Z
2018-04-17T17:43:54Z
dc.type.status.fl_str_mv info:eu-repo/semantics/publishedVersion
dc.type.driver.fl_str_mv info:eu-repo/semantics/masterThesis
format masterThesis
status_str publishedVersion
dc.identifier.uri.fl_str_mv http://repositorio.uem.br:8080/jspui/handle/1/3779
url http://repositorio.uem.br:8080/jspui/handle/1/3779
dc.language.iso.fl_str_mv por
language por
dc.rights.driver.fl_str_mv info:eu-repo/semantics/openAccess
eu_rights_str_mv openAccess
dc.publisher.none.fl_str_mv Universidade Estadual de Maringá
Brasil
Departamento de Engenharia Química
Programa de Pós-Graduação em Engenharia Química
UEM
Maringá, PR
Centro de Tecnologia
publisher.none.fl_str_mv Universidade Estadual de Maringá
Brasil
Departamento de Engenharia Química
Programa de Pós-Graduação em Engenharia Química
UEM
Maringá, PR
Centro de Tecnologia
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instname_str Universidade Estadual de Maringá (UEM)
instacron_str UEM
institution UEM
reponame_str Repositório Institucional da Universidade Estadual de Maringá (RI-UEM)
collection Repositório Institucional da Universidade Estadual de Maringá (RI-UEM)
repository.name.fl_str_mv Repositório Institucional da Universidade Estadual de Maringá (RI-UEM) - Universidade Estadual de Maringá (UEM)
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