Author(s): Pereira, Martinha
Date: 2005
Persistent ID: http://hdl.handle.net/1822/4829
Origin: RepositóriUM - Universidade do Minho
Subject(s): Endo-poligalacturonase; Kluyveromyces marxianus; Sistemas de duas fases aquosas; Polímeros termossensíveis; Endo-polygalacturonase; Aqueous two-phase systems; Thermoseparating polymers; 577.15; 582.282.23; 663.1
Dissertação de doutoramento em Engenharia Química e Biológica.
Com o trabalho presente nesta dissertação pretendeu-se estabelecer procedimentos para a purificação da enzima endo-poligalacturonase, produzida pela levedura Kluyveromyces marxianus. Para tal foram usados os sistemas de duas fases aquosas. As estirpes provenientes da fermentação do cacau foram estudadas para encontrar a melhor produtora de enzima utilizando, como fonte de carbono, a glucose ou a sacarose. Foram seleccionadas as estirpes CH4-1, CH0-1 e CCT 3172. Para esta última estirpe, foram realizados ensaios em que se verificou que a produtividade da enzima era máxima quando somente era utilizada a agitação mecânica, sem introdução de ar. Para os ensaios de purificação foram determinados alguns diagramas de fases, cuja informação foi utilizada nos ensaios de partição e extracção. Foram realizados ensaios de partição para vários sistemas de duas fases aquosas. Verificou-se que os sistemas mais adequadas para a partição da enzima são os sistemas de composição 24 % PEG-5,6 % (NH4)2SO4 e 6,9 % PEG8000-8,7 % PVA (percentagens mássicas). Foram propostos 2 esquemas de separação. O primeiro, constituído por um sistema de composição 24 % PEG-5,6 % (NH4)2SO4, cujo objectivo foi a extracção da enzima, em série com um segundo que, por adição de mais sal, permitia a recuperação do PEG. O factor de purificação obtido foi de 1,25 e factor de concentração entre 5 e 8. Este esquema promovia a concentração da endo-PG. O segundo esquema tem como objectivo a purificação e a concentração. Para tal é utilizado um sistema de composição 6,9 % PEG8000-8,7 % PVA, seguido de outro de composição 24 % PEG-5,6 % (NH4)2SO4. Foram obtidos factores de purificação e concentração de 1,87 e 5,2, respectivamente. A recuperação da enzima foi de 91 %. Os polímeros termossensíveis foram também estudados. Foi proposto um esquema de separação, baseado no sistema 27 % Ucon – 2,2 % (NH4)2SO4 que permitia a purificação e concentração da enzima e, concomitantemente, a recuperação do Ucon, por aumento da temperatura. Foram alcançados factores de concentração de 10 com recuperação de enzima superiores a 95 %. Com este trabalho foram propostos 3 esquemas de extracção, que se verificaram adequados para a purificação da enzima endo-poligalacturonase. . The aim of the work presented in this thesis was the establishment of procedures for the purification of the enzyme endo-polygalacturonase, obtained by Kluyveromyces marxianus fermentation. For that purpose, the aqueous two-phase extraction was used. The strains, isolated from cocoa fermentation, were studied to find the best enzyme producer, using glucose or sucrose as carbon source. The strains CH4-1, CH0-1 e CCT 3172 were selected. For this last strain, some experiments were done, being possible to see that the enzyme productivity was higher when only mechanical stirring was used, without air injection. Partition experiments were done using various aqueous two-phase systems. It was possible to see that the more suitable systems for enzyme extraction were the ones with the following composition: 24 % PEG-5.6 % (NH4)2SO4 and 6.9 % PEG8000-8.7 % PVA. Two separation schemes were proposed. The first one, constructed for the system 24 % PEG-5.6 % (NH4)2SO4, which objective was the enzyme extraction, in series with a second one that, with more salt addition, allows to the PEG recuperation. The purification factor obtained was 1.25 and the concentração factor was between 5 and 8. This scheme allows endo-PG purification. The second scheme has the objective of purification and concentration. For that, a system with composition 6.9 % PEG8000-8.7 % PVA, followed by another of 24 % PEG-5.6 % (NH4)2SO4, was used. Purification and concentration factor of 1.87 and 5.2, respectively, were obtained. The enzyme yield was 91 %. The thermoseparating polymers were also studied. One separation scheme based on the system 27 % Ucon – 2.2 % (NH4)2SO4, was proposed. This scheme allows enzyme purification and concentration and at the same time Ucon recuperation, by temperature increasing. Concentration factor of 10 and enzyme recuperation higher than 95 % were obtained.
The aim of the work presented in this thesis was the establishment of procedures for the purification of the enzyme endo-polygalacturonase, obtained by Kluyveromyces marxianus fermentation. For that purpose, the aqueous two-phase extraction was used. The strains, isolated from cocoa fermentation, were studied to find the best enzyme producer, using glucose or sucrose as carbon source. The strains CH4-1, CH0-1 e CCT 3172 were selected. For this last strain, some experiments were done, being possible to see that the enzyme productivity was higher when only mechanical stirring was used, without air injection. Partition experiments were done using various aqueous two-phase systems. It was possible to see that the more suitable systems for enzyme extraction were the ones with the following composition: 24 % PEG-5.6 % (NH4)2SO4 and 6.9 % PEG8000-8.7 % PVA. Two separation schemes were proposed. The first one, constructed for the system 24 % PEG-5.6 % (NH4)2SO4, which objective was the enzyme extraction, in series with a second one that, with more salt addition, allows to the PEG recuperation. The purification factor obtained was 1.25 and the concentração factor was between 5 and 8. This scheme allows endo-PG purification. The second scheme has the objective of purification and concentration. For that, a system with composition 6.9 % PEG8000-8.7 % PVA, followed by another of 24 % PEG-5.6 % (NH4)2SO4, was used. Purification and concentration factor of 1.87 and 5.2, respectively, were obtained. The enzyme yield was 91 %. The thermoseparating polymers were also studied. One separation scheme based on the system 27 % Ucon – 2.2 % (NH4)2SO4, was proposed. This scheme allows enzyme purification and concentration and at the same time Ucon recuperation, by temperature increasing. Concentration factor of 10 and enzyme recuperation higher than 95 % were obtained.
Fundação para a Ciência e a Tecnologia (FCT) - Praxis XXI BD/18060/98.
CEC - INCO-DC - contract number ERB IC18 CT97 0182.
Fundação Calouste Gulbenkian (FCG).
