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| Author(s): |
Elizama Aguiar Oliveira
Total Authors: 1
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| Document type: | Doctoral Thesis |
| Press: | Campinas, SP. |
| Institution: | Universidade Estadual de Campinas (UNICAMP). Faculdade de Engenharia de Alimentos |
| Defense date: | 2012-02-13 |
| Examining board members: |
Francisco Maugeri Filho;
Eloizio Julio Ribeiro;
Gabriela Alves Macedo;
Jonas Contiero;
Telma Teixeira Franco
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| Advisor: | Francisco Maugeri Filho |
| Abstract | |
The production of fructooligosaccharides (FOS) - a prebiotic sugar with wide industrial application - from immobilized enzymes includes two modern concepts: industrial application of immobilized enzymes and functional food. A strain isolated from flowers from the Brazilian Atlantic Forest identified as Rhodotorula sp. LEB-V10 showed great potential for production of the extracellular fructosyltransferase (FTase), which is capable of producing FOS from sucrose. It can be recovered only by precipitation with ethanol (partial purification), directly from the cell-free culture medium. According to previous studies, the kinetics between purified and partially purified immobilized enzymes showed no significant differences that could justify additional purification steps. Other previous studies have selected the adsorption on particles of a solid-acid support (Nb) an alloy consisting of niobium (~95%) and graphite (~5%) as suggested methodology for the immobilization of FTase for FOS production. Thus, this work presents a series of characterization studies of this immobilized biocatalyst. After the immobilization, it was observed that besides the pH of maximum activity presented by the free enzyme (pH 4.5), a second distinct point of maximum activity (25% lower than at pH 4.5) was also observed, pH 6.0, wich was proved to be the best condition for thermal stability and also better for synthesis with the immobilized enzyme than pH 4.5. The study of thermal stability has also shown that immobilization induces the formation of two distinct phases with different denaturation activation energy (Ead): between 47 and 51°C, the immobilized enzyme is more stable than the free enzyme, and between 52 and 70°C it is less stable. Incubation of free and immobilized FTase at 52°C for 15 min resulted in enzymatic activation, being more pronounced with the immobilized enzyme at pH 4.5 with a 1.8-fold increase in enzyme activity. However, due to other studies carried out in this work, it has been shown that this activation is merely transient and not cumulative regarding other methodologies, indicating that this temperature is a kind of energetic threshold, and also because FTase is a hetero-dimer, which may lead to different interactions with the niobium support. The optimization of the synthesis of FOS carried out by applying the methodology of experimental design with the immobilized enzyme, allowing a yield increase (YFOS = 0.58) of 5%, a reduction in synthesis time (24 h) of 4 times and an increased productivity (12.05 g/L.h) of 6 times. The adsorption on niobium has shown not to involve the FTase active sites, since the addition of sucrose to the dispersion medium for adsorption did not affect the performance of the biocatalyst. On the other hand, the addition of CuSO4, which is a strong thermal stabilizing agent for this enzyme, resulted in a biocatalyst incapable of producing FOS. Considering the industrial application of FTase, it was also evaluated the effects of lyophilization over the free and immobilized enzyme. With the free enzyme, lyophilization caused loss of enzyme activity in dependence on the density of the starting solution, but in general, resulted in a powder with up to 6 times more activity per gram as compared to non-lyophilized free enzyme; the individual effects of cryo-protectants additives presented higher stabilizing effect than with the presence of other protectants. The lyophilization of the immobilized enzyme did not show drastic effects and it was observed, in general, the maintenance of the initial properties and characteristics before lyophilization. For both, free and immobilized enzyme was possible to observe significant changes in the kinetics of FOS production, the yield was increased in different proportions but, especially, there was a high composition of GF4, which was not observed before lyophilization. In addition, two commercial enzymes (inulinase and invertase) were immobilized on niobium but no significant changes were observed as those with FTase. Thus, the immobilization of FTase in niobium proves to have a big potential for application due to operational features. As a result of this work, two articles have been published (Chapters 2 and 4) and one has been accepted for publication (Chapter 5) (AU) | |