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| Author(s): |
Paula Chequer Gouveia Mól
Total Authors: 1
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| Document type: | Doctoral Thesis |
| Press: | São José do Rio Preto. 2021-03-26. |
| Institution: | Universidade Estadual Paulista (Unesp). Instituto de Biociências Letras e Ciências Exatas. São José do Rio Preto |
| Defense date: | 2021-02-22 |
| Advisor: | Roberto Da Silva; Lizzy Ayra Alcântara Veríssimo; Luis Antonio Minim |
| Abstract | |
β-glucosidases are enzymes that hydrolyze terminal β-(1,4) glycosidic bonds, an essential function to many biological processes, that is of great interest in biochemistry and biotechnology. The purpose of this study encompasses the obtaining and posterior purification and immobilization studies of β-glucosidase from thermophilic fungus Thermoascus aurantiacus in supermacroporous supports of polyacrylamide produced in freezing conditions (knows as cryogel). The enzyme was produced by solid state fermentation using corn cob as substrate. The first cryogel column used (Chapter II) was obtained by chemical modification of the cryogel by the introduction of the ionic groups 2-(dimethylamino)ethyl methacrylate. The adsorption experiments were carried out in different pH conditions and the results of yield (%) and purification factors were calculated and submitted to ANOVA at 95% of significance level. The efficiency was considerably affected (p<0.05) by the pH and the best result was achieved at pH 5.0 (82%). Purification factors did not vary and the results were low since isocratic elution was performed (1.25-1.33). However, SDS-PAGE was also realized to investigate purity and it was verified that the crude extract was partially purified, regardless of the pH. The second column synthetized (Chapter III) was obtained by the incorporation of the aminoacid L-phenylalanine on the cryogel surface and was used in β-glucosidase immobilization tests via hydrophobic interaction. The adsorption was initially studied as a function of the pH and the highest purification factor was found at pH 3.0. Then, the effect of the temperature and ionic strength of different saline solutions on adsorption and consequently on the activity of the immobilized enzyme was evaluated. In addition, the reuse of the biocatalyst was investigated for seven consecutive cycles and no decrease on its the specific activity was observed. In both cases, the cryogel columns produced were characterized in terms of their morphological and hydrodynamic properties. The results obtained in this work show that the cryogels produced can be potential supports of protein separation and immobilization. (AU) | |
| FAPESP's process: | 16/17812-6 - Purification and immobilization of beta-glucosidase from Thermoascus aurantiacus using supermacroporous cryogels and application in bioprocesses |
| Grantee: | Paula Chequer Gouveia Mol |
| Support Opportunities: | Scholarships in Brazil - Doctorate |