Imobilização da enzima frutosiltransferase extracelular de Aspergillus oryzae IPT-301 em compósito de poli ácido lático e argila para produção de frutooligossacarídeos

Abstract

Fructooligosaccharides (FOS) are known as “unconventional sugars”. They are functional foods and prebiotics beneficial to human health. They are composed of a sucrose unit elongated by fructosyl units linked together by β-type bonds (2→1). FOS can occur naturally in different types of plants or be produced by different ways. Among the forms of commercial production, the transfructosylation reaction of sucrose, which is carried out by the enzyme fructosyltransferase (FTase) stands out. Among the microorganisms that produce these enzymes, Aspergillus oryzae IPT-301 stands out because it produces fructosyltransferase (FTase) with high transfructosylation activity (𝐴𝑡). The use of free enzyme on a commercial scale is a difficult process due to its high instability and the need for great control of process variables. Enzymatic immobilization consists of a technique aimed at fixing enzymes on organic or inorganic supports in order to provide greater stability and ease of operation for bioprocesses. In this context, this work aims to evaluate the process of immobilization of extracellular FTase from Aspergillus oryzae IPT-301 in poly (lactic acid) support enriched with 10% of refractory clay (PLA – 10AR). For that, tests of immobilization of extracellular FTase in PLA - 10AR were carried out at a temperature of 35 ºC, for 8 hours, with agitation at 175 rpm in a 10:1 proportion of broth from cell culture at pH 5.5 for support and a recovered transfructosylation activity of 17.23 ± 0.87%. A central rotational composite design (DCCR) design was carried out in order to evaluate the influences of temperature and pH on 𝐴𝑡 and to obtain the optimal conditions for the reaction that were achieved at 50ºC and pH 5.5. For characterization studies of the immobilized biocatalyst, tests were carried out to evaluate the influence of substrate concentration on the enzymatic reaction, stability against incubation pH and storage stability tests. The study of concentration effects showed that the highest activities were achieved for concentrations between 400 g.L-1 and 600 g.L-1 of sucrose, with values higher than 85% and that the Hill kinetic model provides a better fit of the data presented by the biocatalyst. Furthermore, stability against incubation pH provides relative activities above 76% for a pH range between 5.0 and 6.5. In view of the evaluation of the thermal stability for temperatures between 30 and 60ºC for 24 hours, it was pointed out that there was no gain in stability of the heterogeneous biocatalyst against the soluble enzyme. Regarding the operational stability, the biocatalyst showed a constant and stable behavior between cycles 4 and 7, decaying in cycle 8 due to the rupture of the bonds between enzyme and support. Also noteworthy is the technological innovation present in this study of immobilization of extracellular FTase in support made from a PLA nanocomposite with 10% clay for the production of FOS.