Imobilização de células íntegras de Aspergillus oryzae IPT-301 visando estudos biocatalíticos em reator de leito fixo para a produção de frutooligossacarídeos

Abstract

Fructooligosaccharides (FOS) are low-calorie prebiotic sugars that have several benefits to human health and nutrition. They are commercially available through synthetic production, by transfructosylation reaction, using microbial enzymes such as fructosyltransferase (FTase, E.C.2.4.1.9) and sucrose as a substrate. In the group of the microorganisms potentially producing these enzymes, Aspergillus oryzae IPT-301 stands out synthesizing mycelial FTase (enzyme attached to microbial cells) with high transfructosylation (AT) activity. Presently, the production of FOS is carried out in batch bioreactors, a slow and costly process. Therefore, it is necessary to implement continuous reaction systems, in fixed bed reactors (PBR) that increase the production volume of FOS and reduce its production costs. Consequently, the use of biocatalysts in the form of intact cells to produce sugar becomes advantageous because they exhibit natural support for the enzyme itself. For that reason, this work aimed to immobilize intact A. oryzae IPT-301 cells by crosslinking with glutaraldehyde and to evaluate their biocatalytic effects when packed in a fixed bed reactor (PBR), point to obtain high enzymatic activity to produce FOS. Firstly, we sought to develop the biocatalyst through the production and immobilization of intact microbial cells and, afterwards, implement the continuous process of sugar production in a PBR reactor, to evaluate the biocatalytic effects of the immobilized biomass. For the development of the biocatalyst, the influence of immobilization variables (pH, temperature, glutaraldehyde concentration and stirring speed) on AT activity was investigated by different experimental design techniques. From the best immobilization conditions achieved (pH 7.9; 25 ºC, 200 rpm and 2.1 % v.v-1 of glutaraldehyde), different reaction times (30, 45, 60, 75 and 90 min) were investigated, for the process, obtaining the highest values of AT when crosslinking the microbial biomass for 45 minutes. The results obtained show the promising development of a biocatalyst with high enzymatic activity. For the implementation of cross-linked intact cells in a PBR reactor, the influence of the height of the catalytic bed (20, 15 and 10 cm), the temperature of the reaction medium (30, 40, 50 and 60 ºC), the concentration of substrate (200, 300, 400, 473, 500 and 600 g L-1) and the volumetric flow rate (1.0, 2.0, 3.0, 4.0- and 5.0-mL min-1) in the AT profiles. The kinetic parameters were also obtained by adjusting the kinetic model to the experimental data and, finally, external mass transfer (TME) studies and operational stability tests were carried out, in the absence and presence of recycle current, in the PBR reactor. The best enzyme activity profiles were achieved for the reactor filled with 20 cm of catalytic bed, containing crosslinked intact cells with an equivalent diameter of 2.58 ± 0.3 mm, operated at 50 ºC and fed with sucrose solution of 473 g L -1, pH 5.5, at a flow rate of 1.0 mL min-1. Enzyme kinetics was better adjusted to the Michaelis-Menten model. It was also noted that the reaction was limited by the effects of TME. The operational stability tests showed that, by implementing the recycle current in the reactor, an increase of 60% in the relative AT was obtained, whose maximum enzymatic activity was maintained after 540 minutes of reaction. In view of the results obtained, it was concluded that the implementation of the continuous process, aiming at the production of FOS, proved to be promising in achieving high profiles of enzymatic activity in a PBR reactor filled with cross-linked intact cells.