Imobilização de β-galactosidase e L-arabinose isomerase em agarose visando a produção integrada de D-tagatose

Abstract

The aim of this thesis was to immobilize the enzymes Kluyveromyces lactis β-galactosidase (LAC) and Enterococcus faecium L-arabinose isomerase expressed in E. coli DH10B (LAI) onto agarose as a proposal for D-tagatose integrated production. Followed by two strategies: immobilization enzymes LAC and LAI onto agarose-based supports, DEAE (diethylaminoethyl) MANAE (Monoaminoethyl-N-Ethyl), and lastly, 6 BCL glyoxyl agarose (6 % cross-linked agarose particles). The first step of the research consisted in immobilizing the enzymes separately by adsorption on DEAE and MANAE supports in 5 mM potassium phosphate buffer, pH 7, supplemented with 0.1 mM MnCl2 at pH 7.0 and room temperature. The biocatalysts presented immobilization yield above 89 %, especially the biocatalysts immobilized in MANAE. This support was also more advantageous when observing a higher rate of bioconversion for both LAC and LAI (98.7 and 6.6 %, respectively). The LAC and LAI biocatalysts presented higher values for Vmax, 1.3 and 0.09 mM.min-1, respectively, when immobilized in MANAE and DEAE, respectively. In the second step, the enzymes LAC and LAI were immobilized via multipoint binding in support of Glyoxyl-agarose 6BCL with concentrations of aldehyde groups of 75 and 200 µmol.mL-1, in 100 mM potassium bicarbonate buffer supplemented with 0.1 M KCl and 0.1 mM MgCl2, at pH 10.0. The methodology manifested the application of biocatalysts in co-immobilization assays, since it presented immobilization yield above 98 %, and t1/2 100 and 900 min and 96 and 5.8 % bioconversions for LAC and LAI enzymes, respectively. The co-immobilized enzymes were analyzed for the integrated production of D-tagatose from D-lactose as well as cheese whey, reaching in both cases 1.4 mM of D-tagatose. Thus, the study reaches new approaches for obtaining D-tagatose.