Preparação de agregados enzimáticos magnéticos (mCLEAs) de β-galactosidase para produção integrada de D-tagatose a partir de lactose

Abstract

The use of immobilized enzymes allows achieving an improvement in its efficiency, enabling the reuse in different processes and easy product recovery. Besides, it also allows the simultaneous use of enzymes in cascade reactions, making it faster. Thus, the present work aims to establish an efficient immobilization protocol for β-galactosidase using the magnetic cross-linked enzyme aggregates (mCLEAs) methodology and its application with L-arabinose isomerase (LAI) mCLEAs, to produce D-tagatose from whey lactose. Different immobilization protocols using mCLEAs were investigated, using different glutaraldehyde concentrations and dextran-aldehyde as enzyme cross-linking agents. The immobilization parameters of the biocatalysts were compared, such as recovered activity compared to the free enzyme, lactose hydrolysis capacity, enzyme kinetics, as well as thermal, operational, and storage stabilities. The biocatalyst that stood out in critical parameters for catalysis was chosen to be used with LAI mCLEAs to produce D-tagatose from lactose. After the studies of recovered activity, thermal stability at 60 °C, and lactose hydrolysis capacity, the biocatalysts mCLEA- GLU-1.5 and mCLEA-DEX were selected between the studied ones as the most efficient to application. mCLEA-GLU-1.5 presented lower substrate affinity when compared to mCLEA-DEX and the free enzyme and lower stability at 50 °C. In contrast, it was the most stable biocatalyst when assayed for operational and storage stabilities. With higher recovered activity and substrate affinity, mCLEA-DEX was used in sequential reactors with mCLEA-LAI, using a lactose solution with the same concentration as whey, yielding 10% conversion of lactose into D-tagatose at the end of the process. This work allowed to certify the viability of mCLEAs to immobilize β-galactosidase, with mCLEA-GLU-1.5 and mCLEA-DEX being viable biocatalysts to industrial use in whey lactose hydrolysis. Besides, it proves the potential of β- galactosidase and LAI mCLEAs joint use to produce D-tagatose from whey.