Preparation of CLEAs and magnetic CLEAs of a recombinant L-arabinose isomerase for D-tagatose synthesis

The insolubilization of a recombinant L-arabinose isomerase (L-AI) from Enterococcus faecium by cross-linked enzyme aggregates (CLEA) was investigated, aiming the biochemical production of D-tagatose from D-galactose. D-tagatose is a functional sweetener that has many health benefits, sweetening properties and lower calorific value. Different precipitants (ammonium sulfate, ethanol, acetone, polyethylene glycol 4000) were used in the first step of the protocol, in order to establish the precipitation conditions, and the best results of yield and activity were achieved with ammonium sulfate. In order to facilitate the recovery of the biocatalyst, a new strategy for immobilization of the multimeric enzyme L-arabinose isomerase was proposed. Magnetic crosslinked enzyme aggregates (m-CLEA) were obtained using ammonium sulfate as precipitant and magnetic nanoparticles (MNP) functionalized with APTES (3- Aminopropyltriethoxysilane). Another immobilization strategy was to immobilize the enzyme onto MNP-APTES, as a control. The best results were achieved when the m-CLEA was produced with 20 mg of MNP, 7.69 U. g(-1) of enzymatic activity, 7.61 % of recovered activity, 99 % of yield of immobilization. On the other hand, the enzyme immobilized onto MNP-APTES, presented only 2.12 U. g(-1) of enzymatic activity, 32.3 % of recovered activity, and 15 % of yield of immobilization.