Abstract
This paper shows the immobilization of the dimeric D-amino acid oxidase (DAAO) following different methodologies. Immobilization on glyoxyl-agarose yielded activity recoveries under 5%, and it was discarded. The anion exchange on aminated supports permits the rapid enzyme immobilization, but its activity decreased and the final biocatalysts is less stable than the free enzyme, suggesting that the near presence of a cationic surface is negative for enzyme performance. Treating this biocatalyst with glutaraldehyde led to the full enzyme inactivation. However, immobilization at pH 7 on glutaraldehyde preactivated agarose beads permitted very high stabilizations (enzyme dissociation is prevented) with expressed activity near 95%. The immobilization at pH 9 produced a slightly higher stabilization, but the expressed activity was 70%. The treatment of the biocatalyst with polyethylenimine produced the slowdown of the initial enzyme inactivation. The use of vinyl sulfone agarose permitted the immobilization of the enzyme, but even immobilizing the enzyme at pH 5 the expressed activity was 50%. The blocking step with aspartic permitted to maintain the activity and stability of the unblocked biocatalyst, while the aminated compounds led to enzyme destabilization/inactivation. Considering all factors, immobilization on glutaraldehyde support at pH 7 seems to be the recommended immobilization protocol for DAAO.
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•The low stability of DAAO at pH 10 prevents its immobilization on glyoxyl-agarose.•Immobilization on aminated supports gave poor stability.•Immobilization in vinyl sulfone gave moderate stabilization and expressed activity.•Optimal immobilization was achieved using preactivated glutaraldehyde supports at pH 7.