Abstract

The enzymatic one-pot synthesis of D-tagatose from lactose was optimized. The beta-galactosidase from Aspergillus oryzae (beta-gal-Ao) and the L-arabinose isomerase from Thermotoga maritima (L-AI-Tm) were used as biocatalyst. Since the enzymes have quite different optimum reaction conditions, operational windows methodology was employed to identify a reaction condition were both express high catalytic activities. In this manner, 65 degrees C and pH 6.0 were determined as the best conditions for the studied enzymes to conduct the one-pot synthesis of Dtagatose. Then, the effect of process configuration was evaluated by comparing the sequential and one-pot syntheses. Simultaneously, the effect of immobilizing the enzymes in glyoxyl-agarose was assessed. To do this, soluble enzymes and immobilized derivatives (beta-gal-AoGA and L-AI-TmGA) were evaluated in both process configurations. Remarkably, one-pot process showed better performance than sequential reaction mode. Depending on the biocatalyst combination, one-pot configuration allowed to reach reaction yields between 40 and 70% higher than the sequential mode. beta-Gal-Ao and L-AI-TmGA was determined as the best biocatalyst combination. beta-Gal-Ao exhibited better hydrolytic performance than beta-gal-AoGA. The latter expressed an enhanced transgalactosylation activity, requiring longer reaction times to complete lactose hydrolysis. On the other hand, immobilizing L-AI-Tm in glyoxyl-agarose enhanced enzyme stability, enabling to achieve maximum D-tagatose yield. In the one-pot synthesis, a maximum isomerization yield of 63.2 +/- 2.2% was reached under optimized conditions.