Imobilização direcionada da L-arabinose isomerase recombinante em suporte agarose

Abstract

The search for healthy eating is a growing concern, with it researching for healthier sweeteners as well, especially for sucrose replacement. D-tagatose is a sugar with 92% of the organoleptic properties of sucrose, but with 1/3 of its calories and it is not cariogenic. This sugar can be produced by enzymatic route through the isomerization of D-galactose by the enzyme L-arabinose isomerase (L-AI) EC 5.3.1.4. However, this enzyme is not yet sold commercially due to bottlenecks present in its production. Therefore, the immobilization of L-arabinose isomerase produced heterologously in Escherichia coli (E. coli) was studied in order to characterize it and compare it in its free state. In addition, computational tools understand the interaction of the enzyme with its substrate D-galactose. For comparative purposes, D-tagatose was produced using the enzyme free and immobilized on 6BCL agarose. The pH and maximum activity temperature, when the enzyme is free, is 5.6 and 50°C, respectively and with the immobilized enzyme is 10 and 50°C. The enzyme characterization assays showed greater stability against temperature and pH when the enzyme is immobilized on agarose support. At the end of storage, the free enzyme immobilized, lost 47% of its catalytic activity. The enzyme immobilized after 10 batch cycles lost only 12% of its activity. The model produced by computational tools was successful and it was possible to understand the interactions of D-galactose with L-AI, giving evidence of the importance of the E306 residue for catalysis.