ENGINEERED TERMOSTABLE INVERTASES AND THEIR APPLICATION IN REDUCING SUCROSE CONTENTS IN COMPLEX MIXTURES

Diabetes is one of the fastest-growing diseases in the world, with projections to affect up to 700 million people till 2045. This illness development is mostly associated with genetic and environmental factors, including the excessive consumption of sugar. In this scenario, this research project aims to develop a simple method to selectively remove and recover sucrose from complex mixtures, such as beverages and obtain products with low sugar content. The invertase from Thermotoga maritima was chosen as the sucrose-sequestering agent due its highly thermostable properties permit the enzyme to be used at high temperatures for a long time without significant activity loss. To avoid the undesirable hydrolysis of sucrose's glycosidic bond, site-directed mutagenesis was performed on the gene coding for the invertase enzyme to replace key residues affecting enzyme activity. With these modifications, three modified enzymes were obtained alongside the native one. Each of the proteins was expressed through heterologous expression in Escherichia coli BL21(DE3) and purified using affinity chromatography. The magnetite nanoparticles were prepared using the reverse co-precipitation method in the presence of sodium silicate, which induced the coating of the magnetic nucleus with a silica layer in situ during the synthesis. These magnetite and silica core-shell nanoparticles (MNPs) were then subjected to steps of functionalization to permit protein immobilization on their surface. The enzymes were subsequently immobilized on the MNPs and were recovered from the media by magnetic separation. Further, the target proteins are chemically and kinetically characterized in both free and immobilized forms. The immobilized native enzyme, which has shown catalytic activity towards sucrose, was applied to produce inverted sugar from sucrose solutions, and its reusability in various catalytic cycles was evaluated. The three immobilized modified enzymes, which did not show catalytic activity, were applied for removal and recovery of sucrose from solutions, complex mixtures, and orange juice. Sucrose concentration in enzymatic assays and during the removal of sucrose from complex mixtures was determined by polarimetry and quantitative proton nuclear magnetic resonance.