Descrizione: The use of recombinant proteins has increased significantly in the last years, as well as the improvement of the expression and purification features; according to their final use it is helpful to plan the nature of the host, vectors, and expression conditions before setting up large scale protein production. A consolidated expertise has been here developed in order to produce recombinant proteins for biochemical and biotechnological applications. This expertise has been applied in several systems among which it is worth mentioning fructosamine oxidase enzymes (FAOXs) and Carbonic Anhydrases (CAs). FAOXs are deglycating enzymes. Glycation is a highly deleterious process that impacts on human health via two major mechanisms. The first is an endogenous process in which glycemia-driven damage to proteins leads to age-related impairment of molecular and tissue function through the formation of glucose-derived protein adducts and crosslinks. The second is an exogenous process that leads to the formation of proinflammatory and carcinogenic modifications in heat-processed foods. Humans are thus exposed to the combined threat of foods modified by glycation products and those formed endogenously. To this aim the use of engineered fungal deglycating enzymes targeting the Amadori product will allow to control and limit hyperglycation in basic molecular mechanisms of tissue aging and hyperglycemia, and in food-derived inflammatory processes. CAs proteins are a widespread class of metalloenzymes able to catalyze the reversible conversion of carbon dioxide to bicarbonate ion and a proton. CAs are expressed in differentiated tissues and play an important physiological role in facilitating ion transport and maintaining pH homeostasis. Several studies have been carried out to obtain stable preparation of these metalloenzymes in order to improve our knowledge on their catalytic properties, oligomerization and post-translational modifications. To this aim protein engineering has been carried out on many of these systems in order to reduce non-covalent aggregation and/or covalent intermolecular cross-linking, improve chemical stability and investigate the punctual role of critical aminoacids in the catalytic mechanisms. Engineered recombinant enzymes have been subsequently characterized by different biophysical and biochemical methods.
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