Supramolecular-mediated thermostabilization of phenylalanine dehydrogenase modified with β-cyclodextrin derivatives

Two different monoactivated β-cyclodextrin derivatives, named mono-6-amino-6-deoxy-β-CD (CD1) and mono-6-(5-carboxypentane-1-carboxamidoyl)-6-deoxy-β-CD (CD2) were evaluated as modifying agents for Bacillus badius phenylalanine dehydrogenase. The enzyme glycosidated with CD1 and CD2 contained about 18 mol and 15 mol oligosaccharide per mol of protein and retained 60% and 81% of the initial activity, respectively. The optimum temperature for the catalytic activity of phenylalanine dehydrogenase was increased in 10 °C after attaching the CDs residues. The enzyme thermostability profile was improved, and its resistance to thermal inactivation at different temperatures ranging from 45 °C to 60 °C was noticeably increased after glycosidation. The activation free energy of thermal inactivation was increased by 16.8 kJ/mol and 12.6 kJ/mol for the enzyme modified with CD1 and CD2, respectively. The influence of supramolecular host–guest associations on the improved thermotolerance showed by the modified enzyme forms was demonstrated by fluorescence spectroscopy and enzymatic measurements.