Stabilization of D-Amino Acid Oxidase via Covalent Immobilization and Mathematical Model of D-Methionine Oxidative Deamination Catalyzed by Immobilized Enzyme

D-Amino acid oxidases (DAAOs) have an important role in several processes of interest in the fine chemical and pharmaceutical industry for drug discovery. They are used in the production of optically pure L-amino acids1 and unnatural amino acids2, and the production of 7-aminocephalosporanic acid (7-ACA)2,3 which is a precursor of antibiotics with annual demand of approximately 100 t worldwide3. Therefore, DAAO stability is an important property for its industrial application. Potential industrial use of enzymes in general is often limited by their instability under operating conditions. Consequently, enzyme properties have to be improved before their implementation at industrial scale4. Available stabilization strategies5 are immobilization6, protein engineering4 or chemical modification7, medium engineering by using additives, screening for enzymes from extremophiles and their isolation, as well as the production of stable enzymes in genetically manipulated mesophilic organisms. Besides stabilization, enzyme immobilization enables several benefits for the industrial process, such as simplified product purification and easy enzyme recovery. It can also enable masking of sensitive enzyme residues against chemical reagents. This can be achieved by immobilization of enzyme on supports having large internal surfaces. In that case, the immobilized enzyme is in close contact with large support surface and may become less accessible to deleterious reagents6. This might also be useful for protection of oxidases against air bubbles4 which may promote enzyme inactivation8,9. The literature data suggests that gas bubbles cannot inactivate the enzymes immobilized on porous support10,11 and that is why such support was chosen for this work12–14. In our previous work14 operational stability of soluble porcine kidney DAAO was investigated during D-methionine oxidative deamination (Fig. 1) in the presence and absence of aeration. It was found that DAAO’s activity decreases during oxidation and that oxygen concentration in the reaction solution affects the rate of enzyme activity decay; even though aeration is crucial to achieve higher reaction rates and to shorten the reaction time. The operational stability decay was the result of the combined effect of shear stress at the air-liquid interface, which may induce an irreversible aggregation of proteins and a negative effect of oxygen, which may lead to oxidation of protein residues.14 The topic of this work was therefore the investigation of the operational stability of immobilized porcine kidney DAAO. DAAOs of various origin were immobilized15,16 on different supports with remarkable results. For example, a 600-fold Stabilization of D-Amino Acid Oxidase via Covalent Immobilization and Mathematical Model of D-Methionine Oxidative Deamination Catalyzed by Immobilized Enzyme