Oxygen influence on amino acid oxidative deamination catalyzed by different amino acid oxidases

Abstract

Amino acid oxidases catalyze oxidative deamination of a wide range of amino acids to their corresponding  -keto acids. In this work two D-amino acid oxidases: from porcine kidney and Arthrobacter protophormiae were compared in their action in the reaction of D-methionine oxidative deamination. Special attention has been paid to the oxygen influence on enzyme activity. Kinetics of the reaction was determined by the initial reaction rate method by the standard peroxidase-o-dianisidine enzyme assay. The oxygen impact on the initial reaction rate was measured by following the oxygen consumption during the reaction at different initial oxygen concentration. Kinetic parameters were estimated from the experimental results by using the non-linear regression analysis implemented in the package program SCIENTIST. It was found that both enzymes have similar affinities towards D-methionine as well as oxygen ( (porcine kidney)=0.13 mM, (A. protophormiae)=0.22 mM). Both enzymes are inhibited by 2-oxo-4-methylthiobutyric acid, and the inhibition constant is lower in the case of porcine kidney enzyme which indicates higher inhibition. Oxygen volume transfer coefficient (kLa) has been determined at different air flow-rates using integral. Mathematical model of the process that consists of kinetic and mass balance equations has been developed in the batch reactor. The experiments without and with continuous oxygen (air) supply were carried out to validate the mathematical model. The reaction solution was initially saturated with air to achieve the maximum oxygen solubility at the experimental conditions. Considering that oxygen is one of the reactants spent in the reaction, the reaction rate in the initial part of the experiments is faster if air is supplied to the reactor. In the case of porcine kidney oxidase the reaction was very slow without the air supply. 100 % D-methionine conversion was achieved after 12 hours. Another two experiments were carried out at the continuous air supply (flow-rates 5 and 10 L/h). The reaction went faster in the initial part of both experiments ; however enzyme deactivation occurred and prevented the complete substrate conversion. In the case of oxidase from A. protophormiae experiments without and with the air supply (flow-rate 10 L/h) were carried. In both cases 100 % D-methionine conversion could be achieved. This enzyme was more tolerant to oxygen since enzyme deactivation constant (kd = 7.9  10-5 min-1) was found to be several orders of magnitude lower than the ones estimated for D-AAO from porcine kidney (kd, 5L/h= 0.0108 and kd, 10L/h=0.0198 min-1).