Study of the thermal stability of D-amino acid oxidase fromTrigonopsis variabilisreveals enzyme inactivation via multiple steps

Abstract

The thermal stability of a highly purified preparation of D-amino acid oxidase from Trigonopsis variabilis (Tv DAO), which does not show microheterogeneity due to the partial oxidation of Cys-108, was studied based on dependence of temperature (20 Á608C) and protein concentration (5 Á100 mmol L (1 ). The time courses of loss of enzyme activity in 100 mmol L (1 potassium phosphate buffer, pH 8.0, are well described by a formal kinetic mechanism in which two parallel denaturation processes, partial thermal unfolding and dissociation of the FAD cofactor, combine to yield the overall inactivation rate. Estimates from global fitting of the data revealed that the first-order rate constant of the unfolding reaction (k a ) increased 10 4 -fold in response to an increase in temperature from 20 to 608C. The rate constants of FAD release (k b ) and binding (k (b ) as well as the irreversible aggregation of the apo-enzyme (k agg ) were less sensitive to changes in temperature, their activation energy (E a ) being about 52 kJ mol (1 in comparison with an E a value of 185 kJ mol (1 for k a . The ratedetermining step of Tv DAO inactivation switched from FAD dissociation to unfolding at high temperatures. The model adequately described the effect of protein concentration on inactivation kinetics. Its predictions regarding the extent of FAD release and aggregation during thermal denaturation were confirmed by experiments. Tv DAO is shown to contain two highly reactive cysteines per protein subunit whose modification with 5,5?-dithio-bis (2-nitrobenzoic acid) was accompanied by inactivation. Dithiothreitol (1 mmol L (1 ) enhanced up to 10-fold the recovery of enzyme activity during ion exchange chromatography of technical-grade Tv DAO. However, it did not stabilize Tv DAO at all temperatures and protein concentrations, suggesting that deactivation of cysteines was not responsible for thermal denaturation.

Evaluation of immobilized enzymes for industrial applications

“…35 An experimental case study on the partial thermal unfolding of D-amino acid oxidase as well as the dissociation of the cofactor flavin adenine dinucleotide shows that the overall inactivation rate is composed of several steps. 56 Therefore one has to consider that the loss of activity is not directly connected to unfolding of the protein structure. Furthermore, deactivation and unfolding for a certain enzyme are even often uncoupled.…”

Encapsulation of Trigonopsis variabilis D‐amino acid oxidase and fast comparison of the operational stabilities of free and immobilized preparations of the enzyme

“…3, panel C) likely reflects stabilization of E-S against thermally induced reversible release of the cofactor. These results therefore suggest that the kinetic mechanisms of thermal inactivation of E-S and E (Dib et al, 2006;Slavica et al, 2006) are similar.…”

Stability and stabilization ofD-amino acid oxidase from the yeastTrigonopsis variabilis

“…It also serves as a mechanism-based tool for the stabilization of the soluble enzyme and carrier-bound immobilized forms of it. The inactivation rate of the enzyme increased with increasing concentrations of the protein subunit in the range 1-20 µM (10 mM Tris buffer, pH 7.5; 50 • C) [31] and 5-100 µM (50 mM potassium phosphate buffer, pH 8.0; 30 • C) [37]. Stabilization by external FAD, added in 100-fold molar excess over native protein, was gradually lost upon raising the concentration of the TvDAO subunit from 1 to 20 µM.…”

“…However, differences in stability of the two TvDAO forms were completely eliminated upon addition of a high concentration of cofactor (1 mM) [31]. Slavica et al [37] showed that the rate-determining step of inactivation of TvDAO changed from FAD dissociation to partial unfolding at high temperature. Therefore the relative stabilizing effect of exogenous FAD is expected to decrease as the incubation temperature increases.…”