Thermal inactivation of D‐amino acid oxidase from Trigonopsis variabilis occurs via three parallel paths of irreversible denaturation

Abstract

Trigonopsis variabilis D-amino acid oxidase (TvDAO) is a long-known flavoenzyme whose most important biocatalytic application is currently the industrial production of 7-amino-cephalosporanic acid (7-ACA) from cephalosporin C. Lacking mechanistic foundation, rational stabilization of TvDAO for improved process performance remains a problem. We report on results of thermal denaturation studies at 50 degrees C in which two purified TvDAO forms were compared: the native enzyme, and a site-specifically oxidized protein variant that had the side chain of cysteine108 converted into a sulfinic acid and lost 75% of original specific activity. Although inactivation time courses for both enzymes are fairly well described by simple single-exponential decays, the underlying denaturation mechanisms are shown by experiments and modeling to be complex. One main path leading to inactivation is FAD release, a process whose net rate is determined by the reverse association rate constant (k), which is 25-fold lower in the oxidized form of TvDAO. Cofactor dissociation is kinetically coupled to aggregation and can be blocked completely by the addition of free FAD. Aggregation is markedly attenuated in the less stable Cys108-SO(2)H-containing enzyme, suggesting that it is a step accompanying but not causing the inactivation. A second parallel path, characterized by a k-value of 0.26/h that is not dependent on protein concentration and identical for both enzymes, likely reflects thermal unfolding reactions. A third, however, slow process is the conversion of the native enzyme into the oxidized form (k < 0.03/h). The results fully explain the different stabilities of native and oxidized TvDAO and provide an inactivation mechanism-based tool for the stabilization of the soluble oxidase.

Fusion to a pull‐down domain: a novel approach of producing Trigonopsis variabilisD‐amino acid oxidase as insoluble enzyme aggregates

“…We chose reaction conditions that were known from earlier studies with the resting enzyme to promote fast inactivation of the soluble oxidase due to contact with a gas-liquid interface (I. Dib and B.N., unpublished results 2006;Fernández-Lafuente et al, 1998) and thermal denaturation (at 508C; Dib et al, 2006). Time courses of loss of activity were in all cases reasonably well described by a single-exponential decay in the initial phase of inactivation (!40% residual activity), justifying the use of half-life time as parameter describing the kinetic stability of TvDAO (Fig.…”

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

“…The chosen buffer conditions were similar to those used before by other groups (Schräder & Andreesen 1993;Fernández-Lafuente et al 1999b;Betancor et al 2003). The evidence from experiments and modelling suggests that the mechanism underlying thermal denaturation of Tv DAO in phosphate buffer is essentially the same as that proposed by Dib et al (2006). However, the relative importance of individual denaturation steps is changed upon replacing the original Tris buffer with phosphate buffer.…”

“…In the course of these studies it was found that the stability of Tv DAO in Tris buffer Dib et al 2006) is quite different from that in phosphate buffer. To determine the source of this ''medium effect'', we performed a detailed analysis of the thermal inactivation of Tv DAO in 100 mmol L (1 sodium phosphate buffer, pH 8.0.…”

“…Therefore, the observed microheterogeneity can seriously impair the study of stability and stabilization of Tv DAO. Dib et al (2006) have shown that thermal inactivation of highly purified native Tv DAO which contains Cys-108 exclusively in the reduced state occurs via two parallel paths of denaturation of which one involves the dissociation of FAD, kinetically coupled to protein aggregation, and another appears to reflect partial protein unfolding at elevated temperatures.…”

High-level expression of Rhodotorula gracilis d-amino acid oxidase in Pichia pastoris

“…Taking into account that loss of FAD appears to be the preponderant path of inactivation of TvDAO under a wide range of conditions (Betancor et al 2003;Dib and Nidetzky 2008;Dib et al 2006;Pollegioni et al 2004), the ability of RgDAO to bind its cofactor by one order of magnitude more tightly than TvDAO made the enzyme from R. gracilis a promising candidate for development of DAO catalysts showing further enhanced robustness. However, exploitation of the extra stability of RgDAO as compared to TvDAO is hampered by relatively inefficient production of the R. gracilis enzyme in both native (Molla et al 2003) and E. coli (Wang et al 2008) host strains.…”