Use of Physicochemical Tools to Determine the Choice of Optimal Enzyme: Stabilization of -Amino Acid Oxidase

Abstract

An evaluation of the stability of several forms (including soluble and two immobilized preparations) of d-amino acid oxidases from Trigonopsis variabilis (TvDAAO) and Rhodotorula gracilis (RgDAAO) is presented here. Initially, both soluble enzymes become inactivated via subunit dissociation, and the most thermostable enzyme seemed to be TvDAAO, which was 3-4 times more stable than RgDAAO at a protein concentration of 30 microg/mL. Immobilization on poorly activated supports was unable to stabilize the enzyme, while highly activated supports improved the enzyme stability. Better results were obtained when using highly activated glyoxyl agarose supports than when glutaraldehyde was used. Thus, multisubunit immobilization on highly activated glyoxyl agarose dramatically improved the stability of RgDAAO (by ca. 15,000-fold) while only marginally improving the stability of TvDAAO (by 15-20-fold), at a protein concentration of 6.7 microg/mL. Therefore, the optimal immobilized RgDAAO was much more stable than the optimal immobilized TvDAAO at this enzyme concentration. The lower stabilization effect on TvDAAO was associated with the inactivation of this enzyme by FAD dissociation that was not prevented by immobilization. Finally, nonstabilized RgDAAO was marginally more stable in the presence of H(2)O(2) than TvDAAO, but after stabilization by multisubunit immobilization, its stability became 10 times higher than that of TvDAAO. Therefore, the most stable DAAO preparation and the optimal choice for an industrial application seems to be RgDAAO immobilized on glyoxyl agarose.

Hydrogen Peroxide in Biocatalysis. A Dangerous Liaison

“…Glutyaraldehyde immobilization on magnetic nanoparticles of the same enzyme also coupled thermal stabilization (Tm was increased from 45 °C to 55 °C) with stabilization in the presence of 20 mM H 2 O 2 , (the immobilized form retained 93% of its activity after 5 h while the free form was completely inactivated after 3.5 h) [254]. Multipoint covalently immobilized D-amino acid oxidase (DAAO) from Rhodotorula gracilis on glyoxyl-agarose is 11-fold more stable than the native enzyme against the deleterious effect of hydrogen peroxide, with an improved thermostabillity as an added benefit [251,255]. However, D-amino acid oxidase from Trigonopsis variabilis was not stabilized by rigidification versus hydrogen peroxide although an improved thermostability was detected.…”

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

“…However, as noticed by other authors 16 , a comprehensive analysis of operational stability of immobilized enzyme is most often missing. Thus, in this report the authors have focused on this topic, and not on the optimization of the immobilization procedure itself, although some groups of authors reported thorough studies on stability of immobilized enzyme 6,15,17,18 . DAAO was immobilized on Eupergit C in this work.…”

Single-Site Oxidation, Cysteine 108 to Cysteine Sulfinic Acid, ind-Amino Acid Oxidase fromTrigonopsis variabilisand Its Structural and Functional Consequences

“…A number of papers have reported the results of experiments designed to analyze and improve the stability of TvDAO (2,14,15). The persistence of soluble, carrier-bound, and physically entrapped forms of TvDAO against inactivation is not fully satisfactory and is seen as a major shortcoming of the enzyme (2,27).…”

“…The persistence of soluble, carrier-bound, and physically entrapped forms of TvDAO against inactivation is not fully satisfactory and is seen as a major shortcoming of the enzyme (2,27). Unfortunately, the available evidence is partly conflicting and cannot be consolidated to a clear focal point for the production of a more suitable TvDAO.…”

“…Unfortunately, the available evidence is partly conflicting and cannot be consolidated to a clear focal point for the production of a more suitable TvDAO. The typically observed time courses of activity loss in the absence and presence of oxidative stress are complex (2,23), suggesting that the denaturation mechanism involves more than a single species of active protein. We show here by combining high-resolution protein and peptide fractionation, structure determination by tandem mass spectrometry, and biochemical methods that sitespecific oxidative conversion of Cys108 in TvDAO is a main source of structural and functional heterogeneity in the enzyme.…”