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Pregled bibliografske jedinice broj: 267551

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


Dib, Iskandar; Slavica, Anita; Riethorst, Waander; Nidetzky, Bernd
Thermal inactivation of D-amino acid oxidase from Trigonopsis variabilis occurs via three parallel paths of irreversible denaturation // Biotechnology & Bioengineering, 94 (2006), 4; 645-654 (međunarodna recenzija, članak, znanstveni)


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

Autori
Dib, Iskandar ; Slavica, Anita ; Riethorst, Waander ; Nidetzky, Bernd

Izvornik
Biotechnology & Bioengineering (0006-3592) 94 (2006), 4; 645-654

Vrsta, podvrsta i kategorija rada
Radovi u časopisima, članak, znanstveni

Ključne riječi
stability; stabilization; (D-amino acid) oxidase; thermal inactivation mechanism; cysteine sulfinic acid; oxidative protein modification

Sažetak
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 508Cin 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_2H-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.

Izvorni jezik
Engleski

Znanstvena područja
Biotehnologija



POVEZANOST RADA


Projekti:
0058011

Ustanove:
Prehrambeno-biotehnološki fakultet, Zagreb

Profili:

Avatar Url Anita Slavica (autor)

Citiraj ovu publikaciju

Dib, Iskandar; Slavica, Anita; Riethorst, Waander; Nidetzky, Bernd
Thermal inactivation of D-amino acid oxidase from Trigonopsis variabilis occurs via three parallel paths of irreversible denaturation // Biotechnology & Bioengineering, 94 (2006), 4; 645-654 (međunarodna recenzija, članak, znanstveni)
Dib, I., Slavica, A., Riethorst, W. & Nidetzky, B. (2006) Thermal inactivation of D-amino acid oxidase from Trigonopsis variabilis occurs via three parallel paths of irreversible denaturation. Biotechnology & Bioengineering, 94 (4), 645-654.
@article{article, year = {2006}, pages = {645-654}, keywords = {stability, stabilization, (D-amino acid) oxidase, thermal inactivation mechanism, cysteine sulfinic acid, oxidative protein modification}, journal = {Biotechnology and Bioengineering}, volume = {94}, number = {4}, issn = {0006-3592}, title = {Thermal inactivation of D-amino acid oxidase from Trigonopsis variabilis occurs via three parallel paths of irreversible denaturation}, keyword = {stability, stabilization, (D-amino acid) oxidase, thermal inactivation mechanism, cysteine sulfinic acid, oxidative protein modification} }
@article{article, year = {2006}, pages = {645-654}, keywords = {stability, stabilization, (D-amino acid) oxidase, thermal inactivation mechanism, cysteine sulfinic acid, oxidative protein modification}, journal = {Biotechnology and Bioengineering}, volume = {94}, number = {4}, issn = {0006-3592}, title = {Thermal inactivation of D-amino acid oxidase from Trigonopsis variabilis occurs via three parallel paths of irreversible denaturation}, keyword = {stability, stabilization, (D-amino acid) oxidase, thermal inactivation mechanism, cysteine sulfinic acid, oxidative protein modification} }

Časopis indeksira:


  • Current Contents Connect (CCC)
  • Web of Science Core Collection (WoSCC)
    • Science Citation Index Expanded (SCI-EXP)
    • SCI-EXP, SSCI i/ili A&HCI
  • Scopus


Uključenost u ostale bibliografske baze podataka:


  • Biological Abstracts
  • Chemical Abstracts
  • Analytical Abstracts
  • International Abstracts in Operations Research (IAOR)
  • IAOR-Online





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