engleski

Selective modification of surface exposed thiol groups in Trigonopsis variabilis D-amino acid oxidase using poly(ethylene glycol) maleimide and its effect on activity and stability of the enzyme

Covalent modification of purified Trigonopsis variabilis D-amino acid oxidase using maleimide-activated poly(ethylene glycol) 5000 yielded a stable bioconjugate in which three surface-exposed cysteine side chains were selectively derivatized. Compared with the native enzyme, the PEGylated variant displayed substantially ( 3.3-fold) slowed dissociation rate of FAD cofactor at 50°C, and this caused a twofold thermostabilization of the enzyme activity. The stability under reaction conditions at 30°C was also markedly enhanced in the PEG-oxidase conjugate. PEGylation did not affect steady-state kinetic parameters for oxidative deamination of D-methionine when 2, 6-dichloroindophenol replaced dioxygen as the cosubstrate while it caused a ninefold decrease in substrate catalytic efficiency for the dioxygen-dependent reaction.

D-amino acid oxidase; PEG-conjugate; (thermo) stabilization; cysteine modification; FAD; cofactor dissociation

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