Selective modification of surface-exposed thiol groups in Trigonopsis variabilisD-amino acid oxidase using poly(ethylene glycol) maleimide and its effect on activity and stability of the enzyme
✍ Scribed by Anita Slavica; Iskandar Dib; Bernd Nidetzky
- Publisher
- John Wiley and Sons
- Year
- 2006
- Tongue
- English
- Weight
- 253 KB
- Volume
- 96
- Category
- Article
- ISSN
- 0006-3592
No coin nor oath required. For personal study only.
✦ Synopsis
Abstract
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. Biotechnol. Bioeng. 2007;96: 9–17. © 2006 Wiley Periodicals, Inc.