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 (approximately 3.3-fold) slowed dissociation rate of FAD cofactor at 50 degrees C, and this caused a twofold thermostabilization of the enzyme activity. The stability under reaction conditions at 30 degrees 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.
Slavica
Anita
A
Research Centre Applied Biocatalysis, Petersgasse 14, A-8010 Graz, Austria.
Dib
Iskandar
I
Nidetzky
Bernd
B
D003605:D-Amino-Acid Oxidase / Q000737:chemistry*; D004789:Enzyme Activation; D004795:Enzyme Stability; D008301:Maleimides / Q000737:chemistry*; D011092:Polyethylene Glycols / Q000737:chemistry*; D004718:Saccharomycetales / Q000201:enzymology*; D013438:Sulfhydryl Compounds / Q000737:chemistry*; D013499:Surface Properties
D008301:Maleimides; D013438:Sulfhydryl Compounds; D011092:Polyethylene Glycols; D003605:D-Amino-Acid Oxidase