Phospholipase D modified with a polyethylene glycol derivative.
Phospholipase D from Streptomyces sp. AA586, PLDP, was modified with methoxypolyethylene glycol succinimidylsuccinate (ss-PEG), an active derivative of polyethylene glycol. By titration with trinitrobenzene sulfonate (TNBS), approximately 70% of the free amino groups in the enzyme protein were shown to be modified by treatment with ss-PEG. By this modification, the molecular weight of the enzyme was increased, judging from the results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis, gel filtration with Toyopearl HW-55F and TNBS titration. Due to loss of cationic charges, the enzyme protein became eluted faster in high performance liquid chromatography with CM-Toyopearl. By modification with ss-PEG, the enzyme became fairly thermostable, while pH-stability and optimal pH were not influenced. The value of Km for phosphatidylcholine of the hydrolytic reaction increased 2-fold, whereas that of the transphosphatidyl reaction was not significantly altered.
Matsuyama
H
H
Faculty of Pharmaceutical Sciences, Nagoya City University, Japan.
Taguchi
R
R
Ikezawa
H
H
D004795:Enzyme Stability; D010739:Phospholipase D / Q000737:chemistry* / Q000378:metabolism; D011092:Polyethylene Glycols / Q000737:chemistry; D013302:Streptomyces / Q000201:enzymology; D013388:Succinimides / Q000737:chemistry
D013388:Succinimides; D011092:Polyethylene Glycols; C069871:methoxypolyethylene glycol succinimidylsuccinate; D010739:Phospholipase D