Abstract
A fumarate hydratase (EC 4.2.1.2, known as fumarase) produced by Lactobacillus delbrueckii subsp. bulgaricus 7235 was purified and its characteristics were examined. The purification of fumarase was carried out by protamine sulfate and ammonium sulfate fractionation and by column chromatographies of Mono P, Hi Load and Mono Q.
The purified enzyme showed 277-fold higher activity than that of cell-free extracts and was observed as a single band by SDS-polyacrylamide gel electrophoresis.
The purified fumarase was shown to have a molecular weight of 163,000 by gel filtration chromatography. Since the molecular weight of subunit was estimated 52,000 by SDS-PAGE, this enzyme was assumed to be composed of three subunits. The Km value was 15.38 mM for fumarate. Optimal pH was 5.5 whereas optimal temperature was 45℃. The activity was found to be stable between 20 and 45℃ after 30 minutes incubation. Although the activity was inhibited in the presence of Cu2+ and Zn2+ it was activated by the addition of Al3+.
