Isolation and characterization of the newly evolved ebg beta-galactosidase of Escherichia coli K-12.

The ebg beta-galactosidase of Escherichia coli K-12 strain LC110 has been purified and characterized. Strain LC110 is a Lac+ revertant of a mutant with a deletion of the lacZ beta-galactosidase gene. Its new ebg beta-galactosidase activity was shown to be due to a discrete protein, immunologically unrelated to lacZ beta-galactosidase. Its kinetics of action conformed to those of a simple conventional enzyme. With o-nitrophenyl-beta-D-galactoside as substrate, the Vmax was 11,200 nmol/min per mg of enzyme, the Km was 5 mM, and the activation energy was 12,400 cal/mol. Corresponding values for lacZ beta-galactosidase of wild-type E. coli K-12 were 350,000 nmol/min per mg of enzyme, 1.3 mM, and 8,000 cal/mol. A series of sugars has been examined as competitive inhibitors of ebg beta-galactosidase. Kinetic analyses suggest that ebg beta-galactosidase has a particularly high affinity for galactosamine and gamma-galactonolactone, binds galatose more tightly than lactose, and shows a general preference for monosaccharides rather than beta-galactosides. We conclude that the ebg beta-galactosidase may have arisen by modification of a gene involved with the metabolism of a monosaccharide, possibly a 2-amino sugar.