Isolation and characterization of the newly evolved ebg β galactosidase of Escherichia coli K 12

Abstract

The ebg β galactosidase of E. 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 β galactosidase gene. Its new ebg β galactosidase activity was shown to be due to a discrete protein, immunologically unrelated to lacZ β galactosidase. Its kinetics of action conformed to those of a simple conventional enzyme. With o nitrophenyl β-D-galactoside as substrate, the V(max) was 11,200 nmol/min per mg of enzyme, the K(m) was 5 mM, and the activation energy was 12,400 cal/mol. Corresponding values for lacZ β 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 β galactosidase. Kinetic analyses suggest that ebg β galactosidase has a particularly high affinity for galactosamine and γ galactonolactone, binds galactose more tightly than lactose, and shows a general preference for monosaccharides rather than β galactosides. It is concluded that the ebg β galactosidase may have arisen by modification of a gene involved with the metabolism of a monosaccharide, possibly a 2 amino sugar.