Characterization of an artificial bifunctional enzyme, beta-galactosidase/galactokinase, prepared by gene fusion

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Abstract
An artificial bifunctional enzyme, β-galactosidase/galactokinase, has been prepared by gene fusion. The hybrid protein catalyzes the hydrolysis of lactose followed by phosphorylation of galactose. The protein has been purified using DEAE-Sephacel chromatography and gel filtration on Sephacryl S-400 Superfine. The configuration of the hybrid protein is mainly tetrameric but also higher aggregates can be detected. The monomer Mr is 160 000 as judged from sodium dodecyl sulfate/polyarcylamide gel electrophoresis and the native Mr has been calculated to be 600 000–650 000 from gel filtration experiments. β-Galactosidase/galactokinase has different thermostability curves, pH/activity profiles and km values as compared with the native enzymes. By using a third enzyme, galactose dehydrogenase, which competes with galactokinase for the galactose formed by β-galactosidase, substrate channeling can be detected. This proximity effect becomes even more pronouned in an assay mixture containing poly(ethylene glycol).