Preparation of artificial bifunctional enzymes by gene fusion

Within the cellular framework operate a number of sequentially acting enzyme systems of varying complexity and spatial organization. In some cases they are found as bi- or even multi-functional enzymes, where each single polypeptide chain carries two or more catalytic functions. To mimic these enzymes, a number of different bifunctional enzymes have been prepared by in-frame gene fusion. Thus, beta-galactosidase and galactokinase as well as beta-galactosidase and galactose dehydrogenase were genetically fused. Different physical and chemical properties of the hybrid proteins have been investigated, such as protein stability, subunit interactions, linker regions and substrate channeling in vitro and in vivo. Important fields of application for artificial bifunctional enzymes can be found in biochemical analysis, protein purification and metabolic engineering.