The in-frame fusion between two oligomeric enzymes, beta-galactosidase and galactose dehydrogenase, is described. The lacZ gene was fused to the 3' end of the galdh gene with a linker encoding only three amino acids. The purified artificial bifunctional enzyme displayed the enzymic activity of both gene products. The hybrid protein was found in two major forms, consisting of four and six subunits, but other forms could also be identified. The molecular weight of each subunit was determined to be 145,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The bifunctional enzyme shows kinetic advantages over the identical native system in conversion of lactose to galactonolactone. A higher steady-state rate and a reduction of the transient time are observed. This phenomenon is especially pronounced at low initial substrate concentrations and when the pH is adjusted to a level at which the galactose dehydrogenase activity is much higher than that of the beta-galactosidase.

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A chemically synthesized DNA linker coding for a peptide fragment that contains four histidines was fused in-frame to the 5'-end of the Bacillus stearothermophilus lactate dehydrogenase gene. The gene product, His4/lactate dehydrogenase, could be purified to homogeneity using either immobilized metal (Zn2+)-affinity chromatography or affinity chromatography on oxamate agarose. The stability against heat and urea for the modified enzymes was decreased as compared to the native lactate dehydrogenase but could be increased if zinc ions were present during the denaturation. In the presence of zinc ions the His4/lactate dehydrogenase could catalyse the sequential reaction from oxaloacetate to L-lactate, hence operating as a semi-synthetic bifunctional enzyme. A small increase in the apparent second-order rate constant (kcat/Km) of the coupled reaction was observed as compared to a corresponding system with native lactate dehydrogenase.

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