Preparation of a Soluble, Bifunctional Enzyme Aggregate and Studies on Its IGnetic Behaviour in Polymer Media

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Abstract
Soluble, bifunctional enzyme aggregates have been prepared by crosslinking sequentially acting enzymes with glutaraldehyde. Aggregates of β‐glucosidase and glucose oxidase with molecular weights in the range 200000–300000 have been characterized from such a preparation. The sequentially working enzymes malate dehydrogenase and citrate synthase have also been aggregated using the same method. The kinetic behaviour of a system comprising two sequentiaily acting enzymes, either separately or as an aggregate, was studied in media containing increasing concentrations of poly(ethyleneglycol). A plot of transition time against polymer concentrations was bell‐shaped in the case of β‐glucosidase and glucose oxidase whereas in the system malate dehydrogenase–citrate synthase almost no lagphase was observed and comequently no transition time was registered. In both enzyme systems the steady‐state rate in the overall reaction was stimulated up to 30% at lower polymer concentrations, whereas reduced rates were obtained in more concentrated polymer solutions. These effects are interpreted in terms of (a) an exclusion effect leading to a relative enrichment of the rate‐limiting intermediate and an apparently higher concentration of the enzymes in the sequence and (b) a decreased diffusion rate which lowers the apparent V.In a similar polymer medium glucose oxidase alone shows decreased apparent Km for glucose on increasing the polymer content.