The interpretation of in vitro measurements of fructosyl transferase activity: an analysis of patterns of fructosyl transfer by fungal invertase

Authors

  • ANDREW J. CAIRNS,

    1. Environmental Biology Department, Institute of Grassland and Environmental Research, Welsh Plant Breeding Station, Plas Gogerddan, Aberystwyth, SY23 3EB, UK
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  • JENNIFER E. ASHTON

    1. Environmental Biology Department, Institute of Grassland and Environmental Research, Welsh Plant Breeding Station, Plas Gogerddan, Aberystwyth, SY23 3EB, UK
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summary

A commercially available invertase preparation from yeast was analysed by native gel electrophoresis and shown to contain the large and small forms of yeast invertase but no specific fructosyl transferase. Both invertase isoforms catalysed the synthesis of the trisaccharides isokestose, neokestose and kestose from 600 mol m−3 sucrose at pH 5.

The hydrolytic and fructosyl transferase properties of the unfractionated invertase preparation were analysed under conditions similar to those used for the analysis of sucrose : sucrose fructosyl transferase (SST) from grasses. The sucrose concentration kinetics of the transferase gave a Km of between 224–308 mol m−33 which is similar to that reported for SST from graminaceous and other plant species. The sucrose Km of the hydrolase was estimated at between 11–34 mol m−3.

The transferase and hydrolase activities of yeast invertase could be distinguished on the basis of their responses to reaction temperature and pH, and in these respects, the data reproduced the results of grass SST studies. These observed differences in properties can be explained in terms of changes in reaction sucrose concentration and the differential kinetic properties of the hydrolase and transferase activities.

The two activities could not be distinguished on the basis of their temperature-stability characteristics or their responses to pyridoxal HCI. Pyridoxal HCI was shown to cause acidification of enzyme reactions and the direct effects of this compound on the transferase and hydrolase activities were thus difficult to interpret.

The results are discussed with respect to the evidence for separate SST and invertase activities in mixed enzyme systems derived from grass tissues.

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