Expression and Purification of SuSyl from E. coli

The comparison of the kinetic data for recombinant SuSyl from yeast and E. coli revealed no significant changes in the substrate affinities (Table 2.2.6.2) (Sauer-zapfe and Elling, unpublished results). The influence of phosphorylation of SuSy on the enzyme s affinity for sucrose and UDP are discussed controversially in the literature. Nakai et al. found an increase in the substrates affinities [16] however, Zhang et al. could not detect changes in the kinetic data for SuSy from soybean nodules [12]. With reference to our work, the expression in a eukaryotic or prokaryotic host influences the protein chemical characteristics of SuSyl. However, we cannot yet decide whether recombinant SuSyl from yeast is phosphorylated. 

The most significant indication that the biocatalytic properties also depend on the expression host comes from our studies on the fructose analogues DMDP 10 (2,5-dihydroxymethyl-(3S,4R)-dihydroxypyrrolidine) and 5-deoxy-D-fructose as inhibitors of SuSyl (Fig. 2.2.6.2). In contrast to SuSyl from yeast, the strongest inhibitor for recombinant SuSyl from E. coli is 10 (IC501.5 mM), suggesting that the enzyme prefers the D-fructofuranose conformation. 

The ketoses D-tagatose 6 (43% relative activity) and D-ribulose 28 (24% relative activity) were identified as new acceptor substrates they are not accepted by recombinant SuSyl from yeast. However the acceptance for D-xylulose 5 is lost. The most significant changes were observed for the aldoses tested L-arabinose 14, D-xylose 12, and D-lyxose 11 are better substrates than D-fructose, with relative activities of 490%, 300%, and 151%, respectively. In the hexose series L-glucose 29 and L-rhamnose 30 were identified as new acceptor substrates, whereas the acceptance for D-and L-mannose, 16 and 21, was improved (Fig. 2.2.6.5) (Sauerzapfe and Elling, unpublished results). 

In summary, our results demonstrate that the biocatalytic properties of SuSyl are broadened and improved by expression in different hosts. So far, four new acceptor substrates have been identified and the acceptance of three aldoses has been improved. 

4 Sucrose Synthase 1 Mutants Expressed in S. cerevisiae and . coli 

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