Isomaltulose synthase(EC. 5. 4. 99. 11) is an intracellular enzyme which converts sucrose to isomaltulose and trehalulose. In this study, mutants of Erwinia rhapontici and recombinant E. coli were developed for the production of isomaltulose with an enhanced activity of isomaltulose synthase.
By NTG treatment, a mutant, E. rhapontici 27B4, was screened and the enzyme activity was increased from 75.5 U/mg to 80.4 U/mg compared to the wild type. The whole cells were immobilized with Ca-alginate as a bead form. The size of Ca-alginate beads did not affect the conversion of sucrose to isomaltulose. However, as the molecular weight of alginate decreased, the reaction rate and conversion yield were slightly increased. When a mutant 27B4 was used for the production of isomaltulose, the operational stability was slightly decreased compared with that of wild strain.
The Isomaltulose synthase gene of E.rhapontici was fully cloned (1,800 bp) from the partial sequences (1,300 bp) that has been reported previously. And a recombinant E. coli was constructed with an expression vector pET16. Using a recombinant E. coli, culture conditions were optimized for the maximization of isomaltulose synthase ctivity. Sucrose was found to be the most suitable carbon source in an aspect of enzyme expression level and activity. Using 2 % sucrose as carbon source with 0.4 mM IPTG for 10 hours culture, the enzyme activity expressed was increased to 77. 4 U/mg, which was comparable to that of original strain, E. rhapontici. It was also found that most of protein expressed was remained as an inclusion body rather than a soluble form. Further study will be carried out in order to increase the soluble form of isomaltulose synthase.
