Effect of a single point mutation on the interaction of glucans with a glucansucrase from Leuconostoc mesenteroides NRRL B-1118

• Mutations of B-1118 glucansucrase at T654 affect activation by soluble dextrans.
• Dextran causes change in Vmax but not in KM for sucrose.
• Low-yielding enzymes give higher yields in the presence of dextran.
• Activation and increased yield depends on unbranched α-(1→6) glucan sequences.
• Increase in yields is only partially due to graft copolymer formation.

Our previous work showed that substitution of an amino acid that is coupled with the +2 subsite adjacent to the transition stabilizer of a glucansucrase, which produces a water-insoluble glucan, resulted in significant changes in the structures and yields of the water-insoluble glucans produced. We now describe how these changes affect the ability of the glucansucrase to bind to exogenous glucans, and how these glucans can influence the yield, product structures, and kinetics of the mutant glucansucrases. The activity of the wild-type enzyme, with threonine at position 654, is not significantly activated by added dextran, and the yield of water-insoluble glucan from sucrose is only slightly increased by dextran. Mutant T654Y is not affected at all by the addition of dextran. However, several mutant enzymes exhibit markedly lower yields of glucan relative to the wild type; these lower yields can be partially or completely overcome by the addition of water-soluble dextran. Although evidence indicates that the soluble dextran is incorporated into water-insoluble glucan, the increased yields cannot be accounted for solely by incorporation of the dextran into insoluble product. Furthermore, these DsrI mutants are significantly activated by exogenous glucans. The addition of dextran does not markedly change the KM for sucrose in the mutant enzymes, but does increase the Vmax of the reaction. These effects apparently depend on the presence of unbranched sequences of α1→6-linked D-glucose units in the glucan.

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