The dsrE gene from Leuconostoc mesenteroides NRRL B-1299 was shown to encode a very large protein with two potentially active catalytic domains (CD1 and CD2) separated by a glucan binding domain (GBD). From sequence analysis, DSR-E was classified in glucoside hydrolase family 70, where it is the only enzyme to have two catalytic domains. The recombinant protein DSR-E synthesizes both ␣-1,6 and ␣-1,2 glucosidic linkages in transglucosylation reactions using sucrose as the donor and maltose as the acceptor. To investigate the specific roles of CD1 and CD2 in the catalytic mechanism, truncated forms of dsrE were cloned and expressed in Escherichia coli. Gene products were then small-scale purified to isolate the various corresponding enzymes. Dextran and oligosaccharide syntheses were performed. Structural characterization by 13 C nuclear magnetic resonance and/or high-performance liquid chromatography showed that enzymes devoid of CD2 synthesized products containing only ␣-1,6 linkages. On the other hand, enzymes devoid of CD1 modified ␣-1,6 linear oligosaccharides and dextran acceptors through the formation of ␣-1,2 linkages. Therefore, each domain is highly regiospecific, CD1 being specific for the synthesis of ␣-1,6 glucosidic bonds and CD2 only catalyzing the formation of ␣-1,2 linkages. This finding permitted us to elucidate the mechanism of ␣-1,2 branching formation and to engineer a novel transglucosidase specific for the formation of ␣-1,2 linkages. This enzyme will be very useful to control the rate of ␣-1,2 linkage synthesis in dextran or oligosaccharide production.From sucrose, Leuconostoc mesenteroides NRRL B-1299 produces a very uncommon dextran containing 61% ␣-1,6 glucosidic linkages in the linear chain and 28% ␣-1,2 linkages at branching points (11). The dextransucrase catalyzing the formation of this unusual dextran also produces glucooligosaccharides (GOS) by acceptor reaction. In the presence of a maltose acceptor, the glucosyl moiety of sucrose is transferred to maltose at the cost of polymer synthesis. This results in the formation of two different GOS families: the ␣-1,6 GOS series and the ␣-1,2 GOS series (5). The ␣-1,6 GOS series includes linear GOS possessing only ␣-1,6 linkages and a maltose residue at the reducing end. These oligosaccharides can also be produced by L. mesenteroides NRRL B-512F dextransucrase (12). The ␣-1,2 GOS series consists of ␣-1,6 GOS bearing an additional glucosyl residue linked by an ␣-1,2 linkage at the nonreducing end of the molecule or at the branching points. GOS containing ␣-1,2 linkages are resistant to enzymatic digestion in animals and humans and have been shown to possess prebiotic properties (3,20,22,25). They are commercialized for application in animal and human nutrition, as well as in dermocosmetics.To investigate the mechanism of highly ␣-1,2 branched dextran and GOS formation, three genes from L. mesenteroides

Recombinant expression of the dextransucrase dsrS gene by Escherichia coli was optimized to produce 5850 U L(-1) (culture) of DSR-S, corresponding to a 30-fold increase compared with previous studies. Rational deletions of the signal peptide, the beginning of the variable region and the last four repeats of the C-terminal end caused no loss of activity. This new variant successfully purified was remarkably stable. With a k(cat) of 584 s(-1), it is the most efficient recombinant glucansucrase described to date. The synthesized polymer possesses more than 95% of alpha-1,6 links, like the dextran produced by the native enzyme, and innovative gel properties were obtained.