Monday, July 25, 2011
Grand Ballroom, 5th fl (Sheraton New Orleans)
A glucansucrase gene, which is responsible for dextran formation using sucrose, was isolated by shotgun cloning of KpnI fragments of Leuconostoc mesenteroides NRRL B-1149 DNA and sequenced. About 5 kb KpnI fragments were ligated with plasmid pGEM-3Zf(+) and transformed into Escherichia coli DH5α. The nucleotide sequence of this glucansucrase gene was determined and found to consist of an open reading frame (ORF) of 3,714 base pairs (bp) coding for a 1,237-amino acid protein with an molecular weight of 136,070 Da. The aa sequence exhibited a high similarity with other glucosyltransferases. The recombinant DSRL synthesized dextran on an agar plate containing 3% (w/v) sucrose. The synthesized dextran was hydrolyzed with Penicillium endo-dextranase and produced glucose, isomaltose, and branched isomaltotrisaccharide, branched isomaltotetrasaccharide. The 13C-NMR spectrum of the soluble glucan prepared by DSRL using 5% sucrose showed nine intense peaks (100.3, 98.29, 82.1, 73.4, 71.93, 70.47, 70.19, 66.17, 60.74 ppm), which corresponded to the relative peak positions for α-(1→6) glucosyl linkage and α-(1→3) glucosyl linkage. Methylation analysis also confirmed that the DSRL dextran was composed of α-(1→6) and α-(1→3) linkages in the value of 83% and 17%, respectively.