| Research Abstract |
Zymomonas mobilis produces an exocellular fructosyltransferase (EC2.4.1.10, sucrose : 2,6-beta-D-fructan 6-beta-D-fructosyltransferase, FTase) that is capable of synthesizing branched fructan (levan) of high molecular mass from sucrose. In addition, the enzyme catalyzes the fructosyl transfer from sucrose to numerous acceptors other than levan, such as water, short-chain acylalcohols, and various mono- and disaccharides. In the present study, molecular genetic approaches have been utilized to confirm the structure of the catalytic site of Z.mobilis FTase (sucZE2), as follows ; 1) Cloning and characterization of the gene cording for FTase, 2) Presumption of function domains in FTase by random mutation, 3) Alteration of transfructosylating activity by site-directed mutagenesis, 4) Alteration of substrate specificities of FTase by site-directed mutagenesis, 5) Identification of catalycally important residues, Glu278, His296, inFTase gene, 6) Presumption of transfructosylating reaction of FTase via a general acid-base catalytic mechanism.
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