Separation of levan-formation and sucrose-hydrolysis catalyzed by levansucrase of Zymomonas mobilis using in vitro mutagenesis

A levansucrase (SacB) of Zymomonas mobilis capable of sucrose hydrolysis bu t not le,tan formation was Isolated through in vitro mutagenesis of cloned sacB gene. When the sacB mutant gene was expressed in Escherichia coli stra ins, only 50% of the sucrose-hydrolysing activity (2.0 U/mg) was produced, compared to the wild type levansucrase (4.0 U/mg). Sequencing of the sacB m utant gene revealed changes of two amino acid residues (Phe-102 to Leu and Trp-261 to Lys in the levansucrase). The absence of mutation at the site of Cys of SacB is contradictory to the inhibition kinetics that demonstrated the involvement of Cys in conferring the levan-forming activity to the SacB . The present finding is useful in understanding the mechanism of selective modulation of levan-forming (polymerase) activity of levansucrase.