Levansucrase (EC 2.4.1.10), an exoenzyme of Pseudomonas syringae pv. phaseolicola, was purified to homogeneity from the cell supernatant by chromatography on TMAE-Fraktogel and butyl-Fraktogel. The
enzyme has molecular masses of 45 kDa under denaturing conditions and 68 kDa during gel filtration of the native form. In isoelectric focusing, active bands appeared at pH 3.55 and 3.6. Maximum
sucrose cleaving activities were measured at pH 5.8 to 6.6 and 60 degrees C. The
enzyme was highly tolerant to denaturing agents,
proteases, and repeated freezing and thawing. The molecular weight of the produced
levan depended on temperature, salinity, and
sucrose concentration. The
enzyme had
levan-degrading activity and did not accept
raffinose as a substrate. Comparison of the N-terminal amino acid sequence with the predicted amino acid sequence of levansucrases from Erwinia amylovora and Zymomonas mobilis showed 88 and 69% similarity, respectively, in
amino acids 5 to 20. No similarity could be detected to levansucrases of gram-positive bacteria in the first 20
amino acids. By comparison of all levansucrases which have been sequenced to date, the
enzyme seems to be conserved in the gram-negative bacteria. The rheological behavior of the product
levan prompted a new assessment of the
enzyme's role in pathogenesis. Depending on formation conditions,
levan solutions exclude other
polymer solutions. This behavior supports the presumption that the
levansucrase is important in the early phase of
infection by creating a separating layer between bacteria and plant cell wall to prevent the pathogen from recognition.