Purification and characterization of an extracellular levansucrase from Pseudomonas syringae pv. phaseolicola

Abstract

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؇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.

Unexpected presence of fructan 6‐exohydrolases (6‐FEHs) in non‐fructan plants: characterization, cloning, mass mapping and functional analysis of a novel ‘cell‐wall invertase‐like’ specific 6‐FEH from sugar beet (Beta vulgaris L.)

“…This behaviour supports the idea that levans ful®l a crucial role during the early phase of infection by creating a separating layer between bacteria and plant cell wall polymers, in a way preventing recognition of the pathogen (Hettwer et al, 1995). Further straightforward evidence for the importance of levans during the plant±pathogen interaction comes from the fact that Erwinia amylovora strains de®cient in levansucrase had reduced virulence (Bereswill et al, 1997;Tharaud et al, 1994).…”

Formation of Levan from Raffinose by Levansucrase of Zymomonas mobilis

“…However, the utilisable substrate range for Z. mobilis is restricted to glucose, fructose, and sucrose [1], and only sucrose should be recognised as an appropriate substrate for levan synthesis. It has been shown that multiform bacterial levansucrases can [1] or cannot [5] perform the polymerisation of fructose moiety from catabolised raffinose. There exists a lack of data concerning the kinetics and the products of such a reaction for Z. mobilis, excepting a few observations on the limited formation of levan on raffinose as compared to sucrose medium by the non-growing Z. mobilis ATCC 10988 [6] as well as on such an above-mentioned ability of a Z. mobilis levansucrase from a recombinant Escherichia coli [7] to perform this polymerisation.…”

Mutational analysis of the role of calcium ions in the Lactobacillus reuteri strain 121 fructosyltransferase (levansucrase and inulosucrase) enzymes

“…Previously, Ohtsuka et al [19] and Hettwer et al [20] have shown that the activity and stability of the levansucrases from the Gram-negative bacteria Rahnella aquatilis and Pseudomonas syringae are not affected by the presence of calcium or EDTA.…”