Purification of a novel fructosyltransferase from Lactobacillus reuteri strain 121 and characterization of the levan produced

Hijum, S van

doi:10.1016/s0378-1097(01)00490-6

Cited by 87 publications

(43 citation statements)

References 15 publications

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“…3; see also van Hijum et al, 2002). Lev synthesized relatively more of a large polymer of the levan type (this study), containing almost exclusively (>95 %) b(2R6) linkages (see van Hijum et al, 2001), plus small amounts of b(2R1) FOS, and a range of unidentified molecules that may constitute b(2R6) FOSs (Table 1, Fig. 2).…”

Section: Ftf Synthesis Of Inulin-and Levan-like Fossmentioning

confidence: 68%

“…After preincubation of the assay mixture at the appropriate assay temperature for 5 min, reactions were started by enzyme addition. Samples were taken every 3 min, and used to determine the amount of glucose and fructose released from sucrose (van Hijum et al, 2001). The amount of glucose formed reflects the total amount of sucrose utilized during the reaction (V G ).…”

Section: Methodsmentioning

confidence: 99%

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The levansucrase and inulosucrase enzymes of Lactobacillus reuteri 121 catalyse processive and non-processive transglycosylation reactions

et al. 2006

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Bacterial fructosyltransferase (FTF) enzymes synthesize fructan polymers from sucrose. FTFs catalyse two different reactions, depending on the nature of the acceptor, resulting in: (i) transglycosylation, when the growing fructan chain (polymerization), or mono-and oligosaccharides (oligosaccharide synthesis), are used as the acceptor substrate; (ii) hydrolysis, when water is used as the acceptor. Lactobacillus reuteri 121 levansucrase (Lev) and inulosucrase (Inu) enzymes are closely related at the amino acid sequence level (86 % similarity). Also, the eight amino acid residues known to be involved in catalysis and/or sucrose binding are completely conserved. Nevertheless, these enzymes differ markedly in their reaction and product specificities, i.e. in b(2R6)-versus b(2R1)-glycosidic-bond specificity (resulting in levan and inulin synthesis, respectively), and in the ratio of hydrolysis versus transglycosylation activities [resulting in glucose and fructooligosaccharides (FOSs)/polymer synthesis, respectively]. The authors report a detailed characterization of the transglycosylation reaction products synthesized by the Lb. reuteri 121 Lev and Inu enzymes from sucrose and related oligosaccharide substrates. Lev mainly converted sucrose into a large levan polymer (processive reaction), whereas Inu synthesized mainly a broad range of FOSs of the inulin type (non-processive reaction). Interestingly, the two FTF enzymes were also able to utilize various inulin-type FOSs (1-kestose, 1,1-nystose and 1,1,1-kestopentaose) as substrates, catalysing a disproportionation reaction; to the best of our knowledge, this has not been reported for bacterial FTF enzymes. Based on these data, a model is proposed for the organization of the sugar-binding subsites in the two Lb. reuteri 121 FTF enzymes. This model also explains the catalytic mechanism of the enzymes, and differences in their product specificities.

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Section: Ftf Synthesis Of Inulin-and Levan-like Fossmentioning

confidence: 68%

Section: Methodsmentioning

confidence: 99%

The levansucrase and inulosucrase enzymes of Lactobacillus reuteri 121 catalyse processive and non-processive transglycosylation reactions

et al. 2006

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“…In general, LAB produce two types of fructans using FS (see also Table 2): levans, consisting mainly of ␤-(236)-linked fructose residues, occasionally containing ␤-(231)-linked branches, and inulin-type fructans, with ␤-(231)-linked fructose residues, with ␤-(236)-linked branches. Levan production has been reported for streptococci (19,70,178), L. mesenteroides (156), L. reuteri 121 (208,210), and Lactobacillus sanfranciscensis (92,93). Lactobacillus frumenti, Lactobacillus pontis, Lactobacillus panis and Weissella confusa were also found to produce fructans, but their fructan binding types have not been determined (92,93,198).…”

Section: Reactions Catalyzed and Fructan Product Synthesismentioning

confidence: 99%

Structure-Function Relationships of Glucansucrase and Fructansucrase Enzymes from Lactic Acid Bacteria

Hijum

Kralj

Ozimek

et al. 2006

Microbiol Mol Biol Rev

393

315

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SUMMARY Lactic acid bacteria (LAB) employ sucrase-type enzymes to convert sucrose into homopolysaccharides consisting of either glucosyl units (glucans) or fructosyl units (fructans). The enzymes involved are labeled glucansucrases (GS) and fructansucrases (FS), respectively. The available molecular, biochemical, and structural information on sucrase genes and enzymes from various LAB and their fructan and α-glucan products is reviewed. The GS and FS enzymes are both glycoside hydrolase enzymes that act on the same substrate (sucrose) and catalyze (retaining) transglycosylation reactions that result in polysaccharide formation, but they possess completely different protein structures. GS enzymes (family GH70) are large multidomain proteins that occur exclusively in LAB. Their catalytic domain displays clear secondary-structure similarity with α-amylase enzymes (family GH13), with a predicted permuted (β/α)8 barrel structure for which detailed structural and mechanistic information is available. Emphasis now is on identification of residues and regions important for GS enzyme activity and product specificity (synthesis of α-glucans differing in glycosidic linkage type, degree and type of branching, glucan molecular mass, and solubility). FS enzymes (family GH68) occur in both gram-negative and gram-positive bacteria and synthesize β-fructan polymers with either β-(2→6) (inulin) or β-(2→1) (levan) glycosidic bonds. Recently, the first high-resolution three-dimensional structures have become available for FS (levansucrase) proteins, revealing a rare five-bladed β-propeller structure with a deep, negatively charged central pocket. Although these structures have provided detailed mechanistic insights, the structural features in FS enzymes dictating the synthesis of either β-(2→6) or β-(2→1) linkages, degree and type of branching, and fructan molecular mass remain to be identified.

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“…After preincubation of the assay mixture at the assay temperature for 5 min, reactions were started by enzyme addition. Samples were taken every 3 min and used to determine the amount of glucose released from sucrose [10]. The amount of glucose formed reflects the total amount of sucrose utilized during the reaction (V G ).…”

Section: Ftf Enzyme Activity Assaysmentioning

confidence: 99%

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

et al. 2005

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Purification of a novel fructosyltransferase from Lactobacillus reuteri strain 121 and characterization of the levan produced

Cited by 87 publications

References 15 publications

The levansucrase and inulosucrase enzymes of Lactobacillus reuteri 121 catalyse processive and non-processive transglycosylation reactions

The levansucrase and inulosucrase enzymes of Lactobacillus reuteri 121 catalyse processive and non-processive transglycosylation reactions

Structure-Function Relationships of Glucansucrase and Fructansucrase Enzymes from Lactic Acid Bacteria

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

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