title: The probiotic Lactobacillus johnsonii NCC 533 produces high-molecular-mass inulin from sucrose by using an inulosucrase enzyme.
abstract: Fructansucrase enzymes polymerize the fructose moiety of sucrose into levan or inulin fructans, with beta(2-6) and beta(2-1) linkages, respectively. The probiotic bacterium Lactobacillus johnsonii strain NCC 533 possesses a single fructansucrase gene (open reading frame AAS08734) annotated as a putative levansucrase precursor. However, (13)C nuclear magnetic resonance (NMR) analysis of the fructan product synthesized in situ revealed that this is of the inulin type. The ftf gene of L. johnsonii was cloned and expressed to elucidate its exact identity. The purified L. johnsonii protein was characterized as an inulosucrase enzyme, producing inulin from sucrose, as identified by (13)C NMR analysis. Thin-layer chromatographic analysis of the reaction products showed that InuJ synthesized, besides the inulin polymer, a broad range of fructose oligosaccharides. Maximum InuJ enzyme activity was observed in a pH range of 4.5 to 7.0, decreasing sharply at pH 7.5. InuJ exhibited the highest enzyme activity at 55 degrees C, with a drastic decrease at 60 degrees C. Calcium ions were found to have an important effect on enzyme activity and stability. Kinetic analysis showed that the transfructosylation reaction of the InuJ enzyme does not obey Michaelis-Menten kinetics. The non-Michaelian behavior of InuJ may be attributed to the oligosaccharides that were initially formed in the reaction and which may act as better acceptors than the growing polymer chain. This is only the second example of the isolation and characterization of an inulosucrase enzyme and its inulin (oligosaccharide) product from a Lactobacillus strain. Furthermore, this is the first Lactobacillus strain shown to produce inulin polymer in situ.
authors.affiliation: Centre for Carbohydrate Bioprocessing, TNO-University of Groningen, PO Box 14, 9750 AA Haren, The Netherlands.
authors.forename: Munir A
authors.initials: MA
authors.lastname: Anwar
authors.forename: Slavko
authors.initials: S
authors.lastname: Kralj
authors.forename: Marc J E C
authors.initials: MJ
authors.lastname: van der Maarel
authors.forename: Lubbert
authors.initials: L
authors.lastname: Dijkhuizen
mesh_terms: D002118:Calcium / Q000494:pharmacology; D002247:Carbon Isotopes / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D003067:Coenzymes / Q000494:pharmacology; D004492:Edetic Acid / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D017353:Gene Deletion; D006602:Hexosyltransferases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007444:Inulin / Q000096:biosynthesis*; D007700:Kinetics; D007778:Lactobacillus / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D009844:Oligosaccharides / Q000096:biosynthesis; D010802:Phylogeny; D019936:Probiotics; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013395:Sucrose / Q000378:metabolism*; D013696:Temperature; D001426:Bacterial Proteins
chemical_list: D002118:Calcium; D002247:Carbon Isotopes; D003067:Coenzymes; D004492:Edetic Acid; D004791:Enzyme Inhibitors; D006602:Hexosyltransferases; D007444:Inulin; D009844:Oligosaccharides; D011994:Recombinant Proteins; D013395:Sucrose; C116580:fructooligosaccharide; C021451:inulosucrase; D001426:Bacterial Proteins