AEM Accepts, published online ahead of print on 11 April 2008

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Appl. Environ. Microbiol. doi:10.1128/AEM.00377-08
Copyright (c) 2008, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

The probiotic Lactobacillus johnsonii NCC 533 produces high molecular mass inulin from sucrose using an inulosucrase enzyme

Munir A. Anwar, Slavko Kralj, Marc J. E. C. van der Maarel, and Lubbert Dijkhuizen^*

Centre for Carbohydrate Bioprocessing (CCB), TNO-University of Groningen, PO Box 14, 9750 AA Haren, The Netherlands; Department of Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute (GBB), University of Groningen, Kerklaan 30, 9751 NN, Haren, The Netherlands

^* To whom correspondence should be addressed. Email: L.Dijkhuizen{at}rug.nl.

Fructansucrase enzymes polymerize the fructose moiety of sucrose into levan or inulin fructans, with (2-6) and (2-1) linkages, respectively. The probiotic bacterium Lactobacillus johnsonii strain NCC 533 possesses a single fructansucrase gene (orf AAS08734) annotated as a putative levansucrase precursor. However, ¹³C 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 ¹³C NMR analysis. TLC analysis of the reaction products showed that InuJ synthesized besides inulin polymer a broad range of fructose oligosaccharides (FOS). Maximum InuJ enzyme activity was observed in the pH range of 4.5 to 7.0, decreasing sharply at pH 7.5. InuJ exhibited highest enzyme activity at 55 °C, with a drastic decrease at 60 °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.