Highly Hydrolytic Reuteransucrase from Probiotic Lactobacillus reuteri Strain ATCC 55730

ABSTRACT Lactobacillus reuteri strain ATCC 55730 (LB BIO) was isolated as a pure culture from a Reuteri tablet purchased from the BioGaia company. This probiotic strain produces a soluble glucan (reuteran), in which the majority of the linkages are of the α-(1→4) glucosidic type (∼70%). This reuteran also contains α-(1→6)- linked glucosyl units and 4,6-disubstituted α-glucosyl units at the branching points. The LB BIO glucansucrase gene (gtfO) was cloned and expressed in Escherichia coli, and the GTFO enzyme was purified. The recombinant GTFO enzyme and the LB BIO culture supernatants synthesized identical glucan polymers with respect to linkage type and size distribution. GTFO thus is a reuteransucrase, responsible for synthesis of this reuteran polymer in LB BIO. The preference of GTFO for synthesizing α-(1→4) linkages is also evident from the oligosaccharides produced from sucrose with different acceptor substrates, e.g., isopanose from isomaltose. GTFO has a relatively high hydrolysis/transferase activity ratio. Complete conversion of 100 mM sucrose by GTFO nevertheless yielded large amounts of reuteran, although more than 50% of sucrose was converted into glucose. This is only the second example of the isolation and characterization of a reuteransucrase and its reuteran product, both found in different L. reuteri strains. GTFO synthesizes a reuteran with the highest amount of α-(1→4) linkages reported to date.

[1]  F. Paul,et al.  Oligosaccharide feed additives , 2007 .

[2]  L. Dijkhuizen,et al.  Glucan synthesis in the genus Lactobacillus: isolation and characterization of glucansucrase genes, enzymes and glucan products from six different strains. , 2004, Microbiology.

[3]  L. Dijkhuizen,et al.  Biochemical and molecular characterization of Lactobacillus reuteri 121 reuteransucrase. , 2004, Microbiology.

[4]  M. Nadkarni,et al.  Quantitative Analysis of Diverse Lactobacillus Species Present in Advanced Dental Caries , 2004, Journal of Clinical Microbiology.

[5]  Frédéric Leroy,et al.  Lactic acid bacteria as functional starter cultures for the food fermentation industry , 2004 .

[6]  N. Valeur,et al.  Colonization and Immunomodulation by Lactobacillus reuteri ATCC 55730 in the Human Gastrointestinal Tract , 2004, Applied and Environmental Microbiology.

[7]  L. Dijkhuizen,et al.  Efficient Screening Methods for Glucosyltransferase Genes in Lactobacillus Strains , 2003 .

[8]  R. Vogel,et al.  In Situ Production of Exopolysaccharides during Sourdough Fermentation by Cereal and Intestinal Isolates of Lactic Acid Bacteria , 2003, Applied and Environmental Microbiology.

[9]  S. Bozonnet,et al.  Molecular Characterization of DSR-E, an α-1,2 Linkage-Synthesizing Dextransucrase with Two Catalytic Domains , 2002, Journal of bacteriology.

[10]  L. Dijkhuizen,et al.  Molecular Characterization of a Novel Glucosyltransferase from Lactobacillus reuteri Strain 121 Synthesizing a Unique, Highly Branched Glucan with α-(1→4) and α-(1→6) Glucosidic Bonds , 2002, Applied and Environmental Microbiology.

[11]  M. de Vrese,et al.  Probiotics, prebiotics, and synbiotics--approaching a definition. , 2001, The American journal of clinical nutrition.

[12]  P. Monsan,et al.  Glucansucrases: molecular engineering and oligosaccharide synthesis , 2000 .

[13]  B. Svensson,et al.  Involvement of Gln937 of Streptococcus downei GTF-I glucansucrase in transition-state stabilization. , 2000, European journal of biochemistry.

[14]  R. Hrabal,et al.  Towards regioselective synthesis of oligosaccharides by use of alpha-glucosidases with different substrate specificity. , 1999, Carbohydrate research.

[15]  L. Dijkhuizen,et al.  Biochemical and Structural Characterization of the Glucan and Fructan Exopolysaccharides Synthesized by theLactobacillus reuteri Wild-Type Strain and by Mutant Strains , 1999, Applied and Environmental Microbiology.

[16]  P. Monsan,et al.  Glucansucrases: mechanism of action and structure-function relationships. , 1999, FEMS microbiology reviews.

[17]  L De Vuyst,et al.  Heteropolysaccharides from lactic acid bacteria. , 1999, FEMS microbiology reviews.

[18]  V. Planchot,et al.  Sequence Analysis of the Gene Encoding Amylosucrase from Neisseria polysaccharea and Characterization of the Recombinant Enzyme , 1999, Journal of bacteriology.

[19]  S. Salminen.  Lactic acid bacteria: microbiology and functional aspects. , 1998 .

[20]  A. Chesson,et al.  Biotechnology in animal feeds and animal feeding. , 1995 .

[21]  H. Kuramitsu,et al.  Identification of amino acid residues in Streptococcus mutans glucosyltransferases influencing the structure of the glucan product , 1994, Journal of bacteriology.

[22]  P. Giffard,et al.  Definition of a Fundamental Repeating Unit in Streptococcal Glucosyltransferase Glucan-binding Regions and Related Sequences , 1994, Journal of dental research.

[23]  Seppo Salminen,et al.  Lactic Acid Bacteria , 2004 .

[24]  Seppo Salminen,et al.  Lactic acid bacteria: classification and physiology. , 1993 .

[25]  E. Myers,et al.  Basic local alignment search tool. , 1990, Journal of molecular biology.

[26]  W. Dobrogosz,et al.  Antagonistic activities of lactic acid bacteria in food and feed fermentations. , 1990, FEMS microbiology reviews.

[27]  D. Kemp,et al.  A procedure for in vitro amplification of DNA segments that lie outside the boundaries of known sequences. , 1988, Nucleic acids research.

[28]  W. Loesche.  Role of Streptococcus mutans in human dental decay. , 1986, Microbiological reviews.

[29]  G. J. Walker,et al.  Application of high-performance liquid chromatography to a study of branching in dextrans , 1985 .

[30]  W. Bowen,et al.  Studies on dextrans and dextranases , 1971 .

[31]  E. J. Bourne,et al.  Studies on dextrans and dextranases. 3. Structures of oligosaccharides from Leuconostoc mesenteroides (Birmingham) dextran. , 1963, The Biochemical journal.

[32]  K. Kleppe,et al.  The hydrolysis of alpha-D-glucosides by amyloglucosidase from Aspergillus niger. , 1962, The Journal of biological chemistry.

[33]  J. Pazur,et al.  The hydrolysis of glucosyl oligosaccharides with alpha-D-(1-4) and alpha-D-(1-6) bonds by fungal amyloglucosidase. , 1960, The Journal of biological chemistry.