title: The Gram-negative bacterium Azotobacter chroococcum NCIMB 8003 employs a new glycoside hydrolase family 70 4,6-α-glucanotransferase enzyme (GtfD) to synthesize a reuteran like polymer from maltodextrins and starch.
abstract: BACKGROUND
  Originally the glycoside hydrolase (GH) family 70 only comprised glucansucrases of lactic acid bacteria which synthesize α-glucan polymers from sucrose. Recently we have identified 2 novel subfamilies of GH70 enzymes represented by the Lactobacillus reuteri 121 GtfB and the Exiguobacterium sibiricum 255-15 GtfC enzymes. Both enzymes catalyze the cleavage of (α1→4) linkages in maltodextrin/starch and the synthesis of consecutive (α1→6) linkages. Here we describe a novel GH70 enzyme from the nitrogen-fixing Gram-negative bacterium Azotobacter chroococcum, designated as GtfD.
  
  
  METHODS
  The purified recombinant GtfD enzyme was biochemically characterized using the amylose-staining assay and its products were identified using profiling chromatographic techniques (TLC and HPAEC-PAD). Glucans produced by the GtfD enzyme were analyzed by HPSEC-MALLS-RI, methylation analysis, 1D/2D [1]H/[13]C NMR spectroscopy and enzymatic degradation studies.
  
  
  RESULTS
  The A. chroococcum GtfD is closely related to GtfC enzymes, sharing the same non-permuted domain organization also found in GH13 enzymes and displaying 4,6-α-glucanotransferase activity. However, the GtfD enzyme is unable to synthesize consecutive (α1→6) glucosidic bonds. Instead, it forms a high molecular mass and branched α-glucan with alternating (α1→4) and (α1→6) linkages from amylose/starch, highly similar to the reuteran polymer synthesized by the L. reuteri GtfA glucansucrase from sucrose.
  
  
  CONCLUSIONS
  In view of its origin and specificity, the GtfD enzyme represents a unique evolutionary intermediate between family GH13 (α-amylase) and GH70 (glucansucrase) enzymes.
  
  
  CONCLUSIONS
  This study expands the natural repertoire of starch-converting enzymes providing the first characterization of an enzyme that converts starch into a reuteran-like α-glucan polymer, regarded as a health promoting food ingredient.
authors: Microbial Physiology, Groningen Biomolecular Sciences and Biotechnology Institute (GBB), University of Groningen, Nijenborgh 7, 9747 AG Groningen, The Netherlands.
authors: Joana
authors: J
authors: Gangoiti
authors: Microbial Physiology, Groningen Biomolecular Sciences and Biotechnology Institute (GBB), University of Groningen, Nijenborgh 7, 9747 AG Groningen, The Netherlands.
authors: Sander S
authors: SS
authors: van Leeuwen
authors: Nestlé Research Center, Vers-Chez-Les-Blanc, Lausanne, Switzerland.
authors: Christina
authors: C
authors: Vafiadi
authors: Microbial Physiology, Groningen Biomolecular Sciences and Biotechnology Institute (GBB), University of Groningen, Nijenborgh 7, 9747 AG Groningen, The Netherlands. Electronic address: L.Dijkhuizen@rug.nl.
authors: Lubbert
authors: L
authors: Dijkhuizen
mesh_terms: D000595:Amino Acid Sequence; D001395:Azotobacter / Q000201:enzymology*; D005936:Glucans / Q000096:biosynthesis*; D006004:Glycogen Debranching Enzyme System / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000096:biosynthesis; D011134:Polysaccharides / Q000378:metabolism*; D013213:Starch / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D005936:Glucans; D006004:Glycogen Debranching Enzyme System; D009844:Oligosaccharides; D011134:Polysaccharides; C008315:maltodextrin; D013213:Starch; C020569:4 alpha-glucanotransferase
keywords: 4,6-α-glucanotransferase; Azotobacter chroococcum; Family GH70; Glucansucrase; Isomalto-/malto-polysaccharide; Reuteran