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

Background: Originally the glycoside hydrolase (GH) family 70 only comprised glucansucrases of lactic acid bacteria which synthesize alpha-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 (alpha(1) -> 4) linkages in maltodextrin/starch and the synthesis of consecutive (alpha(1) -> 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-alpha-glucanotransferase activity. However, the GtfD enzyme is unable to synthesize consecutive (alpha(1) -> 6) glucosidic bonds. Instead, it forms a high molecular mass and branched alpha-glucan with alternating (alpha(1) -> 4) and (alpha(1) -> 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 (alpha-amylase) and GH70 (glucansucrase) enzymes. General significance: This study expands the natural repertoire of starch-converting enzymes providing the first characterization of an enzyme that converts starch into a reuteran-like alpha-glucan polymer, regarded as a health promoting food ingredient. (C) 2016 Elsevier B.V. All rights reserved.