Abstract
Previously we have reported that the Gram-negative bacterium Azotobacter chroococcum NCIMB 8003 uses the 4,6-α-glucanotransferase GtfD to convert maltodextrins and starch into a reuteran-like polymer consisting of (α1→4) glucan chains connected by alternating (α1→4)/(α1→6) linkages and (α1→4,6) branching points. This enzyme constituted the single evidence for this reaction and product specificity in the GH70 family, mostly containing glucansucrases encoded by lactic acid bacteria (http://www.CAZy.org). In this work, 4 additional GtfD-like proteins were identified in taxonomically diverse plant-associated bacteria forming a new GH70 subfamily with intermediate characteristics between the evolutionary related GH13 and GH70 families. The GtfD enzyme encoded by Paenibacillus beijingensis DSM 24997 was characterized providing the first example of a reuteran-like polymer synthesizing 4,6-α-glucanotransferase in a Gram-positive bacterium. Whereas the A. chroococcum GtfD activity on amylose resulted in the synthesis of a high molecular polymer, in addition to maltose and other small oligosaccharides, two reuteran-like polymer distributions are produced by P. beijingensis GtfD: a high-molecular mass polymer and a low-molecular mass polymer with an average Mw of 27 MDa and 19 kDa, respectively. Compared to the A. chroooccum GtfD product, both P. beijingensis GtfD polymers contain longer linear (α1→4) sequences in their structure reflecting a preference for transfer of even longer glucan chains by this enzyme. Overall, this study provides new insights into the evolutionary history of GH70 enzymes, and enlarges the diversity of natural enzymes that can be applied for modification of the starch present in food into less and/or more slowly digestible carbohydrate structures.
Highlights
The Glycoside Hydrolase family 70 (GH70) was originally defined for glucansucrase (GS) enzymes from lactic acid bacteria catalyzing the synthesis of α-glucans with various types of glucosidic linkages from sucrose [1,2]
These searches revealed that the A. chroococcum GtfD shows 71, 48, 46 and 45% amino acid sequence identity to the hypothetical GH70 enzymes encoded by the Dyella-like sp
To gain more insight into the carbohydrate structures of the high molecular mass (HMM) and low molecular mass (LMM) P. beijingensis GtfD products, and to compare them with the A. chroococcum GtfD reuteran-like polymer and L. reuteri 121 GtfB IMMP, these α-glucans were incubated for 48 h with different hydrolytic enzymes: α-amylase, dextranase and pullulanase M1 (Fig 6)
Summary
The Glycoside Hydrolase family 70 (GH70) was originally defined for glucansucrase (GS) enzymes from lactic acid bacteria catalyzing the synthesis of α-glucans with various types of glucosidic linkages from sucrose [1,2]. In vitro digestibility of the products synthesized by the P. beijingensis and A. chroococcum GtfD enzymes from amylose and wheat starch
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