Characterization of the Paenibacillus beijingensis DSM 24997 GtfD and its glucan polymer products representing a new glycoside hydrolase 70 subfamily of 4,6-α-glucanotransferase enzymes

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.

Microbial Starch‐Converting Enzymes: Recent Insights and Perspectives

“…Glucansucrases are members of family GH70. Specific glucansucrases may be termed dextransucrases, mutansucrases, alternansucrases, reuteransucrases, α‐1,2‐branching dextransucrases, α‐4,6‐glucanotransferases, or α‐4,3‐glucanotransferases (Bai et al., ; Brison et al., ; Gangoiti, van Leeuwen et al., ; Gangoiti, Lamothe et al., ; Miao et al., ; Song et al., ). Gluansucrases are evolutionary intermediates between enzymes in the GH13 and GH70 families, but their substrate specificities are completely different (Kralj et al., ).…”

“…Amino acid residues that provide a catalytic nucleophile, an acid/base catalyst, and a transition‐state stabilizer and constitute the catalytic site, are present in all glucansucrases (Vujičić‐Žagar et al., ). However, the conserved amino acid residues in regions I, II, III and IV are different in glucansucrases, especially the residues in regions I and IV for the –1, +1 and +2 donor/acceptor substrate‐binding subsites (Gangoiti, Lamothe et al., ).…”

“…α‐4,6‐Glucanotransferases predominantly cleave an α‐1,4 bond from the nonreducing end of linear maltodextrin molecules and transfer the cleaved glucosyl unit to the nonreducing end of an acceptor molecule, mostly by formation of α‐1,6 bonds and occasionally α‐1,4 linkages (Figure E), forming some small linear maltooligosaccharides, but mostly maltooligosaccharides that have linear α‐1,6‐linked oligosaccharide units attached to their nonreducing ends that are usually longer than are the acceptor chains (Dobruchowska et al., ; Leemhuis et al., ). Compared to the A. chroococcum glucansucrase product, which contained only α‐1,4‐linked maltooligosaccharides (up to DP 5), products formed using Paenibacillus beijingensis glucansucrase had longer (up tp DP 8) α‐1,4‐linked chains and α‐1,6‐linkages, either at the nonreducing end or at branch points (Gangoiti, Lamothe et al., ). The α‐4,6‐glucanotransferase activity on amylose V resulted in the synthesis of a reuteran‐like polymer built‐up from starch or a maltooligosaccharide of DP 3 to 7.…”

“…Gangoiti, Lamothe et al. () reported that 4,6‐α‐glucanotransferase synthesized a high molecular weight and branched glucan with alternating α‐1,4 and α‐1,6 bonds from amylose/starch that was similar to reuteran produced from sucrose by Lactobacillus reuteri glucansucrase, which is regarded as a health‐promoting functional food ingredient. Solabia Group has developed a unique prebiotic glucooligosaccharide via enzymatic synthesis and markets it under the name BioEcolians®.…”

Purification and biochemical characterization of extracellular glucoamylase from Paenibacillus amylolyticus strain

“…The genus Paenibacillus has been reported for production of several industrially important enzymes such as amylase, cellulase, hemicellulase, pectinase, lignin‐modifying enzymes , chitosanase , xylan‐degrading enzymes , 4,6‐α‐glucanotransferase enzymes . The enzymes have been used for many applications in industries such as biofuel, paper, textiles, detergents, food and feed, as well as healthcare.…”