Identification and Characterization of Dextran α-1,2-Debranching Enzyme from <i>Microbacterium dextranolyticum</i>

Abstract

Dextran α-1,2-debranching enzyme (DDE) releases glucose with hydrolyzing α-(1→2)-glucosidic linkages in α-glucans, which are made up of dextran with α-(1→2)-branches and are generated by Leuconostoc bacteria. DDE was isolated from Microbacterium dextranolyticum (formerly known as Flavobacterium sp. M-73) 40 years ago, although the amino acid sequence of the enzyme has not been determined. Herein, we found a gene for this enzyme based on the partial amino acid sequences from native DDE and characterized the recombinant enzyme. DDE had a signal peptide, a glycoside hydrolase family 65 domain, a carbohydrate-binding module family 35 domain, a domain (D-domain) similar to the C-terminal domain of Arthrobacter globiformis glucodextranase, and a transmembrane region at the C-terminus. Recombinant DDE released glucose from α-(1→2)-branched α-glucans produced by Leuconostoc citreum strains B-1299, S-32, and S-64 and showed weak hydrolytic activity with kojibiose and kojitriose. No activity was detected for commercial dextran and Leuconostoc citreum B-1355 α-glucan, which do not contain α-(1→2)-linkages. The removal of the D-domain decreased the affinity for α-(1→2)-branched α-glucans but not for kojioligosaccharides, suggesting that D-domain plays a role in α-glucan binding. Genes for putative dextranases, oligo-1,6-glucosidase, sugar-binding protein, and permease were present in the vicinity of the DDE gene, and as a result these gene products may be necessary for the use of α-(1→2)-branched glucans. Our findings shed new light on how actinobacteria utilize polysaccharides produced by lactic acid bacteria. <Key words> actinobacteria, dextran, exopolysaccharide, GH65, kojibiose, lactic acid bacteria

Bacteroidota polysaccharide utilization system for branched dextran exopolysaccharides from lactic acid bacteria

“…EPSs have been demonstrated to play a crucial role in bacterial colonization, adherence, stress resistance, host–bacterium interactions, and immunomodulation ( 72 ). This study provided insight into the mechanism of α-(1→2)- and α-(1→3)-branched dextran degradation by Gram-negative bacteria, which is different from that of α-(1→2)-branched dextran degradation by the Gram-positive bacterium M. dextranolyticum ( 34 ). M. dextranolyticum removes α-(1→2) branches extracellularly, degrades dextran, and uptakes isomaltooligosaccharides into the cytosol, whereas F. johnsoniae extracellularly degrades branched dextran to oligosaccharides (with branches) and hydrolyzes all linkages, including branches, in the periplasm.…”

“…L. citreum S-32 and S-64 strains, which were isolated from the lines of a sugar-manufacturing factory, synthesize branched α-glucan. 13 C-NMR analysis revealed that S-32 α-glucan contains 30% α-(1→3)-linkages in addition to α-(1→6)-linkages; moreover, enzyme digestion analysis showed that it contains α-(1→2)-linked glucose ( 33 , 34 ). By contrast, S-64 α-glucan contains 24% α-(1→2)-, 24% of α-(1→3)-, and 9% of α-(1→4)-linkages ( 33 , 34 ).…”

“…Amino acid sequence analysis of MdDDE revealed that it is a member of the GH65 family. Recombinant MdDDE could convert α-(1→2)-branched dextran produced by strains B-1299, S-32, and S-64 into glucose ( 34 ). Thus, much remains unknown about how microorganisms completely degrade branched dextran.…”