The C-Terminal Domain of Liquorilactobacillus nagelii Dextransucrase Mediates the Production of Larger Dextrans Compared to Liquorilactobacillus hordei.

Julia Bechtner,Frank Jakob,Matthias Ehrmann,Verena Hassler,Daniel Wefers

doi:10.3390/gels8030171

Abstract

Dextransucrases released by certain lactic acid bacteria form glucose polymers with predominantly α-1,6-linkages and may be exploited biotechnologically for the tailored production of polysaccharides with application potential. Despite releasing two closely related dextransucrases, previous studies showed that water kefir borne Liquorilactobacillus (L.) hordei TMW 1.1822 and L. nagelii TMW 1.1827 produce different amounts of polysaccharides with distinct particle sizes (molecular weight and radius of gyration) and molecular architectures. To investigate where these differences originate and thus to provide deeper insights into the functionally diverse nature of polysaccharide formation during water kefir fermentation, we constructed two variants of the L. nagelii dextransucrase—a full-length enzyme and a truncated variant, devoid of a C-terminal glucan-binding domain that reflects the domain architecture of the L. hordei dextransucrase—and applied them at various enzyme concentrations to form dextran over 24 h. The full-length enzyme exhibited a high activity, forming constant amounts of dextran until a four-fold dilution, whereas the truncated variant showed a gradual decrease in activity and dextran formation at an increasing dilution. The application of the full-length enzyme resulted in higher average particle sizes compared to the truncated variant. However, the dilution of the enzyme extracts also led to a slight increase in the average particle size in both enzymes. Neither the domain architecture nor the enzyme concentration had an impact on the structural architecture of the dextrans. The presented results thus suggest that the comparatively higher processivity of the L. nagelii dextransucrase is predominantly caused by the additional C-terminal glucan-binding domain, which is absent in the L. hordei dextransucrase. The average particle size may be influenced, to some extent, by the applied reaction conditions, whereas the structural architecture of the dextrans is most likely caused by differences in the amino acid sequence of the catalytic domain.

Highlights

Belonging to this group of enzymes, glycoside hydrolase family 70 (GH70) enzymes represent a diverse group of enzymes that can produce large and often branched extracellular polysaccharides (EPS) on the basis of sucrose, starch or maltooligosaccharides [11]
SDS-PAGE with subsequent activity staining of heterologously expressed dextransucrase variants compared to the native extracellular dextransucrases of L. hordei TMW 1.1822 and L. nagelii TMW 1.1827 (B)
Previous research on the fermentative production of dextrans [34], as well as native extracellular dextransucrases of L. hordei and L. nagelii, indicated that dextran formation during water kefir fermentation is functionally diverse, even among closely related species encoding highly similar dextransucrases [30]. This already started with the release of these enzymes, as L. hordei only released its dextransucrase in the presence of sucrose, despite intracellular accumulation at other conditions [30,32]

Summary

Introduction

Glucansucrases use sucrose as a substrate to transfer the glucose moiety to the non-reducing end of an acceptor substrate, which may be water (hydrolysis) or a growing oligo- or polysaccharide chain (transglycosylation), but other acceptor substrates are utilized as well [13] By transglycosylation, these enzymes form α-glucans with different linkage types within the polysaccharide backbone and branches [14]. While single amino acids within and outside the substrate- and acceptorbinding sites were already identified to determine the linkage type of the polysaccharide backbone and branch points [17–20], it is rather scarcely understood where differences in the side-chain length and polymer size originate All of these factors influence the techno-functional properties of the formed polysaccharides. It is necessary to study the factors affecting the molecular and macromolecular properties of the polysaccharides formed by extracellular sucrases in order to lay the basis for the tailored production of such

Methods

Results

Conclusion