Insights into extracellular dextran formation by Liquorilactobacillus nagelii TMW 1.1827 using secretomes obtained in the presence or absence of sucrose

Highlights

• Water-kefir borne Liquorilactobacillus (L.) nagelii extracellularly produces dextran.

• More dextransucrase (activity) was released by L. nagelii in the presence of sucrose.

• The environmental pH did not significantly affect detxransucrase release.

• The dextran amounts increased up to 24 h despite already consumed sucrose.

• The degree of branching of dextran increased despite already consumed sucrose.

Abstract

Dextrans are α-(1,6)-linked glucose polymers, which are exclusively produced by lactic acid bacteria from sucrose via extracellular dextransucrases. Previous studies suggested that the environmental pH and the presence of sucrose can impact the release and activity of these enzymes. To get deeper insight into this phenomenon, the dextransucrase expressed by water kefir borne Liquorilactobacillus (L.) nagelii TMW 1.1827 (formerly Lactobacillus nagelii) was recovered in supernatants of buffered cell suspensions that had been incubated with or without sucrose and at different pH. The obtained secretomes were used to time-dependently produce and recover dextrans, whose molecular and macromolecular structures were determined by methylation analysis and AF4-MALS-UV measurements, respectively. The initial pH of the buffered cell suspensions had solely a minor influence on the released dextransucrase activity. When sucrose was present during incubation, the secretomes contained significantly higher dextransucrase activities, although the amounts of totally released proteins obtained with or without sucrose were comparable. However, the dextransucrase appeared to be released in lower amounts into the environment if sucrose was not present. The amount of isolable dextran increased up to 24 h of production, although the total sucrose was consumed within the first 10 min of incubation. Furthermore, the sucrose isomer leucrose had been formed after 10 min, while its concentrations decreased over time and the portions of longer isomaltooligosaccharides (IMOs) increased. This indicated that leucrose can be used by L. nagelii TMW 1.1827 to produce more elongated and branched dextran molecules from presynthesized IMOs, while disproportionation reactions on short IMOs may appear additionally. This leads to increasing amounts of high molecular weight dextran in a state of sucrose depletion. These findings reveal new insights into the pH- and sucrose-dependent kinetics of extracellular dextran formation and may be useful for optimization of fermentative and enzymatic dextran production processes.

Introduction

Dextransucrases are Glycoside-Hydrolase (GH) family 70 (EC. 2.4.1.5) enzymes, which are produced and released into the extracellular milieu by lactic acid bacteria (LAB) of the genera Streptococcus, Weissella, Leuconostoc, Limosilactobacillus, Liquorilactobacillus and Lentilactobacillus [[1], [2], [3], [4], [5], [6]]. So far, many different types of dextransucrases have been found among LAB, varying in molar mass of 120–200 kDa, amino acid sequence, and domain architecture [7]. Further differences may occur in the presence or absence of a signal peptide and a cell wall anchor determining the enzyme to be freely released into the extracellular milieu or remaining cell-wall bound [[7], [8], [9]]. In general, dextransucrases feature one or two characteristic GH70 catalytic domains, including the essential substrate binding sites. Moreover, dextransucrases commonly exhibit at least one C- or N-terminal glucan-binding domain, most likely involved in holding of a growing polysaccharide chain [3,[10], [11], [12], [13]]. From sucrose, dextransucrases specifically catalyze the synthesis of high molecular weight α-glucans, while fructose is continuously released [2,7,14,15]. The carbohydrate backbone of dextran is mainly composed of α-1,6-glycosidic linkages and may be branched with glucose or glucose oligomers at position O2, O3 or O4 [1,7,[16], [17], [18]]. Particularly, the polymer’s type of branching appears to be highly specific for certain characteristics of the enzymes GH70 active site [[18], [19], [20], [21], [22]]. The dextransucrase reaction occurs by transferring the activated glucosyl residue to the non-reducing end of an exogenous acceptor involving only one catalytic site of the enzyme [7,19,23,24]. However, dextransucrases are not only capable of using dextran molecules as acceptors, but also e.g. maltose, sucrose, fructose or water, resulting in oligosaccharide synthesis, production of sucrose isomers such as leucrose and palatinose or simple hydrolysis of the substrate [19,23,24].

Although these findings laid the basis for the understanding of the complex reaction mechanism, using dextransucrases for directed design of polysaccharides with custom-made properties remains difficult. Indeed, physico-chemical properties were shown to be not only determined by the branching type of a certain dextran, but also from the degree of branching, as well as molecular size distribution. These dimensions are, however, more variable than the branching type and may be influenced by amount and position of glucan-binding domains, as well as general enzyme activity and surrounding factors, e.g. pH, salt, temperature and substrate or enzyme concentrations [7,9,18,[25], [26], [27], [28]]. Therefore, investigations of the production of high molecular weight dextrans is commonly done by using heterologously expressed enzymes in order to keep these factors constant, while fermentative dextran formation undergoes various fluctuations, e.g. pH decrease, enzyme concentration and amount of substrate, that may simultaneously be consumed by the cells [[29], [30], [31]]. Especially extracellular enzyme concentrations may vary at different environmental conditions, which can alter the amount of extracellular dextransucrase, e.g. Liquorilactobacillus (L.) hordei (formerly Lactobacillus hordei [5]) accumulates its dextransucrase independently from sucrose, but releases it solely in the presence of sucrose [9,26]. L. hordei was originally isolated from water kefir, which proved to be a promising reservoir for LAB producing dextrans of different physico-chemical properties [32,33]. Recently, we identified the dextransucrase of water kefir borne L. nagelii TMW 1.1827, which was detectable in supernatants independently of the presence of sucrose. Moreover, we could show its high efficiency of polymerization yielding the dextransucrase in a cell suspension, which was subsequently cleared from the cells to investigate dextran formation under constant reaction conditions. In that study, the L. nagelii dextransucrase showed to be highly efficient in transglycosylation, while consuming the total amount of sucrose within the first 10 min of reaction [9]. Nonetheless, extracellular dextransucrase activity was solely investigated yielding the crude enzyme extract in the presence of sucrose at an initial pH of 6.5. Our aim was therefore to study extracellular native dextran formation more detailed with regards to the presence or absence of sucrose as well as initial release pH of the enzyme extracts, as these environmental factors may not only influence the amount of dextransucrase released, but also the functionality of the enzyme due to e.g. differences in protein folding or aggregation [34,35]. As consumption of total sucrose within the first 10 min of reaction implied that dextran formation was already completed at this point of time, we additionally modulated the experimental setup to allow for the analysis of dextran formation on the product level over time.