Cloning, expression and characterization of a β-D-xylosidase from Lactobacillus rossiae DSM 15814(T)

Abstract

Background: Among the oligosaccharides that may positively affect the gut microbiota, xylo-oligosaccharides (XOS) and arabinoxylan oligosaccharides (AXOS) possess promising functional properties. Ingestion of XOS has been reported to contribute to anti-oxidant, anti-bacterial, immune-modulatory and anti-diabetic activities. Because of the structural complexity and chemical heterogeneity, complete degradation of xylan-containing plant polymers requires the synergistic activity of several enzymes. Endo-xylanases and β-D-xylosidases, collectively termed xylanases, represent the two key enzymes responsible for the sequential hydrolysis of xylan. Xylanase cocktails are used on an industrial scale for biotechnological purposes. Lactobacillus rossiae DSM 15814(T) can utilize an extensive set of carbon sources, an ability that is likely to contribute to its adaptive ability. In this study, the capacity of this strain to utilize XOS, xylan, D-xylose and L-arabinose was investigated.

Results: Genomic and transcriptomic analyses revealed the presence of two gene clusters, designated xyl and ara, encoding proteins predicted to be responsible for XOS uptake and hydrolysis and D-xylose utilization, and L-arabinose metabolism, respectively. The deduced amino acid sequence of one of the genes of the xyl gene cluster, LROS_1108 (designated here as xylA), shows high similarity to (predicted) β-D-xylosidases encoded by various lactic acid bacteria, and belongs to glycosyl hydrolase family 43. Heterologously expressed XylA was shown to completely hydrolyse XOS to xylose and showed optimal activity at pH 6.0 and 40 °C. Furthermore, β-D-xylosidase activity of L. rossiae DSM 15814(T) was also measured under sourdough conditions.

Conclusions: This study highlights the ability of L. rossiae DSM 15814(T) to utilize XOS, which is a very useful trait when selecting starters with specific metabolic performances for sourdough fermentation or as probiotics.

Keywords: Functional foods; Gut microbiota; Prebiotic; Probiotic; Sourdough; Xylo-oligosaccharides.

Fig. 1

Heatmap based on the transcriptome analysis of Lactobacillus rossiae DSM15814^T grown on d-xylose, l-arabinose, XOS and maltose as the sole carbon source. XOS hydrolysis and utilization of the end product d-xylose (a) and the utilization of the l-arabinose (b) operon. The gene expression is expressed as RPKM (Reads per kilobase per million)

Fig. 2

Schematic representation of the genetic organization (ca. 11 kb region) in Lactobacillus rossiae DSM 15814^T. XOS hydrolysis and utilization of the end product d-xylose (a) and the utilization of the l-arabinose (b) operon. The size and orientation of each of the genes were deduced from their DNA sequences. The map was derived by the use of CloneManager Professional software (Scientific and Educational Software; USA)

Fig. 3

Phylogenetic tree showing the relationship between the amino acid sequences of β-d-xylosidase from Lactobacillus rossiae DSM15814^T and reference of sequences of some lactic acid bacteria in GenBank. The tree was constructed using the neighbour-joining software, numbers at the node are the bootstrap values (%). GroEL of Bifidobacterium adolescentis was used as outlier

Fig. 4

Hydrolysis of XOS as assessed by high-performance anion-exchange chromatography (HPAEC). Lane 1, 5 mg ml⁻¹ (wt/vol) standard of XOS; lane 2, Lactococcus lactis subsp. cremoris NZ9000 containing the empty plasmid (pNZ8048) (negative control); lane 3; Lc. lactis subsp. cremoris NZ9000 containing the pNZ8048.1108 construct; lane 4, 5 mg ml⁻¹ (wt/vol) standard of d-xylose. Details on recombinant Lc. lactis subsp. cremoris NZ9000 are reported in “Methods” section