Degradation of beechwood xylan using food-grade bacteria-like particles displaying β-xylosidase from Limosilactobacillus fermentum

Abstract

The display of enzymes on bacterial surfaces is an interesting approach for immobilising industrially important biocatalysts. In recent years, non-recombinant surface display using food-grade bacteria, such as lactic acid bacteria (LAB), have gained interest because of their safety, simplicity, and cost-effectiveness. β-Xylosidase is one of the many biocatalytic enzymes targeted for immobilisation due to its key role in the complete saccharification of lignocellulosic biomass, including xylan hemicellulose. Recently, the xylose-tolerant β-xylosidase, LfXyl43, was identified in Limosilactobacillus fermentum. LfXyl43 is capable of producing xylose from the degradation of xylo-oligosaccharides (XOS) and beechwood xylan. This study aimed to immobilise this new biocatalyst on the surface of LAB-derived bacteria-like particles (BLP) and investigate its applicability and reusability in the degradation of xylan hemicellulose. Additionally, the influence of the anchor position and the presence of linker peptides on the display and activity of the β-xylosidase was investigated. Four expression vectors were constructed to express different anchor-xylosidase fusion proteins. Upon expression and purification, all anchor-xylosidase fusion proteins were active towards the artificial substrate p-nitrophenyl-β-D-xylopyranoside. In addition, all anchor-xylosidase fusion proteins were successfully displayed on the surface of BLP. However, only the β-xylosidases with linker peptide showed hydrolytic activity after immobilisation on BLP. BLP displaying β-xylosidases demonstrated high activity against XOS and beechwood xylan, thereby producing high amounts of xylose. Moreover, the immobilised enzyme demonstrated reusability across several bioconversion cycles. Overall, this study highlights the potential industrial application of surface-displayed β-xylosidase for the effective degradation of lignocellulosic biomass.

Keywords: Beta-xylosidase; Biocatalyst; Immobilisation; Lactic acid bacteria; Surface display; Xylan.

Fig. 1

Construction of the anchor-xylosidase fusion protein expression vectors. Fusion of the anchor, CshA, to the β-xylosidase gene, at either the N- or C-terminus, with or without the (GGGGS)₃ peptide linker (A-B). The linker peptide is denoted by “L”, whereas the 6× histidine tag is denoted by “H”. Expression vectors of the histidine-tagged fusion proteins with and without the linker peptide based on the pET21b (+) vector (C-D)

Fig. 2

SDS-PAGE analysis of the Ni-NTA purified fusion proteins with and without the flexible (GGGGS)₃ linker peptide. N and C denote the N- or C-terminal position of the cell surface anchor CshA, respectively. The expected protein size of the fusion proteins without the linker peptide (CshA-Xyl and Xyl-CshA) is 71.2 kDa, whereas that of the fusion proteins with linker peptide (CshA-L-Xyl and Xyl-L-CshA) is 72.1 kDa. M indicates the protein markers (LPS Solution, Daejeon, Republic of Korea). Arrows indicate the expected sizes of the fusion proteins

Fig. 3

Surface display of β-xylosidase on lactic acid bacteria-derived bacteria-like particles (BLP) detected by immunofluorescence microscopy and flow cytometry. BLP used as a control for immunofluorescence microscopy (A). Non-displaying BLP (B), BLP + CshA-Xyl (C), BLP + Xyl-CshA (D), BLP + CshA-L-Xyl (E), and BLP + Xyl-L-CshA (F) viewed with a 570 nm filter. Flow cytometry measurements of the fluorescence intensity of BLP displaying β-xylosidase than that of non-displaying BLP: BLP + CshA-Xyl (G), BLP + Xyl-CshA (H), BLP + CshA-L-Xyl (I), and BLP + Xyl-L-CshA (J)

Fig. 4

Hydrolysis of xylo-oligosaccharides (XOS) by bacteria-like particles (BLP)-displaying β-xylosidase. Release of reducing sugars equivalent to xylose (in mM) by surface-displayed β-xylosidase from 2 mM xylobiose (A), xylotriose (B), and xylotetraose (C). Thin-layer chromatography analysis of the degradation of XOS using surface-displayed β-xylosidase: xylobiose (D), xylotriose (E), and xylotetraose (F). The enzyme assays were performed in 50 mM sodium phosphate buffer (pH 7.0), at 35 °C, for 5 min. Assays were performed in triplicates. Abbreviations: Std, standard; X₁, xylose; X₂, xylobiose; X₃, xylotriose; and X₄, xylotetraose

Fig. 5

Reusability of bacteria-like particles (BLP)-displaying β-xylosidase and the time course xylose production. The activity of surface-displayed β-xylosidase with N-terminal anchor (BLP + CshA-L-Xyl) or C-terminal anchor (BLP + Xyl-L-CshA) after several bioconversion cycles (A). A washing step in between the cycles was performed before the addition of fresh substrate. Time-course production of xylose from 30 g/L beechwood xylan using non-displaying BLP or BLP displaying either CshA-L-Xyl or Xyl-L-CshA (B). The total concentration of surface-displayed β-xylosidase per mL of reaction was 0.5 mg. The reactions were performed in 50 mM sodium phosphate buffer (pH 7.0), at 35 °C. Data are reported as the mean ± standard deviation of triplicate experiments. Error bars smaller than the symbol are not shown