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

Surface display / Beta-xylosidase / Lactic acid bacteria / Xylan / Immobilisation / Biocatalyst

References

[1] AliA, AbdulameerMK, MahdiMH, et al.. Utilizing a Deformation/Aggregation-Based approach for determination of selenium using plasmonic silver nanoparticles. Plasmonics, 2024, 20: 2797-2805

[2] AraiR, UedaH, KitayamaA, et al.. Design of the linkers which effectively separate domains of a bifunctional fusion protein. Protein Eng Des Sel, 2001, 14: 529-532

[3] Åvall-JääskeläinenS, Kylä-NikkiläK, KahalaM, et al.. Surface display of foreign epitopes on the Lactobacillus brevis S-layer. Appl Environ Microbiol, 2002, 68: 5943-5951

[4] BosettoA, JustoPI, ZanardiB, et al.. Research progress concerning fungal and bacterial β-Xylosidases. Appl Biochem Biotechnol, 2016, 178: 766-795

[5] BosmaT, KanningaR, NeefJ, et al.. Novel surface display system for proteins on non-genetically modified gram-positive bacteria. Appl Environ Microbiol, 2006, 72: 880-889

[6] BoudrantJ, WoodleyJM, Fernandez-LafuenteR. Parameters necessary to define an immobilized enzyme Preparation. Process Biochem, 2020, 90: 66-80

[7] ChenX, ZaroJL, ShenW-C. Fusion protein linkers: property, design and functionality. Adv Drug Deliv Rev, 2013, 65: 1357-1369

[8] CorradiniFAS, MilessiTS, GonçalvesVM, et al.. High stabilization and hyperactivation of a Recombinant β-Xylosidase through immobilization strategies. Enzyme Microb Technol, 2021, 145: 109725

[9] FalckP, Linares-PasténJA, AdlercreutzP, KarlssonEN. Characterization of a family 43 β-xylosidase from the xylooligosaccharide utilizing putative probiotic Weissella Sp. strain 92. Glycobiology, 2016, 26: 193-202

[10] Garcia-GalanC, Berenguer‐MurciaÁ, Fernandez‐LafuenteR, RodriguesRC. Potential of different enzyme immobilization strategies to improve enzyme performance. Adv Synth Catal, 2011, 353: 2885-2904

[11] GlentingJ, BeckHC, VrangA, et al.. Anchorless surface associated glycolytic enzymes from Lactobacillus plantarum 299v bind to epithelial cells and extracellular matrix proteins. Microbiol Res, 2013, 168: 245-253

[12] GordilloTB, PalumboMC, AllieviMC, et al.. Strategies to display heterologous proteins on the cell surface of lactic acid bacteria using as anchor the C-terminal domain of Lactobacillus acidophilus SlpA. World J Microbiol Biotechnol, 2020, 36: 169

[13] GuerfaliM, MaalejI, GargouriA, BelghithH. Catalytic properties of the immobilized Talaromyces thermophilus β-xylosidase and its use for xylose and xylooligosaccharides production. J Mol Catal B Enzym, 2009, 57: 242-249

[14] GuoQ, AnY, YunJ, et al.. Enhanced d-tagatose production by spore surface-displayed l-arabinose isomerase from isolated Lactobacillus brevis PC16 and biotransformation. Bioresour Technol, 2018, 247: 940-946

[15] HincK, IwanickiA, ObuchowskiM. New stable anchor protein and peptide linker suitable for successful spore surface display in B. subtilis. Microb Cell Fact, 2013, 12: 22

[16] HomaeiAA, SaririR, VianelloF, StevanatoR. Enzyme immobilization: an update. J Chem Biol, 2013, 6: 185-205

[17] HuS, KongJ, KongW, et al.. Characterization of a novel lysm domain from lactobacillus fermentam bacteriophage endolysin and its use as an anchor to display heterologous proteins on the surfaces of lactic acid bacteria. Appl Environ Microbiol, 2010, 76: 2410-2418

[18] HwangIC, KimSH, KangDK. Complete genome sequence of Lactobacillus plantarum SK156, a candidate vehicle for mucosal vaccine delivery. J Anim Sci Technol, 2021, 62: 956-958

[19] Hwang IC, Valeriano VD, Song JH et al (2023) Mucosal immunization with Lactiplantibacillus plantarum-displaying Recombinant SARS-CoV-2 epitopes on the surface induces humoral and mucosal immune responses in mice. Microb Cell Fact 22. https://doi.org/10.1186/s12934-023-02100-7

[20] IlievI, VasilevaT, BivolarskiV, et al.. Metabolic profiling of xylooligosaccharides by lactobacilli. Polym (Basel), 2020, 12: 1-18

[21] JangMH, KimMD. β-1,4-Xylosidase activity of leuconostoc lactic acid bacteria isolated from Kimchi. Korean J Food Sci Technol, 2011, 43: 169-175

[22] KwonSJ, JungH-C, PanJ-G. Transgalactosylation in a Water-Solvent biphasic reaction system with β-Galactosidase displayed on the surfaces of Bacillus subtilis spores. Appl Environ Microbiol, 2007, 73: 2251-2256

[23] LagaertS, PolletA, CourtinCM, VolckaertG. β-Xylosidases and α-l-arabinofuranosidases: accessory enzymes for arabinoxylan degradation. Biotechnol Adv, 2014, 32: 316-332

[24] LeeSY, ChoiJH, XuZ. Microbial cell-surface display. Trends Biotechnol, 2003, 21: 45-52

[25] LiG, HuangZ, ZhangC, et al.. Construction of a linker library with widely controllable flexibility for fusion protein design. Appl Microbiol Biotechnol, 2016, 100: 215-225

[26] LiN, ZhangR, ZhouJ, HuangZ. Structures, biochemical characteristics, and functions of β-Xylosidases. J Agric Food Chem, 2023, 71: 7961-7976

[27] LiN, LiQ, GeF, CuiX. Immobilization of β-glucosidase and β-xylosidase on inorganic nanoparticles for glycosylated substances conversion. Int J Biol Macromol, 2025, 292: 139173

[28] LuJ, QiaoY, JiangY, et al.. Preparation of phosphorylated thermoplastic polymeric nanofibrous aerogels for effective protein adsorption. Microchem J, 2025, 209: 112835

[29] MaghrabyYR, El-ShabasyRM, IbrahimAH, AzzazyHME-S. Enzyme immobilization technologies and industrial applications. ACS Omega, 2023, 8: 5184-5196

[30] ManzoorS, Adnan TahirR, Adnan YounisM, et al.. Synthesis, biological and molecular Docking studies of pyrimidine-derived bioactive schiff bases. Bioorg Chem, 2023, 140: 106822

[31] MaoR, WuD, WangY. Surface display on lactic acid bacteria without genetic modification: strategies and applications. Appl Microbiol Biotechnol, 2016, 100: 9407-9421

[32] MariaA, MargaritaT, IiliaI, IskraI. Gene expression of enzymes involved in utilization of xylooligosaccharides by Lactobacillus strains. Biotechnol Biotechnol Equip, 2014, 28: 941-948

[33] MathiesenG, ØverlandL, KuczkowskaK, EijsinkVGH. Anchoring of heterologous proteins in multiple Lactobacillus species using anchors derived from Lactobacillus plantarum. Sci Rep, 2020, 10: 9640

[34] MattossovichR, IaconoR, CangianoG, et al.. Conversion of Xylan by recyclable spores of Bacillus subtilis displaying thermophilic enzymes. Microb Cell Fact, 2017, 16: 218

[35] MichlmayrH, KneifelW. β-Glucosidase activities of lactic acid bacteria: mechanisms, impact on fermented food and human health. FEMS Microbiol Lett, 2014, 352: 1-10

[36] MichlmayrH, HellJ, LorenzC, et al.. Arabinoxylan oligosaccharide hydrolysis by family 43 and 51 glycosidases from Lactobacillus brevis DSM 20054. Appl Environ Microbiol, 2013, 79: 6747-6754

[37] MichonC, LangellaP, EijsinkVGH, et al.. Display of Recombinant proteins at the surface of lactic acid bacteria: strategies and applications. Microb Cell Fact, 2016, 15: 70

[38] MillerGL. Use of Dinitrosalicylic acid reagent for determination of reducing sugar. Anal Chem, 1959, 31: 426-428

[39] Mohammadi MoradianJ, AliA, YangK, et al.. Direct transformation of rice straw to electricity and hydrogen by a single yeast strain: performance and mechanism. Fuel, 2024, 376: 132697

[40] MoranaA, MangioneA, MaurelliL, et al.. Immobilization and characterization of a thermostable β-xylosidase to generate a reusable biocatalyst. Enzyme Microb Technol, 2006, 39: 1205-1213

[41] MouraP, BarataR, CarvalheiroF, et al.. In vitro fermentation of xylo-oligosaccharides from corn cobs autohydrolysis by Bifidobacterium and Lactobacillus strains. Lwt, 2007, 40: 963-972

[42] NaiduDS, HlangothiSP, JohnMJ. Bio-based products from Xylan: A review. Carbohydr Polym, 2018, 179: 28-41

[43] NguyenH-M, LeV, NguyenK-T, et al.. Surface-Displayed mannanolytic and chitinolytic enzymes using peptidoglycan binding lysm domains. J Agric Food Chem, 2024, 72: 12655-12664

[44] OharaH, OwakiM, SonomotoK. Xylooligosaccharide fermentation with Leuconostoc lactis. J Biosci Bioeng, 2006, 101: 415-420

[45] Park M (2020) Surface display technology for biosensor applications: a review. Sens (Switzerland) 20

[46] PereiraM, OhJK, KangD-K, et al.. Hacking commensal Bacteria to consolidate the adaptive mucosal immune response in the Gut–Lung axis: future possibilities for SARS-CoV-2 protection. BioTech, 2022, 11: 3

[47] PhamM-L, TranA-M, KittibunchakulS, et al.. Immobilization of β-Galactosidases on the Lactobacillus cell surface using the Peptidoglycan-Binding motif lysm. Catalysts, 2019, 9: 443

[48] Pham ML, Tran AM, Kittibunchakul S et al (2019b) Immobilization of β-galactosidases on the lactobacillus cell surface using the peptidoglycan-binding motif lysm. Catalysts 9

[49] PhamM-L, TranA-M, MathiesenG, et al.. Cell wall anchoring of a bacterial Chitosanase in Lactobacillus plantarum using a Food-Grade expression system and two versions of an LP × TG anchor. Int J Mol Sci, 2020, 21: 3773

[50] PontonioE, MahonyJ, Di CagnoR, et al.. Cloning, expression and characterization of a β-d-xylosidase from Lactobacillus rossiae DSM 15814T. Microb Cell Fact, 2016, 15: 72

[51] PurichDL Factors influencing enzyme activity, 2010 In: Enzyme Kinetics Catalysis & Control. Elsevier 379-484

[52] Raya-TonettiF, MüllerM, SacurJ, et al.. Novel lysm motifs for antigen display on lactobacilli for mucosal immunization. Sci Rep, 2021, 11: 21691

[53] RohmanA, DijkstraBW, PuspaningsihNNT. β-xylosidases: structural diversity, catalytic mechanism, and Inhibition by monosaccharides. Int J Mol Sci, 2019, 20: 5524

[54] RomeroG, ContrerasLM, Aguirre CéspedesC, et al.. Efficiency assessment between entrapment and covalent bond immobilization of mutant β-Xylosidase onto Chitosan support. Polym (Basel), 2023, 15: 3170

[55] SahaBC. Hemicellulose bioconversion. J Ind Microbiol Biotechnol, 2003, 30: 279-291

[56] Sirisha VL, Jain A, Jain A (2016) Enzyme Immobilization: An Overview on Methods, Support Material, and Applications of Immobilized Enzymes. In: Advances in Food and Nutrition Research. pp 179–211

[57] SuR, ShiZ, LiE, et al.. A Trim-RBD-GEM vaccine candidate protects mice from SARS-CoV-2. Virology, 2023, 585: 145-154

[58] TayPKR, LimPY, OwDSW. A SH3_5 cell anchoring domain for Non-recombinant surface display on lactic acid Bacteria. Front Bioeng Biotechnol, 2021, 8: 1589

[59] TerrasanCRF, Romero-FernándezM, OrregoAH, et al.. Immobilization and stabilization of Beta-Xylosidases from penicillium Janczewskii. Appl Biochem Biotechnol, 2017, 182: 349-366

[60] VasquezR, BagonBB, SongJH, et al.. A novel, non-GMO surface display in Limosilactobacillus fermentum mediated by cell surface hydrolase without anchor motif. BMC Microbiol, 2022, 22: 190

[61] VasquezR, SongJH, ParkYS, et al.. Application of probiotic bacteria in ginsenoside bioconversion and enhancing its health-promoting benefits: a review. Food Sci Biotechnol, 2024

[62] VasquezR, SongJH, LeeJS, et al.. Heterologous expression and characterization of xylose-tolerant GH 43 family β-xylosidase/ α-L-arabinofuranosidase from Limosilactobacillus fermentum and its application in Xylan degradation. Front Bioeng Biotechnol, 2025

[63] Vasquez R, Song JH, Mendoza RM et al (2025b) Oral immunisation with Non-GMO surface displayed SARS-CoV-2 Spike epitopes on Bacteria‐Like particles provokes robust humoral and cellular immune responses, and modulated the gut Microbiome in mice. Microb Biotechnol 18. https://doi.org/10.1111/1751-7915.70073

[64] Yoo D, Bagon BB, Valeriano VDV et al (2017) Complete genome analysis of Lactobacillus fermentum SK152 from Kimchi reveals genes associated with its antimicrobial activity. FEMS Microbiol Lett 364. https://doi.org/10.1093/femsle/fnx185

[65] ZadravecP, ŠtrukeljB, BerlecA. Heterologous surface display on lactic acid bacteria: Non. GMO Alternative?? Bioengineered, 2015, 6: 179-183

[66] Zhou X, Gao M, De X et al (2023) Bacterium-like particles derived from probiotics: progress, challenges and prospects. Front Immunol 14. https://doi.org/10.3389/fimmu.2023.1263586

Funding

Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry and Fisheries(321035052HD020)

National Research Foundation of Korea(NRF-RS-2023-00275307)

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