Skip to main content
SpringerLink
Log in

Screening and identification of an Enterobacter ludwigii strain expressing an active β-xylosidase

  • Original Article
  • Published:
Annals of Microbiology Aims and scope Submit manuscript

Abstract

Researchers have expressed increasing interest in the xylanolytic enzymes used in hemicellulose hydrolysis that convert wood and agricultural residues to second-generation biofuels. In our study, 32 isolates showed clear hydrolysis zones on agar plates containing xylan after Congo red staining. Among these isolates, strain LY-62 exhibited the highest β-xylosidase activity (1.29 ± 0.05 U/mL). According to the phylogenetic analysis of the 16S rDNA, strain LY-62 belongs to the Enterobacter genus. Using a combination of electron microscopy, Gram-staining, and conventional physiological and biochemical examinations, the strain LY-62 was identified as Enterobacter ludwigii. The β-xylosidase gene from Enterobacter ludwigii LY-62 was cloned, and the full-length protein was expressed in Escherichia coli as an N-terminal or C-terminal His-tagged fusions protein. Optimal β-xylosidase activity was achieved at pH 7.0 and 40 °C. The Michaelis constant KM values for His-Xyl62 and Xyl62-His were 1.55 and 2.8 mmol/L, respectively. The kcat values for His-Xyl62 and Xyl62-His were 8.51 and 6.94 s−1, respectively. The catalytic efficiencies of His-Xyl62 and Xyl62-His were 5.49 and 2.48 s−1 × mM−1, respectively. Thus, Xyl62 is a functional β-xylosidase, and our study represents the first report of a β-xylosidase from Enterobacter ludwigii.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8

Similar content being viewed by others

References

  • Ammoneh H, Harba M, Akeed Y, Al-Halabi M, Bakri Y (2014) Isolation and identification of local Bacillus isolates for xylanase biosynthesis. Iran J Microbiol 6(2):127–132

    PubMed  PubMed Central  Google Scholar 

  • Basaran P, Hanq YD, Basaran N, Worobo RW (2001) Cloning and heterologous expression of xylanase gene from Pichia stipitis in Escherichia coli. J Appl Microbiol 90(2):248–255

    Article  CAS  PubMed  Google Scholar 

  • Beg QK, Kapoor M, Mahajan L, Hoondal GS (2001) Microbial xylanases and their industrial applications: a review. Appl Microbiol Biotechnol 56(3–4):326–338

    Article  CAS  PubMed  Google Scholar 

  • Boone DR, Castenholz RW, Garrity GM, Bergey DH (2001) Bergey’s manual of systematic bacteriology. Springer, New York

  • Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254

    Article  CAS  PubMed  Google Scholar 

  • Campos E, Negro MJ, Lorenzo GSD, Gonzalez S, Rorig M, Talia P, Grasso DH, Sáez F, Manzanares Secades P, Ballesteros Perdices M, Cataldi AA (2013) Purification and characterization of a GH43 β-xylosidase from Enterobacter sp. identified and cloned from forest soil bacteria. Microbiol Res 169(2–3):213–220

    PubMed  Google Scholar 

  • Changhao B, Xueli Z, Ingram LO, Preston JF (2009) Genetic engineering of Enterobacter asburiae strain JDR-1 for efficient production of ethanol from hemicellulose hydrolysates. Appl Environ Microbiol 75(18):5743–5749

    Article  Google Scholar 

  • Corrêa JM, Graciano L, Abrahão J, Loth EA, Gandra RF, Kadowaki MK, Henn C, Simão R (2012) Expression and characterization of a GH39 β-xylosidase II from Caulobacter crescentus. Appl Biochem Biotechnol 168(8):2218–2229

    Article  PubMed  Google Scholar 

  • Coutinho PM, Henrissat B (1999) Carbohydrate-active enzymes: an integrated database approach. In: Gilbert HJ, Davies G, Henrissat H, Svensson B (eds) Recent advances in carbohydrate bioengineering. The Royal Society of Chemistry, Cambridge, pp 3–12

    Google Scholar 

  • Falck P, Linares-Pastén JA, Adlercreutz P, Karlsson EN (2015) Characterization of a family 43 β-xylosidase from the xylooligosaccharide utilizing putative probiotic Weissella sp. strain 92. Glycobiology 26(2):193–202

    Article  PubMed  PubMed Central  Google Scholar 

  • Graciano L, Corrêa JM, Gandra RF, Seixas F, Kadowaki MK, Sampaio SC, Silva J, Osaku CA, Simão R (2012) The cloning, expression, purification, characterization and modeled structure of Caulobacter crescentus β-xylosidase I. World J Microbiol Biotechnol 28:2879–2888

    Article  CAS  PubMed  Google Scholar 

  • Haltrich D, Nidetzky B, Kulbe KD, Steiner W, Zupancic S (1996) Production of fungal xylanases. Bioresour Technol 58:137–161

    Article  CAS  Google Scholar 

  • Hao S, Xun L, Huaxiang G, Yu Z, Yingjuan H, Liangliang W, Wang F (2013) Biochemical properties of a novel thermostable and highly xylose-tolerant β-xylosidase/α-arabinosidase from Thermotoga thermarum. Biotechnol Biofuels 6:27

    Article  Google Scholar 

  • Hayashi S, Ohno T, Ito M, Yokoi H (2001) Purification and properties of the cell-associated β-xylosidase from Aureobasidium. J Ind Microbiol Biotechnol 26(5):276–279

    Article  CAS  PubMed  Google Scholar 

  • Hoffmann H, Stindl S, Stumpf A, Mehlen A, Monget D, Heesemann J, Schleifer KH, Roggenkamp A (2005) Description of Enterobacter ludwigii sp. nov., a novel Enterobacter species of clinical relevance. Syst Appl Microbiol 28(3):206–212

    Article  CAS  PubMed  Google Scholar 

  • Huy ND, Thayumanavan P, Kwon TH, Park SM (2013) Characterization of a recombinant bifunctional xylosidase/arabinofuranosidase from Phanerochaete chrysosporium. J Biosci Bioeng 116(2):152–159

    Article  CAS  PubMed  Google Scholar 

  • Jain I, Kumar V, Satyanarayana T (2014) Applicability of recombinant β-xylosidase from the extremely thermophilic bacterium Geobacillus thermodenitrificans in synthesizing alkylxylosides. Bioresour Technol 170:462–469

    Article  CAS  PubMed  Google Scholar 

  • Jordan DB, Braker JD (2011) Opposing influences by subsite −1 and subsite +1 residues on relative xylopyranosidase/arabinofuranosidase activities of bifunctional β-D-xylosidase/α-L-arabinofuranosidase. Biochim Biophys Acta 1814:1648–1657

    Article  CAS  PubMed  Google Scholar 

  • Jordan DB, Li XL, Dunlap CA, Whitehead TR, Cotta MA (2007) Structure-function relationships of a catalytically efficient beta-D-xylosidase. Appl Biochem Biotechnol 141(1):51–76

    Article  CAS  PubMed  Google Scholar 

  • Jordan DB, Stoller JR, Lee CC, Chan VJ, Wagschal K (2017) Biochemical characterization of a GH43 β-xylosidase from Bacteroides ovatus. Appl Biochem Biotechnol 182:250–260

  • Jordan DB, Wagschal K, Grigorescu A, Braker JD (2012) Highly active β-xylosidases of glycoside hydrolase family 43 operating on natural and artificial substrates. App Microbiol Biotechnol 97:4415–4428

    Article  Google Scholar 

  • Khandeparkar R, Bhosle NB (2006) Purification and characterization of thermoalkalophilic xylanase isolated from the Enterobacter sp. MTCC 5112. Res Microbiol 157(4):315–325

    Article  CAS  PubMed  Google Scholar 

  • Lachke AH (1988) 1,4-β-D-Xylan xylohydrolase of Sclerotium rolfsii. Methods Enzymol 160C(C):679–684

    Article  Google Scholar 

  • Lagaert S, Pollet A, Delcour JA, Lavigne R, Courtin CM, Volckaert G (2011) Characterization of two β-xylosidases from Bifidobacterium adolescentis and their contribution to the hydrolysis of prebiotic xylooligosaccharides. Appl Microbiol Biotechnol 92:1179–1185

    Article  CAS  PubMed  Google Scholar 

  • Lajoie MJ, Rovner AJ, Goodman DB, Aerni HR, Haimovich AD, Kuznetsov G, Mercer JA, Wang HH, Carr PA, Mosberg JA, Rohland N, Schultz PG, Jacobson JM, Rinehart J, Church GM, Isaacs FJ (2013) Genomically recoded organisms expand biological functions. Science 342(6156):357–360

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Lama L, Calandrelli V, Gambacorta A, Nicolaus B (2004) Purification and characterization of thermostable xylanase and β-xylosidase by the thermophilic bacterium Bacillus thermantarcticus. Res Microbiol 155(4):283–289

    Article  CAS  PubMed  Google Scholar 

  • Liu F, Yang J, Xiao Y, Li L, Yang F, Jin Q (2016) Complete genome sequence of a clinical isolate of Enterobacter asburiae. Genome Announc 4(3):e00523-16

    Article  PubMed  PubMed Central  Google Scholar 

  • Lombard V, Ramulu HG, Drula E, Coutinho PM, Henrissat B (2014) The carbohydrate-active enzymes database (CAZy) in 2013. Nucleic Acids Res 42:490–495

    Article  Google Scholar 

  • Moraïs S, Alber OS, Barak Y, Hadar Y, Wilson DB, Lamed R, Shoham Y, Bayer EA (2012) Functional association of catalytic and ancillary modules dictates enzymatic activity in glycoside hydrolase family 43 β-xylosidase. J Biol Chem 287(12):9213–9221

    Article  PubMed  PubMed Central  Google Scholar 

  • Moure A, Gullón P, Domínguez H, Parajó JC (2006) Advances in the manufacture, purification and applications of xylo-oligosaccharides as food additives and nutraceuticals. Process Biochem 41(9):1913–1923

    Article  CAS  Google Scholar 

  • Michlmayr H, Hell J, Lorenz C, Böhmdorfer S, Rosenau T, Kneifel W (2013) Arabinoxylan oligosaccharide hydrolysis by family 43 and 51 glycosidases from Lactobacillus brevis DSM 20054. Appl Environ Microbiol 79(21):6747–6754

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Nieto-Domínguez M, LId E, Barriuso J, Prieto A, BFd T, Canales-Mayordomo Á, Martínez MJ (2015) Novel pH-stable glycoside hydrolase family 3 β-xylosidase from Talaromyces amestolkiae: an enzyme displaying regioselective transxylosylation. Appl Environ Microbiol 81(18):6380–6392

    Article  PubMed  PubMed Central  Google Scholar 

  • Polizeli ML, Rizzatti AC, Monti R, Terenzi HF, Jorge JA, Amorim DS (2005) Xylanases from fungi: properties and industrial applications. Appl Microbiol Biotechnol 67(5):577–591

    Article  CAS  PubMed  Google Scholar 

  • Quinlan RJ, Teter S, Xu F (2010) Development of cellulases to improve enzymatic hydrolysis of lignocellulosic biomass. Bioalcohol Production 7:178–201

  • Ratnadewi AAI, Fanani M, Kurniasih SD, Sakka M, Wasito EB, Sakka K, Nurachman Z, Puspaningsih NNT (2013) β-D-Xylosidase from Geobacillus thermoleovorans IT-08: biochemical characterization and bioinformatics of the enzyme. Appl Biochem Biotechnol 170:1950–1964

    Article  CAS  PubMed  Google Scholar 

  • Rizzatti AC, Jorge JA, Terenzi HF, Rechia CG, Polizeli ML (2001) Purification and properties of a thermostable extracellular beta-D-xylosidase produced by a thermotolerant Aspergillus phoenicis. J Ind Microbiol Biotechnol 26:156–160

    Article  CAS  PubMed  Google Scholar 

  • Saitou N, Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4(4):406–425

    CAS  PubMed  Google Scholar 

  • Sanyan Z, Huimin W, Pengjun S, Bo X, Yingguo B, Huiying L, Bin Y (2014) Cloning, expression, and characterization of a thermostable β-xylosidase from thermoacidophilic Alicyclobacillus sp. A4. Process Biochem 49:1422–1428

    Article  Google Scholar 

  • Shallom D, Leon M, Bravman T, Ben-David A, Zaide G, Belakhov V, Shoham G, Schomburg D, Baasov T, Shoham Y (2005) Biochemical characterization and identification of the catalytic residues of a family 43 beta-D-xylosidase from Geobacillus stearothermophilus T-6. Biochemistry 44(1):387–397

    Article  CAS  PubMed  Google Scholar 

  • Sinnott ML (1990) Catalytic mechanisms of enzymic glycosyl transfer. Chem Rev 90(7):1171–1202

    Article  CAS  Google Scholar 

  • Studier FW (2005) Protein production by auto-induction in high density shaking cultures. Protein Expr Purif 41(1):207–234

    Article  CAS  PubMed  Google Scholar 

  • Subramanian S, Prema P (2002) Biotechnology of microbial xylanases: enzymology, molecular biology, and application. Crit Rev Biotechnol 22(1):33–64

    Article  Google Scholar 

  • Techapun C, Poosaran N, Watanabe M, Sasaki K (2003) Thermostable and alkaline-tolerant microbial cellulase-free xylanases produced from agricultural wastes and the properties required for use in pulp bleaching bioprocesses: a review. Process Biochem 38(9):1327–1340

    Article  CAS  Google Scholar 

  • Van Doorslaer E, Kersters-Hilderson H, De Bruyne CK (1985) Hydrolysis of β-D-xylo-oligosaccharides by β-D-xylosidase from Bacillus pumilus. Carbohydr Res 140:342–346

    Article  Google Scholar 

  • Viborg AH, Sørensen KI, Gilad O, Steen-Jensen DB, Dilokpimol A, Jacobsen S, Svensson B (2013) Biochemical and kinetic characterisation of a novel xylooligosaccharide-upregulated GH43 β-D-xylosidase/α-L-arabinofuranosidase (BXA43) from the probiotic Bifidobacterium animalis subsp. lactis BB-12. AMB Express 3(1):56

    Article  PubMed  PubMed Central  Google Scholar 

  • Wagschal K, Heng C, Lee CC, Robertson GH, Orts WJ, Wong DW (2009) Purification and characterization of a glycoside hydrolase family 43 beta-xylosidase from Geobacillus thermoleovorans IT-08. Appl Biochem Biotechnol 155(1–3):304–313

    CAS  PubMed  Google Scholar 

  • Weilan S, Yemin X, Ailian W, Kataeva I, Jianjun P, Huawei W, Wiegel J (2011) Characterization of a novel β-Xylosidase, XylC, from Thermoanaerobacterium saccharolyticum JW/SL-YS485. Appl Environ Microbiol 77(3):719–726

    Article  Google Scholar 

  • Wong KK, Tan LU, Saddler JN (1988) Multiplicity of beta-1,4 xylanase in microorganisms: functions and applications. Microbiol Rev 52(3):305–317

    CAS  PubMed  PubMed Central  Google Scholar 

  • Xinzhou Y, Pengjun S, Huoqing H, Huiying L, Yaru W, Wei Z, Bin Y (2014) Two xylose-tolerant GH43 bifunctional β-xylosidase/α-arabinosidases and one GH11 xylanase from Humicola insolens and their synergy in the degradation of xylan. Food Chem 148:381–387

    Article  Google Scholar 

  • Xu WZ, Shima Y, Negoro S, Urabe I (1991) Sequence and properties of beta-xylosidase from Bacillus pumilus IPO. Contradiction of the previous nucleotide sequence. Eur J Biochem 202(3):1197–1203

    Article  CAS  PubMed  Google Scholar 

  • Yan QJ, Wang L, Jiang ZQ, Yang SQ, Zhu HF, Li LT (2008) A xylose-tolerant β-xylosidase from Paecilomyces thermophila: characterization and its co-action with the endogenous xylanase. Bioresour Technol 99:5402–5410

    Article  CAS  PubMed  Google Scholar 

  • Zanoelo FF, Polizeli Md Mde L, Terenzi HF, Jorge JA (2004) Purification and biochemical properties of a thermostable xylose-tolerant β-D-xylosidase from Scytalidium thermophilum. J Ind Microbiol Biotechnol 31:170–176

    Article  CAS  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. School of Bioscience and Bioengineering, South China University of Technology, Guangzhou, 510006, Guangdong, China

    Jiashi Zhang, Tangbing Cui & Xueqing Li

Authors
  1. Jiashi Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Tangbing Cui
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Xueqing Li
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Tangbing Cui.

Ethics declarations

Conflict of interest

The authors declare that they have no conflicts of interest.

Research involving human participants and/or animals

Not applicable.

Informed consent

Not applicable.

Ethics approval

Not applicable.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Zhang, J., Cui, T. & Li, X. Screening and identification of an Enterobacter ludwigii strain expressing an active β-xylosidase. Ann Microbiol 68, 261–271 (2018). https://doi.org/10.1007/s13213-018-1334-2

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s13213-018-1334-2

Keywords

Search

Navigation