Skip to main content
SpringerLink
Log in

Characterization of a Glucose-, Xylose-, Sucrose-, and d-Galactose-Stimulated β-Glucosidase from the Alkalophilic Bacterium Bacillus halodurans C-125

  • Published:
Current Microbiology Aims and scope Submit manuscript

Abstract

The gene (Bhbgl) encoding a β-glucosidase from the alkalophilic bacterium Bacillus halodurans C-125 was synthesized chemically via the PCR-based two-step DNA synthesis (PTDS) method and expressed in Escherichia coli. Bhbgl contained an open reading frame (ORF) of 1359 bp encoding a 453-amino acid protein belonging to glycoside hydrolase family 1 (GHF1), and the deduced molecular mass of recombinant Bhbgl (52,488 Da) was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme exhibited a high specific activity with o-nitrophenyl-β-d-glucopyranoside (oNPGlu) and an apparent K m value of 0.32 mM. With oNPGlu as the substrate, Bhbgl displayed pH and temperature optima of ~7.0 and 50°C, respectively. The enzyme was relatively stable under alkaline conditions and >50% activity was retained after incubation at pH 9.5 for 24 h at 4°C. Recombinant Bhbgl activity was inhibited by 5 mM Zn2+, Fe3+, or Cd2+, but was enhanced by 1 mM Mg2+ and other metal ions. Enzyme activity was also stimulated by at least four sugars (sucrose, d-galactose, xylose, glucose) at concentrations ranging from 50 to 800 mM.

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

Similar content being viewed by others

References

  1. Hakulinen N, Paavilainen S, Korpela T, Rouvinen J (2000) The crystal structure of β-glucosidase from Bacillus circulans sp. alkalophilus: ability to form long polymeric assemblies. J Struct Biol 129:69–79

    Article  PubMed  CAS  Google Scholar 

  2. Henrissat B (1991) A classification of glycosyl hydrolases based on amino acid sequence similarities. Biochem J 280:309–316

    PubMed  CAS  Google Scholar 

  3. Shipkowski S, Brenchley JE (2005) Characterization of an unusual cold-active β-glucosidase belonging to family 3 of the glycoside hydrolases from the psychrophilic isolate Paenibacillus sp. strain C7. Appl Environ Microbiol 17:4225–4232

    Article  Google Scholar 

  4. Faure D (2002) The family 3 glycoside hydrolases: from housekeeping functions to host–microbe interactions. Appl Environ Microbiol 68:1485–1490

    Article  PubMed  CAS  Google Scholar 

  5. Morrissey JP, Wubben JP, Osbourn AE (2000) Stagonospora avenae secretes multiple enzymes that hydrolyze oat leaf saponins. Mol Plant Microbe Interact 13:1041–1052

    Article  PubMed  CAS  Google Scholar 

  6. Osbourn A, Bowyer P, Lunness P, Clarke B, Daniels M (1995) Fungal pathogens of oat roots and tomato leaves employ closely related enzymes to detoxify different host plant saponins. Mol Plant Microbe Interact 8:971–978

    Article  PubMed  CAS  Google Scholar 

  7. Bhat M, Bhat TS (1997) Cellulose degrading enzymes and their potential industrial applications. Biotechnol Adv 15:583–620

    Article  PubMed  CAS  Google Scholar 

  8. Bhatia Y, Mishra S, Bisaria VS (2002) Microbial β-glucosidases: cloning, properties, and applications. Crit Rev Biotechnol 22:375–407

    Article  PubMed  CAS  Google Scholar 

  9. Hu Y, Luan HW, Zhou K, Ge GB, Yang SL, Yang L (2009) Purification and characterization of a novel glycosidase from the china white jade snail (Achatina fulica) showing transglycosylation activity. Enzyme Microb Technol 43:35–42

    Article  Google Scholar 

  10. Han YJ, Chen HZ (2008) Characterization of β-glucosidase from corn stover and its application in simultaneous saccharification and fermentation. Bioresource Technol 99:6081–6087

    Article  CAS  Google Scholar 

  11. Sue M, Ishihara A, Iwamura H (2000) Purification and characterization of a hydroxamic acid glucoside β-glucosidase from wheat (Triticum aestivum L.) seedlings. Planta 210:432–438

    Article  PubMed  CAS  Google Scholar 

  12. Jiang C, Ma G, Li S, Hu T, Che Z, Shen P, Yan B (2009) Characterization of a novel β-glucosidase-like activity from a soil metagenome. J Microbiol 47:542–548

    Article  PubMed  CAS  Google Scholar 

  13. Benoliel B, Torres MJ, Moraes LM (2010) Expression of a glucose-tolerant β-glucosidase from Humicola grisea var. thermoidea in Saccharomyces cerevisiae. Appl Biochem Biotechnol 9:8732–8737

    Google Scholar 

  14. Hong J, Tamaki H, Kumagai H (2007) Cloning and functional expression of thermostable β-glucosidase gene from Thermoascus aurantiacus. Appl Microbiol Biotechnol 73:1331–1339

    Article  PubMed  CAS  Google Scholar 

  15. Hong MR, Kim YS, Park CS, Lee JK, Oh DK (2009) Characterization of a recombinant β-glucosidase from the thermophilic bacterium Caldicellulosiruptor saccharolyticus. J Biosci Bioeng 108:36–40

    Article  PubMed  CAS  Google Scholar 

  16. Takami H, Nakasone K, Takaki Y, Maeno G, Sasaki R, Masui N, Fuji F (2000) Complete genome sequence of the alkaliphilic bacterium Bacillus halodurans and genomic sequence comparison with Bacillus subtilis. Nucleic Acids Res 28:4317–4331

    Article  PubMed  CAS  Google Scholar 

  17. Ikura Y, Horikoshi K (1979) Isolation and some properties of β-galactosidase producing bacteria. Agric Biol Chem 43:85–88

    CAS  Google Scholar 

  18. Honda H, Kudo T, Horikoshi K (1985) Molecular cloning and expression of the xylanase gene of alkalophilic Bacillus sp. strain C-125 in Escherichia coli. J Bacteriol 161:784–785

    PubMed  CAS  Google Scholar 

  19. Honda Y, Kitaoka M (2004) A family 8 glycoside hydrolase from Bacillus halodurans C-125 (BH2105) is a reducing end xylose-releasing exo-oligoxylanase. J Biol Chem 279:55097–55103

    Article  PubMed  CAS  Google Scholar 

  20. Smaali I, Remond C, O’Donohue MJ (2006) Expression in Escherichia coli and characterization of β-xylosidases GH39 and GH-43 from Bacillus halodurans C-125. Appl Microbiol Biotechnol 73:582–590

    Article  PubMed  CAS  Google Scholar 

  21. Navarro-Fernandez J, Martinez-Martinez I, Montoro-Garcia S, Garcia-Carmona F, Takami H, Sanchez-Ferrer A (2008) Characterization of a new rhamnogalacturonan acetyl esterase from Bacillus halodurans C-125 with a new putative carbohydrate binding domain. J Bacteriol 190:1375–1382

    Article  PubMed  CAS  Google Scholar 

  22. Xiong AS, Yao QH, Peng RH, Li X, Fan HQ, Cheng ZM, Li Y (2004) A simple, rapid, high-fidelity and cost-effective PCR-based two-step DNA synthesis method for long gene sequences. Nucleic Acids Res 32:e98

    Article  PubMed  Google Scholar 

  23. Sambrook S, Russell D (2001) Molecular cloning: a laboratory manual, 3rd edn. CSHL Press, Woodbury, NY

    Google Scholar 

  24. Stanislawski J (1991) Enzyme kinetics, version 1.5. Trinity Software, Fort Pierce, FL

    Google Scholar 

  25. Bradford MM (1976) A rapid and sensitive method for detecting microgram amounts of protein utilizing the principle of protein–dye binding. Anal Biochem 72:248–254

    Article  PubMed  CAS  Google Scholar 

  26. Higgins D, Thompson J, Gibson T (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22:4673–4680

    Article  PubMed  Google Scholar 

  27. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215:403–410

    PubMed  CAS  Google Scholar 

  28. Finn RD, Mistry J, Tate J, Coggill P, Heger A, Pollington JE, Gavin OL (2010) The Pfam protein families database. Nucleic Acids Res 38:211–222

    Article  Google Scholar 

  29. Nielsen H, Engelbrecht J, Brunak S, von Heijne G (1997) A neural network method for identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Int J Neural Syst 8:581–599

    Article  PubMed  CAS  Google Scholar 

  30. Krogh A, Larsson B, von Heijne G, Sonnhammer EL (2001) Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes. J Mol Biol 305:567–580

    Article  PubMed  CAS  Google Scholar 

  31. Peng RH, Xiong AS, Yao QH (2006) A direct and efficient PAGE-mediated overlap extension method for gene multiple-site mutagenesis. Appl Microbiol Biotechnol 73:234–240

    Article  PubMed  CAS  Google Scholar 

  32. Xiong AS, Yao QH, Peng RH, Duan H, Li X, Fan HQ, Cheng ZM (2006) PCR-based accurate synthesis of long DNA sequences. Nat Protoc 1:791–797

    Article  PubMed  CAS  Google Scholar 

  33. Sanz-Aparicio J, Hermoso JA, Martinez-Ripoll M, Lequerica JL, Polaina J (1998) Crystal structure of β-glucosidase A from Bacillus polymyxa: insights into the catalytic activity in family 1 glycosyl hydrolases. J Mol Biol 275:491–502

    Article  PubMed  CAS  Google Scholar 

  34. Saha BC, Shelby NF, Rodney JB (1994) Production, purification, and properties of a thermostable β-glucosidase from a color variant strain of Aureobasidium pullulans. Appl Environ Microbiol 103:774–3780

    Google Scholar 

  35. Kaper T, Lebbink JHG, Pouwels J, Kopp J, Schulz GE, van der Oost J, de Vos WM (2000) Comparative structural analysis and substrate specificity engineering of the hyperthermostable β-glucosidase CelB from Pyrococcus furiosus. Biochemistry 39:4963–4970

    Article  PubMed  CAS  Google Scholar 

  36. Xiong AS, Peng RH, Cheng ZM, Li Y, Liu JG, Zhuang J, Gao F, Xu F, Qiao YS, Zhang Z, Chen JM, Yao QH (2007) Concurrent mutations in six amino acids in β-glucuronidase improve its thermostability. Protein Eng Des Sel 20:319–325

    Article  PubMed  CAS  Google Scholar 

  37. Karnchanatat A, Petsom A, Sangvanich P, Piaphukiew J, Whalley AJ, Reynolds CD, Sihanonth P (2007) Purification and biochemical characterization of an extracellular beta-glucosidase from the wood-decaying fungus Daldinia eschscholzii. FEMS Microbiol Lett 270:162–170

    Article  PubMed  CAS  Google Scholar 

  38. Lymar ES, Li B, Renganathan V (1995) Purification and characterization of a cellulose-binding β-glucosidase from cellulose-degrading cultures of Phanerochaete chrysosporium. Appl Environ Microbiol 10:2976–2980

    Google Scholar 

  39. Bhiri F, Chaabouni SE, Limam F, Ghrir R, Marzouki N (2008) Purification and biochemical characterization of extracellular β-glucosidases from the hypercellulolytic Pol6 mutant of Penicillium occitanis. Appl Biochem Biotechnol 149:169–182

    Article  PubMed  CAS  Google Scholar 

  40. Kaur J, Chadha BS, Kumar BA, Saini HS (2007) Purification and characterization of β-glucosidase from Melanocarpus sp. MTCC 3922. Electron J Biotechnol 10:260–270

    Article  CAS  Google Scholar 

  41. Riou C, Salmon JM, Vallier MJ, Gunata Z, Barre P (1998) Purification, characterization, and substrate specificity of a novel highly glucose-tolerant β-glucosidase from Aspergillus oryzae. Appl Environ Microbiol 103:607–3614

    Google Scholar 

  42. Souza FHM, Nascimento CV, Rosa JC, Masui DC, Leone FA, Jorge JA, Furriel RPM (2010) Purification and biochemical characterization of a mycelial glucose- and xylose-stimulated β-glucosidase from the thermophilic fungus Humicola insolens. Process Biochem 45:272–278

    Article  CAS  Google Scholar 

Download references

Acknowledgments

This research was supported by the Youth Fund of the Shanghai Academy of Agricultural Sciences (Grant No. 2008-6, 2009-19), the Shanghai Basic Research Project (Grant No. 08JC1418000), the Key Project Fund of the Shanghai Municipal Committee of Agriculture (Grant No. 2008-7-5), and the Key Project Fund of the Shanghai Municipal Committee of Agriculture (Grant No. 2009-6-4). We thank Dr John Buswell for linguistic revision of the manuscript.

Author information

Authors and Affiliations

  1. College of Bioscience and Biotechnology, Yangzhou University, Jiangsu, China

    Hu Xu & Jian-Min Chen

  2. Shanghai Key Laboratory of Agricultural Genetics and Breeding, Biotechnological Research Institute, Shanghai Academy of Agricultural Sciences, Shanghai, China

    Hu Xu, Ai-Sheng Xiong, Wei Zhao, Yong-Sheng Tian, Ri-He Peng & Quan-Hong Yao

Authors
  1. Hu Xu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ai-Sheng Xiong
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Wei Zhao
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Yong-Sheng Tian
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Ri-He Peng
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Jian-Min Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Quan-Hong Yao
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Jian-Min Chen or Quan-Hong Yao.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Xu, H., Xiong, AS., Zhao, W. et al. Characterization of a Glucose-, Xylose-, Sucrose-, and d-Galactose-Stimulated β-Glucosidase from the Alkalophilic Bacterium Bacillus halodurans C-125. Curr Microbiol 62, 833–839 (2011). https://doi.org/10.1007/s00284-010-9766-3

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00284-010-9766-3

Keywords

Search

Navigation