Skip to main content
SpringerLink
Log in

Purification, Characterization, and Heterologous Expression of a Thermostable β-1,3-1,4-Glucanase from Bacillus altitudinis YC-9

  • Published:
Applied Biochemistry and Biotechnology Aims and scope Submit manuscript

Abstract

Purification, characterization, gene cloning, and heterologous expression in Escherichia coli of a thermostable β-1,3-1,4-glucanase from Bacillus altitudinis YC-9 have been investigated in this paper. The donor strain B. altitudinis YC-9 was isolated from spring silt. The native enzyme was purified by ammonium sulfate precipitation, diethylaminoethyl-cellulose anion exchange chromatography, and Sephadex G-100 gel filtration. The purified β-1,3-1,4-glucanase was observed to be stable at 60 °C and retain more than 90 % activity when incubated for 2 h at 60 °C and remain about 75 % and 44 % activity after incubating at 70 °C and 80 °C for 10 min, respectively. Acidity and temperature optimal for this enzyme was pH 6 and 65 °C. The open reading frame of the enzyme gene was measured to be 732 bp encoding 243 amino acids, with a predicted molecular weight of 27.47 kDa. The gene sequence of β-1,3-1,4-glucanase showed a homology of 98 % with that of Bacillus licheniformis. After being expressed in E. coli BL21, active recombinant enzyme was detected both in the supernatants of the culture and the cell lysate, with the activity of 102.7 and 216.7 U/mL, respectively. The supernatants of the culture were used to purify the recombinant enzyme. The purified recombinant enzyme was characterized to show almost the same properties to the wild enzyme, except that the specific activity of the recombinant enzyme reached 5392.7 U/mg, which was higher than those ever reported β-1,3-1,4-glucanase from Bacillus strains. The thermal stability and high activity make this enzyme broad prospect for industry application. This is the first report on β-1,3-1,4-glucanase produced by B. altitudinis.

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. Planas, A. (2000). Bacterial 1,3-1,4-beta-glucanases: Structure, function and protein engineering. Biochimica et Biophysica Acta, 1543, 361–382.

    Article  CAS  Google Scholar 

  2. Olsen, O., Borriss, R., Simon, O., & Thomsen, K. K. (1991). Hybrid Bacillus (1–3,1-4)-beta-glucanases: Engineering thermostable enzymes by construction of hybrid genes. Molecular and General Genetics MGG, 225, 177–185.

    Article  CAS  Google Scholar 

  3. Qiao, J., Dong, B., Li, Y., Zhang, B., & Cao, Y. (2009). Cloning of a beta-1,3-1,4-glucanase gene from Bacillus subtilis MA139 and its functional expression in Escherichia coli. Applied Biochemistry and Biotechnology, 152, 334–342.

    Article  CAS  Google Scholar 

  4. Lloberas, J., Perez-Pons, J. A., & Querol, E. (1991). Molecular cloning, expression and nucleotide sequence of the endo-beta-1,3-1,4-d-glucanase gene from Bacillus licheniformis. Predictive structural analyses of the encoded polypeptide. European journal of biochemistry / FEBS, 197, 337–343.

    Article  CAS  Google Scholar 

  5. Teng, D., Wang, J. H., Fan, Y., Yang, Y. L., Tian, Z. G., Luo, J., Yang, G. P., & Zhang, F. (2006). Cloning of beta-1,3-1,4-glucanase gene from Bacillus licheniformis EGW039 (CGMCC 0635) and its expression in Escherichia coli BL21 (DE3). Applied Microbiology and Biotechnology, 72, 705–712.

    Article  CAS  Google Scholar 

  6. Borriss, R., Buettner, K., & Maentsaelae, P. (1990). Structure of the beta-1,3-1,4-glucanase gene of Bacillus macerans: Homologies to other beta-glucanases. Molecular and General Genetics MGG, 222, 278–283.

    Article  CAS  Google Scholar 

  7. Akita, M., Kayatama, K., Hatada, Y., Ito, S., & Horikoshi, K. (2005). A novel beta-glucanase gene from Bacillus halodurans C-125. FEMS Microbiology Letters, 248, 9–15.

    Article  CAS  Google Scholar 

  8. Kim, J. Y. (2003). Overproduction and secretion of Bacillus circulans endo-beta-1,3-1,4-glucanase gene (bglBC1) in B. subtilis and B. megaterium. Biotechnology Letters, 25, 1445–1449.

    Article  CAS  Google Scholar 

  9. Gosalbes, M. J., Perez-Gonzalez, J. A., Gonzalez, R., & Navarro, A. (1991). Two beta-glycanase genes are clustered in Bacillus polymyxa: Molecular cloning, expression, and sequence analysis of genes encoding a xylanase and an endo-beta-(1,3)-(1,4)-glucanase. Journal of Bacteriology, 173, 7705–7710.

    CAS  Google Scholar 

  10. Yang, P., Shi, P., Wang, Y., Bai, Y., Meng, K., Luo, H., Yuan, T., & Yao, B. (2007). Cloning and overexpression of a Paenibacillus beta-glucanase in Pichia pastoris: Purification and characterization of the recombinant enzyme. Journal of Microbiology and Biotechnology, 17, 58–66.

    Google Scholar 

  11. Teather, R. M., & Erfle, J. D. (1990). DNA sequence of a Fibrobacter succinogenes mixed-linkage beta-glucanase (1,3-1,4-beta-d-glucan 4-glucanohyd-rolase) gene. Journal of Bacteriology, 172, 3837–3841.

    CAS  Google Scholar 

  12. Schimming, S., Schwarz, W. H., & Staudenbauer, W. L. (1991). Properties of a thermoactive beta-1,3-1,4-glucanase (lichenase) from Clostridium thermocellum expressed in Escherichia coli. Biochemical and Biophysical Research Communications, 177, 447–452.

    Article  CAS  Google Scholar 

  13. Ekinci, M. S., McCrae, S. I., & Flint, H. J. (1997). Isolation and overexpression of a gene encoding an extracellular beta-(1,3-1,4)-glucanase from Streptococcus bovis JB1. Applied and Environmental Microbiology, 63, 3752–3756.

    CAS  Google Scholar 

  14. Murray, P. G., Grassick, A., Laffey, C. D., Cuffe, M. M., Higgins, T., Savage, A. V., Planas, A., & Tuohy, M. G. (2001). Isolation and characterization of a thermostable endo-beta-glucanase active on 1,3-1,4-beta-d-glucans from the aerobic fungus Talaromyces emersonii CBS 814.70. Enzyme and Microbial Technology, 29, 90–98.

    Article  CAS  Google Scholar 

  15. Wang, J. L., Ruan, H., Zhang, H. F., Zhang, Q., Zhang, H. B., He, G. Q., & Shen, S. R. (2007). Characterization of a thermostable and acidic-tolerable beta-glucanase from aerobic fungi Trichoderma koningii ZJU-T. Journal of Food Science, 72, C452–456.

    Article  CAS  Google Scholar 

  16. Hua, C., Yan, Q., Jiang, Z., Li, Y., & Katrolia, P. (2010). High-level expression of a specific beta-1,3-1,4-glucanase from the thermophilic fungus Paecilomyces thermophila in Pichia pastoris. Applied Microbiology and Biotechnology, 88, 509–518.

    Article  CAS  Google Scholar 

  17. Hong, M. R., Kim, Y. S., Joo, A. R., Lee, J. K., Kim, Y. S., & Oh, D. K. (2009). Purification and characterization of a thermostable beta-1,3-1,4-glucanase from Laetiporus sulphureus var. miniatus. Journal of Microbiology and Biotechnology, 19, 818–822.

    Article  CAS  Google Scholar 

  18. Tang, Y., Yang, S., Yan, Q., Zhou, P., Cui, J., & Jiang, Z. (2012). Purification and characterization of a novel beta-1,3-1,4-glucanase (lichenase) from thermophilic Rhizomucor miehei with high specific activity and its gene sequence. Journal of Agricultural and Food Chemistry, 60, 2354–2361.

    Article  CAS  Google Scholar 

  19. Michel, G., Chantalat, L., Duee, E., Barbeyron, T., Henrissat, B., Kloareg, B., & Dideberg, O. (2001). The kappa-carrageenase of P. carrageenovora features a tunnel-shaped active site: A novel insight in the evolution of Clan-B glycoside hydrolases. Structure, 9, 513–525.

    Article  CAS  Google Scholar 

  20. Wen, T. N., Chen, J. L., Lee, S. H., Yang, N. S., & Shyur, L. F. (2005). A truncated Fibrobacter succinogenes 1,3-1,4-beta-d-glucanase with improved enzymatic activity and thermotolerance. Biochemistry, 44, 9197–9205.

    Article  CAS  Google Scholar 

  21. van Rensburg, P., van Zyl, W. H., & Pretorius, I. S. (1997). Over-expression of the Saccharomyces cerevisiae exo-beta-1,3-glucanase gene together with the Bacillus subtilis endo-beta-1,3-1,4-glucanase gene and the Butyrivibrio fibrisolvens endo-beta-1,4-glucanase gene in yeast. Journal of Biotechnology, 55, 43–53.

    Article  Google Scholar 

  22. Teng, D., Fan, Y., Yang, Y. L., Tian, Z. G., Luo, J., & Wang, J. H. (2007). Codon optimization of Bacillus licheniformis beta-1,3-1,4-glucanase gene and its expression in Pichia pastoris. Applied Microbiology and Biotechnology, 74, 1074–1083.

    Article  CAS  Google Scholar 

  23. Teng, D., Wang, J. H., Yao, Y., Yang, Y. L., & Liu, L. H. (2005). Cloning and expression of Bacillus licheniformis EG039 beta-1,3-1,4-glucanase in Pichia methanolica. In W. Jianhua (Ed.), Research and development of the new safe feed additives (pp. 102–109). Beijing: China Science.

    Google Scholar 

  24. Liu, J. R., Yu, B., Zhao, X., & Cheng, K. J. (2007). Coexpression of rumen microbial beta-glucanase and xylanase genes in Lactobacillus reuteri. Applied Microbiology and Biotechnology, 77, 117–124.

    Article  CAS  Google Scholar 

  25. Cheng, H. L., Tsai, L. C., Lin, S. S., Yuan, H. S., Yang, N. S., Lee, S. H., & Shyur, L. F. (2002). Mutagenesis of Trp(54) and Trp(203) residues on Fibrobacter succinogenes 1,3-1,4-beta-D-glucanase significantly affects catalytic activities of the enzyme. Biochemistry, 41, 8759–8766.

    Article  CAS  Google Scholar 

  26. Sun, J., Wang, H., Lv, W., Ma, C., Lou, Z., & Dai, Y. (2011). Construction and characterization of a fusion beta-1,3-1,4-glucanase to improve hydrolytic activity and thermostability. Biotechnology Letters, 33, 2193–2199.

    Article  CAS  Google Scholar 

  27. Huang, J. W., Cheng, Y. S., Ko, T. P., Lin, C. Y., Lai, H. L., Chen, C. C., Ma, Y., Zheng, Y., Huang, C. H., Zou, P., Liu, J. R., & Guo, R. T. (2012). Rational design to improve thermostability and specific activity of the truncated Fibrobacter succinogenes 1,3-1,4-beta-d-glucanase. Applied Microbiology and Biotechnology, 94, 111–121.

    Article  CAS  Google Scholar 

  28. Zhang, Q., Chen, Q. H., Fu, M. L., Wang, J. L., Zhang, H. B., & He, G. Q. (2008). Construction of recombinant industrial Saccharomyces cerevisiae strain with bglS gene insertion into PEP4 locus by homologous recombination. Journal of Zhejiang University. Science B, 9, 527–535.

    Article  CAS  Google Scholar 

  29. Zhang, X.-y., Ruan, H., Mu, L., He, G.-q., Tang, X.-j., & Chen, Q.-h. (2006). Enhancement of the thermostability of β-1,3-1,4-glucanase by directed evolution. Journal of Zhejiang University SCIENCE A, 7(11), 1948–1955.

    Article  Google Scholar 

  30. Sneath, P.H.A. (1986). Endospore-forming Gram-positive rods and cocci. In: P.H.A. Sneath (Ed.), Bergey’s manual of systematic bacteriology, vol 2. Williams and Wilkins, Baltimore, MD. pp 1104–1138 ISBN 0–683–07893–3.

  31. Weisburg, W. G., Barns, S. M., Pelletier, D. A., & Lane, D. J. (1991). 16S ribosomal DNA amplification for phylogenetic study. Journal of Bacteriology, 173, 697–703.

    CAS  Google Scholar 

  32. Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227, 680–685.

    Article  CAS  Google Scholar 

  33. Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, 72, 248–254.

    Article  CAS  Google Scholar 

  34. Miller, G. L. (1959). Use of dinitrosalicylic acid reagent for determination of reducing sugar. Analytical Chemistry, 31, 426–428.

    Article  CAS  Google Scholar 

  35. Maheshwari, R., Bharadwaj, G., & Bhat, M. K. (2000). Thermophilic fungi: Their physiology and enzymes. Microbiology and Molecular Biology Reviews, 64, 461–488.

    Article  CAS  Google Scholar 

  36. Dubendorff, J. W., & Studier, F. W. (1991). Controlling basal expression in an inducible T7 expression system by blocking the target T7 promoter with Lac repressor. Journal of Molecular Biology, 219, 45–59.

    Article  CAS  Google Scholar 

  37. Celestino, K. R., Cunha, R. B., & Felix, C. R. (2006). Characterization of a beta-glucanase produced by Rhizopus microsporus var. microsporus, and its potential for application in the brewing industry. BMC Biochemistry, 7, 23.

    Article  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. College of Food Science and Technology, Nanjing Agricultural University, Key Laboratory of Food Processing and Quality Control, Ministry of Agriculture of China, 1 Weigang, Nanjing, 210095, People’s Republic of China

    Shurui Mao, Zhaoxin Lu, Chong Zhang, Fengxia Lu & Xiaomei Bie

Authors
  1. Shurui Mao
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Zhaoxin Lu
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Chong Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Fengxia Lu
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Xiaomei Bie
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Xiaomei Bie.

Electronic Supplementary Material

Below is the link to the electronic supplementary material.

ESM 1

(DOC 260 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Mao, S., Lu, Z., Zhang, C. et al. Purification, Characterization, and Heterologous Expression of a Thermostable β-1,3-1,4-Glucanase from Bacillus altitudinis YC-9. Appl Biochem Biotechnol 169, 960–975 (2013). https://doi.org/10.1007/s12010-012-0064-3

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12010-012-0064-3

Keywords

Search

Navigation