Advertisement

Molecular Biotechnology

, Volume 40, Issue 2, pp 195–201 | Cite as

Cloning of the Thermostable Cellulase Gene from Newly Isolated Bacillus subtilis and its Expression in Escherichia coli

  • Wang Li
  • Wei-Wei Zhang
  • Ming-Ming YangEmail author
  • Yu-Lin ChenEmail author
Research

Abstract

A bacterial strain with high cellulase activity (0.26 U/ml culture medium) was isolated from hot spring, and classified and named as B. subtilis DR by morphological and 16SrDNA gene sequence analysis. A thermostable endocellulase, CelDR, was purified from the isolated strain. The optimum temperature of the enzyme reaction was 50°C, and CelDR retained 70% of its maximum activity at 75°C after incubation for 30 min. The putative gene celDR, consisting an open reading frame (ORF) of 1,524 nucleotides and encoding a protein of 508 amino acids with a molecular weight of 55 kDa, was purified from B. subtilis DR and cloned into pET-28a for expression. The cellulase production in E. coli BL21 (DE3) was enhanced to approximately three times that of the wild-type strain.

Keywords

Cellulase Heat-resistance Bacillus subtilis Clone and expression 

Notes

Acknowledgments

We gratefully acknowledge the financial support of the National New Productions Project from the Science and Technology Ministry (P. R. China).

References

  1. 1.
    Lee, R. L., Paul, J. W., Willem, H. V., Zyl, I., & Pretorius, S. (2002). Microbial cellulose utilization: Fundamentals and biotechnology. Microbiology and Molecular Biology Reviews, 66, 506–577. doi: 10.1128/MMBR.66.3.506-577.2002.CrossRefGoogle Scholar
  2. 2.
    Rizzatti, A. C. S., Jorge, J. A., Terenzi, H. F., Rechia, C. G. V., & Polizeli, M. L. T. M. (2001). Purification and properties of a thermostable extracellular β-d-xylosidase produced by a thermotolerant Aspergillus phoenicis. Journal of Industrial Microbiology & Biotechnology, 26, 156–160. doi: 10.1038/sj.jim.7000107.CrossRefGoogle Scholar
  3. 3.
    Masohiro, N., Masahiro, G., Hirofumi, O., & Yasushi, M. (2001). l-Sorbose induces cellulase gene transcription in the cellulolytic fungus Trichoderma reesei. Current Genetics, 38, 329–334. doi: 10.1007/s002940000165.CrossRefGoogle Scholar
  4. 4.
    Watanabe, H., & Tokuda, G. (2001). Animal cellulases, CMLS. Cellular and Molecular Life Sciences, 58, 1167–1178. doi: 10.1007/PL00000931.CrossRefGoogle Scholar
  5. 5.
    Goedegebuur, F., Fowler, T., Phillips, J., Kley, P. V., Solingen, P. V., Dankmeyer, L., et al. (2002). Cloning and relational analysis of 15 novel fungal endoglucanases from family 12 glycosyl hydrolase. Current Genetics, 41, 89–98. doi: 10.1007/s00294-002-0290-2.CrossRefGoogle Scholar
  6. 6.
    Will, F., Bauckhage, K., & Dietrich, H. (2002). Apple pomace liquefaction with pectinases and cellulases: Analytical data of the corresponding juices. European Food Research and Technology, 211, 291–297. doi: 10.1007/s002170000171.CrossRefGoogle Scholar
  7. 7.
    Hiroki, I., Tadanori, A., Keisuke, T., & Yutaka, K. (2005). Heterologous expression and characterization of the endocellulase encoding gene cel3A from the basidiomycete Polyporus arcularius. Mycoscience, 46, 154–161. doi: 10.1007/s10267-005-0225-0.CrossRefGoogle Scholar
  8. 8.
    Johnson, E. A., Madia, A., & Demain, A. C. (1981). Chemically defined minimal medium for growth of the anaerobic cellulolytic thermophile Clostridium thermocellum. Applied and Environmental Microbiology, 41, 1060–1062.Google Scholar
  9. 9.
    Bhat, M. K. (2000). Cellulase and related enzymes in biotechnology. Biotechnology Advances, 18, 355–383. doi: 10.1016/S0734-9750(00)00041-0.CrossRefGoogle Scholar
  10. 10.
    Doyle, J., Pavel, R., Barness, G., & Steinberger, Y. (2006). Cellulase dynamics in a desert soil. Soil Biology & Biochemistry, 38, 371–376.Google Scholar
  11. 11.
    Li, Y. H., Ding, M., Wang, J., Xu, G. J., & Zhao, F. (2006). A novel thermoacidophilic endoglucanase, Ba-EGA, from a new cellulose-degrading bacterium, Bacillus sp. AC-1. Applied Microbiology and Biotechnology, 70, 430–436. doi: 10.1007/s00253-005-0075-x.CrossRefGoogle Scholar
  12. 12.
    Herbert, R. A. (1992). A perspective on the biotechnological potential of extremophiles. Trends in Biotechnology, 10, 395–402. doi: 10.1016/0167-7799(92)90282-Z.CrossRefGoogle Scholar
  13. 13.
    Paula, S. P., Alexandra, M., José, C. D., Maria, R., Aires, B., & Maria, C. F. (2002). Rapid production of thermostable cellulase-free xylanase by a strain of Bacillus subtilis and its properties. Enzyme and Microbial Technology, 30, 924–933. doi: 10.1016/S0141-0229(02)00034-0.CrossRefGoogle Scholar
  14. 14.
    Showale, J. G., & Sadana, J. C. (1978). Cellulase and b-glucosidase production by Basidiomycetes species. Canadian Journal of Microbiology, 24, 1204–1216.CrossRefGoogle Scholar
  15. 15.
    Sambrook, J., Fritsch, E. F., & Maniatis, T. (1989). Molecular cloning: A laboratory manual (2nd ed.). New York: Cold Spring Harbor Laboratory Press.Google Scholar
  16. 16.
    Teather, R. M., & Wood, P. J. (1982). Use of Congo red-polysaccharide interactions in enumeration and characterisation of cellulolytic bacteria from the bovine rumen. Applied and Environmental Microbiology, 43, 777–780.Google Scholar
  17. 17.
    Crawford, D., & Mccoy, E. (1972). Cellulases of Thermomonospora fusca and Streptomyces thermodiastaticus. Applied Microbiology, 24, 150–152.Google Scholar
  18. 18.
    Miller, G. L. (1959). Use of dinitrosalicylic acid reagent for the determination of reducing sugar. Analytical Chemistry, 31, 426–428. doi: 10.1021/ac60147a030.CrossRefGoogle Scholar
  19. 19.
    Kim, C. H., & Kim, D. S. (1992). Production and characterization of crystalline cellulose-degrading cellulase components from a thermophilic and moderately alkalophilic bacterium. Journal of Microbiology and Biotechnology, 2, 7–13.Google Scholar
  20. 20.
    Nazneen, B., & Javed, M. (2003). Characterization of a new Pseudomonas aeruginosa strain NJ-15 as a potential biocontrol agent. Current Microbiology, 46, 324–328. doi: 10.1007/s00284-002-3857-8.CrossRefGoogle Scholar
  21. 21.
    Hollien, J., & Marqusee, S. (1999). Structural distribution of stability in a thermophilic enzyme. Proceedings of the National Academy of Sciences of the USA, 96, 13674–13678. doi: 10.1073/pnas.96.24.13674.CrossRefGoogle Scholar
  22. 22.
    Te’o, V. S. J., Saul, D. J., & Bergquist, P. L. (1995). CelA, another gene coding for a multidomain cellulase from the extreme thermophile Caldocellum saccharolyticum. Applied Microbiology and Biotechnology, 43, 291–296. doi: 10.1007/BF00172827.CrossRefGoogle Scholar
  23. 23.
    Yoshihiro, H., Kenzo, K., Tadashi, Y., Hajimi, M., Tohru, K., & Susumu, I. (1997). Thermostable alkaline cellulase from an alkaliphilic isolate, Bacillus sp. KSM-S237. Extremophiles, 1, 151–156. doi: 10.1007/s007920050028.CrossRefGoogle Scholar
  24. 24.
    Robertson, L. D., & Koehn, R. D. (1978). Characteristics of the cellulase produced by the Ascomycete Poronia punctata. Mycologia, 70, 1113–1121. doi: 10.2307/3759142.CrossRefGoogle Scholar
  25. 25.
    Zeikus, J. G., Vieille, C., & Savchenko, A. (1998). Thermozymes: Biotechnology and structure–function relationships. Extremophiles, 2, 179–183. doi: 10.1007/s007920050058.CrossRefGoogle Scholar

Copyright information

© Humana Press 2008

Authors and Affiliations

  1. 1.College of Animal SciencesNorthwest A&F UniversityYanglingPeople’s Republic of China
  2. 2.Yangguang-Guangji Medical R&D Co. Ltd.WuhanPeople’s Republic of China
  3. 3.College of Animal SciencesHenan S&T UniversityLuoyangPeople’s Republic of China

Personalised recommendations