Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
권호 표지
DOI QR코드

DOI QR Code

Improvement of Cellulase Activity Using Error-Prone Rolling Circle Amplification and Site-Directed Mutagenesis

  • Vu, Van Hanh (Institute of Biotechnology, Vietnam Academy of Science and Technology) ;
  • Kim, Keun (Department of Bioscience and Biotechnology, The University of Suwon)
  • Received : 2011.07.18
  • Accepted : 2012.01.27
  • Published : 2012.05.28
Download PDF
⟨ Previous Next ⟩

Abstract

Improvement of endoglucanase activity was accomplished by utilizing error-prone rolling circle amplification, supplemented with 1.7 mM $MnCl_2$. This procedure generated random mutations in the Bacillus amyloliquefaciens endoglucanase gene with a frequency of 10 mutations per kilobase. Six mutated endoglucanase genes, recovered from six colonies, possessed endoglucanase activity between 2.50- and 3.12-folds higher than wild type. We sequenced these mutants, and the different mutated sites of nucleotides were identified. The mutated endoglucanase sequences had five mutated amino acids: A15T, P24A, P26Q, G27A, and E289V. Among these five substitutions, E289V was determined to be responsible for the improved enzyme activity. This observation was confirmed with site-directed mutagenesis; the introduction of only one mutation (E289V) in the wild-type endoglucanase gene resulted in a 7.93-fold (5.55 U/mg protein) increase in its enzymatic activity compared with that (0.7 U/mg protein) of wild type.

Keywords

References

  1. Arnold, F. H., P. L. Wintrode, K. Miyazaki, and A. Gershenson. 2001. How enzymes adapt: Lessons from directed evolution. Trends Biochem. Sci. 26: 100-106. https://doi.org/10.1016/S0968-0004(00)01755-2
  2. Beckman, R. A., A. S. Mildvan, and L. A. Loeb. 1985. On the fidelity of DNA replication: Manganese mutagenesis in vitro. Biochem. 24: 5810-5817. https://doi.org/10.1021/bi00342a019
  3. Bradford, M. 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. https://doi.org/10.1016/0003-2697(76)90527-3
  4. Coughlan, M. P. 1985. The properties of fungal and bacterial cellulases with comment on their production and application. Biotechnol. Genet. Eng. Rev. 3: 39-109. https://doi.org/10.1080/02648725.1985.10647809
  5. Coughlan, M. P. and L. G. Ljungdahl. 1988. Comparative biochemistry of fungal and bacterial cellulolytic systems, pp. 11-30. In J. P. Aubert, P. Beguin, and J. Millet (eds.). Biochemistry and Genetics for Cellulose Degradation. Academic Press, London & San Diego.
  6. Dean, F. B., J. R. Nelson, T. L. Giesler, and R. S. Lasken. 2001. Rapid amplification of plasmid and phage DNA using Phi29 DNA polymerase and multiply-primed rolling circle amplification. Genome Res. 11: 1095-1099. https://doi.org/10.1101/gr.180501
  7. Devega, M., J. M. Lazaro, and M. Salas. 2000. Phage Phi29 DNA polymerase residues involved in the proper stabilisation of the primer terminus at the 3'-5' exonuclease active site. J. Mol. Biol. 304: 1-9. https://doi.org/10.1006/jmbi.2000.4178
  8. Ding, X., A. K. Snyder, R. Shaw, W. G. Farmerie, and W. Y. Song. 2003. Direct retransformation of yeast with plasmid DNA isolated from single yeast colonies using rolling circle amplification. BioTechniques 35: 774-779.
  9. Fujii, R., M. Kitaoka, and K. Hayashi. 2004. One-step random mutagenesis by error-prone rolling circle amplification. Nucleic Acids Res. 32: e145. https://doi.org/10.1093/nar/gnh147
  10. Greener, A., M. Callahan, and B. Jerpseth. 1996. In M. K. Trower (ed.). In Vitro Mutagenesis Protocols. Humana Press, New Jersey.
  11. Kornberg, A. and T. Baker. 1992. DNA Replication. Freeman WH & Company, New York.
  12. Kunkel, T. A. 1985. Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc. Natl. Acad. Sci. USA 82: 488-492. https://doi.org/10.1073/pnas.82.2.488
  13. Lee, Y. J., B. K. Kim, B. H. Lee, K. I. Jo, N. K. Lee, C. H. Chung, et al. 2008. Purification and characterization of cellulase produced by Bacillus amyoliquefaciens DL-3 utilizing rice hull. Bioresour. Technol. 99: 378-386. https://doi.org/10.1016/j.biortech.2006.12.013
  14. Leung, D. W., E. Chen, and D. W. Goeddel. 1989. A method for random mutagenesis of a defined DNA segment using a modified polymerase chain reaction. Techniques 1: 11-15.
  15. Liu, D. Y., S. L. Daubendiek, M. A. Zillman, K. Ryan, and E. T. Kool. 1996. Rolling circle DNA synthesis: Small circular oligonucleotides as efficient templates for DNA polymerases. J. Am. Chem. Soc. 118: 1587-1594. https://doi.org/10.1021/ja952786k
  16. Lizardi, P. M., X. Huang, Z. Zhu, P. Bray-Ward, D. C. Thomas, and D. C. Ward. 1998. Mutation detection and single-molecule counting using isothermal rolling-circle amplification. Nat. Genet. 19: 225-232. https://doi.org/10.1038/898
  17. Miller, G. L. 1959. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal. Chem. 3: 426-428.
  18. Ohmiya, K., K. Sakka, S. Karita, and T. Kimura. 1997. Structure of cellulases and their applications. Biotechnol. Gen. Eng. Rev. 14: 365-414. https://doi.org/10.1080/02648725.1997.10647949
  19. Wang, T., X. Liu, Q. Yu, X. Zhang, Y. Qu, and P. Gao. 2005. Directed evolution for engineering pH profile of endoglucanase III from Trichoderma reesei. Biomol. Eng. 22: 89-94. https://doi.org/10.1016/j.bioeng.2004.10.003
  20. Zhang, Y. H. P., M. E. Himmel, and J. R. Mielenz. 2006. Outlook for cellulase improvement: Screening and selection strategies. Biotechnol. Adv. 24: 452-481. https://doi.org/10.1016/j.biotechadv.2006.03.003

Cited by

  1. Neue Entwicklungen im Bereich Cellulase‐Engineering vol.85, pp.6, 2012, https://doi.org/10.1002/cite.201200190
  2. Directed co-evolution of an endoglucanase and a β-glucosidase in Escherichia coli by a novel high-throughput screening method vol.49, pp.65, 2012, https://doi.org/10.1039/c3cc42485e
  3. Current Developments in Cellulase Engineering vol.1, pp.1, 2012, https://doi.org/10.1002/cben.201300006
  4. Enzymatischer Abbau von (Ligno)Cellulose vol.126, pp.41, 2014, https://doi.org/10.1002/ange.201309953
  5. Enzymatic Degradation of (Ligno)cellulose vol.53, pp.41, 2012, https://doi.org/10.1002/anie.201309953
  6. Lignocellulosic biomass: Biosynthesis, degradation, and industrial utilization vol.16, pp.1, 2012, https://doi.org/10.1002/elsc.201400196
  7. Development of simple random mutagenesis protocol for the protein expression system in Pichia pastoris vol.9, pp.None, 2012, https://doi.org/10.1186/s13068-016-0613-z
  8. Rational engineering of Cel5E from Clostridium thermocellum to improve its thermal stability and catalytic activity vol.102, pp.19, 2012, https://doi.org/10.1007/s00253-018-9204-1