Journal of Microbiology and Biotechnology

  • 제22권5호
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  • Pages.607-613
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  • 2012
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  • 1017-7825(pISSN)
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  • 1738-8872(eISSN)
한국미생물·생명공학회 (The Korean Society for Microbiology and Biotechnology)
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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)
  • 투고 : 2011.07.18
  • 심사 : 2012.01.27
  • 발행 : 2012.05.28
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초록

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.

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참고문헌

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피인용 문헌

  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
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