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Applied Microbiology and Biotechnology

, Volume 83, Issue 4, pp 649–657 | Cite as

Directed evolution of endoglucanase III (Cel12A) from Trichoderma reesei

  • Hikaru Nakazawa
  • Katsunori Okada
  • Tomoko Onodera
  • Wataru Ogasawara
  • Hirofumi OkadaEmail author
  • Yasushi Morikawa
Biotechnologically Relevant Enzymes and Proteins

Abstract

The stability and specific activity of endo-β-1,4-glucanase III from Trichoderma reesei QM9414 was enhanced, and the expression efficiency of its encoding gene, egl3, was optimized by directed evolution using error-prone PCR and activity screening in Escherichia coli RosettaBlue (DE3) pLacI as a host. Relationship between increase in yield of active enzyme in the clones and improvement in its stability was observed among the mutants obtained in the present study. The clone harboring the best mutant 2R4 (G41E/T110P/K173M/Y195F/P201S/N218I) selected in via second-round mutagenesis after optimal recombinating of first-round mutations produced 130-fold higher amount of mutant enzyme than the transformant with wild-type EG III. Mutant 2R4 produced by the clone showed broad pH stability (4.4–8.8) and thermotolerance (entirely active at 55°C for 30 min) compared with those of the wild-type EG III (pH stability, 4.4–5.2; thermostability, inactive at 55°C for 30 min). kcat of 2R4 against carboxymethyl-cellulose was about 1.4-fold higher than that of the wild type, though the Km became twice of that of the wild type.

Keywords

Directed evolution Endoglucanase Trichoderma reesei Error-prone PCR 

Notes

Acknowledgment

This work was partly supported by a grant from the Research Institute of Innovative Technology for the Earth (RITE) project.

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Copyright information

© Springer-Verlag 2009

Authors and Affiliations

  • Hikaru Nakazawa
    • 1
  • Katsunori Okada
    • 1
  • Tomoko Onodera
    • 1
  • Wataru Ogasawara
    • 1
  • Hirofumi Okada
    • 1
    Email author
  • Yasushi Morikawa
    • 1
  1. 1.Department of BioengineeringNagaoka University of TechnologyNagaokaJapan

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