Enhancement of the thermostability and activity of mesophilic Clostridium cellulovorans EngD by in vitro DNA recombination with Clostridium thermocellum CelE

Chae Yoeng Lee, Kyung Ok Yu, Seung Wook Kim, Sung Ok Han

Research output: Contribution to journalArticle

13 Citations (Scopus)

Abstract

The thermal stability and catalytic activity of endoglucanase (EngD) from mesophilic Clostridium cellulovorans were improved by evolutionary molecular engineering. Thermostable mutants were isolated after staggered extension process (StEP) with celE from thermophilic Clostridium thermocellum performed to conduct family shuffling and overlay screening of the resultant mutant library. The relative activity of the best-evolved clone has been improved of about 2 times higher at 50 °C and showed a higher kcat/Km value than its engD parental clone. We determined that these variants had two amino acid substitutions (L157N, Q158E) and confirmed their effects by substituting these amino acids in the parental gene by site-directed mutagenesis. These substitutions resulted in an increase in hydrophilic or charged residues. Our results demonstrate that in vitro recombination is an effective approach to improve the thermostability and enzymatic activity of a mesophilic enzyme.

Original languageEnglish
Pages (from-to)331-336
Number of pages6
JournalJournal of Bioscience and Bioengineering
Volume109
Issue number4
DOIs
Publication statusPublished - 2010 Apr

Keywords

  • Cellulase
  • Clostridium cellulovorans
  • Clostridium thermocellum
  • Thermostability
  • in vitro DNA recombination

ASJC Scopus subject areas

  • Biotechnology
  • Bioengineering
  • Applied Microbiology and Biotechnology

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