Enhanced activity of Thermotoga maritima cellulase 12A by mutating a unique surface loop

Ya Shan Cheng, Tzu Ping Ko, Jian Wen Huang, Tzu Hui Wu, Cheng Yen Lin, Wenhua Luo, Qian Li, Yanhe Ma, Chun Hsiang Huang, Andrew H.J. Wang, Je Ruei Liu, Rey Ting Guo

Research output: Contribution to journalArticlepeer-review

29 Citations (Scopus)

Abstract

Cellulase 12A from Thermotoga maritima (TmCel12A) is a hyperthermostable ß-1,4-endoglucanase. We recently determined the crystal structures of TmCel12A and its complexes with oligosaccharides. Here, by using sitedirected mutagenesis, the role played by Arg60 and Tyr61 in a unique surface loop of TmCel12A was investigated. The results are consistent with the previously observed hydrogen bonding and stacking interactions between these two residues and the substrate. Interestingly, the mutant Y61G had the highest activity when compared with the wild-type enzyme and the other mutants. It also shows a wider range of working temperatures than does the wild type, along with retention of the hyperthermostability. The kcat and Km values of Y61G are both higher than those of the wild type. In conjunction with the crystal structure of Y61G-substrate complex, the kinetic data suggest that the higher endoglucanase activity is probably due to facile dissociation of the cleaved sugar moiety at the reducing end. Additional crystallographic analyses indicate that the insertion and deletion mutations at the Tyr61 site did not affect the overall protein structure, but local perturbations might diminish the substrate-binding strength. It is likely that the catalytic efficiency of TmCel12A is a subtle balance between substrate binding and product release. The activity enhancement by the single mutation of Y61G provides a good example of engineered enzyme for industrial application.

Original languageEnglish
Pages (from-to)661-669
Number of pages9
JournalApplied Microbiology and Biotechnology
Volume95
Issue number3
DOIs
Publication statusPublished - Aug 2012
Externally publishedYes

Keywords

  • Crystal structure
  • Endoglucanase
  • Enzyme kinetics
  • Protein engineering
  • Thermostability

ASJC Scopus subject areas

  • Biotechnology
  • Applied Microbiology and Biotechnology

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