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Truncation of the cellulose binding domain improved thermal stability of endo-β-1,4-glucanase fromBacillus subtilisJA18

Authors
Journal
Bioresource Technology
0960-8524
Publisher
Elsevier
Publication Date
Volume
100
Issue
1
Identifiers
DOI: 10.1016/j.biortech.2008.06.001
Keywords
  • Bacillus Subtilis
  • Cellulose-Binding Domain
  • Endo-β-1
  • 4-Glucanase
  • Thermal Stability

Abstract

Abstract The C-terminus region of endo-β-glucanase Egl499 from Bacillus subtilis JA18 was suggested to be a putative family 3 cellulose-binding domain (CBD) by computer analysis. To prove this proposal, C-terminus truncation mutant Egl330 was constructed and expressed. Compared with Egl499, Egl330 lost the cellulose binding capability at 4 °C, confirming the C-terminus region was a CBD. Binding of the CBD to Avicel was inhibited by carboxymethylcellulose (CMC), but not by barley β-glucan and glucose at concentration of 0.1% and 0.5%. Kinetic analysis showed both the turnover rate ( k cat) and the catalytic efficiency ( k cat/ K m) of Egl330 increased for the substrate CMC compared to Egl499. A great improvement in thermal stability was observed in Egl330. The half life of Egl330 at 65 °C increased to three folds that of Egl499, from 10 to 29 min. After treated at 80 °C for 10 min, Egl330 could recover more than 60% of its original activity while Egl499 only recovered 12% activity. UV spectrometry analysis showed Egl330 and Egl499 differed in refolding efficiency after heat treatment.

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