Engineering a de novo internal disulfide bridge to improve the thermal stability of xylanase from Bacillus stearothermophilus No. 236

Mi Young Jeong, Sanguk Kim, Cheol Won Yun, Yong Jin Choi, Ssang Goo Cho

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62 Citations (Scopus)


Improvement of thermal stability of the Bacillus stearthermophilus No. 236 endo-β-1,4-xylanase (XynA) was tried by engineering a de novo designed disulfide bridge. Disulfide design was performed firstly using the disulfide bond design program (Disulfide by Design™) to identify residue pairs having the favorable geometric characteristics for disulfide formation. Subsequently, the selected 25 amino acid pairs were filtered with the evolutionarily conserved Cys residues identified by alignment of 34 family 11 mesophilic and thermophilic xylanases, and also by doing inspection of the molecular model of the xylanases. Only one pair (Ser100 and Asn150) was finally chosen, and the respective amino acids were substituted with cysteine residues. The newly designed disulfide bridge increased thermostability of the XynA about 5 °C. This improved thermal stability was supported by the increase in the energy barrier for inactivation. As expected, the mutant XynA SNC demonstrated its better use in the hydrolysis of xylan at substantially higher temperatures than permitted by its native counterpart. The mutation had little influence on the catalytic efficiency and other functional properties of the XynA. In conclusion, it is evident that the strategically placed disulfide bridge has made the XynA be more effective in resisting thermal inactivation.

Original languageEnglish
Pages (from-to)300-309
Number of pages10
JournalJournal of Biotechnology
Issue number2
Publication statusPublished - 2007 Jan 1

Bibliographical note

Funding Information:
This work was supported by the Korea Research Foundation Grant funded by the Korean Government (MOEHRD) (KRF-2005-070-C00095 and KRF2004-F00019) and by a Grant from the ERC program of the Korea Science & Engineering Foundation (No. R11-2002-100-01000-0).


  • Bacillus stearothermophilus No. 236
  • Disulfide bridge
  • Endo-β-l,4-xylanase
  • Thermostability

ASJC Scopus subject areas

  • Biotechnology
  • Bioengineering
  • Applied Microbiology and Biotechnology


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