Engineering the thermostability of a TIM-barrel enzyme by rational family shuffling

Biochem Biophys Res Commun. 2008 Oct 3;374(4):725-30. doi: 10.1016/j.bbrc.2008.07.095. Epub 2008 Jul 27.


A possible approach to generate enzymes with an engineered temperature optimum is to create chimeras of homologous enzymes with different temperature optima. We tested this approach using two family-10 xylanases from Thermotoga maritima: the thermophilic xylanase A catalytic domain (TmxAcat, T(opt)=68 degrees C), and the hyperthermophilic xylanase B (TmxB, T(opt)=102 degrees C). Twenty-one different chimeric constructs were created by mimicking family shuffling in a rational manner. The measured temperature optima of the 16 enzymatically active chimeras do not monotonically increase with the percentage of residues coming from TmxB. Only four chimeras had a higher temperature optimum than TmxAcat, the most stable variant (T(opt)=80 degrees C) being the one in which both terminal segments came from TmxB. Further analysis suggests that the interaction between the N- and C-terminal segments has a disproportionately high contribution to the overall thermostability. The results may be generalizable to other enzymes where the N- and C-termini are in contact.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Catalysis
  • Catalytic Domain
  • Endo-1,4-beta Xylanases / biosynthesis*
  • Endo-1,4-beta Xylanases / genetics
  • Enzyme Stability
  • Hot Temperature*
  • Kinetics
  • Protein Conformation
  • Protein Engineering / methods*
  • Recombinant Fusion Proteins / biosynthesis*
  • Recombinant Fusion Proteins / genetics
  • Thermodynamics
  • Thermotoga maritima / enzymology*


  • Recombinant Fusion Proteins
  • Endo-1,4-beta Xylanases