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

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

Author information

1
Institute of Enzymology, Biological Research Center, Hungarian Academy of Sciences, Pf. 7, H-1518 Budapest, Hungary.

Abstract

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.

PMID:
18667161
DOI:
10.1016/j.bbrc.2008.07.095
[Indexed for MEDLINE]

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