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J Biol Chem. 2008 Aug 15;283(33):22557-64. doi: 10.1074/jbc.M800936200. Epub 2008 May 30.

Engineering hyperthermostability into a GH11 xylanase is mediated by subtle changes to protein structure.

Author information

1
Institute for Cell and Molecular Biosciences, Newcastle University, The Medical School, Newcastle Upon Tyne NE2 4HH, United Kingdom.

Abstract

Understanding the structural basis for protein thermostability is of considerable biological and biotechnological importance as exemplified by the industrial use of xylanases at elevated temperatures in the paper pulp and animal feed sectors. Here we have used directed protein evolution to generate hyperthermostable variants of a thermophilic GH11 xylanase, EvXyn11. The Gene Site Saturation Mutagenesis (GSSM) methodology employed assesses the influence on thermostability of all possible amino acid substitutions at each position in the primary structure of the target protein. The 15 most thermostable mutants, which generally clustered in the N-terminal region of the enzyme, had melting temperatures (Tm) 1-8 degrees C higher than the parent protein. Screening of a combinatorial library of the single mutants identified a hyperthermostable variant, EvXyn11TS, containing seven mutations. EvXyn11TS had a Tm approximately 25 degrees C higher than the parent enzyme while displaying catalytic properties that were similar to EvXyn11. The crystal structures of EvXyn11 and EvXyn11TS revealed an absence of substantial changes to identifiable intramolecular interactions. The only explicable mutations are T13F, which increases hydrophobic interactions, and S9P that apparently locks the conformation of a surface loop. This report shows that the molecular basis for the increased thermostability is extraordinarily subtle and points to the requirement for new tools to interrogate protein folding at non-ambient temperatures.

PMID:
18515360
DOI:
10.1074/jbc.M800936200
[Indexed for MEDLINE]
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