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Thermostable variants of the recombinant xylanase a from Bacillus subtilis produced by directed evolution show reduced heat capacity changes
Author(s) -
Ruller Roberto,
Deliberto Laila,
Ferreira Tatiana Lopes,
Ward Richard J.
Publication year - 2008
Publication title -
proteins: structure, function, and bioinformatics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.699
H-Index - 191
eISSN - 1097-0134
pISSN - 0887-3585
DOI - 10.1002/prot.21617
Subject(s) - thermostability , dna shuffling , bacillus subtilis , directed evolution , mutant , xylanase , protein engineering , context (archaeology) , biochemistry , circular dichroism , chemistry , heat capacity , biology , enzyme , genetics , thermodynamics , bacteria , paleontology , gene , physics
Directed evolution techniques have been used to improve the thermal stability of the xylanase A from Bacillus subtilis (XylA). Two generations of random mutant libraries generated by error prone PCR coupled with a single generation of DNA shuffling produced a series of mutant proteins with increasing thermostability. The most Thermostable XylA variant from the third generation contained four mutations Q7H, G13R, S22P, and S179C that showed an increase in melting temperature of 20°C. The thermodynamic properties of a representative subset of nine XylA variants showing a range of thermostabilities were measured by thermal denaturation as monitored by the change in the far ultraviolet circular dichroism signal. Analysis of the data from these thermostable variants demonstrated a correlation between the decrease in the heat capacity change (ΔC p ) with an increase in the midpoint of the transition temperature (T m ) on transition from the native to the unfolded state. This result could not be interpreted within the context of the changes in accessible surface area of the protein on transition from the native to unfolded states. Since all the mutations are located at the surface of the protein, these results suggest that an explanation of the decrease in Δ C p should include effects arising from the protein/solvent interface. Proteins 2008. © 2007 Wiley‐Liss, Inc.