Phage display based identification of novel stabilizing mutations in glycosyl hydrolase family 11 B. subtilis endoxylanase XynA

Two combinatorial libraries of glycosyl hydrolase family 11 (GH11) Bacillus subtilis endoxylanase XynA were constructed and displayed on phage. Both phage-displayed libraries were subjected to three consecutive biopanning rounds against immobilized endoxylanase inhibitor TAXI, each time preceded by...

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Bibliographic Details
Published in:Biochemical and biophysical research communications 2008-03, Vol.368 (1), p.74-80
Main Authors: Beliën, Tim, Verjans, Priscilla, Courtin, Christophe M., Delcour, Jan A.
Format: Article
Language:English
Subjects:
Bacillus subtilis
Bacillus subtilis - enzymology
Bacillus subtilis - genetics
Endoxylanase
Models, Molecular
Molecular evolution
Mutation - genetics
Peptide Library
Phage display
Protein Denaturation
Protein Structure, Tertiary
Temperature
Thermostability
Xylosidases - chemistry
Xylosidases - genetics
Xylosidases - metabolism
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Summary:Two combinatorial libraries of glycosyl hydrolase family 11 (GH11) Bacillus subtilis endoxylanase XynA were constructed and displayed on phage. Both phage-displayed libraries were subjected to three consecutive biopanning rounds against immobilized endoxylanase inhibitor TAXI, each time preceded by an incubation step at elevated temperature. DNA sequence analysis of enriched phagemid panning isolates allowed identification of mutations conferring enhanced thermal stability. In particular, substitutions T44C, T44Y, F48C, T87D, and Y94C were retained, and their thermostabilizing effect was confirmed by testing site-directed XynA variants. None of these mutations was identified in earlier endoxylanase engineering studies. Each single mutation increased the half-inactivation temperature by 2–3°C over that of the wild-type enzyme. Intriguingly, the three selected cysteine variants generated dimers by formation of intermolecular disulfide bridges.
ISSN:0006-291X
1090-2104
DOI:10.1016/j.bbrc.2008.01.047