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 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 degrees C over that of the wild-type enzyme. Intriguingly, the three selected cysteine variants generated dimers by formation of intermolecular disulfide bridges.
Beliën
Tim
T
Faculty of Applied Bioscience and Engineering, Laboratory of Food Chemistry and Biochemistry, Katholieke Universiteit Leuven, Kasteelpark Arenberg 20, B-3001 Leuven, Belgium.
Verjans
Priscilla
P
Courtin
Christophe M
CM
Delcour
Jan A
JA
D001412:Bacillus subtilis / Q000201:enzymology* / Q000235:genetics; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics; D019151:Peptide Library; D011489:Protein Denaturation; D017434:Protein Structure, Tertiary; D013696:Temperature; D014995:Xylosidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D019151:Peptide Library; D014995:Xylosidases