title: Engineering a thermostable fungal GH10 xylanase, importance of N-terminal amino acids.
abstract: Xylanases are used in many industrial processes including pulp bleaching, baking, detergent, and the hydrolysis of plant cell wall in biofuels production. In this work we have evolved a single domain GH10 xylanase, Xyn10A_ASPNG, from Aspergillus niger to improve its thermostability. We introduced a rational approach involving as the first step a computational analysis to guide the design of a mutagenesis library in targeted regions which identified thermal important residues that were subsequently randomly mutagenized through rounds of iterative saturation mutagenesis (ISM). Focusing on five residues, four rounds of ISM had generated a quintuple mutant 4S1 (R25W/V29A/I31L/L43F/T58I) which exhibited thermal inactivation half-life (t1/2 ) at 60°C that was prolonged by 30 folds in comparison with wild-type enzyme. Whereas the wild-type enzyme retained 0.2% of its initial activity after a heat treatment of 10 min at 60°C and was completely inactivated after 2 min at 65°C, 4S1 mutant retained 30% of its initial activity after 15 min heating at 65°C. Furthermore, the mutant melting temperature (Tm ) increased by 17.4°C compared to the wild type. Each of the five mutations in 4S1 was found to contribute to thermoresistance, but the dramatic improvement of enzyme thermoresistance of 4S1 was attributed to the synergistic effects of the five mutations. Comparison of biochemical data and model structure between 4S1 and the wild-type enzyme suggested that the N-terminal coil of the enzyme is important in stabilizing GH10 xylanase structure. Based on model structure analyses, we propose that enforced hydrophobic interactions within N-terminal elements and between N- and C-terminal ends are responsible for the improved thermostability of Xyn10A_ASPNG.
authors: Institut National de la Recherche Scientifique, INRS-Institut Armand-Frappier, Laval, QC H7V 1B7, Canada.
authors: Letian
authors: L
authors: Song
authors: Adrian
authors: A
authors: Tsang
authors: Michel
authors: M
authors: Sylvestre
mesh_terms: D019943:Amino Acid Substitution; D000596:Amino Acids / Q000235:genetics* / Q000378:metabolism*; D001234:Aspergillus niger / Q000201:enzymology* / Q000235:genetics; D004795:Enzyme Stability / Q000528:radiation effects; D008958:Models, Molecular; D016296:Mutagenesis; D020125:Mutation, Missense; D011487:Protein Conformation; D015202:Protein Engineering; D055550:Protein Stability / Q000528:radiation effects; D013696:Temperature; D044366:Transition Temperature; D014995:Xylosidases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
chemical_list: D000596:Amino Acids; D014995:Xylosidases
keywords: GH10; biocatalysis; directed evolution; glycoside hydrolase; structure analysis; thermostability