Directed evolution to produce an alkalophilic variant from a Neocallimastix patriciarum xylanase.
The catalytic domain of a xylanase from the anaerobic fungus Neocallimastix patriciarum was made more alkalophilic through directed evolution using error-prone PCR. Transformants expressing the alkalophilic variant xylanases produced larger clear zones when overlaid with high pH, xylan-containing agar. Eight amino acid substitutions were identified in six selected mutant xylanases. Whereas the wild-type xylanase exhibited no activity at pH 8.5, the relative and specific activities of the six mutants were higher at pH 8.5 than at pH 6.0. Seven of the eight amino acid substitutions were assembled in one enzyme (xyn-CDBFV) by site-directed mutagenesis. Some or all of the seven mutations exerted positive and possibly synergistic effects on the alkalophilicity of the enzyme. The resulting composite mutant xylanase retained a greater proportion of its activity than did the wild type at pH above 7.0, maintaining 25% of its activity at pH 9.0, and its retention of activity at acid pH was no lower than that of the wild type. The composite xylanase (xyn-CDBFV) had a relatively high specific activity of 10128 micromol glucose x min(-1) x (mg protein)(-1) at pH 6.0. It was more thermostable at 60 degrees C and alkaline tolerant at pH 10.0 than the wild-type xylanase. These properties suggest that the composite mutant xylanase is a promising and suitable candidate for paper pulp bio-bleaching.
Chen
Y L
YL
Institute of BioAgricultural Sciences, Academia Sinica, Taipei, Taiwan, Republic of China.
Tang
T Y
TY
Cheng
K J
KJ
D000940:Antigenic Variation / Q000502:physiology; D019020:Directed Molecular Evolution; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D016297:Mutagenesis, Site-Directed; D020088:Neocallimastix / Q000201:enzymology* / Q000235:genetics; D011897:Random Allocation; D043325:Xylan Endo-1,3-beta-Xylosidase; D014995:Xylosidases / Q000235:genetics* / Q000378:metabolism
D014995:Xylosidases; D043325:Xylan Endo-1,3-beta-Xylosidase