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An evolutionary route to xylanase process fitness
Author(s) -
Palackal Nisha,
Brennan Yali,
Callen Walter N.,
Dupree Paul,
Frey Gerhard,
Goubet Florence,
Hazlewood Geoffrey P.,
Healey Shaun,
Kang Young E.,
Kretz Keith A.,
Lee Edd,
Tan Xuqiu,
Tomlinson Geoffery L.,
Verruto John,
Wong Vicky W.K.,
Mathur Eric J.,
Short Jay M.,
Robertson Dan E.,
Steer Brian A.
Publication year - 2004
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1110/ps.03333504
Subject(s) - thermostability , directed evolution , xylanase , protein engineering , amino acid , chemistry , residue (chemistry) , rendering (computer graphics) , enzyme , amino acid residue , biochemistry , stereochemistry , mutant , biophysics , biology , peptide sequence , computer science , computer graphics (images) , gene
Directed evolution technologies were used to selectively improve the stability of an enzyme without compromising its catalytic activity. In particular, this article describes the tandem use of two evolution strategies to evolve a xylanase, rendering it tolerant to temperatures in excess of 90°C. A library of all possible 19 amino acid substitutions at each residue position was generated and screened for activity after a temperature challenge. Nine single amino acid residue changes were identified that enhanced thermostability. All 512 possible combinatorial variants of the nine mutations were then generated and screened for improved thermal tolerance under stringent conditions. The screen yielded eleven variants with substantially improved thermal tolerance. Denaturation temperature transition midpoints were increased from 61°C to as high as 96°C. The use of two evolution strategies in combination enabled the rapid discovery of the enzyme variant with the highest degree of fitness (greater thermal tolerance and activity relative to the wild‐type parent).