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An evolutionary route to xylanase process fitness

2004; Wiley; Volume: 13; Issue: 2 Linguagem: Inglês

10.1110/ps.03333504

1469-896X

Nisha Palackal, Yali Brennan, Walter Callen, Paul Dupree, Gerhard Frey, Florence Goubet, Geoffrey P. Hazlewood, Shaun Healey, Young E. Kang, Keith A. Kretz, Edd Lee, Xuqiu Tan, Geoffery L. Tomlinson, John Verruto, Vicky W.K. Wong, Eric J. Mathur, Jay M. Short, Dan E. Robertson, Brian A. Steer,

Enzyme Production and Characterization

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 degrees 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 degrees C to as high as 96 degrees 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).