title: An evolutionary route to xylanase process fitness.
abstract: 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).
authors.affiliation: Diversa Corp., 4955 Directors Place, San Diego, CA 92121, USA.
authors.forename: Nisha
authors.initials: N
authors.lastname: Palackal
authors.forename: Yali
authors.initials: Y
authors.lastname: Brennan
authors.forename: Walter N
authors.initials: WN
authors.lastname: Callen
authors.forename: Paul
authors.initials: P
authors.lastname: Dupree
authors.forename: Gerhard
authors.initials: G
authors.lastname: Frey
authors.forename: Florence
authors.initials: F
authors.lastname: Goubet
authors.forename: Geoffrey P
authors.initials: GP
authors.lastname: Hazlewood
authors.forename: Shaun
authors.initials: S
authors.lastname: Healey
authors.forename: Young E
authors.initials: YE
authors.lastname: Kang
authors.forename: Keith A
authors.initials: KA
authors.lastname: Kretz
authors.forename: Edd
authors.initials: E
authors.lastname: Lee
authors.forename: Xuqiu
authors.initials: X
authors.lastname: Tan
authors.forename: Geoffery L
authors.initials: GL
authors.lastname: Tomlinson
authors.forename: John
authors.initials: J
authors.lastname: Verruto
authors.forename: Vicky W K
authors.initials: VW
authors.lastname: Wong
authors.forename: Eric J
authors.initials: EJ
authors.lastname: Mathur
authors.forename: Jay M
authors.initials: JM
authors.lastname: Short
authors.forename: Dan E
authors.initials: DE
authors.lastname: Robertson
authors.forename: Brian A
authors.initials: BA
authors.lastname: Steer
mesh_terms: D019943:Amino Acid Substitution; D019020:Directed Molecular Evolution / Q000379:methods*; D043364:Endo-1,4-beta Xylanases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D004795:Enzyme Stability; D014644:Genetic Variation / Q000235:genetics; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D017386:Sequence Homology, Amino Acid; D044366:Transition Temperature
chemical_list: D043364:Endo-1,4-beta Xylanases