An evolutionary route to xylanase process fitness.
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).
Palackal
Nisha
N
Diversa Corp., 4955 Directors Place, San Diego, CA 92121, USA.
Brennan
Yali
Y
Callen
Walter N
WN
Dupree
Paul
P
Frey
Gerhard
G
Goubet
Florence
F
Hazlewood
Geoffrey P
GP
Healey
Shaun
S
Kang
Young E
YE
Kretz
Keith A
KA
Lee
Edd
E
Tan
Xuqiu
X
Tomlinson
Geoffery L
GL
Verruto
John
J
Wong
Vicky W K
VW
Mathur
Eric J
EJ
Short
Jay M
JM
Robertson
Dan E
DE
Steer
Brian A
BA
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
D043364:Endo-1,4-beta Xylanases