Protein Science
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH
QUICK SEARCH:   [advanced]


     

Published online before print January 10, 2004
Protein Science, DOI: 10.1110/ps.03333504
 Copyright © 2004 The Protein Society
This Article
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
ps.03333504v1
13/2/494    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by PALACKAL, N.
Right arrow Articles by STEER, B. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by PALACKAL, N.
Right arrow Articles by STEER, B. A.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

An evolutionary route to xylanase process fitness

NISHA PALACKAL1, YALI BRENNAN1, WALTER N. CALLEN1, PAUL DUPREE2, GERHARD FREY1, FLORENCE GOUBET2, GEOFFREY P. HAZLEWOOD1, SHAUN HEALEY1, YOUNG E. KANG1, KEITH A. KRETZ1, EDD LEE1, XUQIU TAN1, GEOFFERY L. TOMLINSON1, JOHN VERRUTO1, VICKY W.K. WONG2, ERIC J. MATHUR1, JAY M. SHORT1, DAN E. ROBERTSON1 and BRIAN A. STEER1

1 Diversa Corp., San Diego, California 92121, USA
2 Department of Biochemistry, University of Cambridge, Cambridge CB2 1QW, UK

(RECEIVED July 29, 2003; FINAL REVISION September 22, 2003; ACCEPTED October 27, 2003)



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°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).

Keywords: Directed evolution; thermal stability; thermophilic xylanase

Reprint requests to: Brian A. Steer, Diversa Corp., 4955 Directors Place, San Diego, CA 92121, USA; e-mail: bsteer{at}diversa.com; fax: (858) 526-5764.

Abbreviations: ANTS, 8-aminonaphthalene-1,3,6-trisulfonic acid; CP, citrate-phosphate; Tm , denaturation temperature transition midpoint; GSSM, Gene Site Saturation Mutagenesis; PACE, polysaccharide analysis using carbohydrate electrophoresis.

Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.03333504


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 J. Biol. Chem.Home page
C. Dumon, A. Varvak, M. A. Wall, J. E. Flint, R. J. Lewis, J. H. Lakey, C. Morland, P. Luginbuhl, S. Healey, T. Todaro, et al.
Engineering Hyperthermostability into a GH11 Xylanase Is Mediated by Subtle Changes to Protein Structure
J. Biol. Chem., August 15, 2008; 283(33): 22557 - 22564.
[Abstract] [Full Text] [PDF]

Home page
 Protein Eng Des SelHome page
J. Rogers, R.J. Schoepp, O. Schroder, T.L. Clements, T.F. Holland, J.Q. Li, J. Li, L.M. Lewis, R.P. Dirmeier, G.J. Frey, et al.
Rapid discovery and optimization of therapeutic antibodies against emerging infectious diseases
Protein Eng. Des. Sel., August 1, 2008; 21(8): 495 - 505.
[Abstract] [Full Text] [PDF]

Home page
 Poult. Sci.Home page
M. A. Leslie, E. T. Moran Jr., and M. R. Bedford
The Effect of Phytase and Glucanase on the Ileal Digestible Energy of Corn and Soybean Meal Fed to Broilers
Poult. Sci., November 1, 2007; 86(11): 2350 - 2357.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
S. J. J. Brouns, H. Wu, J. Akerboom, A. P. Turnbull, W. M. de Vos, and J. van der Oost
Engineering a Selectable Marker for Hyperthermophiles
J. Biol. Chem., March 25, 2005; 280(12): 11422 - 11431.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
A. I. Solbak, T. H. Richardson, R. T. McCann, K. A. Kline, F. Bartnek, G. Tomlinson, X. Tan, L. Parra-Gessert, G. J. Frey, M. Podar, et al.
Discovery of Pectin-degrading Enzymes and Directed Evolution of a Novel Pectate Lyase for Processing Cotton Fabric
J. Biol. Chem., March 11, 2005; 280(10): 9431 - 9438.
[Abstract] [Full Text] [PDF]


HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH
Copyright © 2004 by The Protein Society.