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J. Biol. Chem., Vol. 279, Issue 52, 54369-54379, December 24, 2004

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The Use of Forced Protein Evolution to Investigate and Improve Stability of Family 10 Xylanases

THE PRODUCTION OF Ca²⁺-INDEPENDENT STABLE XYLANASES^*

Simon R. Andrews, Edward J. Taylor, Gavin Pell, Florence Vincent, Valérie M.-A. Ducros, Gideon J. Davies, Jeremy H. Lakey, and Harry J. Gilbert¶

From the Structural Biology Laboratory, Department of Chemistry, University of York, Heslington, York YO10 5YW and Institute of Cell and Molecular Biosciences, University of Newcastle upon Tyne, Newcastle upon Tyne NE2 4HH, United Kingdom

Metal ions such as calcium often play a key role in protein thermostability. The inclusion of metal ions in industrial processes is, however, problematic. Thus, the evolution of enzymes that display enhanced stability, which is not reliant on divalent metals, is an important biotechnological goal. Here we have used forced protein evolution to interrogate whether the stabilizing effect of calcium in an industrially relevant enzyme can be replaced with amino acid substitutions. Our study has focused on the GH10 xylanase CjXyn10A from Cellvibrio japonicus, which contains an extended calcium binding loop that confers proteinase resistance and thermostability. Three rounds of error-prone PCR and selection identified a treble mutant, D262N/A80T/R347C, which in the absence of calcium is more thermostable than wild type CjXyn10A bound to the divalent metal. D262N influences the properties of the calcium binding site, A80T fills a cavity in the enzyme, increasing the number of hydrogen bonds and van der Waals interactions, and the R347C mutation introduces a disulfide bond that decreases the free energy of the unfolded enzyme. A derivative of CjXyn10A (CfCjXyn10A) in which the calcium binding loop has been replaced with a much shorter loop from Cellulomonas fimi CfXyn10A was also subjected to forced protein evolution to select for thermostablizing mutations. Two amino acid substitutions within the introduced loop and the A80T mutation increased the thermostability of the enzyme. This study demonstrates how forced protein evolution can be used to introduce enhanced stability into industrially relevant enzymes while removing calcium as a major stability determinant.

Received for publication, August 6, 2004 , and in revised form, September 27, 2004.

^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The atomic coordinates and structure factors (codes 1w2p (wild type CjXyn10A), 1w32 (D262N mutant of CjXyn10A), and 1w3h (D262N/A80T/R347C mutant of CjXyn10A)) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

The on-line version of this article (available at http://www.jbc.org) contains Supplemental Tables 1S and 2S.

^¶ To whom correspondence should be addressed. Tel.: 44-(0)191-2226962; Fax: 44-(0)191-2227424; E-mail: h.j.gilbert{at}ncl.ac.uk.

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