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Applied and Environmental Microbiology, April 2004, p. 2429-2436, Vol. 70, No. 4
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.4.2429-2436.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Exploring Nitrilase Sequence Space for Enantioselective Catalysis{dagger}

Dan E. Robertson,* Jennifer A. Chaplin, Grace DeSantis, Mircea Podar, Mark Madden, Ellen Chi,{ddagger} Toby Richardson, Aileen Milan, Mark Miller, David P. Weiner, Kelvin Wong, Jeff McQuaid, Bob Farwell, Lori A. Preston, Xuqiu Tan, Marjory A. Snead, Martin Keller, Eric Mathur, Patricia L. Kretz, Mark J. Burk, and Jay M. Short

Diversa Corporation, San Diego, California 92121

Received 18 September 2003/ Accepted 22 December 2003

Nitrilases are important in the biosphere as participants in synthesis and degradation pathways for naturally occurring, as well as xenobiotically derived, nitriles. Because of their inherent enantioselectivity, nitrilases are also attractive as mild, selective catalysts for setting chiral centers in fine chemical synthesis. Unfortunately, <20 nitrilases have been reported in the scientific and patent literature, and because of stability or specificity shortcomings, their utility has been largely unrealized. In this study, 137 unique nitrilases, discovered from screening of >600 biotope-specific environmental DNA (eDNA) libraries, were characterized. Using culture-independent means, phylogenetically diverse genomes were captured from entire biotopes, and their genes were expressed heterologously in a common cloning host. Nitrilase genes were targeted in a selection-based expression assay of clonal populations numbering 106 to 1010 members per eDNA library. A phylogenetic analysis of the novel sequences discovered revealed the presence of at least five major sequence clades within the nitrilase subfamily. Using three nitrile substrates targeted for their potential in chiral pharmaceutical synthesis, the enzymes were characterized for substrate specificity and stereospecificity. A number of important correlations were found between sequence clades and the selective properties of these nitrilases. These enzymes, discovered using a high-throughput, culture-independent method, provide a catalytic toolbox for enantiospecific synthesis of a variety of carboxylic acid derivatives, as well as an intriguing library for evolutionary and structural analyses.

* Corresponding author. Mailing address: Diversa Corporation, 4955 Directors Place, San Diego, CA 92121. Phone: (858) 526-5149. Fax: (858) 526-5649. E-mail: drobertson{at}diversa.com.

{dagger} We dedicate this paper to Mark Madden, whose enthusiasm inspired and propelled our nitrilase research.

{ddagger} Present address: Syrrx Corporation, San Diego, CA 92121.

Applied and Environmental Microbiology, April 2004, p. 2429-2436, Vol. 70, No. 4
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.4.2429-2436.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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