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United States Department of Agriculture

Agricultural Research Service

Title: Creation of a Productive, Highly Enantioselective Nitrilase Through Gene Site Saturation Mutagenesis (Gssm)

Authors
item Desantis, Grace - DIVERSA CORP
item Wong, Kevin - DIVERSA CORP
item Farwell, Bob - DIVERSA CORP
item Chatman, Kelly - DIVERSA CORP
item Zhu, Zoulin - DIVERSA CORP
item Tomlinson, Geoff - DIVERSA CORP
item Huang, Hongjun - DIVERSA CORP
item Tan, Xuqiu - DIVERSA CORP
item Bibbs, Lisa - DIVERSA CORP
item Chen, Pei
item Kretz, Keith - DIVERSA CORP
item Burk, Mark - DIVERSA CORP

Submitted to: Meeting Proceedings
Publication Type: Proceedings
Publication Acceptance Date: January 10, 2004
Publication Date: January 20, 2004
Citation: Greenberg, W.A., Varvak, A., Hanson, S.R., Wong, K., Huang, H., Chen, P., Burk, M.J. 2004. Creation of a productive, highly enantioselective nitrilase through gene site saturation mutagenesis (GSSM). Proceedings of the National Academy of Sciences. 101(16):5788-5793.

Technical Abstract: A process is reported for efficient, enantioselective production of key intermediates for the common chiral side chain of statin-type cholesterol-lowering drugs such as Lipitor (atorvastatin) and Crestor (rosuvastatin). The process features a one-pot tandem aldol reaction catalyzed by a deoxyribose-5-phosphate aldolase (DERA) to form a 6-carbon intermediate with installation of two stereogenic centers from 2-carbon starting materials. An improvement of almost 400-fold in volumetric productivity relative to the published enzymatic reaction conditions has been achieved, resulting in a commercially attractive process that has been run on up to a 100-g scale in a single batch at a rate of 30.6 g/liter per h. Catalyst load has been improved by 10-fold as well, from 20 to 2.0 wt % DERA. These improvements were achieved by a combination of discovery from environmental DNA of DERAs with improved activity and reaction optimization to overcome substrate inhibition. The two stereogenic centers are set by DERA with enantiomeric excess at >99.9% and diastereomeric excess at 96.6%. In addition, down-stream chemical steps have been developed to convert the enzymatic product efficiently to versatile intermediates applicable to preparation of atorvastatin and rosuvastatin.

Last Modified: 4/25/2014
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