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Directed evolution of a para-nitrobenzyl esterase for aqueous-organic solvents

Moore, Jeffrey C. and Arnold, Frances H. (1996) Directed evolution of a para-nitrobenzyl esterase for aqueous-organic solvents. Nature Biotechnology, 14 (4). pp. 458-467. ISSN 1087-0156. doi:10.1038/nbt0496-458.

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Through sequential generations of random mutagenesis and screening, we have directed the evolution of an esterase for deprotection of an antibiotic p-nitrobenzyl ester in aqueous-organic solvents. Because rapid screening directly on the desired antibiotic (loracarbef) nucleus p-nitrobenzyl ester was not feasible, the p-nitrophenyl ester was employed. Catalytic performance on the screening substrate was shown to reasonably mimic enzyme activity toward the desired ester. One p-nitrobenzyl esterase variant performs as well in 30% dimethylformamide as the wildtype enzyme in water, reflecting a 16-fold increase in esterase activity. Random pairwise gene recombination of two positive variants led to a further two-fold improvement in activity. Considering also the increased expression level achieved during these experiments, the net result of four sequential generations of random mutagenesis and the one recombination step is a 50-60-fold increase in total activity. Although the contributions of individual effective amino acid substitutions to enhanced activity are small (< 2-fold increases), the accumulation of multiple mutations by directed evolution allows significant improvement of the biocatalyst for reactions on substrates and under conditions not already optimized in nature. The positions of the effective amino acid substitutions have been identified in a pNB esterase structural model developed based on its homology to acetylcholinesterase and triacylglycerol lipase. None appear to interact directly with the antibiotic substrate, further underscoring the difficulty of predicting their effects in a 'rational' design effort.

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Arnold, Frances H.0000-0002-4027-364X
Additional Information:© 1996 Nature Publishing Group. Received 13 December 1995; accepted 24 January 1996. The authors are grateful to Stephen Quener and Cathleen Cantwell of Eli Lilly & Co. for materials and for many helpful discussions. They also acknowledge the able assistance of Tao Long, Kejian Wu, Janice Lau and Roopa Ramamoorthi. This work was supported by the US Office of Naval Research (N00014-91-J-1397) and the US Department of Energy's program of Biological and Chemical Technologies Research within the Office of Industrial Technologies, Energy Efficiency and Renewables. J. M. is grateful to Merck & Co. for the support of a Merck Graduate Fellowship.
Funding AgencyGrant Number
Office of Naval Research (ONR)N00014-91-J-1397
Department of Energy (DOE)UNSPECIFIED
Subject Keywords:random mutagenesis, antibiotic synthesis, enzymatic deprotection
Issue or Number:4
Record Number:CaltechAUTHORS:20150618-143020949
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Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:58353
Deposited On:18 Jun 2015 22:09
Last Modified:10 Nov 2021 22:03

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