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

ABSTRACT 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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(e-mail: [email protected]). AUTHORS AND AFFILIATIONS * Division of Chemistry and Chemical Engineering 210-41, California Institute of Technology, Pasadena, CA, 91125 Jeffrey C.

Moore Authors * Jeffrey C. Moore View author publications You can also search for this author inPubMed Google Scholar * Frances H. Arnold View author publications You can also search for

this author inPubMed Google Scholar RIGHTS AND PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Moore, J., Arnold, F. Directed evolution of a _para-nitrobenzyl_

esterase for aqueous-organic solvents. _Nat Biotechnol_ 14, 458–467 (1996). https://doi.org/10.1038/nbt0496-458 Download citation * Received: 13 December 1995 * Accepted: 24 January 1996 *

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