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Optimizing industrial enzymes by directed evolution.

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Division of Chemistry and Chemical Engineering 210-41, California Institute of Technology, Pasadena 91125, USA.


Enzymes can be tailored for optimal performance in industrial applications by directing their evolution in vitro. This approach is particularly attractive for engineering industrial enzymes. We have created an efficient para-nitrobenzyl esterase over six generations of random point mutagenesis and recombination coupled with screening for improved variants. The best clones identified after four generations of sequential random mutagenesis and two generations of random recombination display more than 150 times the p-nitrobenzyl esterase activity of wild type towards loracarbef-p-nitrobenzyl ester in 15% dimethylformamide. 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. None appear to interact directly with the antibiotic substrate, further underscoring the difficulty of predicting their effects in a 'rational' design effort.

[Indexed for MEDLINE]

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