esterase for aqueous-organic solvents

来自 Nature

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214

作者：

JC Moore，FH Arnold

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摘要：

Through sequential generations of random mutagenesis and screening, we have directed the evolution of an esterase for deprotection of an antibioticester in aqueous-organic solvents. Because rapid screening directly on the desired antibiotic (loracarbef) nucleusester was not feasible, theester was employed. Catalytic performance on the screening substrate was shown to reasonably mimic enzyme activity toward the desired ester. Oneesterase 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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关键词：

Bacillus subtilis Escherichia coli Water DNA Primers DNA, Recombinant Recombinant Proteins Anti-Bacterial Agents Directed Molecular Evolution Amino Acid Substitution Polymerase Chain Reaction