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.
journal_name
Nat Biotechnoljournal_title
Nature biotechnologyauthors
Moore JC,Arnold FHdoi
10.1038/nbt0496-458subject
Has Abstractpub_date
1996-04-01 00:00:00pages
458-67issue
4eissn
1087-0156issn
1546-1696journal_volume
14pub_type
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journal_title:Nature biotechnology
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pub_type: 杂志文章,评审
doi:10.1038/nbt1324
更新日期:2007-08-01 00:00:00
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更新日期:2009-10-01 00:00:00
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doi:10.1038/74455
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更新日期:2014-05-01 00:00:00
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