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Focusing mutations into the P. fluorescens esterase binding site increases enantioselectivity more effectively than distant mutations
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2005 (English)In: Chemistry and Biology, ISSN 1074-5521, E-ISSN 1879-1301, Vol. 12, no 1, 45-54 p.Article in journal (Refereed) Published
Abstract [en]

Rational design of enzymes with improved properties, such as enantioselectivity, usually focuses mutations within the substrate binding site. On the other hand, directed evolution of enzymes usually targets the entire protein and discovers beneficial mutations far from the substrate binding site. In this paper, we propose an explanation for this discrepancy and show that a combined approach-random mutagenesis within the substrate binding site-is better. To increase the enantioselectivity (E) of a Pseudomonas fluorescens esterase (PFE) toward methyl 3-bromo-2-methylpropionate, we focused mutagenesis into the substrate binding site at Trp28, Val121, Phe198, and Val225. Five of the catalytically active mutants (13%) showed better enantioselectivity than wild-type PFE. The increases in enantioselectivity were higher (up to 5-fold, reaching E = 61) than with mutants identified by random mutagenesis of the entire enzyme.

Place, publisher, year, edition, pages
2005. Vol. 12, no 1, 45-54 p.
Keyword [en]
burkholderia-cepacia kwi-56, directed evolution, pseudomonas-fluorescens, inverting enantioselectivity, saturation mutagenesis, recombinant esterase, rational evolution, epoxide hydrolase, serine-protease, lipase
URN: urn:nbn:se:kth:diva-14505DOI: 10.1016/j.chembiol.2004.10.012ISI: 000226798200009ScopusID: 2-s2.0-12344337569OAI: diva2:332546
QC 20100525Available from: 2010-08-05 Created: 2010-08-05Bibliographically approved

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