| A novel genetic selection system for improved enantioselectivity of Bacillus subtilis lipase A. | |
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MedLine Citation:
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PMID: 18383241 Owner: NLM Status: MEDLINE |
Abstract/OtherAbstract:
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In directed evolution experiments, success often depends on the efficacy of screening or selection methods. Genetic selections have proven to be extremely valuable for evolving enzymes with improved catalytic activity, improved stability, or with altered substrate specificity. In contrast, enantioselectivity is a difficult parameter to select for. In this study, we present a successful strategy that not only selects for catalytic activity, but for the first time also for enantioselectivity, as demonstrated by the selection of Bacillus subtilis lipase A variants with inverted and improved enantioselectivity. A lipase mutant library in an aspartate auxotroph Escherichia coli was plated on minimal medium that was supplemented with the aspartate ester of the desired enantiomer (S)-(+)-1,2-O-isopropylidene-sn-glycerol. To inhibit growth of less enantioselective variants, a covalently binding phosphonate ester of the opposite (R)-(-)-1,2-O-isopropylidene-sn-glycerol enantiomer was added as well. After three selection rounds in which the selection pressure was increased by raising the phosphonate ester concentration, a mutant was selected with an improved enantioselectivity increased from an ee of -29.6 % (conversion 23.4 %) to an ee of +73.1 % (conversion 28.9 %) towards the (S)-(+)-enantiomer. Interestingly, its amino acid sequence showed that the acid of the catalytic triad had migrated to a position further along the loop that connects beta7 and alphaE; this shows that the position of the catalytic acid is not necessarily conserved in this lipase. |
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Authors:
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Ykelien L Boersma; Melloney J Dröge; Almer M van der Sloot; Tjaard Pijning; Robbert H Cool; Bauke W Dijkstra; Wim J Quax |
Publication Detail:
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Type: Journal Article; Research Support, Non-U.S. Gov't |
Journal Detail:
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Title: Chembiochem : a European journal of chemical biology Volume: 9 ISSN: 1439-7633 ISO Abbreviation: Chembiochem Publication Date: 2008 May |
Date Detail:
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Created Date: 2008-05-05 Completed Date: 2008-07-09 Revised Date: - |
Medline Journal Info:
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Nlm Unique ID: 100937360 Medline TA: Chembiochem Country: Germany |
Other Details:
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Languages: eng Pagination: 1110-5 Citation Subset: IM |
Affiliation:
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Department of Pharmaceutical Biology, University of Groningen, Groningen, The Netherlands. |
Export Citation:
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| MeSH Terms | |
Descriptor/Qualifier:
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Alkenes
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metabolism Aspartic Acid / metabolism Bacillus subtilis / enzymology*, genetics Directed Molecular Evolution / methods* Escherichia coli / growth & development, metabolism Gene Expression Regulation, Bacterial Gene Library Glycerol / analogs & derivatives, metabolism Hydrolysis Lipase / genetics*, metabolism* Mutation Periplasm / genetics, metabolism Stereoisomerism Substrate Specificity |
| Chemical | |
Reg. No./Substance:
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0/1,2-O-isopropylidene glycerol; 0/Alkenes; 56-81-5/Glycerol; 56-84-8/Aspartic Acid; EC 3.1.1.3/Lipase |
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine
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