Document Detail

A novel genetic selection system for improved enantioselectivity of Bacillus subtilis lipase A.
MedLine Citation:
PMID:  18383241     Owner:  NLM     Status:  MEDLINE    
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.
Ykelien L Boersma; Melloney J Dröge; Almer M van der Sloot; Tjaard Pijning; Robbert H Cool; Bauke W Dijkstra; Wim J Quax
Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  Chembiochem : a European journal of chemical biology     Volume:  9     ISSN:  1439-7633     ISO Abbreviation:  Chembiochem     Publication Date:  2008 May 
Date Detail:
Created Date:  2008-05-05     Completed Date:  2008-07-09     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  100937360     Medline TA:  Chembiochem     Country:  Germany    
Other Details:
Languages:  eng     Pagination:  1110-5     Citation Subset:  IM    
Department of Pharmaceutical Biology, University of Groningen, Groningen, The Netherlands.
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MeSH Terms
Alkenes / 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
Lipase / genetics*,  metabolism*
Periplasm / genetics,  metabolism
Substrate Specificity
Reg. No./Substance:
0/1,2-O-isopropylidene glycerol; 0/Alkenes; 56-81-5/Glycerol; 56-84-8/Aspartic Acid; EC

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