Premium
A Novel Genetic Selection System for Improved Enantioselectivity of Bacillus subtilis Lipase A
Author(s) -
Boersma Ykelien L.,
Dröge Melloney J.,
van der Sloot Almer M.,
Pijning Tjaard,
Cool Robbert H.,
Dijkstra Bauke W.,
Quax Wim J.
Publication year - 2008
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.200700754
Subject(s) - lipase , bacillus subtilis , enantioselective synthesis , enantiomer , phosphonate , chemistry , stereochemistry , catalytic triad , enantiomeric excess , directed evolution , mutant , active site , biochemistry , escherichia coli , enzyme , catalysis , biology , genetics , bacteria , gene
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 β7 and αE; this shows that the position of the catalytic acid is not necessarily conserved in this lipase.