A novel genetic selection system for improved enantioselectivity of Bacillus subtilis lipase A

Ykelien L. Boersma, Melloney J. Droge, Almer M. van der Sloot, Tjaard Pijning, Robbert H. Cool, Bauke W. Dijkstra, Wim J. Quax*, Melloney J. Dröge

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review

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Abstract

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 enontiomer 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 28.9 916) towards the (S)(+)-enantiomer. Interestingly, its amino acid sequence showed that the acid of the catalytic triad hod migrated to a position further along the loop that connects beta 7 and alpha E; this shows that the position of the catalytic acid is not necessarily conserved in this lipase.

Original languageEnglish
Pages (from-to)1110-1115
Number of pages6
JournalChemBioChem
Volume9
Issue number7
DOIs
Publication statusPublished - 5-May-2008

Keywords

  • Bacillus subtilis lipase A
  • directed evolution
  • dual selection
  • enantioselectivity
  • genetic selection
  • ALPHA/BETA-HYDROLASE FOLD
  • DIRECTED EVOLUTION
  • INDUSTRIAL BIOCATALYSIS
  • SUBSTRATE-SPECIFICITY
  • ESCHERICHIA-COLI
  • TOPOFIT METHOD
  • PHAGE DISPLAY
  • ENZYMES
  • LIBRARIES
  • ACYLASE

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