A generic system for the <i>Escherichia coli</i> cell‐surface display of lipolytic enzymes

Becker, Stefan; Theile, Sebastian; Heppeler, Nele; Michalczyk, Anja; Wentzel, Alexander; Wilhelm, Susanne; Jaeger, Karl‐Erich; Kolmar, Harald

doi:10.1016/j.febslet.2004.12.087

Cited by 80 publications

(82 citation statements)

References 27 publications

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“…Furthermore, this assumption is nicely supported by our recent finding that S. marcescens LipA can be autodisplayed on the surface of E. coli cells by fusing it to an autotransporter protein from P. aeruginosa. The autotransporter domain efficiently inserts into the bacterial outer membrane with the lipase domain facing the extracellular medium, where it folds into its enzymatically active conformation (52).…”

Section: Discussionmentioning

confidence: 99%

A Calcium-gated Lid and a Large β-Roll Sandwich Are Revealed by the Crystal Structure of Extracellular Lipase from Serratia marcescens

Meier

Drepper

Svensson

et al. 2007

Journal of Biological Chemistry

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Lipase LipA from Serratia marcescens is a 613-amino acid enzyme belonging to family I.3 of lipolytic enzymes that has an important biotechnological application in the production of a chiral precursor for the coronary vasodilator diltiazem. Like other family I.3 lipases, LipA is secreted by Gram-negative bacteria via a type I secretion system and possesses 13 copies of a calcium binding tandem repeat motif, GGXGXDXUX (U, hydrophobic amino acids), in the C-terminal part of the polypeptide chain. The 1.8-Å crystal structure of LipA reveals a close relation to eukaryotic lipases, whereas family I.1 and I.2 enzymes appear to be more distantly related. Interestingly, the structure shows for the N-terminal lipase domain a variation on the canonical ␣/␤ hydrolase fold in an open conformation, where the putative lid helix is anchored by a Ca 2؉ ion essential for activity. Another novel feature observed in this lipase structure is the presence of a helical hairpin additional to the putative lid helix that exposes a hydrophobic surface to the aqueous medium and might function as an additional lid. The tandem repeats form two separated parallel ␤-roll domains that pack tightly against each other. Variations of the consensus sequence of the tandem repeats within the second ␤-roll result in an asymmetric Ca 2؉ binding on only one side of the roll. The analysis of the properties of the ␤-roll domains suggests an intramolecular chaperone function.

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Section: Discussionmentioning

confidence: 99%

A Calcium-gated Lid and a Large β-Roll Sandwich Are Revealed by the Crystal Structure of Extracellular Lipase from Serratia marcescens

Meier

Drepper

Svensson

et al. 2007

Journal of Biological Chemistry

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“…Recently, several bacterial esterases have been subjected to laboratory evolution to obtain enzymes with improved properties, including substrate specificity or enantioselectivity (2,33,35). The primary aim of the present study was to combine the advantages of cellular surface display with the attractive features of a bacterial esterase (4). Cellular esterase display would result in a whole-cell biocatalyst that should be easily applicable to industrial applications or organic synthesis processes (10).…”

mentioning

confidence: 99%

Esterase Autodisplay: Enzyme Engineering and Whole-Cell Activity Determination in Microplates with pH Sensors

Schultheiss

Weiss

Winterer

et al. 2008

Appl Environ Microbiol

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Among the GDSL family of serine esterases/lipases is a group of bacterial enzymes that posses C-terminal extensions involved in outer membrane anchoring or translocation. ApeE from Salmonella enterica serovar Typhimurium, a member of this group, has been expressed in Escherichia coli and was resistant to protease digestion when the protease was added to whole cells, indicating a periplasmic localization. The five consensus blocks conserved within all GDSL esterases were identified in ApeE by multiple sequence alignment and separated from the C-terminal extension. The DNA sequence spanning the four invariant residues Ser, Gly, Asn, and His, and hence representing the catalytic domains of ApeE, was amplified by PCR and fused in frame to the transport domains of the autodisplay system. The resulting artificial esterase, called EsjA, was overexpressed in the cell envelope of E. coli and was shown to be active by the use of ␣-naphthyl acetate (␣-NA) as a substrate in an in-gel activity stain after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Surface exposure of EsjA was indicated by its accessibility to protease added to whole cells. The esterase activity of whole cells displaying EsjA was determined by a pH agar assay and by the use of microplates with integrated pH-dependent optical sensors. ␣-NA, ␣-naphthyl butyrate, and ␣-naphthyl caproate were used as substrates, and it turned out that the substrate preferences of artificial EsjA were altered in comparison to original ApeE. Our results indicate that autodisplay of esterase in combination with pH sensor microplates can provide a new platform technology for the screening of tailor-made hydrolase activities.

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“…EstA outer membrane esterase [48,49]. Functional display of OPH-GFP has been demonstrated recently using the AIDA-1 transporter [50].…”

Section: New Cell-surface Anchorsmentioning

confidence: 99%

Versatile microbial surface-display for environmental remediation and biofuels production

Wu¹,

Mulchandani²,

Chen³

2008

Trends in Microbiology

101

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Surface display is a powerful technique that utilizes natural microbial functional components to express proteins or peptides on the cell exterior. Since the reporting of the first surface-display system in the mid-1980s, a variety of new systems have been reported for yeast, Gram-positive and Gram-negative bacteria. Non-conventional display methods are emerging, eliminating the generation of genetically modified microorganisms. Cells with surface display are used as biocatalysts, biosorbents and biostimulants. Microbial cell-surface display has proven to be extremely important for numerous applications ranging from combinatorial library screening and protein engineering to bioremediation and biofuels production.3

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A generic system for the Escherichia coli cell‐surface display of lipolytic enzymes

Cited by 80 publications

References 27 publications

A Calcium-gated Lid and a Large β-Roll Sandwich Are Revealed by the Crystal Structure of Extracellular Lipase from Serratia marcescens

A Calcium-gated Lid and a Large β-Roll Sandwich Are Revealed by the Crystal Structure of Extracellular Lipase from Serratia marcescens

Esterase Autodisplay: Enzyme Engineering and Whole-Cell Activity Determination in Microplates with pH Sensors

Versatile microbial surface-display for environmental remediation and biofuels production

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