Directed Evolution of<i>Bacillus subtilis</i>Lipase A by Use of Enantiomeric Phosphonate Inhibitors: Crystal Structures and Phage Display Selection

Dröge, Melloney J.; Boersma, Ykelien L.; Pouderoyen, Gertie van; Vrenken, Titia E.; Rüggeberg, Carsten J.; Reetz, Manfred T.; Dijkstra, Bauke W.; Quax, Wim J.

doi:10.1002/cbic.200500308

Cited by 65 publications

(39 citation statements)

References 45 publications

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“…Furthermore, LipA of B. subtilis, a lipase with a twin-arginine signal peptide that results in partial dependence on TatC (17), was used as a control protein for display, because functional phage display of this enzyme with the help of the g3p signal peptide was recently demonstrated (13). Importantly, the functional phage display of both CesA and LipA is of particular biotechnological interest as the corresponding displayed fusion proteins can be used for selection of improved variants for the enantioselective conversion of several interesting pharmaceutical compounds, as recently shown for selection of an S-(ϩ)-1,2-O-ispropylidene-sn-glycerol-specific LipA mutant (10,11).…”

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confidence: 99%

Phage Display of an Intracellular Carboxylesterase of Bacillus subtilis : Comparison of Sec and Tat Pathway Export Capabilities

Dröge

Boersma

Braun

et al. 2006

Appl Environ Microbiol

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Using the phage display technology, a protein can be displayed at the surface of bacteriophages as a fusion to one of the phage coat proteins. Here we describe development of this method for fusion of an intracellular carboxylesterase of Bacillus subtilis to the phage minor coat protein g3p. The carboxylesterase gene was cloned in the g3p-based phagemid pCANTAB 5E upstream of the sequence encoding phage g3p and downstream of a signal peptide-encoding sequence. The phage-bound carboxylesterase was correctly folded and fully enzymatically active, as determined from hydrolysis of the naproxen methyl ester with K m values of 0.15 mM and 0.22 mM for the soluble and phage-displayed carboxylesterases, respectively. The signal peptide directs the encoded fusion protein to the cell membrane of Escherichia coli, where phage particles are assembled. In this study, we assessed the effects of several signal peptides, both Sec dependent and Tat dependent, on the translocation of the carboxylesterase in order to optimize the phage display of this enzyme normally restricted to the cytoplasm. Functional display of Bacillus carboxylesterase NA could be achieved when Sec-dependent signal peptides were used. Although a Tat-dependent signal peptide could direct carboxylesterase translocation across the inner membrane of E. coli, proper assembly into phage particles did not seem to occur.In the past decade, the most remarkable successes of protein engineering have been the result of combining random mutagenesis and screening by means of a high-throughput assay (6). Unfortunately, for many enzymes, such as esterases and lipases, no high-throughput methods are available; consequently, evaluation of the enantioselectivity of lipases and esterases is dependent on time-consuming assays (7). Thus, it would be highly advantageous if the screening process could be combined with a rapid selection method that limits the amounts of mutants to be assayed. Phage display (30) is a well-defined technique that has led to a breakthrough in selection methodology for enzymes with desirable properties from a pool of mutants.Derivatives of M13 filamentous phages that are phage particles with a single-stranded genome encapsulated by the phage coat proteins (30) are most commonly used for display in Escherichia coli. Enzymes can be expressed as fusions to one of the M13 phage coat proteins, such as the g3p protein (30). As phage particles are assembled in the cell envelope of E. coli, translocation of the g3p fusion protein across the inner membrane of E. coli is a prerequisite for proper phage display. The g3p protein is synthesized with an 18-residue amino-terminal signal peptide that targets this protein to the E. coli Sec machinery for membrane insertion (26). Theoretically, any protein fused to the amino-terminal region of the g3p protein that is efficiently translocated across the inner membrane and that is able to enter the phage assembly site can be presented as a fusion protein on M13 phages (30).Today, phage display can be used for selection of s...

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Phage Display of an Intracellular Carboxylesterase of Bacillus subtilis : Comparison of Sec and Tat Pathway Export Capabilities

Dröge

Boersma

Braun

et al. 2006

Appl Environ Microbiol

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“…[13] The B. subtilis lipase A was successfully displayed on a bacteriophage and the reaction product was covalently attached to the active site of the enzyme with a phosphonate inhibitor used as a suicide substrate. [14,15] The major advantage of coupled bacterial synthesis, cell-surface display of enzyme libraries and covalent product attachment lies in the fact that surface-exposed enzymes can perform multiple catalytic turnovers since no suicide substrate is used. Enzymatic activity is manifested as accumulation of biotin or any other easily detectable tag on the cell surface of the respective enzyme-producing clone within a large bacterial population.…”

Section: Discussionmentioning

confidence: 99%

Ultrahigh‐Throughput Screening to Identify E. coli Cells Expressing Functionally Active Enzymes on their Surface

et al. 2007

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We show here that E. coli bacteria that display esterases or lipases on their cell surface together with horseradish peroxidase (HRP) are capable of hydrolysing carboxylic acid esters of biotin tyramide. The tyramide radicals generated by the coupled lipase-peroxidase reaction were short-lived and therefore became covalently attached to reactive tyrosine residues that were located in close vicinity on the surface of a bacterial cell that displayed lipase activity. Up to 120 000 biotinylated tyramide derivatives could be covalently coupled through HRP activation to the surface of a single living E. coli cell. Differences in cellular esterase activity were found to correlate with the amount of biotin tyramide deposited on the cell surface. Selective biotin tyramide labelling of cells that had lipase activity allowed their isolation by magnetic cell sorting from a 1:10(6) mixture of control cells. This strategy of covalently attaching a biotin label to esterase-proficient bacteria might open new avenues to ultrahigh-throughput screening of enzyme libraries for hydrolytic enzymes with enhanced activities or enantioselectivities.

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“…22 The acyl part binding pocket in AFL is much larger when compared with that of BSL, and consequently, the enzyme-substrate interactions are more extensive than those in BSL. In addition, the ligand binding direction in the active site of AFL and BSL is opposite to each other.…”

Section: Acyl-binding Sitementioning

confidence: 99%

Structure of the Alkalohyperthermophilic Archaeoglobus fulgidus Lipase Contains a Unique C-Terminal Domain Essential for Long-Chain Substrate Binding

Chen¹,

Lee²,

Ko³

et al. 2009

Journal of Molecular Biology

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Directed Evolution ofBacillus subtilisLipase A by Use of Enantiomeric Phosphonate Inhibitors: Crystal Structures and Phage Display Selection

Cited by 65 publications

References 45 publications

Phage Display of an Intracellular Carboxylesterase of Bacillus subtilis : Comparison of Sec and Tat Pathway Export Capabilities

Phage Display of an Intracellular Carboxylesterase of Bacillus subtilis : Comparison of Sec and Tat Pathway Export Capabilities

Ultrahigh‐Throughput Screening to Identify E. coli Cells Expressing Functionally Active Enzymes on their Surface

Structure of the Alkalohyperthermophilic Archaeoglobus fulgidus Lipase Contains a Unique C-Terminal Domain Essential for Long-Chain Substrate Binding

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