title: Ultrahigh-throughput screening to identify E. coli cells expressing functionally active enzymes on their surface.
abstract: 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.
authors: Department of Biochemistry, Clemens-Schöpf Institute, Darmstadt University of Technology, Petersenstrasse 22, Darmstadt, Germany.
authors: Stefan
authors: S
authors: Becker
authors: Anja
authors: A
authors: Michalczyk
authors: Susanne
authors: S
authors: Wilhelm
authors: Karl-Erich
authors: KE
authors: Jaeger
authors: Harald
authors: H
authors: Kolmar
mesh_terms: D001710:Biotin / Q000031:analogs & derivatives / Q000737:chemistry; D004789:Enzyme Activation; D004926:Escherichia coli / Q000166:cytology / Q000201:enzymology*; D004950:Esterases / Q000032:analysis / Q000737:chemistry*; D005609:Free Radicals / Q000737:chemistry; D006735:Horseradish Peroxidase / Q000737:chemistry; D008049:Lipase / Q000032:analysis / Q000737:chemistry*; D015394:Molecular Structure; D013499:Surface Properties
chemical_list: D005609:Free Radicals; D001710:Biotin; D006735:Horseradish Peroxidase; D004950:Esterases; D008049:Lipase