EstA is an outer membrane-anchored esterase from Pseudomonas aeruginosa. An inactive EstA variant was used as an anchoring motif for the Escherichia coli cell-surface display of lipolytic enzymes. Flow cytometry analysis and measurement of lipase activity revealed that Bacillus subtilis lipase LipA, Fusarium solani pisi cutinase and one of the largest lipases presently known, namely Serratia marcescens lipase were all efficiently exported by the EstA autotransporter and also retained their lipolytic activities upon cell surface exposition. EstA provides a useful tool for surface display of lipases including variant libraries generated by directed evolution thereby enabling the identification of novel enzymes with interesting biological and biotechnological ramifications. 25

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.