title: Display of lipase on the cell surface of Escherichia coli using OprF as an anchor and its application to enantioselective resolution in organic solvent.
abstract: We have developed a new cell surface display system using a major outer membrane protein of Pseudomonas aeruginosa OprF as an anchoring motif. Pseudomonas fluorescens SIK W1 lipase gene was fused to the truncated oprF gene by C-terminal deletion fusion strategy. The truncated OprF-lipase fusion protein was successfully displayed on the surface of Escherichia coli. Localization of the truncated OprF-lipase fusion protein was confirmed by western blot analysis, immunofluorescence microscopy, and whole-cell lipase activity. To examine the enzymatic characteristics of the cell surface displayed lipase, the whole-cell enzyme activity and stability were determined under various conditions. Cell surface displayed lipase showed the highest activity at 37 degrees C and pH 8.0. It retained over 80% of initial activity after incubation for a week in both aqueous solution and organic solvent. When the E. coli cells displaying lipases were used for enantioselective resolution of racemic 1-phenylethanol in hexane, (R)-phenyl ethyl acetate was successfully obtained with the enantiomeric excess of greater than 96% in 36 h of reaction. These results suggest that E. coli cells displaying lipases using OprF as an anchoring motif can be employed for various biotechnological applications both in aqueous and nonaqueous phases.
authors.affiliation: Metabolic and Biomolecular Engineering National Research Laboratory, Department of Chemical & Biomolecular Engineering, and BioProcess Engineering Research Center, Korea Advanced Institute of Science and Technology, Yuseong-gu, Daejeon , Republic of Korea.
authors.forename: Seung Hwan
authors.initials: SH
authors.lastname: Lee
authors.forename: Jong-Il
authors.initials: JI
authors.lastname: Choi
authors.forename: Mee-Jung
authors.initials: MJ
authors.lastname: Han
authors.forename: Jong Hyun
authors.initials: JH
authors.lastname: Choi
authors.forename: Sang Yup
authors.initials: SY
authors.lastname: Lee
mesh_terms: D001425:Bacterial Outer Membrane Proteins / Q000378:metabolism*; D001592:Benzyl Alcohols / Q000737:chemistry; D015153:Blotting, Western; D004269:DNA, Bacterial; D004926:Escherichia coli / Q000201:enzymology*; D008049:Lipase / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D016133:Polymerase Chain Reaction; D012997:Solvents / Q000737:chemistry; D013237:Stereoisomerism
chemical_list: D001425:Bacterial Outer Membrane Proteins; D001592:Benzyl Alcohols; D004269:DNA, Bacterial; C121501:OprG protein, Pseudomonas aeruginosa; D012997:Solvents; C002017:methylphenyl carbinol; D008049:Lipase