Display of lipase on the cell surface of Escherichia coli using OprF as an anchor and its application to enantioselective resolution in organic solvent.
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.
Lee
Seung Hwan
SH
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.
Choi
Jong-Il
JI
Han
Mee-Jung
MJ
Choi
Jong Hyun
JH
Lee
Sang Yup
SY
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
D001425:Bacterial Outer Membrane Proteins; D001592:Benzyl Alcohols; D004269:DNA, Bacterial; C121501:OprG protein, Pseudomonas aeruginosa; D012997:Solvents; C002017:methylphenyl carbinol; D008049:Lipase