title: Cell surface display of lipase in Pseudomonas putida KT2442 using OprF as an anchoring motif and its biocatalytic applications.
abstract: We developed a new cell surface display system in Pseudomonas putida KT2442 using OprF, an outer membrane protein of Pseudomonas aeruginosa, as an anchoring motif in a C-terminal deletion-fusion strategy. The Pseudomonas fluorescens SIK W1 lipase gene was fused to two different C-terminal truncated OprF genes, and the fusion genes were cloned into the broad-host-range plasmid pBBR1MCS2 to make pMO164PL and pMO188PL. Plasmid pMO188PL allowed better display of lipase and thus was chosen for further study. The display of lipase on the surface of P. putida KT2442 was confirmed by Western blot analysis, immunofluorescence microscopy, and measurement of whole-cell lipase activity. The whole-cell lipase activity of recombinant P. putida KT2442 harboring pMO188PL was more than fivefold higher than that of recombinant Escherichia coli displaying lipase in the same manner. Cell surface-displayed lipase exhibited the highest activity at 47 degrees C and pH 9.0, and the whole-cell lipase activity was greater than 90% of the initial activity in organic solvents at 47 degrees C for 1 week. In a biocatalytic application, enantioselective resolution of 1-phenyl ethanol was carried out in an organic solvent. (R)-Phenyl ethyl acetate was successfully produced with 41.9% conversion and an enantiomeric excess of more than 99% in a 36-h reaction. These results suggest that the OprF anchor can be used for efficient display of proteins in P. putida KT2442 and consequently for various biocatalytic applications.
authors.affiliation: Department of Chemical & Biomolecular Engineering, Korea Advanced Institute of Science and Technology, 373-1 Guseong-dong, Yuseong-gu, Daejeon 305-701, Republic of Korea.
authors.forename: Seung Hwan
authors.initials: SH
authors.lastname: Lee
authors.forename: Sang Yup
authors.initials: SY
authors.lastname: Lee
authors.forename: Byoung Chul
authors.initials: BC
authors.lastname: Park
mesh_terms: D002384:Catalysis; D002462:Cell Membrane / Q000201:enzymology; D005434:Flow Cytometry; D007700:Kinetics; D008049:Lipase / Q000378:metabolism*; D010957:Plasmids; D018272:Porins / Q000235:genetics / Q000378:metabolism*; D016958:Pseudomonas putida / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D018272:Porins; D011994:Recombinant Proteins; D008049:Lipase