scholarly journals Cell Surface Display of Lipase in Pseudomonas putida KT2442 Using OprF as an Anchoring Motif and Its Biocatalytic Applications

2005 ◽  
Vol 71 (12) ◽  
pp. 8581-8586 ◽  
Author(s):  
Seung Hwan Lee ◽  
Sang Yup Lee ◽  
Byoung Chul Park
Keyword(s):  
Cell Surface ◽  
Pseudomonas Putida ◽  
Lipase Activity ◽  
Surface Display ◽  
Cell Surface Display ◽  
Broad Host Range ◽  
Lipase Gene ◽  
Whole Cell ◽  
Enantioselective Resolution ◽  
Anchoring Motif

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°C and pH 9.0, and the whole-cell lipase activity was greater than 90% of the initial activity in organic solvents at 47°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.

scholarly journals Construction of Yeast Strains with High Cell Surface Lipase Activity by Using Novel Display Systems Based on the Flo1p Flocculation Functional Domain

2002 ◽  
Vol 68 (9) ◽  
pp. 4517-4522 ◽  
Author(s):  
Takeshi Matsumoto ◽  
Hideki Fukuda ◽  
Mitsuyoshi Ueda ◽  
Atsuo Tanaka ◽  
Akihiko Kondo
Keyword(s):  
Cell Surface ◽  
Lipase Activity ◽  
Fusion Proteins ◽  
Surface Display ◽  
Cell Surface Display ◽  
Yeast Cells ◽  
Upstream Region ◽  
Functional Domain ◽  
Display System ◽  
Cell Surface Display System

ABSTRACT We constructed a novel cell-surface display system, using as a new type of cell-wall anchor 3,297 or 4,341 bp of the 3′ region of the FLO1 gene (FS or FL gene, respectively), which encodes the flocculation functional domain of Flo1p. In this system, the N terminus of the target protein was fused to the FS or FL protein and the fusion proteins were expressed under the control of the inducible promoter UPR-ICL (5′ upstream region of the isocitrate lyase of Candida tropicalis). Using this new system, recombinant lipase with a pro sequence from Rhizopus oryzae (rProROL), which has its active site near the C terminus, was displayed on the cell surface. Cell-surface display of the FSProROL and FLProROL fusion proteins was confirmed by immunofluorescence microscopy and immunoblotting. Lipase activity reached 145 IU/liter (61.3 IU/g [dry cell weight]) on the surface of the yeast cells, which successfully catalyzed the methanolysis reaction. Using these whole-cell biocatalysts, methylesters synthesized from triglyceride and methanol reached 78.3% after 72 h of reaction. To our knowledge, this is the first example of cell-surface display of lipase with high activity. Interestingly, the yeast cells displaying the FLProROL protein showed strong flocculation, even though the glycosylphosphatidylinositol anchor attachment signal and cell-membrane-anchoring region of Flo1p had been deleted from this gene. The cell-surface display system based on FL thus endows the yeast strain with both novel enzyme display and strong flocculation ability.

scholarly journals Display of Polyhistidine Peptides on theEscherichia coli Cell Surface by Using Outer Membrane Protein C as an Anchoring Motif

1999 ◽  
Vol 65 (11) ◽  
pp. 5142-5147 ◽  
Author(s):  
Zhaohui Xu ◽  
Sang Yup Lee
Keyword(s):  
Membrane Protein ◽  
Cell Surface ◽  
Outer Membrane ◽  
Outer Membrane Protein ◽  
Protein C ◽  
Surface Display ◽  
Dry Weight ◽  
Cell Surface Display ◽  
Recombinant Cells ◽  
Anchoring Motif

ABSTRACT A novel cell surface display system was developed by employingEscherichia coli outer membrane protein C (OmpC) as an anchoring motif. Polyhistidine peptides consisting of up to 162 amino acids could be successfully displayed on the seventh exposed loop of OmpC. Recombinant cells displaying polyhistidine could adsorb up to 32.0 μmol of Cd2+ per g (dry weight) of cells.

Yeast cell surface display for lipase whole cell catalyst and its applications

2014 ◽  
Vol 106 ◽  
pp. 17-25 ◽  
Author(s):  
Yun Liu ◽  
Rui Zhang ◽  
Zhongshuai Lian ◽  
Shihui Wang ◽  
Aaron T. Wright
Keyword(s):  
Cell Surface ◽  
Yeast Cell ◽  
Surface Display ◽  
Cell Surface Display ◽  
Whole Cell ◽  
Yeast Cell Surface

scholarly journals Enhanced Display of Lipase on the Escherichia coli Cell Surface, Based on Transcriptome Analysis

2009 ◽  
Vol 76 (3) ◽  
pp. 971-973 ◽  
Author(s):  
Jong Hwan Baek ◽  
Mee-Jung Han ◽  
Seung Hwan Lee ◽  
Sang Yup Lee
Keyword(s):  
Escherichia Coli ◽  
Cell Surface ◽  
Transcriptome Analysis ◽  
Surface Display ◽  
Cell Surface Display ◽  
Display System ◽  
E Coli ◽  
A Cell ◽  
Anchoring Motif ◽  
Cell Surface Display System

ABSTRACT A cell surface display system was developed using Escherichia coli OmpC as an anchoring motif. The fused Pseudomonas fluorescens SIK W1 lipase was successfully displayed on the surface of E. coli cells, and the lipase activity could be enhanced by the coexpression of the gadBC genes identified by transcriptome analysis.

scholarly journals Display of Bacterial Lipase on the Escherichia coli Cell Surface by Using FadL as an Anchoring Motif and Use of the Enzyme in Enantioselective Biocatalysis

2004 ◽  
Vol 70 (9) ◽  
pp. 5074-5080 ◽  
Author(s):  
Seung Hwan Lee ◽  
Jong-Il Choi ◽  
Si Jae Park ◽  
Sang Yup Lee ◽  
Byoung Chul Park
Keyword(s):  
Escherichia Coli ◽  
Cell Surface ◽  
Kinetic Resolution ◽  
Surface Display ◽  
Active Form ◽  
Enantiomeric Excess ◽  
Fatty Acid Transport ◽  
Whole Cell ◽  
E Coli ◽  
Anchoring Motif

ABSTRACT We have developed a novel cell surface display system by employing FadL as an anchoring motif, which is an outer membrane protein involved in long-chain fatty acid transport in Escherichia coli. A thermostable Bacillus sp. strain TG43 lipase (44.5 kDa) could be successfully displayed on the cell surface of E. coli in an active form by C-terminal deletion-fusion of lipase at the ninth external loop of FadL. The localization of the truncated FadL-lipase fusion protein on the cell surface was confirmed by confocal microscopy and Western blot analysis. Lipase activity was mainly detected with whole cells, but not with the culture supernatant, suggesting that cell lysis was not a problem. The activity of cell surface-displayed lipase was examined at different temperatures and pHs and was found to be the highest at 50°C and pH 9 to 10. Cell surface-displayed lipase was quite stable, even at 60 and 70°C, and retained over 90% of the full activity after incubation at 50°C for a week. As a potential application, cell surface-displayed lipase was used as a whole-cell catalyst for kinetic resolution of racemic methyl mandelate. In 36 h of reaction, (S)-mandelic acid could be produced with the enantiomeric excess of 99% and the enantiomeric ratio of 292, which are remarkably higher than values obtained with crude lipase or cross-linked lipase crystal. These results suggest that FadL may be a useful anchoring motif for displaying enzymes on the cell surface of E. coli for whole-cell biocatalysis.

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