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

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 degrees C and pH 9 to 10. Cell surface-displayed lipase was quite stable, even at 60 and 70 degrees C, and retained over 90% of the full activity after incubation at 50 degrees 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.

FIG. 1.

Reaction scheme for enantioselective resolution of racemic methyl mandelate by using lyophilized cells of recombinant E. coli XL10-Gold displaying the FadL_t-lipase fusion protein.

FIG. 2.

Plasmids used for the display of lipase: pTrc99A (Pharmacia Biotech, Uppsala, Sweden), pTrcFadL, truncated fadL of E. coli; pTrcFadLE, truncated fadL of E. coli containing the stop codon; pTrcFadLBL, truncated fadL-Bacillus sp. strain TG43 lipase gene; pTrcFadLBLH, truncated fadL-Bacillus sp. strain TG43 lipase-His₆ fusion gene at the C terminal; pTrcBL, Bacillus sp. strain TG43 lipase. Abbreviations: E, EcoRI; X, XbaI; H, HindIII; P_trc, trc promoter; Ap^r, β-lactamase gene.

FIG. 3.

SDS-PAGE analysis (A) and immunoblotting (B) of recombinant E. coli XL10-Gold cells expressing FadL_t and FadL_t-lipase-His₆ fusion proteins. Lane 1, molecular mass standards; lane 2, whole-cell lysates of E. coli XL10-Gold harboring pTrcFadLE; lane 3, whole-cell lysates of E. coli XL10-Gold harboring pTrcFadLBLH; lane 4, outer membrane fraction of E. coli XL10-Gold harboring pTrcFadLBLH.

FIG. 4.

Differential interference micrographs (upper) and immunofluorescence micrographs (lower) of XL10-Gold cells harboring pTrcFadLE (A) and pTrcFadLBLH (B). Cells were incubated with rabbit anti-His probe antibody followed by probing with goat anti-rabbit IgG-FITC conjugate.

FIG. 5.

Effect of temperature on lipase activity of E. coli XL10-Gold (pTrcFadLBL). The enzyme activity was determined at pH 8.0 by using p-nitrophenyl decanoate as the substrate. Relative activity was calculated by assuming the activity obtained at 60°C was 100%.

FIG. 6.

Effect of pH on lipase activity of E. coli XL10-Gold (pTrcFadLBL). The enzyme activity was determined at 37°C by using p-nitrophenyl decanoate as the substrate. Buffers used were 50 mM potassium phosphate buffer (▿) and 50 mM Tris-HCl (▵). Relative activity was calculated by assuming the activity obtained at pH 9.0 was 100%.

FIG. 7.

Stability of lipase displayed on the cell surface of E. coli XL10-Gold (pTrcFadLBL) cells during prolonged incubation at pH 8.0 and 37°C (○) or 50°C (□). The enzyme activity was determined at 37°C by using p-nitrophenyl decanoate as the substrate. Relative activity was calculated by assuming the initial activity was 100%.