Development of a whole-cell biocatalyst for diisobutyl phthalate degradation by functional display of a carboxylesterase on the surface of Escherichia coli

Abstract

Background: Phthalic acid esters (PAEs) are widely used as plasticizers or additives during the industrial manufacturing of plastic products. PAEs have been detected in both aquatic and terrestrial environments due to their overuse. Exposure of PAEs results in human health concerns and environmental pollution. Diisobutyl phthalate is one of the main plasticizers in PAEs. Cell surface display of recombinant proteins has become a powerful tool for biotechnology applications. In this current study, a carboxylesterase was displayed on the surface of Escherichia coli cells, for use as whole-cell biocatalyst in diisobutyl phthalate biodegradation.

Results: A carboxylesterase-encoding gene (carEW) identified from Bacillus sp. K91, was fused to the N-terminal of ice nucleation protein (inpn) anchor from Pseudomonas syringae and gfp gene, and the fused protein was then cloned into pET-28a(+) vector and was expressed in Escherichia coli BL21(DE3) cells. The surface localization of INPN-CarEW/or INPN-CarEW-GFP fusion protein was confirmed by SDS-PAGE, western blot, proteinase accessibility assay, and green fluorescence measurement. The catalytic activity of the constructed E. coli surface-displayed cells was determined. The cell-surface-displayed CarEW displayed optimal temperature of 45 °C and optimal pH of 9.0, using p-NPC₂ as substrate. In addition, the whole cell biocatalyst retained ~ 100% and ~ 200% of its original activity per OD₆₀₀ over a period of 23 days at 45 °C and one month at 4 °C, exhibiting the better stability than free CarEW. Furthermore, approximately 1.5 mg/ml of DiBP was degraded by 10 U of surface-displayed CarEW cells in 120 min.

Conclusions: This work provides a promising strategy of cost-efficient biodegradation of diisobutyl phthalate for environmental bioremediation by displaying CarEW on the surface of E. coli cells. This approach might also provide a reference in treatment of other different kinds of environmental pollutants by displaying the enzyme of interest on the cell surface of a harmless microorganism.

Keywords: Carboxylesterase; Cell surface display; Diisobutyl phthalate; Phthalic acid esters; Whole-cell biocatalyst.

Fig. 1

Multiple sequence alignment between CarEW and some previously reported esterases with PAEs biodegradation capacities. Sequences retrieved from the NCBI database and were aligned by CLUSTAL W and were rendered using ESPript output. Sequences are grouped according to similarity. Esterase with a known three-dimensional structure (PDB: 1QE3) from Bacillus subtilis; KMW28714.1, Carboxylesterase from Sphingobium yanoikuyae; AGY55960.1, DphB from metagenomics library; AEW03609.1, EstS1 from Sulfobacillus acidophilus DSM 10332; AFK31309.1, PE-hydrolase from Acinetobacter sp. M673; WP_023629646.1, alpha/beta hydrolase from Pseudomonas mosselii; ABH00399.1, PatE from Rhodococcus jostii RHA1. Conserved amino acids are highlighted in a yellow font on a white background. The analysis revealed the presence of tripeptide HGG (red dots on top of the sequences) and PVMVW (underline in red) in most of test strains. Symbols above sequences represent the secondary structure, springs represent helices, and arrows represent β-strands

Fig. 2

Expression of recombinant fusion proteins: CarEW, CarEW-GFP and INPN-CarEW-GFP. (a) SDS-PAGE and (b) western blot of lysates of E. coli harboring pET-28a(+) series plasmids. Lane M, protein marker; Lane 1, E. coli cells harboring pET-28a(+); Lane 2, E. coli cells harboring pET-28a(+)/carEW; Lane 3, E. coli cells harboring pET-28a(+)/carEW/gfp; Lane 4, 5, 6, cytoplasmic fraction, inner membrane, and outer membrane of E. coli cells harboring pET-28a(+)/inpn/carEW/gfp, respectively. Anti-His monoclonal antibody was used as a 1:1000 dilution

Fig. 3

Fluorescence micrographs of recombinant E. coli BL21 (DE3) strain. aE. coli BL21(DE3) cells carrying pET-28a(+)/carEW/gfp and b pET-28a(+)/inpn/carEW/gfp, respectively. Left panel, microphotographs were taken under visible light; Right panel, fluorescence microphotographs

Fig. 4

a The optimum temperature, b pH, and c Long-term stability of the whole cell biocatalyst. Residual activities were determined periodically for over a month. Values are the means of three replicates ± the standard deviation

Fig. 5

DiBP degradation by purified CarEW and the whole cell biocatalyst. The error bars represent the mean ± SD (n = 3)

Fig. 6

The schematic diagram for CarEW surface display engineered strain construction and its applications for PAEs biodegradation