Weon Bae

Enhanced bioaccumulation of heavy metals by bacterial cells displaying synthetic phytochelatins

A novel strategy using synthetic phytochelatins is described for the purpose of developing microbial agents for enhanced bioaccumulation of toxic metals. Synthetic genes encoding for several metal-chelating phytochelatin analogs (Glu-Cys)(n)Gly (EC8 (n = 8), EC11 (n = 11), and EC20 (n = 20)) were synthesized, linked to a lpp-ompA fusion gene, and displayed on the surface of E. coli. For comparison, EC20 was also expressed periplasmically as a fusion with the maltose-binding protein (MBP-EC20). Purified MBP-EC20 was shown to accumulate more Cd(2+) per peptide than typical mammalian metallothioneins with a stoichiometry of 10 Cd(2+)/peptide. Cells displaying synthetic phytochelatins exhibited chain-length dependent increase in metal accumulation. For example, 18 nmoles of Cd(2+)/mg dry cells were accumulated by cells displaying EC8, whereas cells exhibiting EC20 accumulated a maximum of 60 nmoles of Cd(2+)/mg dry cells. Moreover, cells with surface-expressed EC20 accumulated twice the amount of Cd(2+) as cells expressing EC20 periplasmically. The ability to genetically engineer ECs with precisely defined chain length could provide an attractive strategy for developing high-affinity bioadsorbents suitable for heavy metal removal.

Novel synthetic phytochelatin-based capacitive biosensor for heavy metal ion detection.

A novel capacitance biosensor based on synthetic phytochelatins for sensitive detection of heavy metals is described. Synthetic phytochelatin (Glu-Cys)(20)Gly (EC20) fused to the maltose binding domain protein was expressed in Escherichia coli and purified for construction of the biosensor. The new biosensor was able to detect Hg(2+), Cd(2+), Pb(2+), Cu(2+) and Zn(2+) ions in concentration range of 100 fM-10 mM, and the order of sensitivity was S(Zn)>S(Cu)>S(Hg)>>S(Cd) congruent with S(Pb). The biological sensing element of the sensor could be regenerated using EDTA and the storage stability of the biosensor was 15 days.

Enhanced Mercury Biosorption by Bacterial Cells with Surface-Displayed MerR

ABSTRACT The metalloregulatory protein MerR, which exhibits high affinity and selectivity toward mercury, was exploited for the construction of microbial biosorbents specific for mercury removal. Whole-cell sorbents were constructed with MerR genetically engineered onto the surface of Escherichia coli cells by using an ice nucleation protein anchor. The presence of surface-exposed MerR on the engineered strains enabled sixfold-higher Hg2+ biosorption than that found in the wild-type JM109 cells. Hg2+ binding via MerR was very specific, with no observable decline even in the presence of 100-fold excess Cd2+ and Zn2+. The Hg2+ binding property of the whole-cell sorbents was also insensitive to different ionic strengths, pHs, and the presence of metal chelators. Since metalloregulatory proteins are currently available for a wide variety of toxic heavy metals, our results suggest that microbial biosorbents overexpressing metalloregulatory proteins may be used similarly for the cleanup of other important heavy metals.

Genetic engineering of Escherichia coli for enhanced uptake and bioaccumulation of mercury.

ABSTRACT Synthetic phytochelatins (ECs) are a new class of metal-binding peptides with a repetitive metal-binding motif, (Glu-Cys)nGly, which were shown to bind heavy metals more effectively than metallothioneins. However, the limited uptake across the cell membrane is often the rate-limiting factor for the intracellular bioaccumulation of heavy metals by genetically engineered organisms expressing these metal-binding peptides. In this paper, two potential solutions were investigated to overcome this uptake limitation either by coexpressing an Hg2+ transport system with (Glu-Cys)20Gly (EC20) or by directly expressing EC20 on the cell surface. Both approaches were equally effective in increasing the bioaccumulation of Hg2+. Since the available transport systems are presently limited to only a few heavy metals, our results suggest that bioaccumulation by bacterial sorbents with surface-expressed metal-binding peptides may be useful as a universal strategy for the cleanup of heavy metal contamination.

Properties of glutathione- and phytochelatin-capped CdS bionanocrystallites

Abstract The incorporation of inorganic sulfide into cadmium–glutathione (GSH) led to the formation of a variety of GSH-capped CdS (GSH–CdS) complexes that differed in sulfide/Cd(II) ratios, optical spectroscopic properties and Cd(II)-binding capacity of GSH. The size-fractionation of GSH–CdS complexes indicated that the Cd(II)/GSH molar ratio increased from a minimum of 0.3 to a maximum of 25 as sulfide/Cd(II) molar ratio increased from 0 to ∼1.0 equivalent. The absorption shoulders in the 290–400 nm range, photoluminescence in the 400–550 nm range and the ability to reduce methylviologen indicated that these GSH–CdS complexes behaved like semiconductor nanocrystallites (NCs). The predicted radii of (GSH–CdS) NCs varied from 10.8 to 17.3 A. Unlike GSH, phytochelatins (PCs) formed CdS crystallites that appeared uniform in size as was indicated by their similar optical properties. Although the properties of PC-capped CdS (PC-CdS) complexes were controllable by altering the amounts of sulfide titrated, sulfide-induced transitions in the electronic absorption spectrum (indicative of the crystallite size) were limited to the blue of 318 nm. Thus, the maximum predicted radius of PC-capped crystallites was 11.8 A. The titration of PCs into GSH–CdS led to the replacement of GSH with PCs. Interestingly, the displacement of GSH by PCs did not alter the size of CdS particles as indicated by lack of changes in emission λ max or in the characteristic absorption shoulder at 358 nm. However, emission yields were quantitatively decreased upon displacement of GSH with PCs.

Cysteine-capped ZnS nanocrystallites: Preparation and characterization

Abstract Cysteine-capped ZnS nanocrystalline semiconductors (NCs) were prepared by titrating sodium sulfide into preformed Zn–cysteine complexes. Only a maximum of ∼40% of Zn(II) in Zn–cysteine complex was converted into ZnS NCs when the reaction was carried out at room temperature for 30 min. However, incubation of the reaction mixture at 45°C for 60 min significantly enhanced the production of ZnS NCs as a maximum of ∼75% of Zn(II) was converted into NCs. Cysteine capping produced NCs that were smaller than those capped by glutathione. Furthermore, cysteine-capped ZnS exhibited a narrow range of size distribution as suggested by UV/VIS spectroscopy of the NCs separated on a size-fractionation column. Unreacted Zn–cysteine complex could be removed from ZnS NC preparations by selective precipitation with ethanol. The precipitation procedure also led to the isolation of ZnS NCs that appeared more uniform by gel-filtration analysis. Ethanol precipitation procedures allowed preparation of large quantities of powdered NCs which retained their colloidal nature upon resuspension in water or buffers. pH titration experiments indicate that the average size of the particles was smallest in the pH range 7–10, but the size increased as the samples were made acidic or alkaline. Cysteine-capped NCs were capable of causing photoreduction of methylviologen, basic fuchsin and naphthol blue black.

Cell surface display of synthetic phytochelatins using ice nucleation protein for enhanced heavy metal bioaccumulation

Synthetic phytochelatins (ECs) composed of (Glu-Cys)nGly are protein analogs of phytochelatin that exhibit improved metal-binding capacity over metallothioneins (MTs). Expression of EC20 on the surface of E. coli using the Lpp-OmpA anchor resulted in improved bioaccumulation of cadmium and mercury, providing a new method for treating heavy metal contamination. To further improve the whole-cell accumulation of heavy metals, EC20 was expressed on the surface of Moraxella sp., a bacterium known to survive in contaminated environments, using the truncated ice nucleation protein (INPNC) anchor. Production of EC20 was approximately three-fold higher in Moraxella sp. than E. coli. As a consequence, the mercury-binding capacity of the recombinant Moraxella sp. was increased by more than 10-fold. Owing to the very high level of surface expression, the use of Moraxella sp. and INPNC anchor may prove to be useful for the remediation of other environmental contaminants.

METAL-BINDING CHARACTERISTICS OF A PHYTOCHELATIN ANALOG (GLU-CYS)2GLY

Abstract The present studies were undertaken to explore the possibility of using synthetically designed genes encoding phytochelatin analog (Glu-Cys) n Gly peptides in transgenic plants for phytoremediation. We studied the metal-chelating characteristics of a synthetically prepared phytochelatin analog peptide (Glu-Cys) 2 Gly to determine if a gene encoding such a peptide might be useful in phytoremediation, as a first step. Studies with Cd(II), Hg(II), and Pb(II) show that the synthetic (Glu-Cys) 2 Gly peptide exhibits metal-chelating properties similar to the phytochelatin (γ Glu-Cys) 2 Gly. GSH-bound metals were also shown to be quantitatively transferred to (Glu-Cys) 2 Gly. The Cd(II)-form of the synthetic (Glu-Cys) 2 Gly peptide-like PCs was able to form stable complexes with sulfide. The spectroscopic properties of (Glu-Cys) 2 Gly-coated complexes of CdS were comparable to those exhibited by (γ Glu-Cys) 2 Gly-coated CdS particles. Both (γ Glu-Cys) 2 Gly and (Glu-Cys) 2 Gly exhibited a Cd-binding stoichiometry of 0.5 Cd per peptide molecule. UV-visible, HPLC, and mass-spectral analyses indicated that one Hg(II) ion was chelated by each molecule of (γ Glu-Cys) 2 Gly or (Glu-Cys) 2 Gly. Each molecule of (γ Glu-Cys) 2 Gly or (Glu-Cys) 2 Gly bound to one atom of Pb(II).

Biomolecularly capped uniformly sized nanocrystalline materials: glutathione-capped ZnS nanocrystals

Micro-organisms such as bacteria and yeasts form CdS to detoxify toxic cadmium ions. Frequently, CdS particles formed in yeasts and bacteria were found to be associated with specific biomolecules. It was later determined that these biomolecules were present at the surface of CdS. This coating caused a restriction in the growth of CdS particles and resulted in the formation of nanometre-sized semiconductors (NCs) that exhibited typical quantum confinement properties. Glutathione and related phytochelatin peptides were shown to be the biomolecules that capped CdS nanocrystallites synthesized by yeasts Candida glabrata and Schizosaccharomyces pombe. Although early studies showed the existence of specific biochemical pathways for the synthesis of biomolecularly capped CdS NCs, these NCs could be formed in vitro under appropriate conditions. We have recently shown that cysteine and cysteine-containing peptides such as glutathione and phytochelatins can be used in vitro to dictate the formation of discrete sizes of CdS and ZnS nanocrystals. We have evolved protocols for the synthesis of ZnS or CdS nanocrystals within a narrow size distribution range. These procedures involve three steps: (1) formation of metallo-complexes of cysteine or cysteine-containing peptides, (2) introduction of stoichiometric amounts of inorganic sulfide into the metallo-complexes to initiate the formation of nanocrystallites and finally (3) size-selective precipitation of NCs with ethanol in the presence of Na+. The resulting NCs were characterized by optical spectroscopy, high-resolution transmission electron microscopy (HRTEM), x-ray diffraction and electron diffraction. HRTEM showed that the diameter of the ZnS-glutathione nanocrystals was 3.45?0.5 nm. X-ray diffraction and electron diffraction analyses indicated ZnS-glutathione to be hexagonal. Photocatalytic studies suggest that glutathione-capped ZnS nanocrystals prepared by our procedure are highly efficient in degrading a test model compound.

Cysteine-mediated synthesis of CdS bionanocrystallites

Abstract The synthesis of CdS bionanocrystallites has been studied by reacting Cd(II)-cysteine complexes with inorganic sulfide. Cysteine-mediated synthesis of CdS bionanocrystallites proceeded slightly faster at 45°C than at room temperature. The sizes of these CdS bionanocrystallites, as determined by optical spectroscopy, increased with increasing initial sulfide/Cd(II) ratios. Analyses of size-distribution showed significant heterogeneity in sizes only at sulfide/Cd(II) ratios of 0.25. An ethanol precipitation procedure was developed to remove unreacted Cd(II)-cysteine complexes. This procedure also resulted in the isolation of CdS bionanocrystallites (bioNCs) that appeared uniform in size and chemical composition. Cysteine behaved like glutathione insofar as the size-range of CdS particles was concerned. However, cysteine-mediated synthesis of CdS bioNCs resulted in uniformly sized-particles as has been observed previously with phytochelatins. Cysteine-capped CdS particles exhibited pH-dependent changes in their properties. pH-induced changes were more pronounced in emission than in absorption spectra. Photocatalytic activities of these bioNCs were indicated by reduction of three dyes. Irradiation of methylviologen, basic fuchsin and naphthol blue black at 366 nm in the presence of cysteine-capped CdS bioNCs caused reduction of these dyes. Samples exhibiting higher luminescence were also more active in the photocatalysis experiments.