Wilfred Chen

Biosensors for direct determination of organophosphate pesticides.

Direct, selective, rapid and simple determination of organophosphate pesticides has been achieved by integrating organophosphorus hydrolase with electrochemical and opitical transducers. Organophosphorus hydrolase catalyzes the hydrolysis of a wide range of organophosphate compounds, releasing an acid and an alcohol that can be detected directly. This article reviews development, characterization and applications of organophosphorus hydrolase-based potentiometric, amperometric and optical biosensors.

Engineering Plant-Microbe Symbiosis for Rhizoremediation of Heavy Metals

ABSTRACT The use of plants for rehabilitation of heavy-metal-contaminated environments is an emerging area of interest because it provides an ecologically sound and safe method for restoration and remediation. Although a number of plant species are capable of hyperaccumulation of heavy metals, the technology is not applicable for remediating sites with multiple contaminants. A clever solution is to combine the advantages of microbe-plant symbiosis within the plant rhizosphere into an effective cleanup technology. We demonstrated that expression of a metal-binding peptide (EC20) in a rhizobacterium, Pseudomonas putida 06909, not only improved cadmium binding but also alleviated the cellular toxicity of cadmium. More importantly, inoculation of sunflower roots with the engineered rhizobacterium resulted in a marked decrease in cadmium phytotoxicity and a 40% increase in cadmium accumulation in the plant root. Owing to the significantly improved growth characteristics of both the rhizobacterium and plant, the use of EC20-expressing P. putida endowed with organic-degrading capabilities may be a promising strategy to remediate mixed organic-metal-contaminated sites.

Bacterial cell surface display of organophosphorus hydrolase for selective screening of improved hydrolysis of organophosphate nerve agents.

ABSTRACT Organophosphorus hydrolase (OPH) is a bacterial enzyme that has been shown to degrade a wide range of neurotoxic organophosphate nerve agents. However, the effectiveness of degradation varies dramatically, ranging from highly efficient with paraoxon to relatively slow with methyl parathion. Sequential cycles of DNA shuffling and screening were used to fine-tune and enhance the activity of OPH towards poorly degraded substrates. Because of the inaccessibility of these pesticides across the cell membrane, OPH variants were displayed on the surface of Escherichia coli using the truncated ice nucleation protein in order to isolate novel enzymes with truly improved substrate specificities. A solid-phase top agar method based on the detection of the yellow product p-nitrophenol was developed for the rapid prescreening of potential variants with improved hydrolysis of methyl parathion. Two rounds of DNA shuffling and screening were carried out, and several improved variants were isolated. One variant in particular, 22A11, hydrolyzes methyl parathion 25-fold faster than does the wild type. Because of the success that we achieved with directed evolution of OPH for improved hydrolysis of methyl parathion, we believe that we can easily extend this method in creating other OPH variants with improved activity against poorly degraded pesticides such as diazinon and chlorpyrifos and nerve agents such as sarin and soman.

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.

Functional Assembly of Minicellulosomes on the Saccharomyces cerevisiae Cell Surface for Cellulose Hydrolysis and Ethanol Production

ABSTRACT We demonstrated the functional display of a miniscaffoldin on the Saccharomyces cerevisiae cell surface consisting of three divergent cohesin domains from Clostridium thermocellum (t), Clostridium cellulolyticum (c), and Ruminococcus flavefaciens (f). Incubation with Escherichia coli lysates containing an endoglucanase (CelA) fused with a dockerin domain from C. thermocellum (At), an exoglucanase (CelE) from C. cellulolyticum fused with a dockerin domain from the same species (Ec), and an endoglucanase (CelG) from C. cellulolyticum fused with a dockerin domain from R. flavefaciens (Gf) resulted in the assembly of a functional minicellulosome on the yeast cell surface. The displayed minicellulosome retained the synergistic effect for cellulose hydrolysis. When a β-glucosidase (BglA) from C. thermocellum tagged with the dockerin from R. flavefaciens was used in place of Gf, cells displaying the new minicellulosome exhibited significantly enhanced glucose liberation and produced ethanol directly from phosphoric acid-swollen cellulose. The final ethanol concentration of 3.5 g/liter was 2.6-fold higher than that obtained by using the same amounts of added purified cellulases. The overall yield was 0.49 g of ethanol produced per g of carbohydrate consumed, which corresponds to 95% of the theoretical value. This result confirms that simultaneous and synergistic saccharification and fermentation of cellulose to ethanol can be efficiently accomplished with a yeast strain displaying a functional minicellulosome containing all three required cellulolytic enzymes.

Biosensor for direct determination of organophosphate nerve agents. 1. Potentiometric enzyme electrode

A potentiometric enzyme electrode for the direct measurement of organophosphate (OP) nerve agents was developed. The basic element of this enzyme electrode was a pH electrode modified with an immobilized organophosphorus hydrolase (OPH) layer formed by cross-linking OPH with bovine serum albumin (BSA) and glutaradehyde. OPH catalyses the hydrolysis of organophosphorus pesticides to release protons, the concentration of which is proportional to the amount of hydrolysed substrate. The sensor signal and response time was optimized with respect to the buffer pH, ionic concentration of buffer, temperature, and units of OPH immobilized using paraoxon as substrate. The best sensitivity and response time were obtained using a sensor constructed with 500 IU of OPH and operating in pH 8.5, 1 mM HEPES buffer. Using these conditions, the biosensor was used to measure as low as 2 microM of paraoxon, ethyl parathion, methyl parathion and diazinon. The biosensor was completely stable for at least one month when stored in pH 8.5, 1 mM HEPES + 100 mM NaCl buffer at 4 degrees C.

Amperometric Thick-Film Strip Electrodes for Monitoring Organophosphate Nerve Agents Based on Immobilized Organophosphorus Hydrolase

An amperometric biosensor based on the immobilization of organophosphorus hydrolase (OPH) onto screen-printed carbon electrodes is shown useful for the rapid, sensitive, and low-cost detection of organophosphate (OP) nerve agents. The sensor relies upon the sensitive and rapid anodic detection of the enzymatically generated p-nitrophenol product at the OPH/Nafion layer immobilized onto the thick-film electrode in the presence of the OP substrate. The amperometric signals are linearly proportional to the concentration of the hydrolyzed paraoxon and methyl parathion substrates up to 40 and 5 μM, showing detection limits of 9 × 10(-)(8) and 7 × 10(-)(8) M, respectively. Such detection limits are substantially lower compared to the (2-5) × 10(-)(6) M values reported for OPH-based potentiometric and fiber-optic devices. The high sensitivity is coupled to a faster and simplified operation, and the sensor manifests a selective response compared to analogous enzyme inhibition biosensors. The applicability to river water sampling is illustrated. The attractive performance and greatly simplified operation holds great promise for on-site monitoring of OP pesticides.

Enhanced Arsenic Accumulation in Engineered Bacterial Cells Expressing ArsR

ABSTRACT The metalloregulatory protein ArsR, which offers high affinity and selectivity toward arsenite, was overexpressed in Escherichia coli in an attempt to increase the bioaccumulation of arsenic. Overproduction of ArsR resulted in elevated levels of arsenite bioaccumulation but also a severe reduction in cell growth. Incorporation of an elastin-like polypeptide as the fusion partner to ArsR (ELP153AR) improved cell growth by twofold without compromising the ability to accumulate arsenite. Resting cells overexpressing ELP153AR accumulated 5- and 60-fold-higher levels of arsenate and arsenite than control cells without ArsR overexpression. Conversely, no significant improvement in Cd2+ or Zn2+ accumulation was observed, validating the specificity of ArsR. The high affinity of ArsR allowed 100% removal of 50 ppb of arsenite from contaminated water with these engineered cells, providing a technology useful to comply with the newly approved U.S. Environmental Protection Agency limit of 10 ppb. These results open up the possibility of using cells overexpressing ArsR as an inexpensive, high-affinity ligand for arsenic removal from contaminated drinking and ground water.

Surface Display of a Functional Minicellulosome by Intracellular Complementation Using a Synthetic Yeast Consortium and Its Application to Cellulose Hydrolysis and Ethanol Production

ABSTRACT In this paper, we report the surface assembly of a functional minicellulosome by using a synthetic yeast consortium. The basic design of the consortium consisted of four different engineered yeast strains capable of either displaying a trifunctional scaffoldin, Scaf-ctf (SC), carrying three divergent cohesin domains from Clostridium thermocellum (t), Clostridium cellulolyticum (c), and Ruminococcus flavefaciens (f), or secreting one of the three corresponding dockerin-tagged cellulases (endoglucanase [AT], exoglucanase [EC/CB], or β-glucosidase [BF]). The secreted cellulases were docked onto the displayed Scaf-ctf in a highly organized manner based on the specific interaction of the three cohesin-dockerin pairs employed, resulting in the assembly of a functional minicellulosome on the yeast surface. By exploiting the modular nature of each population to provide a unique building block for the minicellulosome structure, the overall cellulosome assembly, cellulose hydrolysis, and ethanol production were easily fine-tuned by adjusting the ratio of different populations in the consortium. The optimized consortium consisted of a SC:AT:CB:BF ratio of 7:2:4:2 and produced almost twice the level of ethanol (1.87 g/liter) as a consortium with an equal ratio of the different populations. The final ethanol yield of 0.475 g of ethanol/g of cellulose consumed also corresponded to 93% of the theoretical value. This result confirms the use of a synthetic biology approach for the synergistic saccharification and fermentation of cellulose to ethanol by using a yeast consortium displaying a functional minicellulosome.

Single-walled carbon nanotube-based chemiresistive affinity biosensors for small molecules: ultrasensitive glucose detection.

We report for the first time single-walled carbon nanotube (SWNT)-based chemiresistive affinity sensors for highly sensitive and selective detection of small and/or weakly charged or uncharged molecules using a displacement format. The detection of glucose, a small, weakly charged molecule, by displacement of plant lectin (concavalin A) bound to a polysaccharide (dextran) immobilized on SWNTs with picomolar sensitivity and selectivity over other sugars and human serum proteins is demonstrated as a proof of concept.