Development of bisphenol A-removing recombinant Escherichia coli by monomeric and dimeric surface display of bisphenol A-binding peptide.

Peptide-displaying Escherichia coli cells were investigated for use in adsorptive removal of bisphenol A (BPA) both in Luria-Bertani medium including BPA or ATM thermal paper eluted wastewater. Two recombinant strains were constructed with monomeric and dimeric repeats of the 7-mer BPA-binding peptide (KSLENSY), respectively. Greater than threefold increased adsorption of BPA [230.4 µmol BPA per g dry cell weight (DCW)] was found in dimeric peptide-displaying cells compared to monomeric strains (63.4 µmol per g DCW) in 15 ppm BPA solution. The selective removal of BPA from a mixture of BPA analogs (bisphenol F and bisphenol S) was verified in both monomeric and dimeric peptide-displaying cells. The binding chemistry of BPA with the peptide was assumed, based on molecular docking analysis, to be the interaction of BPA with serine and asparagine residues within the 7-mer peptide sequence. The peptide-displaying cells also functioned efficiently in thermal paper eluted wastewater containing 14.5 ppm BPA.

Construction of malate-sensing Escherichia coli by introduction of a novel chimeric two-component system.

In an attempt to develop a high-throughput screening system for screening microorganisms which produce high amounts of malate, a MalKZ chimeric HK-based biosensor was constructed. Considering the sequence similarity among Escherichia coli (E. coli) MalK with Bacillus subtilis MalK and E. coli DcuS, the putative sensor domain of MalK was fused with the catalytic domain of EnvZ. The chimeric MalK/EnvZ TCS induced the ompC promoter through the cognate response regulator, OmpR, in response to extracellular malate. Real-time quantitative PCR and GFP fluorescence studies showed increased ompC gene expression and GFP fluorescence as malate concentration increased. By using this strategy, various chimeric TCS-based bacteria biosensors can be constructed, which may be used for the development of biochemical-producing recombinant microorganisms.

Construction of a high efficiency copper adsorption bacterial system via peptide display and its application on copper dye polluted wastewater.

For the construction of an efficient copper waste treatment system, a cell surface display strategy was employed. The copper adsorption ability of recombinant bacterial strains displaying three different copper binding peptides were evaluated in LB Luria-Bertani medium (LB), artificial wastewater, and copper phthalocyanine containing textile dye industry wastewater samples. Structural characteristics of the three peptides were also analyzed by similarity-based structure modeling. The best binding peptide was chosen for the construction of a dimeric peptide display and the adsorption ability of the monomeric and dimeric peptide displayed strains were compared. The dimeric peptide displayed strain showed superior copper adsorption in all three tested conditions (LB, artificial wastewater, and textile dye industry wastewater). When the strains were exposed to copper phthalocyanine dye polluted wastewater, the dimeric peptide display [543.27 µmol/g DCW dry cell weight (DCW)] showed higher adsorption of copper when compared with the monomeric strains (243.53 µmol/g DCW).

The Selective Removal of Bisphenol A Using a Magnetic Adsorbent Fused with Bisphenol A-Binding Peptides.

The potential of bisphenol A (BPA)-binding peptides fused to magnetic beads is demonstrated as novel adsorbents that are reusable and highly selective for BPA removal from aqueous environments, in which various interfering substances coexist. Magnetic beads harboring peptides (peptide beads) showed a higher BPA removal capacity (8.6 mg/g) than that of bare beads without peptides (2.0 mg/g). The BPA adsorption capacity of peptide beads increased with the number of peptides fused onto the beads, where monomeric, dimeric, or trimeric repeats of a BPA-binding peptide were fused to magnetic beads. The BPA-adsorbing beads were regenerated using a methanol-acetic acid mixture, and after six regeneration cycles, the adsorption capacity remained above 87% of its initial capacity. The selective removal of BPA was confirmed in the presence of BPA analogs with high structural similarity (bisphenol F and bisphenol S) or in synthetic wastewater. The present work is a pioneering study that investigates the selective affinity of peptides to remove specific organics with high selectivity from complex environmental matrices.

Modification of response behavior of zinc sensing HydHG two-component system using a self-activation loop and genomic integration.

Characterizing the dynamics of HydHG-a two-component transcriptional regulatory network for exogenous zinc in E. coli-is essential in understanding the biology of these regulatory and signaling pathways. Here, we used a synthetic biology strategy to modify the dynamic characteristics of the HydHG network in two ways. First, a self-activation loop for HydHG network was created under the control of zraP promoter, after which the threshold Zn(2+) concentration for the self-activated HydHG network significantly decreased from 200 to 10 µM. Second, the self-activation loop was integrated into the E. coli genome allowing the threshold Zn(2+) concentration to be elevated to 500 µM. As the threshold Zn(2+) concentration could be modified in both directions, the introduction of a self-activation loop and the entire genomic integration strategy may prove useful for the creation of a two-component bacterial biosensor with varying sensitivities.

Modification of CusSR bacterial two-component systems by the introduction of an inducible positive feedback loop.

The CusSR two-component system (TCS) is a copper-sensing apparatus of E. coli that is responsible for regulating the copper-related homeostatic system. The dynamic characteristics of the CusSR network were modified by the introduction of a positive feedback loop. To construct the feedback loop, the CusR, which is activated by the cusC promoter, was cloned downstream of the cusC promoter and reporter protein. The feedback loop system, once activated by environmental copper, triggers the activation of the cusC promoter, which results in the amplification of a reporter protein and CusR expression. The threshold copper concentration for the activation of the modified CusSR TCS network was lowered from 2,476.5 µg/l to 247.7 µg/l, which indicates a tenfold increase in sensitivity. The intensity of the output signal was increased twofold, and was maintained for 16 h. The strategy proposed in this study can also be applied to modify the dynamic characteristics of other TCSs.

Partial uncompetitive inhibition of horseradish peroxidase by a water-miscible ionic liquid [BMIM][MeSO4].

The ionic liquid, 1-butyl-3-methylimidazolium methylsulfate ([BMIM][MeSO(4)]), was used to investigate the catalytic mechanism of horseradish peroxidase (HRP). The ionic liquid decreased both K(m) and k(cat) values for the HRP-catalyzed oxidation of guaiacol (2-methoxyphenol) by H(2)O(2). These studies imply that [BMIM][MeSO(4)] inhibits the enzyme in an uncompetitive manner. The incorporation of substrate stabilization effects measured by a thermodynamic method into the partial uncompetitive inhibition scheme successfully describes HRP-catalysis in the presence of [BMIM][MeSO(4)], which participates as the inhibitor. The inhibition constant of the ionic liquid was 0.051 M. The turn-over number of the native HRP was almost 14-times higher than that of the HRP-ionic liquid complex indicating that [BMIM][MeSO(4)] does not form a dead-end complex with HRP.

A study on the dynamics of the zraP gene expression profile and its application to the construction of zinc adsorption bacteria.

Zinc ion plays essential roles in biological chemistry. Bacteria acquire Zn(2+) from the environment, and cellular concentration levels are controlled by zinc homeostasis systems. In comparison with other homeostatic systems, the ZraSR two-component system was found to be more efficient in responding to exogenous zinc concentrations. To understand the dynamic response of the bacterium ZraSR two-component system with respect to exogenous zinc concentrations, the genetic circuit of the ZraSR system was integrated with a reporter protein. This study was helpful in the construction of an E. coli system that can display selective metal binding peptides on the surface of the cell in response to exogenous zinc. The engineered bacterial system for monitoring exogenous zinc was successfully employed to detect levels of zinc as low as 0.001 mM, which directly activates the expression of chimeric ompC(t)--zinc binding peptide gene to remove zinc by adsorbing a maximum of 163.6 µmol of zinc per gram of dry cell weight. These results indicate that the engineered bacterial strain developed in the present study can sense the specific heavy metal and activates a cell surface display system that acts to remove the metal.

Optimization of enantioselective synthesis of methyl (R)-2-chloromandelate by whole cells of Saccharomyces cerevisiae.

Methyl (R)-2-chloromandelate, a key intermediate in the synthesis of clopidogrel, was obtained by the reduction of methyl-2-chlorobenzoylformate using whole cells of Saccharomyces cerevisiae. A 100% conversion and 96.1% of enantiomeric excess (ee) value was obtained when 17 methyl-2-chlorobenzoylformate/l was reacted with 8 g S. cerevisiae/l and 83 g glucose/l at pH 7.

Development of fenitrothion adsorbing recombinant Escherichia coli by cell surface display of pesticide-binding peptide.

In this study, constructed Escherichia coli could efficiently adsorb fenitrothion by displaying a pesticide-binding peptide on it using the anchoring motif OmpC. A codon-optimized, pesticide-binding peptide was attached to the C-terminus of OmpC at loop 7 (993 bp). The efficiency of fenitrothion binding by the monomer peptide was evaluated under different temperatures, pH levels, and fenitrothion concentrations. To enhance fenitrothion adsorption, a dimer of pesticide-binding peptide was also constructed and displayed. Compared with the peptide monomer, the dimer-displaying strain showed superior fenitrothion-binding ability. The performance of the strains was evaluated in artificial polluted soil, and their morphology was analyzed by FE-SEM. The results showed that these two kinds of constructed strains can adsorb fenitrothion in contaminated environments with no cellular activity reduction. ARTICLE INFO.

Effects of structural difference of ionic liquids on the catalysis of horseradish peroxidase.

The dependence of the catalytic properties of horseradish peroxidase on the structural changes of ionic liquids was investigated with two water-miscible ionic liquids, N-butyl-3- methypyridinium tetrafluoroborate ([BMPy][BF4]) and 1-butyl- 3-methylimidazolium methylsulfate ([BMIM][MeSO4]), each of which shares an anion (BF4 -) or a cation (BMIM+) with 1-butyl- 3-methylimidazolium tetrafluoroborate ([BMIM][BF4]), respectively. The oxidation of guaiacol (2-methoxyphenol) with H2O2 was used as a model reaction. In order to minimize the effect of solution viscosity on the kinetic constants of the enzymatic catalysis, the enzymatic reactions for the kinetic study were performed in water-ionic liquid mixtures containing 25% (v/v) ionic liquid at maximum. Similarly to the previously reported results for [BMIM][BF4], as the concentration of [BMPy][BF4] increased, the Km value increased with a decrease in the kcat value: the Km value increased markedly from 2.8 mM in 100% water to 12.6 mM in 25% (v/v) ionic liquid, indicating that ionic liquid significantly weakens the binding affinity of guaiacol to the enzyme. On the contrary, [BMIM][MeSO4] decreased the Km value to 1.4 mM in 25% (v/v) ionic liquid. [BMIM][MeSO4] also decreased kcat more than 3- folds [from 13.8 s-1 in 100% water to 4.1 s-1 in 25% (v/v) ionic liquid]. These results indicate that the ionic liquids interact with the enzyme at the molecular level as well as at a macroscopic thermodynamic scale. Specifically, the anionic component of the ionic liquids influenced the catalysis of horseradish peroxidase in different ways.

Chaperone-assisted column refolding of gloshedobin with the use of refolding cocktail.

Gloshedobin, a recently isolated thrombin-like enzyme from the snake venom of Gloydius shedaoensis, is expressed mainly in the form of inclusion bodies (IBs) in Escherichia coli due to its cysteine-rich nature. Following extraction and solubilization of the IBs, one-step immobilized metal affinity chromatography purification produces highly purified (>99%) denatured-solubilized gloshedobin ready to enter the subsequent refolding process. However, the traditional dilution or column refolding strategy, based on gradual denaturant removal, is found to be inefficient for the recovery of protein activity. In this study, a new refolding strategy harnessing the ClpB and DnaK/DnaJ/GrpE bichaperone system is demonstrated to be superior to the conventional refolding methods in either batch dilution or column refolding mode. It is noted that the efficacy of bichaperone-mediated column refolding strategy is further highlighted especially when refolding reaction is attempted at a higher protein concentration with the recirculation of the refolding cocktail containing the bichaperone system. This is evidenced by an uncompromised refolding efficiency (ca. 21.4%) achieved at 2000 microg/mL of initial protein concentration, which is comparable to the refolding efficiency (ca. 22.5%) obtained at 20 times lower protein concentration (i.e. 100 microg/mL) in the conventional batch dilution refolding technique. The demonstrated chaperone-assisted column refolding strategy thus provides an effective tool for refolding-recalcitrant proteins whose reactivation is otherwise difficult to achieve.

Effect of ionic liquid on the kinetics of peroxidase catalysis.

The effect of a water-miscible ionic liquid, 1-butyl-3-methylimidazolium tetrafluoroborate ([BMIM][BF4]), on the horseradish peroxidase (HRP)-catalyzed oxidation of 2-methoxyphenol (guaiacol) with hydrogen peroxide (H2O2) was investigated. HRP maintains its high activity in the aqueous mixtures containing various concentrations of the ionic liquid and even in 90% (v/v) ionic liquid. In order to minimize the effect of solution viscosity on the kinetic constants of HRP catalysis, the enzymatic reactions in the subsequent kinetic study were performed in water-ionic liquid mixtures containing 25% (v/v) ionic liquid at maximum. As the concentration of [BMIM][BF4] increased for the oxidation of guaiacol by HRP, the K(m) value increased with a slight decrease in the k(cat) value: The K(m) value increased from 2.8 mM in 100% (v/v) water to 22.5 mM in 25% (v/v) ionic liquid, indicating that ionic liquid significantly weakens the binding affinity of guaiacol to HRP.

Adsorptive interaction of bisphenol A with mesoporous titanosilicate/reduced graphene oxide nanocomposite materials: FT-IR and Raman analyses.

Nanocomposite materials containing graphene oxide have attracted tremendous interest as catalysts and adsorbents for water purification. In this study, mesoporous titanosilicate/reduced graphene oxide composite materials with different Ti contents were employed as adsorbents for removing bisphenol A (BPA) from water systems. The adsorptive interaction between BPA and adsorption sites on the composite materials was investigated by Fourier transform infrared (FT-IR) and Raman spectroscopy. Adsorption capacities of BPA at equilibrium, q e (mg/g), decreased with increasing Ti contents, proportional to the surface area of the composite materials. FT-IR observations for fresh and spent adsorbents indicated that BPA adsorbed onto the composite materials by the electrostatic interaction between OH functional groups contained in BPA and on the adsorbents. The electrostatic adsorption sites on the adsorbents were categorized into three hydroxyl groups: Si-OH, Ti-OH, and graphene-OH. In Raman spectra, the intensity ratios of D to G band were decreased after the adsorption of BPA, implying adsorptive interaction of benzene rings of BPA with the sp(2) hybrid structure of the reduced graphene oxide.

Highly sensitive and selective determination of bisphenol-A using peptide-modified gold electrode.

Fast and accurate determination of bisphenol A (BPA) in varying matrices has become important in recent years. In this study, a cysteine-flanked heptapeptide sequence Cys-Lys-Ser-Leu-Glu-Asn-Ser-Tyr-Cys (CKSLENSYC), which is capable of recognizing BPA with high specificity, was isolated using a phage display technique. A novel electrochemical biosensor harnessing this affinity peptide as a BPA detection probe, was constructed and its performance was assessed. The formation of a self-assembled peptide monolayer on the gold electrode was confirmed by attenuated total reflection infrared spectroscopy (ATR-IR), cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). Following the exploration of the optimum sensing condition, differential pulse voltammetry (DPV) was used to determine the varying concentrations of BPA in the solution. The developed sensor conveyed excellent performance in view of sensing speed, sensitivity and selectivity by detecting BPA in less than 5 min with a broad dynamic detection range of 1-5000 nM of BPA, despite the presence of several interfering species, such as phenolic compounds and inorganic ions.

Selective lead adsorption by recombinant Escherichia coli displaying a lead-binding peptide.

A highly specific lead-binding peptide ThrAsnThrLeuSerAsnAsn was displayed on Escherichia coli, and lead adsorption characteristics of the recombinant bacteria were investigated. Cell surface-displayed peptide was expressed under the control of an arabinose promoter using outer membrane protein C (OmpC(t)) as an anchoring motif. The optimal induction period and arabinose concentration for the expression of peptide-fused OmpC(t) were determined to be 2 h and 0.001 g/L, respectively. Selective adsorption of Pb(2+) onto recombinant cells was verified with individual or combinatory use of four metal ions, Pb(2+), Ni(2+), Co(2+), and Cu(2+); the amount of bound Pb(2+) onto the biosorbents was significantly higher than the other metal ions. The adsorption isotherm of recombinant cells for Pb(2+) followed the Langmuir isotherm with a maximum adsorption loading (q (max)) of 526 µmol/g dry cell weight.

Construction of copper removing bacteria through the integration of two-component system and cell surface display.

Synthetic biological systems are becoming more and more feasible for commercial and medical purposes through the genetic engineering of several components. The simple assembly of a genetic circuit was shown to stimulate the removal of copper by bacteria through the engineering of a two-component system. The CusSR two-component systems is a regulator of Escherichia coli copper homeostatic system. In this system, genetic circuits of CusSR were fused to a cell surface display system for metal adsorption; this system is suitable for the display of a copper binding peptide through outer membrane protein C (OmpC). E. coli ompC codes for an outer membrane pore protein (porin) are induced at high osmolarity and temperature, which can also be used as an anchoring motif to accept the passenger proteins. The bacteria that produce the chimeric OmpC containing the copper binding peptide adsorbed maximum concentrations of 92.2 µmol of Cu(2+)/gram dry weight of bacterial cells. This synthetic bacterial system senses the specific heavy metal and activates a cell surface display system that acts to remove the metal.

Chromatographic biopanning for the selection of peptides with high specificity to Pb2+ from phage displayed peptide library.

Toxic heavy metal pollution is a global problem occurring in air, soil as well as water. There is a need for a more cost effective, renewable remediation technique, but most importantly, for a recovery method that is selective for one specific metal of concern. Phage display technology has been used as a powerful tool in the discovery of peptides capable of exhibiting specific affinity to various metals or metal ions. However, traditional phage display is mainly conducted in batch mode, resulting in only one equilibrium state hence low-efficiency selection. It is also unable to monitor the selection process in real time mode. In this study, phage display technique was incorporated with chromatography procedure with the use of a monolithic column, facilitating multiple phage-binding equilibrium states and online monitoring of the selection process in search of affinity peptides to Pb2+. In total, 17 candidate peptides were found and their specificity toward Pb2+ was further investigated with bead-based enzyme immunoassay (EIA). A highly specific Pb2+ binding peptide ThrAsnThrLeuSerAsnAsn (TNTLSNN) was obtained. Based on our knowledge, this is the first report on a new chromatographic biopanning method coupled with monolithic column for the selection of metal ion specific binding peptides. It is expected that this monolith-based chromatographic biopanning will provide a promising approach for a high throughput screening of affinity peptides cognitive of a wide range of target species.