Organophosphorus Hydrolase Activity Research Articles

Multifunctional Fe-doped carbon dots and metal-organic frameworks nanoreactor for cascade degradation and detection of organophosphorus pesticides

Organophosphorus pesticides (OPs) are the most widely used pesticides, hence it is crucial to effectively prevent and control OPs residues for agricultural product safety and ecological development. In this work, the nanoreactor consisting of Fe-doped carbon dots (Fe-CDs) and metal-organic framework (MOF-808) were developed for selective degradation as well as sensitive detection of paraoxon and parathion. MOF-808 was selected as the host and used to rapidly degrade paraoxon due to its excellent organophosphorus hydrolase activity. Fe-CDs were severed as guest molecules not only for photocatalytic degradation of parathion, but also for sensing to detect OPs. Through host–guest interaction, Fe-CDs/MOF-808 and Fe-CDs@MOF-808 were synthesized for cascade degradation and detection of paraoxon and parathion, respectively. Paraoxon and parathion were detoxified by catalytic hydrolysis and photocatalytic degradation by Fe-CDs/MOF-808 and Fe-CDs@MOF-808, respectively. Meanwhile, the fluorescence of the nanocomposites was quenched by the internal filtering effect with 4-nitrophenol (4-NP), the degradation product of OPs. Fe-CDs/MOF-808 has a wide linear range for detection of paraoxon from 0.001 to 360 μM, with a limit of detection (LOD) as low as 0.3 nM. Fe-CDs@MOF-808 exhibited a linear range of 0.01–100 μM for parathion, with an LOD about 3.3 nM. The reliability of the nanoreactor was verified by testing the recovery rate of OPs spiked water sample. Meanwhile, the nanoreactors were applied for degradation and detection of OPs in pakchoi. The OPs in pakchoi can be fully degraded within 5 min, and the analytical results are consistent with those by standard method (HPLC). Therefore, the proposed nanoreactor provides a feasible alternative for effective degradation and timely detection of OPs during agricultural production.

Zn-ZrMOF containing Lewis acid-base catalytic sites for hydrolysis and visual detection of profenofos

A colorimetric method for the catalysis hydrolysis of profenofos (C11H15BrClO3PS) was designed based on Zn-ZrMOF. Zn-ZrMOF has the catalytic activity of organophosphorus hydrolase and contains Lewis acid-base catalytic sites, which can catalyze the cleavage of P-O bond of profenofos to obtain hydrolysate (4-bromo-2-chlorophenol). As a phenolic substance, 4-bromo-2-chlorophenol can react with the chromogenic agent 4-aminoantipyrine (4-AAP) and produce a pink product (4-((3-chloro-4-oxocyclohexa-2,5-dien-1-ylidene)amino)− 1,5-dimethyl-2-phenyl-1,2-dihydro-3H-pyrazol-3-one) in the presence of oxidizer K3[Fe(CN)6] under the weak alkalinity. The pink product has a strong UV absorption peak at 508 nm. The solution color gradually changed from colorless to pink with the increase of the concentration of profenofos. The concentration of profenofos could be semi-quantitatively analyzed by naked eye. The linear range of this method was 0.01–1000 μg mL−1, and the detection limit was 6.33 × 10−3 μg mL−1. Additionally, the method has good selectivity and accuracy for the determination of profenofos. This study provides a feasible strategy for the design of a metal-organic framework material with organophosphorus hydrolase-like catalytic activity for the determination of profenofos in vegetables.

Molecular Diversity of Chlorpyrifos Degrading Bacteria Isolated from Apple Orchard Soils of Himachal Pradesh

A total of seventy-two bacterial isolates were obtained employing enrichment culture technique from apple orchard soils contaminated with chlorpyrifos. Pure cultures of bacterial isolates were obtained using streak plate method on mineral salt medium. Bacterial isolates were characterized on the basis of morphology, culture and biochemical properties. Six bacterial isolates exhibited high extracellular organophosphorus hydrolase activity along with high tolerance towards high concentrations of chlorpyrifos. Genomic DNA extraction from bacterial isolates was done with phenol/chloroform method. Molecular Diversity of six chlorpyrifos degrading bacterial isolates was done employing RAPD-PCR technique by using 25 decamer primers, where amplification was showed by only 20 primers. A total of 337 amplified bands and 64 unique bands ranging in size from 100 to 4900bps were produced after RAPD analysis. The similarity coefficient estimated by Jaccard’s coefficient for these bacterial isolates was found to range between 31 to 64 percent.

Rational Design of a Synthetic Peg-Like Polymer for Protein Stabilization

Organophosphorus hydrolase (OPH) is an enzyme that can deteriorate acutely toxic chemicals known as organophosphates, which are ubiquitously employed as pesticides and are thus considerable environmental hazards. However, the proclivity of OPH to aggregate in aqueous media severely hinders its biological activity. Inspired by natural chaperones, we have designed and synthesized heteropolymers to encapsulate and stabilize OPH. When OPH is complexed with the designed heteropolymer, it exhibits a drastic increase in biological activity in a buffer solution - approximately 15 times greater than the native biological activity of pure OPH in the same environment. We have confirmed via dynamic light scattering that while pure OPH aggregates in the buffer solution, the heteropolymer prevents aggregation from occurring.Two model proteins, bovine serum albumin and lysozyme, have also been studied with the heteropolymer to aid our understanding of the protein-polymer interactions that occur in our system. The structure and particle size of the different protein-polymer mixtures at several temperatures have been probed via dynamic light scattering, circular dichroism, and Fourier transform infrared spectroscopy. These characterization techniques have elucidated the mechanisms of interactions between the polymer and proteins.

High-level extracellular secretion of organophosphorous hydrolase of Flavobacterium sp. in Escherichia coli BL21(DE3)pLysS.

Organophosphorus (OPs) compounds are widely used in many pesticides, insecticides, and chemical nerve agents. These compounds are hazardous for humans and the environment. There are many reports on detoxification of these compounds, among them enzymatic cleavage of these compounds with organophosphorus hydrolase (OPH) has been taken into more consideration. Several studies have been performed to improve OPH secretion in Escherichia coli by different signal peptides, but have not been successful. In this study, to achieve the extracellular secretion of OPH in E. coli, the complete opd gene along with its native signal peptide was codon optimized and expressed in E. coli BL21(DE3)pLysS. The culture medium showed OPH activity after 2, 4, and 6H of induction time. The extracellular secretion of OPH was also confirmed by SDS-PAGE and Western blot analysis. The effects of different factors in growth medium were also investigated regarding expression and extracellular secretion of OPH. It appears that the secretion of OPH into the extracellular medium is highly affected by culture conditions. Therefore, our results revealed that the recombinant OPH was successfully secreted into the extracellular medium. This secretion system can be considered as a high efficiency biocatalyst for detoxification of OPs compounds.

Effect of culture conditions on the whole cell activity of recombinant Escherichia coli expressing periplasmic organophosphorus hydrolase and cytosolic GroEL/ES chaperone

Specific whole cell activity strongly affects sensitivity and detection limit of whole cell-based biosensors. Previously, we developed recombinant Escherichia coli coexpressing periplasmic organophosphorus hydrolase (OPH) and cytosolic chaperone GroEL-GroES (GroEL/ES). In present work, we investigated the effect of culture conditions on whole cell OPH activity. Especially, the whole cell OPH activity was significantly affected by the concentration of tetracycline that is an inducer for chaperone GroEL/ES. When cultured at 20°C for 31 h in M9 medium containing 1 mM IPTG, 50 ng/mL tetracycline, and 500 µM CoCl2, the recombinant E. coli exhibited a specific whole cell OPH activity (U/OD600) of ~0.55, which is 2.6-fold higher than that of recombinant E. coli cultured as previously described conditions. In addition, recombinant cells showed adequate storage stability for 1 week with 100% of original response. Finally, the improved activity and adequate stability in the whole cell biocatalyst will contribute to sensitivity, detection time, and stability of a whole cell-based biosensor for the detection of toxic organophosphates.

The Cytoplasmic and Periplasmic Expression Levels and Folding of Organophosphorus Hydrolase Enzyme in Escherichia coli.

Background:Organophosphorus hydrolase (OPH) is a type of organophosphate-degrading enzyme which is widely used in the bioremediation process.Objectives:In this study, the periplasmic and cytoplasmic productions and the activity of recombinant OPH in Escherichia coli were investigated and compared using two pET systems (pET21a and pET26b).Materials and Methods:The sequence encoding the opd gene was synthesized and expressed in the form of inclusion body using pET21a-opd and in the periplasmic space in pET26b-opd.Results:Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed a band of about 37 kDa with a maximum expression level at 30°C from pET21a-opd.However, the obtained results of the periplasmic space extraction of OPH (pET26b-opd) showed a very weak band, while the cytoplasmic expression of OPH (pET21a-opd) produced a strong protein band.Conclusions:The activities studied by the production of PNP were determined by following the increase at 410 nm. The maximum PNP was produced at 30°C with an optical density of 10.62 in the presence of cytoplasmic expression of OPH (pET21a-opd). Consequently, our results suggest cytoplasmic expression system as an appropriate candidate with a high amount of OPH in spite of inclusion body formation, which needs an additional refolding step.

Marked enhancement of Acinetobacter sp. organophosphorus hydrolase activity by a single residue substitution Ile211Ala

The activity of organophosphorus hydrolase (OPH) that catalyzes the hydrolysis of neurotoxic organophosphates (OPs) was reported to evolve from lactonase. In this study, a putative OPH from Acinetobacter sp. (AbOPH) exhibited high lactonase activity with latent OPH activity. Sequence alignment and phylogenetic tree analysis revealed the unique status of AbOPH in evolution. The crystal structure of AbOPH was determined at 2.0 Å resolution and a semi-rational design was performed to enhance the OPH activity of AbOPH through a consensus sequence approach. Compared with wild-type AbOPH, which exhibited undetectable activity toward methyl-parathion (MP), the best variant AbOPHI211A showed markedly improved catalytic efficiency (1.1 μmol min−1 mg protein −1 ) toward MP. Docking studies suggested that the mutation Ile211Ala affects substrate recognition and stabilizes substrate conformation. This result presents the emergence of new enzyme function by a simple mutation strategy and confirms the high possibility that OPH was evolved from its lactonase ancestor.

Improvement of organophosphorus hydrolase activity toward nerve agents by amino acid substitutions

The hydrolytic activities of organophosphorus hydrolase (OPH) toward nerve agents were investigated by using mutant OPH enzymes constructed by site-directed mutagenesis of the gene. The amino acids at position 136, 254, and 257 are known to influence the enzyme activity. The nucleotide sequencing of the OPH gene cloned in our previous study revealed that the deduced amino acid at position 254 was tyrosine (Tyr) instead of histidine (His) that was reported to reside at this position by other research groups. In the present study, such key amino acids were substituted to construct five mutant enzymes, and their hydrolytic activities were compared with that of the wild type enzyme. The activity assays proved that the substitution of Tyr at 254 to His led to the remarkable enhancement of the activities toward paraoxon and also toward VX, which could be decomposed by the wild type OPH only slightly. This mutant enzyme hydrolyzed most of the nerve agents almost completely, and approximately half of the VX during 20 min when the enzyme was activated with 10 mM CoCl2. The cobalt ion could activate the mutant OPH more efficiently than zinc ion, but the difference of the activation ability was not significant when other mutant OPH enzymes were activated with these divalent metal ions. The present results suggest that a practical decontamination system for nerve agents could be established using the mutant OPH with enhanced activity.

Structural and Enzymatic Characterization of the Phosphotriesterase OPHC2 from Pseudomonas pseudoalcaligenes

BackgroundOrganophosphates (OPs) are neurotoxic compounds for which current methods of elimination are unsatisfactory; thus bio-remediation is considered as a promising alternative. Here we provide the structural and enzymatic characterization of the recently identified enzyme isolated from Pseudomonas pseudoalcaligenes dubbed OPHC2. OPHC2 belongs to the metallo-β-lactamase superfamily and exhibits an unusual thermal resistance and some OP degrading abilities.Principal findingsThe X-ray structure of OPHC2 has been solved at 2.1 Å resolution. The enzyme is roughly globular exhibiting a αβ/βα topology typical of the metallo-β-lactamase superfamily. Several structural determinants, such as an extended dimerization surface and an intramolecular disulfide bridge, common features in thermostable enzymes, are consistent with its high Tm (97.8°C). Additionally, we provide the enzymatic characterization of OPHC2 against a wide range of OPs, esters and lactones.SignificanceOPHC2 possesses a broad substrate activity spectrum, since it hydrolyzes various phosphotriesters, esters, and a lactone. Because of its organophosphorus hydrolase activity, and given its intrinsic thermostability, OPHC2 is an interesting candidate for the development of an OPs bio-decontaminant. Its X-ray structure shed light on its active site, and provides key information for the understanding of the substrate binding mode and catalysis.

Cell surface display of organophosphorus hydrolase for sensitive spectrophotometric detection of p-nitrophenol substituted organophosphates

Organophosphates (OPs) widely exist in ecosystem as toxic substances, for which sensitive and rapid analytical methods are highly requested. In the present work, by using N-terminal of ice nucleation protein (INP) as anchoring motif, a genetically engineered Escherichia coli (E. coli) strain surface displayed mutant organophosphorus hydrolase (OPH) (S5) with improved enzyme activity was successfully constructed. The surface location of INP-OPH fusion was confirmed by SDS-PAGE analysis and enzyme activity assays. The OPH-displayed bacteria facilitate the hydrolysis of p-nitrophenol (PNP) substituted organophosphates to generate PNP, which can be detected spectrometrically at 410nm. Over 90% of the recombinant protein present on the surface of microbes demonstrated enhanced enzyme activity and long-term stability. The OPH activity of whole cells was 2.16U/OD600 using paraoxon as its substrate, which is the highest value reported so far. The optimal temperature for OPH activity was around 55°C, and suspended cultures retained almost 100% of its activity over a period of one month at room temperature, exhibiting the better stability than free OPH. The recombinant E. coli strain could be employed as a whole-cell biocatalyst for detecting PNP substituted OPs at wider ranges and lower detection limits. Specifically, the linear ranges of the calibration curves were 0.5–150μM paraoxon, 1–200μM parathion and 2.5–200μM methyl parathion, and limits of detection were 0.2μM, 0.4μM and 1μM for paraoxon, parathion and methyl parathion, respectively (S/N=3). These results indicate that the engineered OPH strain is a promising multifunctional bacterium that could be used for further large-scale industrial and environmental applications.

Construction of genetically engineered bacteria that degrades organophosphorus pesticide residues and can be easily detected by the fluorescence

Organophosphorus compounds (OPs) are widely used in agriculture and industry and there is increased concern about their toxicological effects in the environment. Bioremediation can offer an efficient and cost-effective option for the removal of OPs. Herein, we describe the construction of a genetically engineered microorganism (GEM) that can degrade OPs and be directly detected and monitored in the environment using an enhanced green fluorescent protein (EGFP) fusion strategy. The coding regions of EGFP, a reporter protein that can fluoresce by itself, and organophosphorus hydrolase (OPH), which has a broad substrate specificity and is able to hydrolyse a number of organophosphorus pesticides, were cloned into the expression vector pET-28b. The fusion protein of EGFP–OPH was expressed in E. coli BL21 (DE3) and the protein expression reached the highest level at 11h after isopropyl β -d-thiogalactopyranoside induction. The fluorescence of the GEM was detected by fluorescence spectrophotometry and microscopy, and its ability to degrade OPs was determined by OPH activity assay. Those GEM that express the fusion protein (EGFP and OPH) exhibited strong fluorescence intensity and also potent hydrolase activity, which could be used to degrade organophosphorus pesticide residues in the environment and can also be directly monitored by fluorescence.

The rrnA promoter as a tool for the improved expression of heterologous genes in cyanobacteria

The regulatory sequence of ribosomal RNA A (rrnA) operon from Synechococcus PCC7942 was characterized using green fluorescent protein gene (gfp) as a reporter. The PR promoter (nt. −83 to +2) including upstream promoter element and P1 promoter of rrnA exhibited GFP fluorescence intensity about 30-fold higher than full length sequence (nt. −147 to +79). The effects of PR promoter arranged in tandem with consensus-σ70 promoter (PS) of Escherichia coli on the expression of gfp and opd gene encoding organophosphorus hydrolase (OPH) in Synechococcus were investigated. The PS-PR tandem promoter was superior to all of the other promoters; its GFP fluorescence intensity was a 1.8-fold increase when compared to PR-PR tandem promoter, a 2.5-fold, 9.5-fold and a 15-fold increase compared to PR, PS and promoter of tRNApro, respectively. The GFP from PS-PR tandem promoter accounted for about 12% of its total extracted proteins. OPH activity of Synechococcus harboring opd gene under the control of PS-PR tandem promoter was 738±128units/OD730. We demonstrated that the tandem promoters remarkably enhanced the GFP and OPH production which were detected on SDS-PAGE stained with Coomassie blue. The promoter system in this study could be generally applied to production of valuable organic products from cyanobacteria.

Expression pattern of recombinant organophosphorus hydrolase from Flavobacterium sp. ATCC 27551 in Escherichia coli

Concerned with the influence of tagging system on the expression of heterogeneous protein in Escherichia coli, we attempted to express the organophosphorus hydrolase (OPH) of Flavobacterium sp. ATCC 27551 in E. coli. Recombinant OPH was overproduced successfully in E. coli when modified without the use of a tobacco etch virus (TEV) protease cleavage sequence. In addition, though there has never been a report on the extracellular secretion of recombinant OPH harboring native Tat signal peptides in E. coli, the produced protein was observed to be secreted extracellularly. Through the use of reverse transcriptional quantitative real-time PCR and comparison of the predicted folding rate, it was determined that OPH expression may be affected by the existence of a TEV protease cleavage sequence at the C-terminus during the process of translated protein folding, leading to the suppressed OPH activity. With the potential compatibility between native Tat signal peptides of OPH and E. coli Tat pathway secretion system, we report a successful expression of recombinant OPH harboring native Tat signal peptides in E. coli, for the first time.

An efficacious degradation of pesticide by salt tolerant Streptomyces venezuelae ACT 1

Degradation of the organophosphorus pesticide has been studied using the marine isolate, Streptomyces venezuelae ACT1. The organism exhibited a specific growth rate of 0.371h−1 and the organophosphorus hydrolase activity rate as 0.273h−1. Hence the organism was found to be very effective towards the pesticide degradation. Further the substrate assimilation and inhibition model of the organism were demonstrated using Monod and Haldane model equations which depicted that the inhibition model fits well for both the cell growth and enzyme activity. The maximum specific growth rate and the enzyme activity rate were found to be 0.571h−1 and 0.472h−1, respectively. Effect of P0/X0 ratio on degradation and COD reduction rate revealed that higher these ratios raise the degradation rate and the COD reduction rate.

Identification and characterization of novel catalytic bioscavengers of organophosphorus nerve agents

In an effort to discover novel catalytic bioscavengers of organophosphorus (OP) nerve agents, cell lysates from a diverse set of bacterial strains were screened for their capacity to hydrolyze the OP nerve agents VX, VR, and soman (GD). The library of bacterial strains was identified using both random and rational approaches. Specifically, two representative strains from eight categories of extremophiles were chosen at random. For the rational approach, the protein sequence of organophosphorus hydrolase (OPH) from Brevundimonas diminuta was searched against a non-redundant protein database using the Basic Local Alignment Search Tool to find regions of local similarity between sequences. Over 15 protein sequences with significant sequence similarity to OPH were identified from a variety of bacterial strains. Some of these matches were based on predicted protein structures derived from bacterial genome sequences rather than from bona fide proteins isolated from bacteria. Of the 25 strains selected for nerve agent testing, three bacterial strains had measurable levels of OP hydrolase activity. These strains are Ammoniphilus oxalaticus, Haloarcula sp., and Micromonospora aurantiaca. Lysates from A. oxalaticus had detectable hydrolysis of VR; Haloarcula sp. had appreciable hydrolysis of VX and VR, whereas lysates from M. aurantiaca had detectable hydrolysis of VR and GD.

Simultaneous degradation of organophosphate and organochlorine pesticides by Sphingobium japonicum UT26 with surface-displayed organophosphorus hydrolase

A genetically engineered microorganism (GEM) capable of simultaneously degrading organophosphate and organochlorine pesticides was constructed for the first time by display of organophosphorus hydrolase (OPH) on the cell surface of a hexachlorocyclohexane (HCH)-degrading Sphingobium japonicum UT26. The GEM could potentially be used for removing the two classes of pesticides that may be present in mixtures at contaminated sites. A surface anchor system derived from the truncated ice nucleation protein (INPNC) from Pseudomonas syringae was used to target OPH onto the cell surface of UT26, reducing the potential substrate uptake limitation. The surface localization of INPNC-OPH fusion was verified by cell fractionation, western blot, proteinase accessibility, and immunofluorescence microscopy. Furthermore, the functionality of the surface-exposed OPH was demonstrated by OPH activity assays. Surface display of INPNC-OPH fusion (82kDa) neither inhibited cell growth nor affected cell viability. The engineered UT26 could degrade parathion as well as γ-HCH rapidly in minimal salt medium. The removal of parathion and γ-HCH by engineered UT26 in sterile and non-sterile soil was also studied. In both soil samples, a mixture of parathion (100mgkg(-1)) and γ-HCH (10mgkg(-1)) could be degraded completely within 15days. Soil treatment results indicated that the engineered UT26 is a promising multifunctional bacterium that could be used for the bioremediation of multiple pesticide-contaminated environments.

Enhanced Enzymatic Activity of OPH in Ammonium-Functionalized Mesoporous Silica: Surface Modification and Pore Effects

Organophosphorus hydrolase (OPH) is an enzyme capable of rapidly degrading organophosphorus toxins, such as phosphorus-based nerve agents and pesticides. In these studies, OPH was immobilized within the pores of spherical mesoporous silica particles and the rate of hydrolysis of diethyl-4-nitrophenyl phosphate (paraoxon) was correlated to surface functionalization (acid, amine, or ammonium) and pore diameter. Conversion rates were measured by using a novel 31P NMR method developed by our group. After immobilization within ammonium-modified particles, OPH was more active for paraoxon degradation than free OPH, while amine- or acid-modified particles led to decreased activities. OPH immobilized within the pores of intermediate diameter (6 nm) also showed the highest activities. We also varied the amount of ammonium coverage and found that 14–20% w/w of the ammonium organosilane per particle was sufficient to observe enhancement of OPH activity.

Stabilization of Organophosphorus Hydrolase by Entrapment in Silk Fibroin: Formation of a Robust Enzymatic Material Suitable for Surface Coatings

Organophosphates are some of the most acutely toxic compounds synthesized on an industrial scale, and organophosphorus hydrolase (OPH) has the ability to hydrolyze and inactivate a number of these chemicals. However, OPH activity is vulnerable to harsh environmental conditions that would accompany its practical utility in the field; a limitation that can also be extended to conditions required for incorporation of OPH into useful materials. Here we present evidence that entrapment of OPH in silk fibroin leads to stabilization of OPH activity under a variety of conditions that would otherwise reduce free enzyme activity, such as elevated temperature, UV light exposure and the presence of detergent. Silk fibroin entrapment of OPH also allowed for its dispersal into a polyurethane-based coating that retained organophosphate hydrolysis activity after formulation, application and drying. Together, the data presented here demonstrate the utility of silk fibroin entrapment for the protection of OPH activity under a variety of environmental conditions.

Coexpression of molecular chaperone enhances activity and export of organophosphorus hydrolase in Escherichia coli

Periplasmic secretion has been used in attempts to construct an efficient whole-cell biocatalyst with greatly reduced diffusion limitations. Previously, we developed recombinant Escherichia coli that express organophosphorus hydrolase (OPH) in the periplasmic space using the twin-arginine translocation (Tat) pathway to degrade environmental toxic organophosphate compounds. This system has the advantage of secreting protein into the periplasm after folding in the cytoplasm. However, when OPH was expressed with a Tat signal sequence in E. coli, we found that the predominant OPH was an insoluble premature form in the cytoplasm, and thus, the whole-cell OPH activity was significantly lower than its cell lysate activity. In this work, we, for the first time, used a molecular chaperone coexpression strategy to enhance whole-cell OPH activity by improving the periplasmic translocation of soluble OPH. We found that the effect of GroEL-GroES (GroEL/ES) assistance on the periplasmic localization of OPH was secretory pathway dependent. We observed a significant increase in the amount of soluble mature OPH when cytoplasmic GroEL/ES was expressed; this increase in the amount of mature OPH might be due to enhanced OPH folding in the cytoplasm. Importantly, the whole-cell OPH activity of the chaperone-coexpressing cells was ∼5.5-fold greater at 12 h after induction than that of cells that did not express the chaperone as a result of significant Tat-based periplasmic translocation of OPH in the chaperone-coexpressing cells. Collectively, these data suggest that molecular chaperones significantly enhance the whole-cell activity of periplasmic OPH-secreting cells, yielding an effective whole-cell biocatalyst system with highly reduced diffusion limitations.