Elsevier

Journal of Hazardous Materials

Volume 365, 5 March 2019, Pages 261-269
Journal of Hazardous Materials

Organophosphorus hydrolase-poly-β-cyclodextrin as a stable self-decontaminating bio-catalytic material for sorption and degradation of organophosphate pesticide

https://doi.org/10.1016/j.jhazmat.2018.10.094Get rights and content

Highlights

  • Synthesis of non-toxic, sorptive reinforced self-decontaminating material is reported.

  • Organophosphorus hydrolase (OPH) enzyme is immobilized onto poly-β-cyclodextrin (PCD) by physical entrapment.

  • PCD provides a unique and robust support for OPH enzyme and acts as a regenerative sorption material.

  • OPH-PCD system shows high sorption and remarkably fast degradation of MPO.

  • OPH-PCD is extremely stable for long term usage.

Abstract

A region suffering from an attack of a nerve agent requires not only a highly sorptive material but also a fast-acting catalyst to decontaminate the lethal chemical present. The product should be capable of high sorptive capacity, selectivity and quick response time to neutralize the long lasting harmful effects of nerve agents. Herein, we have utilized organophosphorus hydrolase (OPH) as a non-toxic bio-catalytic material held in with the supporting matrix of poly-β-cyclodextrin (PCD) as a novel sorptive reinforced self-decontaminating material against organophosphate intoxication. OPH coated PCD (OPH-PCD) will not only be providing support for holding enzyme but also would be adsorbing methyl paraoxon (MPO) used as a simulant, in a host-guest inclusion complex formation. Sorption trend for PCD revealed preference towards the more hydrophobic MPO against para-nitrophenol (pNP). The results show sorption capacity of 1.26 mg/g of 100 μM MPO with PCD which was 1.7 times higher compared to pNP. The reaction rate with immobilized OPH-PCD was found to be 23% less compared to free enzyme. With the help of OPH-PCD, continuous hydrolysis (100%) of MPO into pNP was observed for a period of 24 h through packed bed reactor with good reproducibility and stability of enzyme. The long-term stability also confirmed its stable nature for the investigation period of 4 days where it maintained activity. Combined with its fast and reactive nature, the resulting self-decontaminating regenerating material provides a promising strategy for the neutralization of nerve agents and preserving the environment.

Introduction

Detection and decontamination of hazardous chemical warfare agents (CWAs) are gaining attention in light of their use in recent wars in Iraq and Syria [[1], [2], [3], [4], [5]]. Especially organophosphates (OPs) used as nerve agents are the most toxic compounds synthesized capable of not only entering the bloodstream by inhalation but also can diffuse through the skin [6]. Hence, the primary defense mechanism against such highly potent CWAs is a self-decontamination material which can alert the presence of a harmful gas and potentially protect the human body by neutralizing the nerve agent before it enters the bloodstream.

Cyclodextrins (CDs) are nontoxic cyclic oligosaccharides (sugar molecules) produced from enzymatic conversion of starch. Cyclodextrins possess a hydrophobic cavity surrounded by hydrophilic surface giving it the ability to form host-guest inclusion complexes (non-covalent bonding) leading to their wide array of applications such as pharmaceutical industry [[7], [8], [9], [10], [11], [12], [13]], environmental protection [[14], [15], [16], [17]], protein stabilization and refolding [18,19], foods and flavors [20,21], cosmetics [22], and entrapment of essential oils [23]. Additionally, CDs have been studied as detoxification systems against a few of OP compounds due to their benign nature [24,25]. In the 1970s, α-CD first showed enzyme inhibition characteristics for sarin and increased hydrolysis of malathion [26,27]. Later in 1980s work, CDs degraded sarin and soman with β-CD showing a greater affinity to the nerve agents as compared to α- or γ-CD [28,29]. Another study in late 1990s demonstrated catalytic effects of α-, β- and γ-CD on organophosphorus pesticides in neutral aqueous media [30]. A recent study showed the capability of β-CD derivative to detoxify cyclosarin, sarin and tabun in this decreasing order of effectiveness [31]. CD cavities have a higher affinity to organic molecules due to hydrophobic interaction. The varying size of the CD bucket gives it unique capability to form specific binding with hydrophobic molecules depending on their size, geometry and polarity as opposed to non-specific binding of activated carbon [32]. In terms of sorption capacity, cross-linked cyclodextrin polymers are highly absorptive materials with inclusion formation constants K = 108-109 M−1 as compared to the monomeric CD value of K = 10–103 M−1 [33]. Inclusion of organophosphates into β-CD with alkyl spacers was demonstrated to cause its hydrolytic catalysis followed by sequestration of hydrophilic phosphonic acids upon prolonged exposure to PCD [34]. This increased sorption of poly-CD can be explained by the porous structure caused by the spacer (hexamethylene diisocyanate (HDI)) and also the remaining solvent (Dimethylformamide (DMF)) acting as a porogen remaining in between the ring-shaped structures during the polymerization creating its enhanced sorptive nature [16,32,34,35]. β-CD is the most studied and valuable among the CDs with its cheaper cost and ease of accessibility. The advantages of poly-β-CD (PCD) to form strong host-guest inclusion complexes, with stable nature and reversible inclusion complex formation in a stronger organic solvent combined with its detoxification nature makes it a suitable candidate for a self-decontamination material [36].

In order to achieve a high turnover rate for detoxification of phosphate esters, an environmentally friendly catalytic material is needed. Several methods for catalytic degradation using metallic systems [37,38] or Lewis-acidic cation complexes [[39], [40], [41]] have been reported. Although these studies are promising, their application as a decontamination agent for protection on the field would be challenging since these are mostly solution-phase catalysts, require longer time and often need high temperature for optimum performance [[42], [43], [44]]. In this category, enzymes show preferential affinity, higher catalytic activity at ambient conditions to passivate OP compounds and are non-toxic in nature [45]. Especially organophosphorus hydrolase (OPH) has grabbed significant attention as a widely studied non-toxic decontamination enzyme for hydrolyzing nerve agents [[46], [47], [48], [49]].

Several methods have been employed so far for the immobilization of OPH enzyme. It is well-known that once OPH is in solution form, it starts to degrade and eventually losses its activity. Hence, it is paramount to bind it onto a dry solid substrate for long term stability and activity. Various enzyme immobilization methods have been investigated recently such as physical entrapment, covalent attachment, or encapsulation. Few immobilization techniques using physical entrapment or encapsulation developed until now are by using polyurethane foam matrix [48,50], screen printed carbon electrodes [51], mesoporous silica [52,53], polymerizable phospholipids encapsulation [54], layer-by-layer assembly [55,56], single-walled carbon nanotubes [57], silk fibroin [58], and cross-linked poly(c-glutamic acid)/gelatin hydrogel [59]. Recent advances for covalent binding of OPH enzyme include immobilization on ferric magnetic nanoparticles [60], epoxy modified cellulose microfibers [61], and on spore of Bacillus subtilis [62]. An amyloid fibril nanoscaffold was also able to support the enzyme and exhibited higher thermal stability [63]. In other works, mesoporous titania thin films demonstrated good activity with 6-tagged OPH enzyme immobilized to form a biocatalyst film [64]. Even though these methods successfully immobilize the enzyme, they sometimes suffer from low enzymatic activity and in some cases, the loss of enzyme during the fabrication process leading to an unproductive, slow and inefficient product.

Herein, we report a novel, environmentally friendly sorptive reinforced self-decontamination material by immobilizing OPH onto PCD (OPH-PCD) by physical entrapment. PCD will not only serve as a support for the enzyme but also act as a highly sorptive specific binding and detoxifying agent at various pH ranges. The effect of pH on sorption of PCD is also studied. Methyl paraoxon (MPO) is used as the simulant for soman. The synthesized OPH-PCD product is expected to be remarkably stable with potential of long term storage capability, reusability, and fast degradation properties.

Section snippets

Materials

Unless otherwise stated, all chemicals and reagents were used as received. Organophosphorus hydrolase (EC 3.1.8.1) enzyme was received as freeze-dried powder from GenoFocus Inc. (Daejeon, South Korea). β-Cyclodextrin (β-CD), anhydrous N,N-dimethylformamide (DMF), 2-(cyclohexylamino)ethanesulfonic acid (CHES), bis-tris propane (BTP), para-nitrophenol (pNP), methyl paraoxon (O,O-dimethyl O-(4-nitrophenyl) phosphate), were purchased from Sigma-Aldrich (St. Louis, MO, USA). The cross linker,

Preparation and characterization of PCD and OPH-PCD

PCD was synthesized by the reaction of β-CD with HDI with a 1:9 equivalence ratio in the presence of DMF in a round bottom flask connected to a cooling condenser at 70 ℃ followed by rapid quenching. The initial attempts for preparation of PCD powder resulted in gelation or aggregation of the mixture. Hence, a comprehensive investigation was carried out to find the effect of the variables involved such as β-CD, HDI, reaction time, temperature, and quenching agents (see Table S1 Supplementary

Conclusion

In conclusion, the synthesized self-decontaminating biocatalytic OPH-PCD system is extremely stable and shows remarkably fast degradation of MPO, hydrolyzing it completely within 10 min. of exposure time. PCD behaves not only as a unique and robust support for the enzyme but also as a regenerative sorption material for further purifying the organophosphate contamination. In synthesizing PCD, using acetone as the quenching agent of reaction, revealed less hydrophobic PCD. Instead, if only DI

Conflict of interest

The authors declare no competing financial interest.

Acknowledgments

This work was supported by the National Research Council of Science & Technology (NST) grant by the Korean government (MSIT) [No. CMP-16-04-KITECH] and by Brain Pool Program through the Korean Federation of Science and Technology Societies (KOFST) funded by the Ministry of Science and ICT [No. 171S-2-3-1807].

References (67)

  • S. Müller et al.

    In vitro detoxification of cyclosarin (GF) by modified cyclodextrins

    Toxicol. Lett.

    (2011)
  • T. Irie et al.

    Pharmaceutical applications of cyclodextrins. III. Toxicological issues and safety evaluation

    J. Pharm. Sci.

    (1997)
  • B. Desire et al.

    Interaction of soman with β-cyclodextrin

    Fundam. Appl. Toxicol.

    (1986)
  • S. Ishiwata et al.

    Cyclodextrin inclusion: catalytic effects on the degradation of organophosphorus pesticides in neutral aqueous solution

    Chemosphere

    (1999)
  • T. Wille et al.

    Detoxification of nerve agents by a substituted β-cyclodextrin: application of a modified biological assay

    Toxicology

    (2009)
  • A.L. Nielsen et al.

    Self-assembling microparticles with controllable disruption properties based on cyclodextrin interactions

    Colloids Surf. B Biointerfaces

    (2009)
  • E.M.M. Del Valle

    Cyclodextrins and their uses: a review

    Process Biochem.

    (2004)
  • K.L. Klinkel et al.

    Effect of ligand modifications and varying metal-to-ligand ratio on the catalyzed hydrolysis of phosphorus triesters by bimetallic tetrabenzimidazole complexes

    J. Mol. Catal. A Chem.

    (2007)
  • R.A. Moss et al.

    Zirconium and hafnium cations rapidly cleave model phosphodiesters in acidic aqueous solutions

    Tetrahedron Lett.

    (1998)
  • K. Lai et al.

    Characterization of P-S bond hydrolysis in organophosphorothioate pesticides by organophosphorus hydrolase

    Arch. Biochem. Biophys.

    (1995)
  • B. Xu et al.

    Enhanced expression of a bacterial gene for pesticide degradation in a common soil fungus

    J. Ferment. Bioeng.

    (1996)
  • R.M. Dawson et al.

    Degradation of nerve agents by an organophosphate-degrading agent (OpdA)

    J. Hazard. Mater.

    (2008)
  • S.R. Forrest et al.

    Activity and lifetime of organophosphorous hydrolase (OPH) immobilized using layer-by-layer nano self-assembly on silicon microchannels

    Catal. Today

    (2007)
  • M. Sharifi et al.

    Covalent immobilization of organophosphorus hydrolase enzyme on chemically modified cellulose microfibers: statistical optimization and characterization

    React. Funct. Polym.

    (2018)
  • H. Li et al.

    Hyper-crosslinked β-cyclodextrin porous polymer: an adsorption-facilitated molecular catalyst support for transformation of water-soluble aromatic molecules

    Chem. Sci.

    (2016)
  • H. Sekiguchi et al.

    On-site determination of nerve and mustard gases using a field-portable gas chromatograph-mass spectrometer

    Forensic Toxicol.

    (2006)
  • R. Pita et al.

    The use of chemical weapons in the syrian conflict

    Toxics

    (2014)
  • A. E.-B. AA et al.

    2-hydroxypropyl-ß-Cyclodextrin complex with Ketotifen Fumerate for eye drops preparations

    Int. J. Drug Deliv.

    (2011)
  • L. Cui et al.

    Effect of β-Cyclodextrin complexation on solubility and enzymatic conversion of naringin

    Int. J. Mol. Sci.

    (2012)
  • A.B. Nair et al.

    Enhanced oral bioavailability of acyclovir by inclusion complex using hydroxypropyl-β-cyclodextrin

    Drug Deliv.

    (2014)
  • L. Udrescu et al.

    Physicochemical characterization of zofenopril inclusion complex with hydroxypropyl-β-cyclodextrin

    J. Serb. Chem. Soc.

    (2015)
  • L. Tan et al.

    Synthesis and characterization of β-cyclodextrin-conjugated alginate hydrogel for controlled release of hydrocortisone acetate in response to mechanical stimulation

    J. Bioact. Compat. Polym.

    (2015)
  • S. Petralito et al.

    Supportive use of cyclodextrins as decontamination agents for herbicides: the case of fenoxaprop-p-ethyl, Asia-Pac

    J. Chem. Eng.

    (2012)
  • Cited by (38)

    View all citing articles on Scopus
    View full text