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

Accepted Manuscript Title: Organophosphorus Hydrolase-Poly-␤-Cyclodextrin as a Stable Self-Decontaminating Bio-catalytic Material for Sorption and Degradation of Organophosphate Pesticide Authors: Youngkwang Moon, Ali Turab Jafry, Soon Bang Kang, Jin Young Seo, Kyung-Youl Baek, Eui-Joong Kim, Jae-Gu Pan, Jae-Youl Choi, Hyunji Kim, Kang Han Lee, Keunhong Jeong, Seunghan Shin, Jinkee Lee, Yongwoo Lee PII: DOI: Reference:

S0304-3894(18)31012-4 https://doi.org/10.1016/j.jhazmat.2018.10.094 HAZMAT 19915

To appear in:

Journal of Hazardous Materials

Received date: Revised date: Accepted date:

28 July 2018 5 October 2018 31 October 2018

Please cite this article as: Moon Y, Jafry AT, Bang Kang S, Young Seo J, Baek K-Youl, Kim E-Joong, Pan J-Gu, Choi J-Youl, Kim H, Han Lee K, Jeong K, Shin S, Lee J, Lee Y, Organophosphorus Hydrolase-Poly-␤-Cyclodextrin as a Stable Self-Decontaminating Bio-catalytic Material for Sorption and Degradation of Organophosphate Pesticide, Journal of Hazardous Materials (2018), https://doi.org/10.1016/j.jhazmat.2018.10.094 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Organophosphorus Hydrolase-Poly-β-Cyclodextrin as a Stable Self-

Degradation of Organophosphate Pesticide

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Decontaminating Bio-catalytic Material for Sorption and

Youngkwang Moona, Ali Turab Jafrya, Soon Bang Kangb, Jin Young Seob, Kyung-Youl Baekb, Eui-Joong Kimc, Jae-Gu Panc, Jae-Youl Choic, Hyunji Kimb, Kang Han Leeb, Keunhong Jeongd,

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School of Mechanical Engineering, Sungkyunkwan University, Suwon, Gyeonggi-do, 16419,

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Seunghan Shine, Jinkee Leea,*, Yongwoo Leea,*

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Republic of Korea

Korea Institute of Science and Technology, Seoul, Republic of Korea

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GenoFocus, Daejeon, Republic of Korea

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Korea Military Academy, Seoul, Republic of Korea

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Korea Institute of Industrial Technology, Cheonan, Republic of Korea

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*Corresponding Authors at School of Mechanical Engineering, Sungkyunkwan University, Suwon, Gyeonggi-do, 16419, Republic of Korea

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Prof. Jinkee Lee, Email Address: [email protected] Prof. Yongwoo Lee, Email Address: [email protected]

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Graphical abstract

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Highlights:

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

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Organophosphorus hydrolase (OPH) enzyme is immobilized onto poly-β-cyclodextrin

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(PCD) by physical entrapment.

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

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

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OPH-PCD is extremely stable for long term usage.

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ABSTRACT

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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-

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cyclodextrin (PCD) as a novel sorptive reinforced self-decontaminating material against

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organophosphate intoxication. OPH coated PCD (OPH-PCD) will not only be providing support

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for holding enzyme but also would be adsorbing methyl paraoxon (MPO) used as a simulant, in a

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host-guest inclusion complex formation. Sorption trend for PCD revealed preference towards the

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more hydrophobic MPO against para-nitrophenol (pNP). The results show sorption capacity of

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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.

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With the help of OPH-PCD, continuous hydrolysis (100 %) of MPO into pNP was observed for a period of 24 hours through packed bed reactor with good reproducibility and stability of enzyme.

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The long-term stability also confirmed its stable nature for the investigation period of 4 days

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where it maintained activity. Combined with its fast and reactive nature, the resulting selfdecontaminating regenerating material provides a promising strategy for the neutralization of nerve agents and preserving the environment.

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Keywords: Self-decontamination, biocatalytic sorptive material, Organophosphorus hydrolase, Poly-β-Cyclodextrin

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1. 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 [1-5]. 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

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primary defense mechanism against such highly potent CWAs is a self-decontamination material

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which can alert the presence of a harmful gas and potentially protect the human body by

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neutralizing the nerve agent before it enters the bloodstream.

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Cyclodextrins (CDs) are nontoxic cyclic oligosaccharides (sugar molecules) produced from enzymatic conversion of starch. Cyclodextrins possess a hydrophobic cavity surrounded by

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hydrophilic surface giving it the ability to form host-guest inclusion complexes (non-covalent

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bonding) leading to their wide array of applications such as pharmaceutical industry [7-13],

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environmental protection [14-17], protein stabilization and refolding [18, 19], foods and flavors [20, 21], cosmetics [22], and entrapment of essential oils [23]. Additionally, CDs have been

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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

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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

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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

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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

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phosphonic acids upon prolonged exposure to PCD [34]. This increased sorption of poly-CD can

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be explained by the porous structure caused by the spacer (hexamethylene diisocyanate (HDI))

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and also the remaining solvent (Dimethylformamide (DMF)) acting as a porogen remaining in

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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

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cost and ease of accessibility. The advantages of poly-β-CD (PCD) to form strong host-guest

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inclusion complexes, with stable nature and reversible inclusion complex formation in a stronger

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organic solvent combined with its detoxification nature makes it a suitable candidate for a selfdecontamination material [36].

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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

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using metallic systems [37, 38] or Lewis-acidic cation complexes [39-41] 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 [42-44]. In this category,

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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

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hydrolyzing nerve agents [46-49]. 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

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physical entrapment, covalent attachment, or encapsulation. Few immobilization techniques

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using physical entrapment or encapsulation developed until now are by using polyurethane foam

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matrix [48, 50], screen printed carbon electrodes [51], mesoporous silica [52, 53], polymerizable

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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].

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Recent advances for covalent binding of OPH enzyme include immobilization on ferric magnetic

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nanoparticles [60], epoxy modified cellulose microfibers [61], and on spore of Bacillus subtilis

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[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

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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

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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

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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

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is expected to be remarkably stable with potential of long term storage capability, reusability, and fast degradation properties.

2. Experimental section

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2.1. Materials

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Unless otherwise stated, all chemicals and reagents were used as received. Organophosphorus

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hydrolase (EC 3.1.8.1) enzyme was received as freeze-dried powder from GenoFocus Inc.

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(Daejeon, South Korea). β-Cyclodextrin (β-CD), anhydrous N,N-dimethylformamide (DMF), 2(cyclohexylamino)ethanesulfonic acid (CHES), bis-tris propane (BTP), para-nitrophenol (pNP),

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methyl paraoxon (O,O-dimethyl O-(4-nitrophenyl) phosphate), were purchased from Sigma-

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Aldrich (St. Louis, MO, USA). The cross linker, hexamethylene diisocyanate (HDI) was

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purchased from Alfa Aesar (MA, USA). Methanol purchased from Daejung Chemicals (South Korea) was used without any distillation. Deionized water (Milli-Q Millipore, 18.2 MΩcm

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resistivity) was used in all experiments.

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2.2. Material characterizations Scanning auger electron spectroscopy (AES) nanoprobe was performed to take SEM image

with PHI-700 (ULVAC-PHI, Japan). The functional groups for synthesis of PCD were confirmed by Fourier transform infrared (FTIR) spectrometer (Nicolet iS10, Thermo Fisher

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Scientific, USA). UV-Vis spectrophotometer (Agilent 8453) was used for absorbance measurements in the range of 200 – 900 nm.

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2.3. Synthesis of PCD β-Cyclodextrin (β-CD) (0.88 mmol) was dissolved in 5 mL anhydrous N,N-dimethylformamide (DMF) with mechanical stirring (Fig. 1). The mixture was heated to 70 ℃followed by a drop-wise addition of 1.274 mL (7.93 mmol, 9 eq.) of the bifunctional linker, hexamethylene diisocyanate (HDI). The mixture was stirred at 70 ℃oil bath under an argon atmosphere for 1.5 h. Reaction

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mixture was quenched using excess amount (10 mL) of quenching agents. Two types were

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studied; 1. DI water (more hydrophobic PCD) 2. Acetone (less hydrophobic PCD) and filtered

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under aspirator to avoid gelation. The filtered PCD particles were dried at room temperature.

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Sieving was performed to attain a monodispersed particle size range of 32 - 63 µm. The PCD microparticles were purified by agitating in hot dichloromethane to unclog the binding sites

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followed by drying in oven at 80 ℃for 2 h to evaporate any remaining solvent.

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Fig. 1. Synthesis of PCD from β-CD polymers using HDI as crosslinker.

2.4. Sorption of PCD with MPO and pNP

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Kinetics sorption of more hydrophobic PCD was conducted using 20 mL pNP (50 µM) added to 200 mg PCD. Samples were taken at different time intervals, filtered in aspirator, and analyzed under UV-Vis spectrophotometer.

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For a comparative study, 10 mL of 100 µM of both MPO and pNP were each added to 100 mg PCD microparticles and stirred for 2 h. The resulting mixture was filtered, and absorptions were measured in UV-Vis spectrophotometer at 275 nm for MPO and 400 nm for pNP.

The effect of pH on sorption trend of PCD was studied using 50 µM pNP in 85% v/v CHES

buffer (pH 2, 6.6, 8.6, 10; 0.1 mM) containing 15 % v/v methanol. 10 mL pNP was added to 100

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mg of PCD placed in a scintillation vial, the mixture was stirred for 2 h and filtered through a

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0.45 µm disc syringe filter and its absorption was recorded at 315 nm (acidic) and 400 nm

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(basic).

2.5. Preparation of OPH-PCD

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300 mg of OPH powder was added to 5 mL CHES buffer (pH 8.6) with gentle mixing. The

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solution was passed through a 0.45 µm membrane filter to remove any solid residue. PCD was

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added to the OPH solution and left for freeze-drying overnight to obtain OPH enzyme immobilized onto PCD support (OPH-PCD). The obtained OPH-PCD was rinsed with DI water

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to remove any extraneous protein not absorbed by PCD and stored in a refrigerator. In order to assess its immobilization efficiency the activity measurement of OPH before and after its

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immobilization onto PCD were used as described in following section.

2.6. Protein content and enzymatic activity of OPH and OPH-PCD

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Protein content was determined from Bio-Rad kit (CA, USA). Detection solution was made by mixing 1:4 dye reagent to DI water and then filtered to remove particulates. Before measuring the protein content of OPH and OPH-PCD, the absorbance of bovine serum albumin (BSA) was

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measured to get the standard (reference) curve for extinction coefficient. Afterwards, 100 µL at 5 mg/mL of both OPH and OPH-PCD were each mixed with 5 mL of distilled dye reagent. The

resulting absorbance of the protein was measured at 595 nm and compared with the standard to attain the amount of protein present.

MPO as pesticide was employed to demonstrate the catalytic activity of OPH enzyme by

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converting it into para-nitrophenol and phosphonic acid (Fig. 2). Enzymatic activity was

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determined by measuring the amount of pNP produced from hydrolysis of MPO.

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Fig. 2. Degradation of MPO into pNP and phosphonic acid in the presence of OPH enzyme.

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For measurement of enzymatic activity, 0.25 M MPO was prepared in methanol. OPH aqueous solution (9.9 mg/ 0.9 mL) were prepared in CHES buffer with sonication for 10 min.

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For the activity check, 0.1 mL MPO solution was added followed by addition of 0.3 mL of varying concentrations of OPH solution directly under strong stirring. Aliquots of 20 μL were

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removed at subsequent time intervals. UV-Vis spectrum of diluted sample was taken by JAVSO V-670 spectrophotometer and peak at 400 nm was observed and concentration calculated by Beer-Lambert’s Law.

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Reaction kinetics of OPH enzyme were also studied using various substrate concentrations at pH 8.5. Using the Lineweaver-Burk plot, the maximum rate (Vmax) and Michaelis-Menten constant (Km) values were determined.

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OPH-PCD (200 mg) was mixed with 20 mL of 50 µM MPO for 2 h with stirring. Aliquots (1 mL) were removed at subsequent time intervals, filtered, and absorptions were determined. The initial velocity (M/s) of enzyme was calculated for both cases from the quantity of pNP produced with respect to time.

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2.7. Stability of PCD and OPH-PCD

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PCD microparticles (less hydrophobic) contained in two separate packed bed reactors with a

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bed volume of 97 mm3 were each exposed to 50 µM pNP and 50 µM MPO respectively, at a flow

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rate of 1 mL/h. The absorbance was determined at different time intervals for a period of 24 h. Similarly, OPH-PCD was tested with 50 µM MPO at 1 mL/h. The concentration of both MPO

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and degraded pNP were measured and plotted with respect to time.

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Reusability of PCD was determined in batch mode by exposing it to 10 mL of 100 µM pNP

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for 2 h with stirring. The adsorbed pNP to the PCD was rinsed down using methanol. The recovered PCD microparticles was tested again using the same amount of pNP. The process was

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repeated for 4 times.

Stability of OPH-PCD was investigated against 100 µM MPO degradation for 4 consecutive

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days to determine its activity. Using the same condition of packed bed reactor mentioned above, MPO was passed through packed OPH-PCD microparticles at 1 mL/h. The setup was run for 6 h each day and placed in refrigerator after use.

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Fig. 3. Characterization of PCD microparticles. A) SEM image of PCD B) FTIR of β-CD, HDI

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and PCD.

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3. Results and discussion

3.1. Preparation and characterization of PCD and OPH-PCD

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PCD was synthesized by the reaction of β-CD with HDI with a 1:9 equivalence ratio in the

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presence of DMF in a round bottom flask connected to a cooling condenser at 70 ℃followed by

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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

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effect of the variables involved such as β-CD, HDI, reaction time, temperature, and quenching agents (see Table S1 Supplementary information). Compared to β-CD, the β-CD hydrate has

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water which assists in polymerizing to HDI. Various equivalent ratios, quenching times and quenching fluids were tested to optimize the recipe. Our own previous study indicated 8 mol equivalent of HDI at 80 ℃for 4 h [34]. Dequan Li also used 1:8 molar ratio with a temperature of 70 ℃for 16-24 h [33]. Trotta et al. utilized three different molar ratios at 90 ℃for 3 h [36]. A

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recent attempt by Kawano et al. also tried with various molar ratios at 70 ℃for 12 h [17]. Different methods produced PCD particles with various particle size and porosity. In this report, we gave 1.5 h reaction time followed by addition of large amount of water as a quenching agent.

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The solution was easily and rapidly filtered through aspirator, hence avoiding further reaction and gelation of PCD particles. This resulted in producing nano-sized particles of PCD as shown in Fig. 3A.

The FTIR spectroscopy shows HDI peak at 2250 cm-1 (Fig. 3B). After reaction with β-CD, the peak for isocyanate group disappears and the vibration bands at 1530, 1715, and 3370 cm-1

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appear confirming the presence of NH-CO, C=O, and N-H groups respectively. The presence of

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amide group confirms the formation of cyclodextrin polymer. The broad peak at 3370 also

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indicates the presence of hydroxyl group due to water from hydrogen bonding.

3.2. Sorption of PCD towards MPO and pNP

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Adsorption profile for continuous mode for pNP with PCD was plotted as a function of time

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as shown in Fig. 4A. Observing from the graph, it is safe to say that the absorption plateau is

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reached within 20 min of contact time. For ensuring maximum adsorption for batch mode,

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prolonged exposure of 2 h was chosen.

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Fig. 4. A) Sorption of pNP by PCD in continuous mode. B) Sorption capacities for MPO and

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pNP in batch mode with PCD.

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MPO and pNP were tested individually (batch mode) to investigate the sorption behavior of

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PCD. Interior of CD and alkyl chains are both hydrophobic in nature. The exterior of CD is surrounded by –OH group. During the polymerization, the –OH group is bound to alkyl group.

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This bonding can only cover a few of those groups due to structure of CD causing molecular

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crowding. The extinction coefficient of MPO and pNP were found to be 7800 and 18600 a.u./M

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respectively. Both MPO and pNP showed reversible binding with PCD. MPO, being more hydrophobic than pNP, shows preferential binding towards PCD due to the hydrophobic-

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hydrophobic (HB-HB) interaction with the interior and exterior of PCD by host-guest inclusion complex formation. The results indicate sorption capacities of 1.261 mg/g PCD for MPO as

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compared to 0.746 mg/g PCD for pNP revealing a higher ratio of 1.7:1 for adsorption of the more hydrophobic MPO by the PCD microparticles (Fig. 4B). Additional information on the adsorption isotherms of PCD can be found from previous literature [65-67].

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MPO resembles the family of organophosphates primarily used as nerve agents in chemical warfare such as sarin and soman. It is less toxic compared to CWAs and degrades into lesser toxic by-product (pNP); both of which are UV-active compounds. Phosphonic acid, which is

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hydrophilic, is also released during the hydrolytic degradation which can be traced by 31P NMR. The breakdown of CWAs, for example sarin and soman show a similar nature of by-products of hydrophobic and hydrophilic nature. Hence, PCD can serve as sorptive substrate for toxic

hydrophobic compounds by inclusion formation into its cavity. Moreover, we also experimented with different quenching techniques for preparing PCD. Interestingly, using acetone as a

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quenching agent produces slightly hydrophilic PCD which tends to prefer pNP than MPO (See

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Supplementary Information Table S1). Hence, it is possible to modify the binding nature of PCD

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3.3. Effect of pH on sorption of PCD

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to suit the objective for forming the inclusion complex.

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Fig. 5. A) Reversible sorption of PCD with pNP B) Effect of pH on sorption behavior of PCD. Inset showing two forms of pNP in acidic and basic media.

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Fig. 5 demonstrates the pH influence on the sorption capability of pNP into the cyclodextrin domain. The hydrophobic part of pNP is able to reversibly adsorb onto the PCD particle (Fig.

5A). This host-guest formation utilizes exclusively HB-HB interaction for the temporary sorption phenomenon. Moreover, pNP demonstrates two isomers under different pH conditions; in acidic condition it becomes p-nitrophenol (hydrophobic), while in basic conditions it turns to p-

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nitrophenolate (hydrophilic) (Fig. 5B). Absorbance wavelength shifts showing a visual color

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change from yellow to colorless as the pH is reduced to 4 and below (see Fig. S1 Supplementary

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Information). Hence, both phenol and phenolate will exist at different ratios at different pH

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conditions. The sorption is highest at pH 8.6 and decreases in alkaline conditions. Due to its hydrophilic nature and higher solubility, the presence of phenolate at high pH decreases

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adsorption to PCD since it is more likely to remain in solution. At near neutral pH condition, the

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equilibrium between the two molecules brings ideal conditions for the adsorption of phenol into

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the PCD molecule to achieve the maximum sorption capacity. However, in strong acidic conditions, the solubility of phenol may decrease marginally, rendering it unavailable for

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molecular interaction with PCD. Therefore, a slight decrease is observed in the sorption trend.

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3.4. Protein content and enzymatic activity of OPH-PCD The schematic illustration of PCD and OPH-PCD as a sorption and decontamination system

can be seen from Fig. 6. PCD is utilized to remove toxic chemicals such as MPO and pNP as a highly preferential sorptive agent. Toxins can be imbibed into the interior of cyclodextrin via

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host-guest complex formation. This bonding is reversible with the help of organic solvents such as methanol, which can help rinse out sorbed pNP from PCD for safe disposal and help

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regenerate PCD.

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Fig. 6. A) Schematic illustration for PCD as a sorptive and regenerative system with the ability

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to be functionalized by adsorption of enzyme onto its structure. B) Schematic illustration for

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organophosphates.

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OPH-PCD as a stable self-decontaminating bio-catalytic system for sorption and degradation of

PCD can stabilize OPH enzyme in an irreversible absorption (Fig. 6A). Here it should be

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noted that the molecular weight of OPH is 36 kDa while the diameter of β-CD interior is 7.5 Å. While OPH is comparably larger in size, it was partially included near hydrophobic pockets and

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locked in when included. Few immobilization techniques have been known to destabilize the enzyme; however, the uniqueness of PCD to OPH is its capability to provide a stable platform. It is important to note that some enzymes may get denatured during the freeze drying. The possibility to refold it back into its original shape has been shown in previous literature [18].

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Hence, it’s worth mentioning that the same techniques can be used to revive the catalytic activity of enzyme using a similar process. The measured protein content was found to be 294 mg/g OPH-PCD. PCD harbors the

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structure of OPH enzyme. As the enzyme does not cover the entire PCD molecule, it will have partial space as binding sites available for sorption. Overall, a small amount of OPH will be present on PCD. The presence of OPH in the system demonstrates a dual role of OPH-PCD

system as sorption as well as catalytic degradation of toxic chemicals (Fig. 6B). Methyl paraoxon (MW: 247.14) is 9.79 Å in length and is comparably small molecule which can be easily

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included as a guest into the interior of β-cyclodextrin. The irreversible harboring of OPH enzyme

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will greatly improve the stability for long term storage purpose. Moreover, the system shows

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regenerative capability considering the enzyme does not get involved in the chemical reaction

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and neither does PCD which shows reversible sorption characteristics as mentioned earlier.

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Fig. 7. Reaction kinetics of A) OPH and B) OPH-PCD for hydrolysis of MPO in batch mode.

The results of enzymatic hydrolysis of MPO into pNP and phosphonic acid by OPH can be seen from Fig. 7A. Different mass concentrations were used to analyze initial velocity (Vo) of

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OPH enzyme. The resulting rates were 1.61×10-7 M/s (7.5 mg), 1.57×10-7 M/s (3.3 mg), 1.38×10-7 M/s (1.65 mg), and 1.10×10-7 M/s (0.825 mg) for OPH enzyme. The initial rate of OPH-PCD was slightly higher than the 0.825 mg OPH, with a value of 1.24×10-7 M/s (10 mg).

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This rate was also higher compared to previous attempt of deposition of OPH on silica beads (5.0×10-8 M/s). This rate could have been even higher considering the amount of pNP and MPO that’s initially adsorbed by PCD. From the concentration-velocity plot of OPH enzyme (Fig. S2), maximum rate of reaction (Vmax) and Michalis-Menten constant (Km) was found to be 1.43×10-2 mmol/(L∙min) and 2.06×10-2 mmol/L respectively. However, for OPH-PCD, the same curve is

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insufficient to describe the overall kinetics since sorption and enzyme degradation are both

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affecting the concentration of pNP. Additional dynamics must be incorporated to obtain accurate

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reaction kinetics of OPH-PCD. An estimated specific activity of OPH and OPH-PCD from the

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protein content was determined to be 12 U/mg and 5.23 U/mg respectively (see supplementary information for details).

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Complete hydrolysis occurred within 10 mins of exposure to MPO for both cases. When MPO

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encounters OPH-PCD, it naturally meets the multilayers of OPH first, where it is instantly

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hydrolyzed. Phosphonic acid is hydrophilic and will directly go into the bulk solution. Compared to MPO, the produced pNP is less hydrophobic. These two molecules will now compete to take

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the sorption site. Meanwhile, another incoming MPO molecule will attach to the enzyme resulting in catalytic degradation and the cycle will continue in this dynamic scenario. The

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generated pNP can either get adsorbed by PCD or enter the bulk solution. We believe that pNP produced near the innermost layer of sorption will get adsorbed and the ones produced on the outer layer will remain in the bulk solution. The presence of green-yellow color in PCD harboring OPH as well as in the bulk solution confirms the hypothesis. Therefore, the catalytic

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hydrolysis shows a plateau before reaching the maximum concentration. Moreover, we can see that initially MPO is degraded at a fast rate, and it slows down as more pNP (measurand) gets

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adsorbed into the PCD cavity.

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Fig. 8. Continuous flow packed bed column reactor for investigating A) sorption of PCD against MPO and pNP, and B) sorption and degradation properties of OPH-PCD against MPO

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intoxication. C) Reusability of PCD in batch mode by determining sorption rate (%) with pNP for each cycle. D) Daily enzymatic activity of packed bed column of OPH-PCD against MPO.

Continuous sorption and degradation properties were studied using a packed bed column (Fig. S3 Supplementary Information) under constant flow-through conditions. PCD-packed column

20

performed analogous to batch mode revealing a sharp breakthrough for both MPO and pNP (Fig. 8A). The breakthrough time taken for the less hydrophobic pNP is slightly more compared to MPO, which is evident from the fact that the PCD used has a slightly higher preference for the

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less hydrophobic material (see supplementary information Table S1). When MPO was passed through packed column of OPH-PCD, initially nothing comes out since both MPO and

hydrolyzed pNP are absorbed by the PCD microparticles (Fig. 8B). Eventually, the onset of pNP confirms the degradation of MPO as well as the limit of adsorption evident from previous results. Since pNP is a byproduct to degrading MPO, we decided to take advantage of the less

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hydrophobic PCD with a slightly higher preference to pNP instead. As both are hydrophobic in

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nature, our final biocatalyst OPH-PCD will serve to degrade the harmful pesticide and also

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adsorb as much of the by-product as possible while it is in a dynamic state in between both MPO

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and pNP. The immobilized enzyme on PCD exhibited continuous activity for the investigating period of 24 h demonstrating 100% hydrolyzing capability.

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Reusability of PCD was tested in batch mode using pNP sorption (Fig. 8C). After first

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exposure, the yellow colored microparticles were rinsed with methanol and reused for additional

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four cycles. The results indicated a similar performance for first four cycles showing ~ 53 % adsorption of pNP. The fifth cycle caused a decline revealing 42 % sorption rate of PCD.

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Reusability of OPH-PCD was observed under stressful environment of organic solvent methanol with MPO. Under such conditions, OPH alone would degrade rapidly against MPO. However,

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denaturation of OPH became retarded significantly over cycled use through its adherence to the hydrophobic β-CD when immobilized (Fig. S4 Supplementary Information). Thus, without providing barrier protection against delamination and applying polymeric liposome to encase OPH within the polymerized system, we’re able to demonstrate its reusability toward MPO.

21

Finally, the long-term stability of packed-OPH-PCD column was investigated daily for four consecutive days using MPO (Fig. 8D). As indicated earlier, the first four hours of purified solution was completely decontaminated. Hence, only a small amount of pNP is observed for the

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first day experimental run of 6 h. Afterwards, the catalytic activity remained constant for the investigation period of four days. Since both OPH and PCD do not get involved in the reaction, the synthesized biocatalytic material is remarkably stable not only for long-term activity but also for storage purpose. Thus, enhanced reusability and stability of OPH-PCD were demonstrated

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over cycled use when in presence of Poly-β-CD.

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4. Conclusion

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In conclusion, the synthesized self-decontaminating biocatalytic OPH-PCD system is

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extremely stable and shows remarkably fast degradation of MPO, hydrolyzing it completely within 10 mins of exposure time. PCD behaves not only as a unique and robust support for the

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enzyme but also as a regenerative sorption material for further purifying the organophosphate

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contamination. In synthesizing PCD, using acetone as the quenching agent of reaction, revealed

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less hydrophobic PCD. Instead, if only DI water was used, the produced PCD showed higher preference towards the more hydrophobic MPO. Sorption capacity for both PCDs was different

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under pH of 8.5 at room temperature conditions. MPO showed 1.7 times higher preference compared to pNP using more hydrophobic PCD. The reaction rate of OPH-PCD to OPH were

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1.24×10-7 M/s (10 mg) and 1.61×10-7 M/s (7.5 mg) respectively. OPH-PCD showed slightly less (~23 %) degradation rate compared to free OPH enzyme. The reusability of PCD revealed constant adsorption capability for 4 cycles (53 %) and a slight decrease (42 %) on the fifth cycle.

22

The long term storage and stability of OPH-PCD revealed continuous degradation properties for 4 consecutive days hydrolyzing MPO. We believe this work will be valuable to agricultural and industrial sector for purification of

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pesticides. For future applications, the OPH-PCD system will prove to be a beneficial environmental friendly material for sorption and decontamination of nerve agents.

Conflict of interest

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The authors declare no competing financial interest.

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Acknowledgments

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This work was supported by the National Research Council of Science & Technology (NST)

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grant by the Korean government (MSIT) [No. CMP-16-04-KITECH] and by Brain Pool Program

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through the Korean Federation of Science and Technology Societies (KOFST) funded by the

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Ministry of Science and ICT [No. 171S-2-3-1807].

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Appendix A. Supplementary data

Supplementary material related to this article can be found, in the online version or from the

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authors.

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