Immobilized Biocatalyst for Detection and Destruction of the

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Immobilized biocatalyst for detection and destruction of the insensitive explosive, 2,4-dinitroanisole (DNAN) Smruthi Karthikeyan, Zohre Kurt, Gunjan Pandey, and Jim C Spain Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.6b03044 • Publication Date (Web): 12 Sep 2016 Downloaded from http://pubs.acs.org on September 16, 2016

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Environmental Science & Technology

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Immobilized biocatalyst for detection and destruction of the insensitive explosive,

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2,4-dinitroanisole (DNAN)

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Smruthi Karthikeyan1, Zohre Kurt1, Gunjan Pandey3, Jim C. Spain1,2*

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311 Ferst Drive, Atlanta, Georgia 30332, USA

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11000 University Parkway, Pensacola, FL 32514-5751, USA

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Department of Civil and Environmental Engineering, Georgia Institute of Technology,

Center for Environmental Diagnostics & Bioremediation, University of West Florida,

CSIRO Land and Water, Clunies Ross Street, Acton, ACT 2615, Australia

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*Corresponding author:

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Jim C. Spain

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University of West Florida

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Center for Environmental Diagnostics and Bioremediation

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11000 University Parkway,

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Pensacola, FL 32514-5751

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Phone: 770 851-0007

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Email: [email protected]

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1

ACS Paragon Plus Environment


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ABSTRACT

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Accurate and convenient detection of explosive components is vital for a wide spectrum

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of applications ranging from national security and demilitarization to environmental

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monitoring and restoration. With the increasing use of DNAN as a replacement for 2,4,6-

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trinitrotoluene (TNT) in insensitive explosive formulations, there has been a growing

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interest in strategies to minimize its release and to understand and predict its behavior in

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the environment. Consequently, a convenient tool for its detection and destruction could

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enable development of more effective decontamination and demilitarization strategies.

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Biosensors and biocatalysts have limited applicability to the more traditional explosives

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because of the inherent limitations of the relevant enzymes. Here we report a highly

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specific, convenient and robust biocatalyst based on a novel ether hydrolase enzyme,

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DNAN demethylase (that requires no cofactors) from a Nocardioides strain that can

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mineralize DNAN. Biogenic silica encapsulation was used to stabilize the enzyme and

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enable it to be packed into a model microcolumn for application as a biosensor or as a

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bioreactor for continuous destruction of DNAN. The immobilized enzyme was stable and

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not inhibited by other insensitive munitions constituents. An alternative method for

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DNAN detection involved coating the encapsulated enzyme on cellulose filter paper. The

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hydrolase- based biocatalyst could provide the basis for a wide spectrum of applications

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including detection, identification, destruction or inertion of explosives containing

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DNAN (demilitarization operations) and for environmental restorations.

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TOC/Graphic abstract

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Introduction

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Traditionally, military explosives were designed for performance properties with little

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regard for environmental impact or susceptibility to biodegradation. Several common

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explosives components including nitroglycerin 1, dinitrotoluenes 2, and 1,3,5-

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trinitroperhydro-1,3,5-triazine (RDX) 3 appear to have stimulated the evolution of

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degradation pathways and can serve as growth substrates for bacteria. Most of the other

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predominant explosives are subject to partial biotransformation by microorganisms-

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typically reduction of the nitro groups, but such transformations do not support growth of

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the microbes and require the addition of large amounts of supplementary growth

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substrates. Furthermore, the oxygenase and reductase enzymes that catalyze such 3



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transformations require stoichiometric amounts of external cofactors as electron donors

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and thus function poorly if at all outside the microbial cells. The requirement for

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diffusible cofactors and low specificity essentially limits the applicability of free enzymes

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as biocatalysts for practical applications. The recent development of insensitive

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munitions (IM) involves replacement of 2,4,6-trinitrotoluene (TNT) and RDX with 2,4-

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dinitroanisole (DNAN) and 3-nitro-l,2,4-triazol-5-one (NTO). DNAN has proven to be

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not only less sensitive to heat and shock than TNT, but it is also biodegradable by

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bacteria 4. With its increasing use, industrial wastewaters and soil near the manufacturing

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sites and firing ranges are susceptible to DNAN contamination. Consequently, there has

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been considerable effort to study the environmental fate and transport of DNAN over the

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past few years 5-7. Several studies evaluating the fate of DNAN in the environment

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suggest that it is resistant to natural attenuation and persists in natural systems 5, 6, 8, 9,

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hence development of strategies for its detection and degradation are essential. High

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pressure liquid chromatography is effective for detection of DNAN in the laboratory and

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alkaline hydrolysis is effective for its destruction but biocatalysts for rapid detection and

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destruction of the compound have not been developed.

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Recently, a Nocardioides strain, JS1661, capable of biodegrading DNAN under a variety

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of conditions was shown to be effective for decontamination of soil 4. The bacterium uses

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DNAN as the sole carbon, nitrogen and energy source. The key to its versatility appears

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to lie in a novel ether hydrolase (EC 3.3.2.14) that catalyzes cleavage of the otherwise

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stable ether bond. The hydrolase converts the explosive compound to methanol and

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dinitrophenol, which are degraded by a variety of soil bacteria including JS1661. The

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enzyme, which is expressed constitutively, is a hexamer of two subunits that are co-

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transcribed 10 and does not require external cofactors, which raises the possibility that it

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could serve as a biocatalyst for detection and destruction of DNAN in the absence of

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bacterial cells.

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Cell-free biocatalysts can have significant advantages over living cells because they do

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not require growth substrates and they are amenable to in vitro evolution11. The main

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challenge faced in using isolated enzymes as biocatalysts is their limited stability and

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activity over extended periods 12. Immobilization of biomolecules can markedly improve

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their stability and thereby overcome their inherent inadequacies 13. A number of

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techniques have been devised for enzyme immobilization. The major ones include

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covalent bonding, adsorption (which involves hydrogen bonding or hydrophobic

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interaction), aggregation and physical entrapment 14, 15.

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Silica encapsulation has been one of the most widely used techniques for enzyme

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immobilization owing to the favorable physical properties of silica 15, 16. Biomimetic

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silica particles can be synthesized in-vitro in a process very similar to the mechanism

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used by diatoms, which have the remarkable ability to incorporate biogenic silica as a

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template/scaffold in biosilicification 17-20. Lysozyme directed formation of mesoporous

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silica nanoparticles for the immobilization of enzymes is well established 16, 21. The

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immobilization of enzymes using a silica template can provide a versatile platform for

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microfluidic and biocatalytic applications 22, 23. Recently, silica encapsulated atrazine

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chlorohydrolase (AtzA) and cyanuric acid hydrolase were developed for applications in

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water treatment 24, 25.

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Numerous bio-inspired systems have been proposed for the detection of more traditional

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explosives. Such systems range from immunosensors that utilize in-vitro production of

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antibodies to enzymatic and whole-cell biosensors 26, 27.More recently, Escherichia coli

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bioreporters which host a fusion of the yqjF gene promoter and luxCDABE or gfp genes

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that incorporate bioluminescene have been employed for detection of 2,4-DNT and 2,4,6-

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TNT vapors. However, their response times were relatively long and detection limits

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were relatively high 28 . Here we examined the potential to use DNAN hydrolase as a

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colorimetric biomarker to detect the presence of DNAN and also as a biocatalyst for

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destruction of DNAN. The hydrolase- based biocatalyst could provide the basis for a

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wide spectrum of applications including detection, identification, destruction or inertion

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of explosives containing DNAN (demilitarization operations) and for environmental

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restoration.

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Materials and methods

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Reagents

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Lysozyme from hen egg white (lyophilized powder, protein ≥90 %, ≥40,000 units/mg

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protein) and TMOS (tetramethyl orthosilicate) were purchased from Sigma-Aldrich.

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DNAN was from Alfa Aesar (Ward Hill, MA, USA), nitroguanidine (NQ) and 2,4-DNP

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were from Sigma-Aldrich (St, Louis, MO, USA). 3-nitro-1,2,4-triazol-5-one (NTO) was

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provided by the US Army Picatinny Arsenal, NJ. All other chemicals used were reagent

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grade or better. IMX-101 mixtures were prepared by dissolving DNAN, NQ and NTO in

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the ratio 2.25:1.75:1 (weight basis) in BLK medium and the composition was normalized

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to 100 µM DNAN.

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Analytical Methods

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DNAN and 2,4-DNP were analyzed by HPLC on a 4.6 x 100 mm Merck Chromolith C-

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18 reverse phase column and NTO and NQ were analyzed on a 4.6 x 150 mm Agilent

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Eclipse C-18 reverse phase column using an Agilent 1100 HPLC system as described

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previously 4.

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DNAN hydrolase purification and immobilization

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The hydrolase was partially purified by a modification of the method described by Fida

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et.al 10. Cells of JS1661 were grown to late exponential phase in ½ strength trypticase soy

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broth, harvested by centrifugation and suspended in phosphate buffer (20 mM, pH 7).

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The harvested cells were lysed by two passes through a French pressure cell at 40,000

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psi. The exudate was clarified by ultracentrifugation at 50,000 rpm for 45 min at 4°C and

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the supernatant was subjected to precipitation with ammonium sulfate at 0-28%, 28-42%

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and 42-60%. The 28-42% ammonium sulfate fraction was then dissolved in phosphate

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buffer and applied to a fast flow phenyl sepharose column (HiPrep Phenyl FF, GE

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Healthcare) and eluted with a 1.0 to 0 M gradient of ammonium sulfate in phosphate

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buffer (pH 7.0). At each stage of purification DNAN hydrolase activity was monitored

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spectrophotometrically as described previously 10. The active fractions were combined

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and subjected to ultra-filtration (100 kDa Amicon Ultra-15 Centrifugal Filter Units, EMD

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Millipore, Billerica, MA). The retentate was stored at 4°C until used for subsequent

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experiments. The partially purified enzyme was immobilized by the method of Johnson et

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al 16 using lysozyme (100 mg/ml) as a template for precipitating silica and tetramethyl

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orthosilicate (TMOS) as the silica precursor.

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Immobilized enzyme reactor (IMER) column studies

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To test the ability of the enzyme to detect/transform DNAN, the immobilized enzyme

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was packed in a microreactor consisting of a 2 cm x 2 mm stainless steel column (with 5

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µm frits) as described by Berne et al 22. The reactor, which was considerably larger than

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typical microfluidic devices, was operated at room temperature in multiple configurations

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to test the stability and detection limit of the system. The microreactor was also

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integrated into a biosensor system for rapid enzyme based detection of DNAN in water.

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Detection was based on the enzyme catalyzed hydrolysis of DNAN and formation of 2,4-

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dinitrophenol as indicated by the appearance of yellow color which was monitored

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spectrophotometrically in the exit stream.

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Continuous flow system

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The IMER column was used in conjunction with a syringe pump (PHD 22/2000, Harvard

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Apparatus, Holliston, Massachusetts) to enable it to be used as a continuous flow system

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for DNAN hydrolysis. The column was washed initially with HEPES (4-(2-

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hydroxyethyl)-1-piperazineethanesulfonic acid ) buffer (20 mM, pH 8), after which

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solutions of various concentrations of DNAN in HEPES were pumped at flow rates

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ranging from 2 µL/min to 10 µL/min corresponding to retention times of 1.8 to 0.36 min.

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Typical flow rates for microfluidic applications in the literature vary from 1-5 µL/min 22.

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Here, flow rates up to 10 µL/min were used in order to determine breakthrough for a

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given concentration of the influent. The DNAN and DNP concentrations in the effluent

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were measured by HPLC analysis of samples from the exit stream of the column. After

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each change in flow rate, the system was equilibrated for five column volumes prior to

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sample collection and analysis. In order to test the robustness of the enzyme in

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unbuffered solutions and mixtures, IMX mixtures in tap water (pH 6.5) were pumped

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through the system.

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Enzyme based detection system

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The IMER column was incorporated in a detection system by connecting it to an Agilent

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1100 series HPLC system equipped with a diode array detector that facilitated direct

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detection of DNAN. HEPES buffer (pH 8) was used as the mobile phase a flow rate of

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0.1 ml/min and various volumes (1 to 4 µL) of DNAN (100 µM) were injected prior to

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the column. DNAN and 2,4-DNP in the column effluent were monitored at 295 nm and

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400 nm respectively. The efficiency of DNAN detection /conversion was quantified

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based on the ratio of absorbance at 295 nm to 400 nm. DNAN and DNP were

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distinguished based on their UV spectra as established previously 61. Calibration curves

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were generated by injecting known amounts of DNAN and DNP to the system. To

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determine the conversion ratio, DNAN and DNP were injected on an empty column

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(without enzyme) and the peak area ratios (peak area at 400 nm/ peak area at 295 nm)

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were calculated. Then the same amounts of DNAN and DNP were injected on the column

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packed with the immobilized enzyme and the peak area ratios were calculated to

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determine the percentage of DNP vs. DNAN in the exit stream of the active column. All

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of the above microcolumn experiments were done at room temperature. All calibrations

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were done in replicates.

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Paper-based biosensor system

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Samples of the immobilized enzyme suspended in 5 µL of phosphate buffer (0.1 M, pH-

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8) were applied to 0.635 cm blank filter discs (BBL Sensi-Disc Susceptibility Test Discs,

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Becton, Dickinson and Company, New Jersey) which were then air dried for 2-3 h.

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Control discs were prepared by depositing 5 µL of the free enzyme preparation on the

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paper discs.

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Stability tests

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Stability experiments were done on free and immobilized enzyme preparations at 4°C and

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at room temperature. At appropriate intervals, the enzyme activities were quantified

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spectrophotometrically at 400 nm (Varian Cary 50 UV-Vis spectrophotometer, Palo Alto,

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CA) as described previously 7. Assays for the residual activity of the bioactive paper were

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done by suspending the cellulose filter discs in 250 µL of HEPES buffer containing

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DNAN (100 µM). Assay mixtures were incubated with shaking for 45 min after which

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the reactions were stopped by the addition of equal volumes of acetonitrile. Reaction

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mixtures were centrifuged and supernatants were analyzed by HPLC. The residual

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activity was quantified based on the conversion of DNAN to 2,4-DNP.

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Results and discussion

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Enzyme immobilization

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The hydrolase enzyme that was partially purified from the wild type cells of JS1661 had

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a specific activity of 8.8 µmol/min/mg of protein which compared favorably with

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previous highly purified preparations (6.4 umol/min/mg of protein) 10. It was not purified

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to homogeneity. The two major bands corresponding to DNAN hydrolase were

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accompanied by several other unidentified bands from the cell extract when the

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preparation was examined by sodium dodecyl sulfate polyacrylamide gel electrophoresis

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(data not shown). Thus high purity is not necessary for the effectiveness of the

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immobilized biocatalyst. During the immobilization process, the enzyme retained over

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88% of the initial activity (7.8 µmol/min/mg of protein) and the apparent loss in activity

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could be the result of mass transfer limitations in the silica matrix 22, 25, 29. No activity was

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detected in the supernatant fluid after the silica- immobilized enzyme was removed by

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centrifugation.

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Stability tests

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Immobilization is known increase the stability of enzymes used as biocatalysts 25, 30-32.

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Here, the heterologous activity of the immobilized DNAN demethylase remained stable

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while the residual activity for the soluble enzyme declined rapidly at room temperature

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(Fig. 1). Such stabilization has been attributed to the microenvironment provided by the

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silica encapsulation matrix and the prevention of contamination, which preserves enzyme

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activity even under non-physiological conditions 33. Silica matrices can also enhance the

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thermal stability of enzymes by preventing unfolding of the protein 34, 35.

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The decreased activity of the soluble enzyme at room temperature could be due to

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conformation changes in the protein at elevated temperatures, loss/inactivation of an

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essential metal or microbial contamination/degradation. The soluble enzyme lost activity

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even at 4°C, perhaps because of microbial or proteolytic degradation, protein aggregation

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or suboptimal buffer conditions. Moreover, very dilute proteins are more susceptible to

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inactivation in the absence of other proteins 12, 17, 36 . No attempt was made to add

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stabilizing agents such as glycerol or optimize redox conditions during storage 36.

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IMER column studies

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Continuous flow-through systems for DNAN destruction

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Immobilized enzyme biocatalysts are effective in a wide range of reactor configurations

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such a system could be effective in DNAN mitigation strategies. The hydrolase-

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catalyzed biotransformation of DNAN could enable treatment of waste streams resulting

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from demilitarization operations or environmental remediation.

. Since DNAN undergoes little natural attenuation and would persist in the environment

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The IMER column microreactor was tested initially in buffered systems based on the

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optimal conditions for the hydrolase 10. The efficiency of DNAN conversion to DNP at

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all influent DNAN concentrations approached 100% when the flow rate was maintained

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at 4 µL/min (Figs. 2,3). The lowest efficiency (75.05%) was observed at a DNAN feed

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concentration of 500 µM (Fig. 3). In order to determine whether the efficiency was lower

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because of substrate inhibition, the system was operated at different flow rates with the

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same DNAN concentration of 500 µM. At a flow rate of 4 µL/min, the retention time on

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the column was estimated to be 0.9 min. At lower flow rates almost all the DNAN was

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converted to DNP and the conversion efficiency dropped with an increase in the flow rate

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suggesting that the insufficient residence time rather than substrate inhibition was

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responsible for the lower efficiency. Furthermore, concentrations as low as 10 µM were

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converted stoichiometrically to DNP which is consistent with the reported KM of 19.02

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µM 10.

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All of the above experiments were performed at the optimum pH for the enzyme-

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catalyzed reaction in a well- buffered system. When solutions of DNAN (100 µM) in tap

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water (pH = 6.5) were pumped through the column at similar flow rates, the DNAN

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conversion efficiency was greater than 90% (Fig. 5). The above results indicate that

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although the enzyme is most efficient in a system buffered to optimal pH, the HEPES

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buffer is not necessary. The enzyme is active over a pH range of 6.2-9.5 10.

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Continuous flow through systems with IMX mixtures

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Though enzymes are highly specific, they can be inhibited or influenced by the presence

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of other compounds along with the target substrate. Insensitive melt cast explosive

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formulations like IMX-101, IMX-104, PAX-48, PAX-21 and PAX-41 contain DNAN

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along with other insensitive explosives including 3-nitro-1,2,4-triazol-5-one (NTO) and

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nitroguanidine (NQ) in various proportions 9. In practice, therefore, DNAN is likely to be

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found in conjunction with the other munitions constituents in waste streams or

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contaminated sites. In order to assess the consequence of the presence of other

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compounds in conjunction with DNAN, IMX mixtures containing DNAN (100 µM) in

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tap water were pumped through the column. DNAN was destroyed at all flow rates

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although the conversion efficiency dropped slightly at high flow rates (8, 10 µL/min)

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(Fig. 4). NTO and NQ concentrations were similar in the influent and effluent streams.

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The results indicate that the other insensitive munitions components did not inhibit the

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enzyme and were not transformed, which is consistent with the previous observations

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with intact cells 4.

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In the above studies with the continuous flow through system, DNAN destruction was

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achieved over a range of conditions. Previous studies conducted on immobilized enzyme

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microreactors as continuous flow through systems also showed substrate conversion that

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declined at higher loading rates 22, 38. We did not attempt to optimize the operating

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parameters in this study but other studies suggest that using monolithic polymers or

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biocatalytic nanofibers can overcome mass transfer losses and wall effects 39-41.

315 316

Enzyme based online detection system

317 318

Enzymatic biosensors are increasingly gaining prominence due to their stability,

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specificity, reusability and ease of integration in analytical systems. Recently, the field

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has expanded greatly with widespread applications ranging from high throughput enzyme

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mapping to ‘lab on a chip’ medical diagnostic tools 42-44. The above experiments

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suggested the potential for the use of the immobilized enzyme not only for destruction of

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DNAN but also for detection in a real time system 45. A model online enzyme biosensor

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consisted of an HPLC with a continuous flow of buffer through the IMER column and

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known amounts of DNAN injected at intervals. DNP production was monitored

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continuously at 400 nm with a diode array detector (molar extinction coefficients for

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DNAN at 295 nm was estimated to be 4400 whereas for DNP at 400 it was 5300). In all

328

cases, the DNP detected was proportional to the amount of DNAN injected. The

329

efficiency of conversion of DNAN to DNP decreased slightly when DNAN injection

330

volumes increased (Fig. 6) which was consistent with results from the continuous flow

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column described above. The rate of DNAN conversion did not change when HEPES

332

buffer was pumped through the IMER column for 12 h at room temperature (data not

333

shown) and the same column was used repeatedly over the course of several days. The

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334

response time of the sensor for the quantitative detection of DNAN was 0.5- 0.8 min. The

335

results indicated that, as in the continuous flow system, the immobilized enzyme was

336

stable in the column. The decline in conversion efficiency at high concentrations is an

337

issue for treatment of waste streams, but not relevant for the biosensor where detection

338

limits are the key concern. In the configuration described here, with no attempt at

339

optimization, 19.8 ng of DNAN was readily detectable in the 1.0 µl injection volume

340

which corresponds to 19.8 µg/ml. Optimization of the configuration and larger injection

341

volumes would be expected to produce correspondingly lower detection limits. Previous

342

literature on biosensors for the detection of explosive components TNT and DNT varied

343

from 0.01 ng/ml to 23.245 µg/ml 46-52. However even some of the highly sensitive

344

optimized biosensors had inherent drawbacks. Most of them incorporated complex or

345

expensive fabrication techniques, some were for tracking only the volatile explosives in

346

the gas phase and the others had low specificity. The specificity of DNAN hydrolase

347

along with its stability and sensitivity could provide the basis for simple online

348

monitoring and detection systems.

349 350

Bioactive paper

351 352

Paper based biosensors can serve as rapid, cost-efficient and portable systems for the

353

detection of biomarkers of clinical importance 53. They have been used in detecting

354

certain toxins, biological substances and various organic substrates owing to their

355

portability, versatility and low cost 53-55. Paper based microfluidic analytical devices

356

incorporate the capabilities of microfluidic devices onto a simple sheet of paper where

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the cellulose capillary channels aid in transport. The simplicity of paper- based

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microfluidics greatly increases their ease for application and they have been explored

359

extensively for point of care (POC) diagnostics 44, 56, 57. Furthermore, the coated paper

360

discs can be transported and stored at ambient temperature without loss of activity or

361

structural integrity. Analogous low cost, biocompatible systems for detection of

362

explosives could have manifold applications. If such a system could be developed with

363

DNAN hydrolase it could be used for rapidly detecting the presence of DNAN

364

contamination in the field which could aid in screening for explosives.

365 366

When solutions containing various concentrations of DNAN were added to the filter discs

367

containing the immobilized DNAN hydrolase, they turned yellow within seconds

368

indicating conversion to DNP. The color intensity varied in proportion to the

369

concentration of DNAN added (Fig. 7). The most important parameter governing the

370

application of paper- based biosensors is their sensitivity 58. The above preliminary

371

results indicate that concentrations of DNAN as low as 15 µM can be detected (Fig. 7).

372

Coating the paper with soluble enzyme gave similar initial results but the enzyme was

373

substantially less stable during storage (Fig. 8). The response of the soluble enzyme was

374

not linear at higher concentrations probably due to loss of activity of the enzyme. The

375

soluble enzyme lost activity rapidly during storage. It might be possible to stabilize the

376

soluble enzyme by addition of stabilization agents such as chitosan, which can be added

377

to preserve enzymes coated onto paper 59, 60.

378

The simple technique used to coat the filter discs in this study was not optimized.

379

Sophisticated coating techniques such as inkjet printing, wax screen printing,

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380

electrochemical deposition and photolithography have been implemented in the

381

fabrication of paper based microfluidic devices 55, 56. Such strategies could greatly

382

enhance the stability, sensitivity and accuracy of DNAN detection.

383 384

The results presented here indicate that DNAN demethylase provides the basis for

385

biocatalytic applications owing to its stability and specificity coupled with the lack of

386

requirement for additional cofactors. Lysozyme mediated silica encapsulation of the

387

enzyme was effective in preserving its stability and activity. The immobilized hydrolase

388

was resilient and proved to be a robust biosensor for the detection and destruction of

389

DNAN. Additional studies to optimize the configuration of IMER systems or improve the

390

properties of the enzyme could enhance the performance of the system. Furthermore, the

391

microcolumn reactor could be scaled down for microfluidic applications or scaled up for

392

larger decontamination and clean up applications. Field tests conducted with the

393

biosensor system and the paper based test kits at contaminated sites would open more

394

avenues for practical applications.

395 396

Although the enzyme is effective in detection and destruction of DNAN, conversion to

397

2,4- DNP is not a means of detoxification. 2,4-DNP, however, is much more readily

398

biodegradable than DNAN. The Nocardioides strain from which DNAN hydrolase was

399

derived is also capable of complete mineralization of DNAN as well as DNP. The intact

400

bacterial cells could be immobilized if a biocatalyst for complete destruction of DNAN is

401

required for use in related applications. The native DNAN hydrolase demonstrates very

402

high specificity and amenability to non- physiological conditions. The availability of the

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genes encoding the enzyme will facilitate future strategies for improvement of the

404

properties through directed evolution and site directed mutagenesis.

405 406 407

Acknowledgements

408 409

The authors thank Dr. Bettina Bommarius of Georgia Tech for help with the FPLC

410

analyses and for valuable suggestions and comments on the manuscript. We also thank

411

Andreas Schmid and Gregg Whited for helpful comments on the manuscript.

412 413

Figure legends

414 415

Fig 1: Stability of free and immobilized enzyme at room temperature and 4°C.

416 417

Fig 2: DNAN conversion in a continuous flow-through system with buffered feed (flow

418

rate of 4 µL/min).

419 420

Fig 3: Effect of residence time for higher concentrations of DNAN feed.

421 422

Fig. 4: Conversion of DNAN in the packed microcolumn reactor for IMX mixtures.

423 424

Fig. 5: DNAN conversion for an unbuffered system.

425

19



426

Fig. 6: DNAN conversion in the IMER based enzyme biosensor.

427 428

Fig 7: DNAN conversion with various concentrations of DNAN added to the filter discs

429

coated with immobilized and soluble enzyme.

430 431

Fig. 8: Stability of soluble and immobilized enzyme coated filter discs at RT and 4°C.

432 433 434 435 436

FIGURES

437 Free enzyme (4°C)

% Residual activity

150

Free enzyme (RT) Immobilized enzyme (4°C) Immobilized enzyme (RT)

100

50

0 0

438

10

20

30

40

Time(d)

439 440

Fig 1: Stability of free and immobilized enzyme at room temperature and 4°C.

441

20


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600

Influent DNAN

86.6%

Concentration (µM)

Effluent DNP

400 Conversion efficiency 99.12%

200 98.03% 99.88% 94.28 %

99.22%

0 10

25

50

100

200

500

Initial Influent (DNAN) concentrations (µM)

442 443

Fig 2: DNAN conversion in a continuous flow through system with HEPES pH 8.0 (flow

444

rate of 4 µL/min).

445 Influent DNAN

800 Concentration (µM)

Effluent DNP

600

98.68%

87.06%

74.94%

400

200

0 2

446 447

4

8

Flow (µL/min)

Fig 3: Effect of residence time for higher influent concentrations of DNAN feed.

448 449 450

21



Influent DNAN

140 Concentration (µM)

120

Effluent DNP 97.34%

93.86%

74.6%

83.2%

100 80 60 40 20 0 2

4

8

10

Flow (µL/min)

451 452 453

Fig. 4: Conversion of DNAN in the packed microcolumn reactor with IMX mixtures.

454 Influent DNAN

Concentration (µM)

150

Effluent DNP 97.48%

93.30%

91.60%

100

50

0 2

455 456

4

8

Flow (µL/min)

Fig. 5: DNAN conversion in an unbuffered system.

457 458

22


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Page 23 of 30


% Conversion

120

90

60

30

0 0.1

459 460

1.0

2.0

3.0

4.0

Injected DNAN (µL)

Fig. 6: DNAN conversion in the IMER based Enzyme biosensor.

461 462 463

Fig 7: DNAN conversion with various concentrations of DNAN added to filter discs

464

coated with immobilized and soluble enzyme.

23



% conversion of DNAN

150

Disc coated with free enzyme (4°C) Disc coated with free enzyme (R T) Disc coated with immobilized enzyme (4°C)

100

Disc coated with immobilized enzyme (R T)

50

0 0

465 466

10

20

30

40

Time(d)

Fig. 8: Stability of soluble and immobilized enzyme coated filter discs at RT and 4°C.

467 468 469 470 471 472 473 474 475 476 477 478 479 480 481

24


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482


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Immobilized Biocatalyst for Detection and Destruction of the

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