# Patent

## Title
Modified arsenite oxidase and a biosensor for detecting arsenite

## Abstract

The present invention provides an arsenite oxidase enzyme modified to prevent translocation by modification of a translocation signal sequence. A microorganism modified to express the heterologous arsenite oxidase enzyme is also provided by the invention, together with a device for detecting the presence of arsenite in a sample.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a divisional of U.S. application Ser. No. 14/352,437, filed on Apr. 17, 2014, which is a National Stage Application of PCT No. PCT/GB2012/052609, filed on Oct. 19, 2012.
The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, was submitted Apr. 17, 2014, in U.S. application Ser. No. 14/352,437 (National Stage of PCT/GB2012/052609). Said ASCII copy, created on Apr. 15, 2014, is named Sequence Listing for GILJEN 3.3-012 (E) ST25.5XT, is 16,442 bytes in size, and was transferred to the present application.


FIELD OF THE INVENTION
The present invention relates to modified enzymes for detecting the presence of arsenic derivatives, in particular arsenite, and methods for expressing such enzymes in heterologous organisms. The invention is also directed to devices for detecting arsenic, and derivatives thereof, in a sample.
BACKGROUND OF THE INVENTION
It is said that the third major challenge for a sustainable future (together with food security and energy) will be making the best use of limited supplies of pure water for both agricultural use and human consumption, and the remediation of marginal and contaminated water will be essential in achieving this. Already groundwater contamination, resulting from either natural geochemical processes or industrial activities such as mining, is a major problem in many countries.
Arsenic (As) is a groundwater contaminant that is ubiquitous in the environment and the two soluble forms, arsenite (AsIII) and arsenate (Asv), are toxic. Anthropogenic activity has resulted in widespread contamination of both soluble forms but AsIII is prevalent in anoxic environments, including most sources of drinking water. Countries affected include India, Bangladesh, Vietnam, USA, Germany, France, Hungary, Australia, Argentina, Mexico, Canada.
An important aspect of remediation is assessment and monitoring, and whilst laboratory methods exist that demonstrate high specificity and sensitivity (e.g. ICP-MS or HPLC) it is also possible, and indeed desirable, to measure analytes such as arsenite in the field using sensors. Ideally, the sensors should be low-cost, disposable and able to be readily adapted to multiple analytes that are commonly found together in contaminated water.
Many As filed test kits are commercially available (e.g. from Industrial Systems, Inc. Hydrodyne) but these only detect total As, rather than the most toxic form, AsIII, which is dominant in anoxic drinking waters. Moreover, because As remediation preferentially removes Asv (e.g. by binding to iron hydroxide) and requires the pre-oxidation of AsIII, it is crucial to determine whether any AsIII remains in the water. The chemically based arsenic field kits rely on a colorimetric method which reduces the AsIII and Asv to the gas arsine which reacts with the mercuric bromide test strips. These kits require the training of personnel, are expensive (e.g. Arsenic, Quick II Hydrodyne kit US$4.24 per test and Ultra Low Quick II, Industrial Test Systems, Inc. US$6 per test) and have low sensitivity and reproducibility.
Whole cell biosensors have been developed for the detection of AsIII by a number of groups (e.g. Stocker et al. (2003) Environ. Sci. Technol. 37, 4743-4750). These methods are all based on colorimetric assays that sometimes require the use of a luminometer. They all use the regulatory mechanism of the Escherichia coli arsenic resistance system which detects both AsIII and antimonite (SbIII). The regulatory gene in this system, arsR, is fused to a reporter gene (e.g. luciferase gene, luxB) that when expressed after induction with AsIII produces a visible signal (e.g. fluorescence).
There are many problems with whole cell based AsIII biosensors, including: 1) the system is too complex and because of this has a slow response time (i.e. AsIII must enter cells followed by induction of regulator-reporter gene protein—this can take up to 24 hours for a response); 2) lack of specificity as the system does not discriminate between AIII and SbIII; 3) incubation temperatures of 30° C. are often required; 4) colorimetric assay often requires use of a luminometer, which is not feasible at most field sites; and 5) use of genetically modified organisms always presents an additional problem. No whole cell biosensors for the detection of AsIII are commercially available.
A biosensor for AsIII has been developed using molybdenum-containing arsenite oxidase (known as “Aio” and also previously known as Aro and Aso; see Lett et al., Unified Nomenclature for Genes Involved in Prokaryotic Aerobic Arsenite Oxidation; J. Bacteriology, 4 Nov. 2011; p.20′7-208) which is a member of the DMSO reductase family, prepared from chemolithoautotrophic Alphaproteobacterium Rhizobium sp. strain NT-26 (Santini et al. (2000) Appl. Environ. Microbiol. 66, 92-97).
AsIII oxidase catalyses the oxidation of AsIII to Asv and the suitability of this native enzyme for use as a biosensor has been tested and shown to detect down to 1 ppb AsIII, which is 10 times lower than the recommended WHO MCL (maximum contaminant level) of As in drinking water. Furthermore the native enzyme shows specificity for AsIII (Male et al. (2007) Anal. Chem. 79, 7831-7837). The biosensor comprises the enzyme directly linked to the surface of a mulitwalled carbon nanotube-modified electrode, in which electron transfer proceeds directly from enzyme to electrode. The authors noted, however, that certain commonly-used electrode materials, in particular glassy carbon (GC), were not suitable for direct electron transfer in this configuration.
Heterologous expression of molybdenum-containing enzymes, especially members of the DMSO reductase family, is notoriously difficult. Recently, the dissimilatory arsenate reductase from Shewanella sp. str. ANA-3 was expressed in Escherichia coli but comparisons with the native enzyme were not made (Malasarn et al. (2008) J. Bacteriol. 190, 135-142). Expression was optimal when E. coli was grown anaerobically with DMSO although other electron acceptors for anaerobic growth were not tested and neither were different strains.
Since the entire native Aio is poorly expressed in a heterologous expression system, such as E. coli, use of this enzyme in routine detection of AsIII is not commercially viable.
As such, there is a need for improved methods and sensors for cheap and effective detection of AsIII in liquids such as drinking-water, waste-water and biological samples.
BRIEF SUMMARY OF THE INVENTION
According to a first embodiment, the present invention provides an arsenite oxidase enzyme modified to prevent translocation to the periplasm, wherein the enzyme comprises the native AioA subunit from NT-26 and the native AioB subunit from NT-26, wherein a portion of the native AioB subunit corresponding to the translocation signal sequence, or a functional fragment thereof, is modified.
According to a second embodiment, the present invention is directed to the use of a modified AsIII oxidase according to the first aspect of the invention as a biosensor to detect the presence of AsIII in a sample.
According to a third embodiment, the present invention provides a microorganism, modified to express a heterologous AsIII oxidase according to the first aspect of the invention.
According to a fourth embodiment, the present invention provides a method of producing recombinant AsIII oxidase, comprising expressing the modified enzyme heterologously according to the first aspect of the invention in a microorganism.
According to a fifth embodiment, the present invention provides a device for detecting the presence of AsIII in a sample, comprising at least one electrode, an arsenite oxidase enzyme and a redox mediator.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows the micro-structured surface of a device according to the invention;
FIG. 2 is an AsIII dose-response curve showing the comparable results of the sensor of the invention over two consecutive days;
FIG. 3 is a comparison of AsIII oxidase activities in total cell extracts of E. coli strains DH5α and JM109λpir grown with different electron acceptors;
FIG. 4 shows a SDS-Polyacrylamide gel (12%) of purified recombinant NT-26 AsIII oxidase (M: Molecular weight marker: phosphorylase b (94 kDa), albumin (67 kDa), oval albumin (43 kDa), carbonic anhydrase (30 kDa), trypsin inhibitor (20 kDa), β-lactalbumin (14 kDa) (GE Healthcare) 1: Purified recombinant AioBA, two subunits AioA ( ̃94 kDa) AioB with N-terminal His-tag ( ̃19 kDa));
FIG. 5(a-j) show cyclic voltammogram measurements and chrono amperometry for a device according to the invention including ferrocene methanol as the mediator;
FIG. 6(a-d) shows show cyclic voltammogram measurements for a device according to the invention including Tris(2,2′-bipyridine) dichlororuthenium(II) as the redox mediator;
FIGS. 7a, 7b and 7c show cyclic voltammogram and chrono amperometry measurements for a device according to the invention including feccocene carboxylic acid as the mediator;
FIG. 8(a-f) show cyclic voltammograms measurements for a device according to the invention including 2,6-dichlorophenolindophenol as the redox mediator; and
FIG. 9 shows cyclic voltammograms of TTF as the redox mediator.



DETAILED DESCRIPTION OF THE INVENTION
The present inventors have developed a modified version of the native AsIII oxidase from Rhizobium sp. NT-26 (GenBank Accession Number AY345225) which can be successfully expressed in heterologous expression systems such as E. coli. This has been achieved by modifying a translocation signal sequence present in the native enzyme, or a functional fragment thereof, to prevent translocation.
The native AsIII oxidase consists of two heterologous subunits: AioB is the small subunit largely composed of beta sheets and AioA is the large subunit largely composed of alpha-helices (Santini & vanden Hoven (2004) J. Bacteriology. 186(6):1614-1619). The polypeptide sequence of wild-type AioB from Rhizobium sp. NT-26 is shown in SEQ ID No. 1 and SEQ ID No. 5 shows the corresponding nucleotide sequence. The AioB subunit comprises a Tat leader sequence (also referred to herein interchangeably as a Tat translocation signal sequence) which corresponds to the first 25 amino acids of SEQ ID No 1. This translocation signal sequence is shown as SEQ ID No. 2 and SEQ ID No. 6 shows the nucleotide sequence of the portion of the aioB gene encoding the translocation signal sequence. The signal sequence directs the transport of the protein to the periplasm using the Twin Arginine Translocation (Tat) pathway.
The present inventors have modified the native AsIII oxidase by modifying the translocation signal sequence in the AioB subunit. As a result of the modification, the modified enzyme is not exported from the host cytoplasm, and as a result can be expressed in large, commercially-viable quantities.
The translocation signal sequence can be modified by various methods which will be apparent to a person skilled in the art, including frame-shift mutations, substitution mutations or deletion. Any modification that results in loss of function of the native translocation signal sequence is within the scope of the invention, however deletion of the translocation signal sequence or a functional fragment thereof, is preferred.
Accordingly, a first aspect of the invention provides an AsIII oxidase modified to prevent translocation by modification of a translocation signal sequence, or a functional fragment thereof.
The modified AsIII oxidase comprises two subunits. The first subunit corresponds to the native AioA subunit from NT-26, or a variant, derivative or homologue thereof. The second subunit corresponds to the native AioB subunit from NT-26, or a variant, derivative or homologue thereof; however a portion of the native AioB subunit which corresponds to the translocation signal sequence, or a functional fragment of the translocation signal sequence, is modified in the enzyme of the invention. Preferably, the portion of the native AioB subunit which corresponds to the translocation signal sequence, or a functional fragment thereof, is modified by deletion.
In one embodiment, at least a portion of the aioB gene which encodes the translocation signal sequence, or a portion thereof encoding a functional fragment of the translocation signal sequence, is modified, preferably by deletion.
The portion of the aioB gene sequence encoding the translocation signal sequence is identified herein as SEQ ID NO. 6. Either the complete sequence identified as SEQ ID NO. 6 or a homologue of this sequence encoding a functional fragment of the translocation signal sequence may be modified according to the invention. As a result of the modification to the nucleotide sequence, the modified enzyme of the invention does not comprise the amino acid sequence identified herein as SEQ ID No. 2, or does not comprise a portion thereof that is required for a functional translocation signal sequence.
As used herein, the term ‘functional fragment’ means that the portion of the nucleotide sequence that is modified (e.g. by deletion) encodes a polypeptide having Tat translocation signal sequence activity, preferably having at least the same activity of the polypeptide shown as SEQ ID NO: 2. As a result of the modification, the recombinant enzyme of the invention lacks such Tat translocation signal sequence activity.
As used herein, the term “homologue” refers to a nucleotide sequence that encodes a polypeptide which has Tat translocation signal sequence activity. With respect to sequence similarity, preferably there is at least 60%, more preferably at least 70%, more preferably at least 75%, more preferably at least 85%, more preferably at least 90% sequence similarity between SEQ ID NO. 6 and the sequence of the native aioB gene that is modified according to the invention. More preferably there is at least 95%, more preferably at least 98%, sequence similarity. These terms also encompass allelic variations of the sequences.
In another embodiment, the portion of the aioB gene that is modified according to the invention comprises or consists of the sequence identified herein as SEQ ID NO. 6.
SEQ ID No. 3 corresponds to the amino acid sequence of the AioB subunit excluding the entire native leader region. SEQ ID No. 7 shows the corresponding nucleotide sequence. If the entire AioB leader region is deleted, then the AioB subunit of the modified enzyme of the invention will correspond to SEQ ID No. 3. However, it is within the scope of the invention for a portion of the leader region within the AioB subunit to remain unmodified, provided that the portion of the leader region that is modified renders the remaining portion non-functional.
SEQ ID No. 4 shows the amino acid sequence of the AioA subunit and SEQ ID No. 8 shows the corresponding nucleotide sequence.
It is within the scope of the invention for the modified AsIII oxidase to comprise homologues, variants or derivatives of SEQ ID Nos. 3 and 4.
The terms “variant”, “homologue”, “derivative” or “fragment” as used herein include any substitution, variation, modification, replacement, deletion or addition of one (or more) amino acid from or to a sequence. The variant may have a deletion, insertion or substitution variation. The variation may produce a silent change and a functionally equivalent polypeptide, or may result in improved catalytic function or other characteristics of the resulting enzyme. Deliberate amino acid substitutions may be made on the basis of similar physio-chemical properties such as size, charge and hydrophobicity in order to alter the catalytic function or other properties or characteristics of the enzyme.
Unless the context admits otherwise, references to “AioA” and “AioB” include references to such variants, homologues and derivatives of the native subunits. The term “homologue” covers identity with respect to structure and/or function and is used to refer to peptides that share levels of sequence identity or similarity to SEQ ID Nos. 3 and 4 and retain at least the functionality of the native amino acid sequences. The variants may result in improvements in the catalytic activity or other properties of the resulting enzyme. These terms also encompass polypeptides derived from amino acids which are allelic variations of the aioA and/or aioB nucleic acid sequences (SEQ ID Nos. 7 and 8).
Levels of identity or similarity between amino acid sequences can be calculated using known methods. Publicly available computer based methods include BLASTP, BLASTN and FASTA (Atschul et al., Nucleic Acids Res., 25: 3389-3402 (1997)), the BLASTX program available from NCBI, and the GAP program from Genetics Computer Group, Madison Wis.
It is preferable if there is at least 60% sequence identity or similarity to the specified peptides of SEQ ID Nos. 3 and 4, preferably 70%, more preferably 80% and most preferably greater than 90%, e.g. at least 95% to the sequences of SEQ ID Nos. 3 and 4. The removal of the Tat leader sequence or a functional fragment thereof to prevent export to the periplasm is a known technique, and has been used previously in the production of heterodimeric molybdenum-containing enzymes (Malasarn et al. (2008), J Bacteriol, 190, 135-142). However, AsIII oxidase differs from other molybdenum-containing enzymes in that it is a much larger α2β2 heterotetramer containing additional co-factors (i.e. a 3Fe-4S cluster and a Rieske 2Fe-2S cluster) not present in other molybdenum-containing enzymes). It has been demonstrated that the native enzyme is localised to the periplasm (Santini et al. (2000), J Bacteriol, 66, 92-97), given size of the assembled heterotetrameric complex it is therefore reasonable to speculate that two αβ heterodimers formed in the cytoplasm are exported (via the Tat system) to the periplasm prior to heterotetrameric complex formation. Therefore, the present inventors were surprised to find that that following removal of the Tat leader sequence, the modified AsIII oxidase could achieve complete assembly (both subunit assembly and cofactor addition) to form a heterotetramer in the cytoplasm of the various E. coli strains.
For the avoidance of doubt, the abbreviations “aio”, “Aio”, “aro”, “Aro”, “aso” and “Aso” can be used interchangeably and all refer to the arsenite oxidase enzyme (gene or protein). The different abbreviations are the result of different nomenclature that has been used in the art (Lett et al., J. Bacteriology, 4 Nov. 2011; p.207-208).
The advantage of modifying the native AsIII oxidase according to the invention is that is can be expressed successfully in heterologous expression systems such as E. coli at high, commercially-viable yields. Once expressed, the enzyme can be used to detect the presence of AsII. Therefore, a second aspect of the invention is directed to the use of the modified arsenite oxidase enzyme according to the first aspect of the invention as a biosensor to detect the presence of AsIII in a sample. The modified enzyme of the invention has been found to be equally effective as the native enzyme in catalysing the oxidation of AsIII to arsenate Asv (Warelow & Santini, unpublished data). The absence of the translocation sequence does not impact upon the catalytic activity of the enzyme. The modified enzyme is therefore suitable for use in biosensors to detect the presence of AsIII.
Preferably the sample is a liquid sample. The liquid sample may be any type of liquid that is susceptible to AsIII contamination, including, but not limited to, ground-water, drinking-water, environmental liquids such as mining effluent and sewage, waste-water, biological samples.
Preferably the modified enzyme of the invention may be used in laboratory-based biosensors or, preferably, in low-cost disposable biosensor suitable for field use (i.e. detecting AsIII in a sample at the source of the sample rather than in a laboratory).
The advantage of utilising the modified enzyme according to the first aspect of the invention in a biosensor for detecting AsIII in a sample is that it can be expressed in commercially-viable quantities in a variety of heterologous expression systems, including, unexpectedly, host strains that are normally used for cloning rather than protein expression.
Therefore, according to a third aspect the present invention provides a microorganism modified to express the heterologous AsIII oxidase of the first aspect of the invention. The wild-type microorganism may be selected from a range of species including, but not limited to, E. coli. Preferably, the wild-type microorganism is E. coli, in particular E. coli K12 strains DH5α and JM109λpir.
It is surprising that these strains have been found to be the most suitable for expressing the recombinant enzyme of the invention, since they are usually used in the art for cloning, rather than protein expression. As detailed in Example 1 below, the inventors found that unexpectedly high yields were achieved using E. coli strains DH5α and JM109λpir rather than strains BL21 (protease-deficient) and Origami, which are commonly used for heterologous expression of recombinant enzymes.
E. coli strain DH5α is a Hoffman-Berling 1100 strain derivative and can be purchased from InvitrogenTM. The properties of this strain are described by the following standard nomenclature:

    F-endAl glnV44 thi-1 recAl relAl gyrA96 deoR nupG Φ80dlacZAM15 Δ(lacZYA-argF)U169, hsdR17(rK−mK+), λ−.


The DH5α strain is described in the following publications: FOCUS (1986) 8:2, 9.; Hanahan, D. (1985) in DNA Cloning: A Practical Approach (Glover, D. M., ed.), Vol. 1, p. 109, IRL Press; Grant, S. G. N. et al. (1990) Proc. Natl. Acad. Sci. USA 87: 4645-4649 PMID 2162051; and Meselson M. and Yuan R. (1968) Nature 217:1110 PMID. 4868368.
E. coli strain JM109λpir can be purchased from Promega and has the following standard nomenclature properties:

    endAl glnV44 thi-1 relAl gyrA96 recAl mcrB+ Δ(lac-proAB) e14-[F′ traD36 proAB+ laclq 1acZAM15] hsdR17(rK−mK+).


This strain is described in Yanisch-Perron et al (1985) Gene, 33(1):103-19; and Penfold et al (1992) Gene 118:145-146.
According to a fourth embodiment, the present invention provides a method of producing recombinant AsIII oxidase, comprising expressing the modified enzyme according to the first aspect of the invention in a heterologous microorganism. According to an embodiment of this aspect of the invention, novel primers identified herein as SEQ ID NO. 9 and SEQ ID NO. 10 can be used to clone the NT-26 aioB and aioA genes (termed aioBA) into the commercially-available pPROEX Htb vector (Invitrogen). As described in relation to the first aspect of the invention, the aioB gene is modified to delete at least the portion of the nucleotide sequence (SEQ ID NO. 6) encoding at least the functional portion of the TAT leader sequence shown herein as SEQ ID NO. 2.
Preferably the heterologous microorganism is E. coli, and preferably E. coli strains DH5α or JM109λpir. The present inventors have found that strain DH5α provides optimal expression of the heterologous arsenite oxidase enzyme when grown aerobically and JM109λpir provides optimal expression of the heterologous enzyme when grown anaerobically with nitrate as the electron acceptor. Furthermore the inventors have found that use of an affinity tag at the N-terminus of AioB allows for simple purification. This embodiment is described in more detail in Example 1 below.
According to a fifth aspect, the present invention provides a device for detecting the presence of AsIII in a sample. The device comprises an electrode (termed the “test electrode”) incorporating an AsIII oxidase enzyme and a redox mediator.
The device may comprise wild-type AsIII oxidase or the modified AsIII oxidase according to the first aspect of the present invention. Preferably, the enzyme is the modified AsIII oxidase. As discussed above, high yields of the modified enzyme can be obtained through heterologous expression in E. coli. Therefore, it is preferable to use the modified enzyme to keep the manufacturing cost of the device low and to enable the device to be manufactured in commercially-viable quantities.
In one embodiment, the device comprises a test strip made of polymer or ceramic materials. Preferably, the device comprises two or more planar electrodes. Preferably the device comprises a “reference electrode” in addition to the test electrode. At least the test electrode incorporates the AsIII oxidase.
The device includes a redox mediator. The term “redox mediator” is defined as any moiety capable of transferring electrons from the enzyme to the electrode surface. Artificial redox mediators such as 2,6-dichlorophenolindophenol (DCPIP) are often used in solution in laboratory-based spectrophotometric measurements; however spectrophotometric measurements are not routinely used in field test equipment. The inclusion of a redox mediator is advantageous in a number of different ways, compared with existing devices in which the enzyme is directly linked to an electrode. The presence of a redox mediator improves the efficiency of electron transfer and reduces the effects of electrode surface chemistry on the currents measured. This enables a much wider range of electrode materials to be used. As the background currents due to sample components are dependent on the electrode material this gives greater versatility in optimising signal to background.
As shown in Example 2, a wide range of mediators can be used in the in the electrochemical AsIII-sensing device of the invention. Examples of suitable redox mediators include, but are not limited to, metal complexes where the metal exists in two or more different redox states, for example iron, ruthenium or osmium complexes, organic molecules that can exist in two or more accessible redox states, for example cytochromes, conducting organic polymers, conducting organic salts, 2,6-dichlorophenolindophenol, ferrocene and ferrocene derivates including ferrocene carboxylic acid and hydroxymethyl ferrocene (ferrocene methanol), Tris(2,2′-bipyridine)dichlororuthenium(II), tetrathiafulvalene (TTF) and quinones including, but not limited to, benzoquinone and hydroquinone. However not all redox mediators are effective with Aio (arsenite oxidase), for example tetraammine ruthenium (III) chloride, methylene blue, ferricyanide and oxygen have been found not to be effective.
Advantageously, the device of the invention is versatile and has been shown to work with a variety of test electrode materials (see Example 2 below). Preferably, the test electrode is made of one or more conducting materials including, but not limited to, carbon, carbon nanotubes, graphene, graphite, gold, platinum, palladium, glassy carbon, nanostructured metal oxides or nanostructured metal.
Preferably, the reference electrode comprises a reference redox couple, such as Ag/AgCl.
The electrode materials can be deposited on the test strip by a variety methods including, but not limited to, screen-printing or evaporation. The electrodes may be open or covered by a lid so forming a defined volume cell. There may, or may not, be a membrane covering the electrodes.
The test strip is suitable for laboratory use and, preferably, field-based use (i.e. AsIII can be detected in a sample at the source using the test strip or device). The device may be suitable for multiple uses or a single use, and may be disposable.
Preferably, the device comprises a micro-structured surface, with the enzyme entrapped thereon with a mediator (see FIG. 1). A micro-structured surface, for example pillars rising from the base of the electrode, improves the performance of the electrode.
Preferably the sample in which AsIII is detected using the device of the invention is a liquid sample. The liquid sample may be any type of liquid that is susceptible to AsIII contamination, including, but not limited to, ground-water, drinking-water, environmental liquids such as mining effluent and sewage, waste-water, biological samples.
In use, the test sample is brought into direct contact with the test strip. Operation of the sensor device involves applying an electrical potential between the test and reference electrodes and measuring the current. A number of methods would be apparent to those skilled in the art, and include, but are not limited to, chronoamperomerty, square wave voltammetry and coulometry. The response to AsIII can be measured from 5 μM to 40 μM with the same sensor showing comparable results the next day (see FIG. 2).
The advantages of using the device of the invention as described above to test for the presence of AsIII in a sample are that the sensitivity for the analyte is high and the test is specific for AsIII (i.e. the most toxic soluble form of arsenic) due to the presence of the enzyme; the test is quick and results are obtained almost instantaneously; the device is simple to use; the results are reproducible; and the device can be produced and purchased cheaply (particularly important for use in developing countries).
The present invention is further described with reference to the following examples.
EXAMPLE 1
Experimental Procedures
Bacterial Strains, Plasmid and Growth Conditions
Escherichia coli strains DH5α, JM109λpir, RK4353, C43, BL21 and Origami were used to test expression of the NT-26 arsenite oxidase. The expression vector pPROEX-HTb (Invitrogen) was used for expression purposes. All expression conditions involved growing E. coli in LB containing 100 μg/ml ampicillin either aerobically (with aeration at 170 rpm (1:5 ratio liquid to head space) or anaerobically with nitrate (14 mM) or DMSO (14 mM) as electron acceptors and sodium lactate (20 mM) as the electron donor.
Cloning and Expression
The NT-26 aioB and aioA (aioBA) genes were amplified without the native Tat leader sequence using the following primers:









Forward:



PROEXAroBFHis



(SEQ ID No. 9)



5′-GCGAATTCAAGCTACCGCGGCGGCAGGGGTC-3′



 



Reverse:



PROEXAroAR



(SEQ ID No. 10)



5′-GCCTGCAGTCAAGCCGACTGGTATTCTTTCGA-3′






The restriction enzymes EcoRI and Pstl (underlined above) were used to facilitate cloning into the expression vector, pPROEX-HTb. The pPROEX-HTb carrying the aioBA genes was transformed into a variety of E. coli strains to determine which one gave optimal expression. A variety of IPTG (Isopropyl β-D-1-thiogalactopyranoside) and sodium molybdate (Mo) concentrations as well as time of induction were tested. The final optimum expression conditions used for purification of the Aio involved growing DH5α aerobically at 21° C. for 24 hrs in LB containing 40 μM IPTG and 1 mM Mo.
Purification of Recombinant Arsenite Oxidase
Recombinant Aio was purified from DH5α using a combination of affinity and size exclusion chromatography. Cells were harvested by centrifugation at 9,700×g for 10 minutes. The cell pellets were pooled and washed by suspending in binding buffer (10 ml/gm wet weight cells) (20 mM potassium phosphate, 500 mM sodium chloride, 20 mM imidazole, pH 7.2) and centrifuged at 12,000×g for 15 minutes. The cell pellet was then re-suspended in binding buffer (10 ml/gm wet weight cells). The E. coli cells were disrupted by a single pass through a French pressure cell (12,000 psi) and the cell debris removed by centrifugation at 30,000×g for 30 minutes. The supernatant was loaded onto a GraviTrap pre-packed Ni charged affinity chromatography column (GE Healthcare) as per the manufacturer's instructions except with one minor modification; the wash volume used was 120 ml. The eluent was desalted in 50 mM 2-(N-morpholino)ethanesulfonic acid (MES) (pH 5.5) buffer resulting in the precipitation of non-target co-eluted protein(s) which were removed by centrifugation at 10,000×g for 5 minutes. The supernatant was filtered through a 0.22 μm filter (Millipore), concentrated using a Vivaspin 20 (MWCO 100,000) (GE Healthcare) centrifugal concentrator and loaded onto a Superdex 200 gel filtration column (GE Healthcare) pre-equilibrated with 50 mM MES, 100 mM NaCl, pH 5.5 buffer. Chromatography was carried out at a flow rate of 0.3 ml/min. The 0.25 ml fractions containing Aio activity were pooled and concentrated using a Vivaspin 20 centrifugal concentrator (MWCO 100,000).
Confirmation of the native molecular mass of the recombinant Aio was done using a Superdex 200 gel filtration (GE Healthcare) chromatography column. A calibration curve was created using a gel filtration calibration kit (GE Healthcare) of known molecular mass proteins (Thyroglobulin 669 kDa, Ferritin 440 kDa, Aldolase 158 kDa, Conalbumin 75 kDa, Ovalbumin 43 kDa) and the void volume of the column was determined using Blue dextran 2000 (2,000 kDa). Chromatography conditions used were as per the manufacturers' instructions with a flow rate of 0.3 ml/min.
Results and Discussion
Heterologous Expression and Purification of the Recombinant Arsenite Oxidase
In this study the aioB and aioA (designated aioBA) genes were cloned without the aioB Tat leader sequence allowing for expression in the E. coli cytoplasm. A combination of different strains and growth conditions were tested to optimise Aio expression. Surprisingly, the highest Aio specific activity was obtained with E. coli K12 strains DH5α and JM109λpir.
Interestingly, there was also a variation of Aio activity when different electron acceptors were used (FIG. 3), with nitrate the optimal electron acceptor. Given the greater cell yield obtained from aerobic growth, DH5α and oxic conditions were chosen for further studies. Recombinant AsIII oxdase was purified from E. coli using a combination of Ni-NTA and size exclusion chromatography. Based on SDS polyacrylamide gel electrophoresis, the recombinant enzyme was >99% pure and contained the two known Aio subunits, AioA (93 kDa) and AioB (21 kDa) (see FIG. 4). Based on size exclusion chromatography the native molecular mass of the enzyme was 219 kDa which is consistent with the α2β2 oligomeric state of the native enzyme purified from NT-26 (Santini & vanden Hoven, 2004, J. Bacteriology. 186(6):1614-1619).
EXAMPLE 2
A number of experiments were performed to demonstrate that the enzyme described within the patent application can be used in an enzyme mediated electrochemical device to detect arsenite in water. The results demonstrate that a range of redox mediator molecules can be used in the device and that the electrode material can also be varied.
Materials & Methods
Screen printed electrodes were purchased from Dropsens (Oviedo, Spain). Carbon screen printed electrodes have a carbon working electrode (4mm diameter) and counter electrode and silver/silver chloride reference electrode (product ref. DRP-C 110). Carbon nanotube screen printed electrodes have a multi-walled carbon nanotube working electrode (4 mm diameter), carbon counter electrode and silver/silver chloride reference electrode (produce reference DRP-110CNT). Gold screen printed electrode have a gold working electrode (4 mm diameter) and counter electrode and a silver/silver chloride reference electrode (product reference DRP-220AT). Electrochemical measurements (cyclic voltammetry and chrono amperometry) were recorded using a CompactStat.e potentiostat instrument (Ivium Technologies) and IviumSoft software (Ivium Technologies). 2,6-dichlorophenolindophenol, Ferrocene Carboxylic acid, hydroxymethyl ferrocene (ferrocene methanol), tetrathiafulvalene (TTF) and Tris(2,2′-bipyridine)dichlororuthenium(II) mediators were all purchased from Sigma Aldrich. 50 mM solution of sodium arsenite (Fluka) was also purchased from Sigma Aldrich.
In all experiments the working, counter and reference electrodes were completely covered with a 200 μl buffered solution containing the required amount of mediator, arsenite oxidase enzyme and arsenite for each experiment.
Cyclic voltammograms were recorded using carbon, carbon nanotube and gold electodes with a 0.5 mM hydroxymethyl ferrocene solution (10 mM PBS, pH 7.1, 100 mM KCl) containing 0.05 U arsenite oxidase (0.17 nanomoles) with and without 500 micromolar sodium arsenite. A scan rate of 5 mV/s was used. Chrono amperometry was performed using a 0.25 mM hydroxymethyl ferrocene solution (10 mM PBS, pH 7.1, 100 mM KCl) containing 0.05 U arsenite oxidase (0.17 nanomoles) with 0 and 50 micromolar sodium arsenite when using a carbon electrode and 0, 12.5, 25, 50 and 100 micromolar sodium arsenite when using a gold electrode. The solution was mixed between each chrono amperometry run. Triplicate runs were recorded for each concentration of arsenite and the average values plotted. A one step potential of 0.35 V (vs Ag/AgCl) was applied for each run.
Cyclic voltammograms were recorded using carbon and carbon nanotube electodes with a 1 mM Tris(2,2′-bipyridine)dichlororuthenium(II) solution (10 mM PBS, pH 7.1, 100 mM KCl) containing 0.05 U arsenite oxidase (0.17 nanomoles) with and without 500 micromolar sodium arsenite. A scan rate of 5 mV/s was used.
Cyclic voltammograms were recorded using carbon and gold electodes with a 1 mM ferrocene carboxylic acid solution (10 mM PBS, pH 7.1, 100 mM KCl) containing 0.1 U arsenite oxidase (0.34 nanomoles) with and without 1 mM sodium arsenite. A scan rate of 5 mV/s was used. Chrono amperometry was performed using a 1 mM ferrocene carboxylic acid solution (10 mM PBS, pH 7.1, 100 mM KCl) containing 0.1 U arsenite oxidase (0.34 nanomoles) with 0, 50 and 100 micromolar sodium arsenite with a carbon electrode. A single step run was performed applying a potential of 0.38 V (vs Ag/AgCl reference electrode). The solution was mixed between each chrono amperometry run. Duplicate runs were recorded for each concentration of arsenite.
Cyclic voltammograms were recorded using carbon, carbon nanotube and gold electodes with a 1 mM 2,6-dichlorophenolindophenol (DCPIP) solution (50 mM MES, pH 5.5, 100 mM KCl) containing 0.05 U arsenite oxidase (0.17 nanomoles) with and without 500 micromolar sodium arsenite. A scan rate of 5 mV/s was used.
Cyclic voltammograms were recorded using carbon nanotube electode by first depositing 6 μl of 2 mM tetrathiafulvalene (TTF) in acetone onto the working electrode and allowing to dry for 10 minutes. The electrode screen printed electrode was then immersed in 200 μl solution (10 mM PBS, pH 7.1, 100 mM KCl) containing 0.05 U arsenite oxidase (0.17 nanomoles) with and without 500 micromolar sodium arsenite. A scan rate of 50mV/s was used.
Results
2,6-dichlorophenolindophenol, Ferrocene Carboxylic acid, Ferrocene Methanol and Tris(2,2′-bipyridine)dichlororuthenium(II) mediators all showed reversible redox behaviour in the presence of the enzyme (very similar cathodic and anodic peak currents) and an increased cathodic peak current relative to the anodic peak current in the presence of the enzyme and arsenite. This is a typical response of an enzyme mediated electrochemical device.
FIGS. 5(a-f) show cyclic voltammograms of ferrocene methanol as the mediator with carbon (a and b), carbon nanotube (c and d) and gold (e and f) screen printed electrodes without (a, c, e) and with 500 micromolar arsenite (b,d,f). FIG. 5g shows chronoamperometry plots of ferrocene methanol plus enzyme, and ferrocene methanol plus enzyme plus 50 μM arsenite recorded using a carbon electrode. FIGS. 5h and 5i show chronoamperometry plots of ferrocene methanol plus enzyme, and ferrocene methanol plus enzyme plus 0, 12.5, 25, 50 and 100 micromolar arsentie recorded using a gold electrode. FIG. 5i is a zoomed portion of FIG. 5h. FIG. 5j is a plot showing a linear dose response of current to arsenite concentration and refers to the Chrono amperometry plots in FIGS. 5h and 5i, plotting current values taken at four seconds from the chrono amperometry plots.
FIGS. 6(a-d) show cyclic voltammograms of Tris(2,2′-bipyridine) dichlororuthenium(II) as the mediator with carbon (a and b) and carbon nanotube (c and d) screen printed electrodes, without (a and c) and with (b and d) 500 micromolar arsenite.
FIGS. 7a and 7b show cyclic voltammograms of feccocene carboxylic acid as the mediator with carbon (2a) and gold (2b) screen printed electrodes. FIG. 7c shows chrono amperometry plots of ferrocene carboxylic acid plus enzyme with 0, 50 micromolar and 100 micromolar arsenite.
FIGS. 8(a-f) show cyclic voltammograms of 2,6-dichlorophenolindophenol as the mediator with carbon (a and b), gold (c and d) and carbon nanotube (e and f) screen printed electrodes, without (a,c,e) and with (b,d,f) 500 micromolar arsenite.
FIG. 9 shows cyclic voltammograms of TTF as the mediator with carbon nanotube screen printed electrodes, without and with 500 micromolar arsenite.
Chronoamperometry plots show a shift to high current values in the presence of arsenite and there was a linear dose response in the current shift to the arsenite concentration.

## Claims

1. An arsenite oxidase enzyme modified to prevent translocation to the periplasm, wherein the enzyme comprises [1]the native AioA subunit from NT-26, or a variant, homologue or derivative thereof having at least 90% sequence homology to the amino acid sequence of SEQ ID NO:4, and [2]the native AioB subunit from NT-26, or a variant, homologue or derivative thereof having at least 90% sequence homology to the amino acid sequence of SEQ ID NO:3, wherein a portion of the native AioB subunit corresponding to the translocation signal sequence or a functional fragment thereof, is modified, and wherein said enzyme has arsenite oxidase activity.

2. The modified enzyme according to claim 1, wherein the portion of the native aioB gene is identified as SEQ ID NO. 6, or a homologue of SEQ ID NO. 6 encoding a functional fragment of the translocation signal sequence identified as SEQ ID NO. 2.

3. The modified enzyme according to claim 1, wherein the modification is deletion.

4. The modified enzyme according to claim 1, wherein the AioB subunit is encoded by the polynucleotide sequence of SEQ ID NO. 7, or a variant, homologue or derivative thereof.

5. The modified enzyme according to claim 1, wherein the AioA subunit is encoded by the polynucleotide sequence of SEQ ID NO. 8, or a variant, homologue or derivative thereof.