CSIRO ECOSYSTEM SCIENCES

TM
Landguard A900
An enzyme-based remediant for the detoxification of
organophosphate insecticides in animal dips

Dr. Colin Scott

CSIRO Ecosystem Science, GPO Box 1700, Canberra, ACT2601, Australia
8th June 2012

Prepared for: Groundwater and Contaminated Land Environment Agency

Olton Court 10, Warwick Road, Olton, Solihull, B92 7HX

2
Copyright and disclaimer
© 2012 CSIRO To the extent permitted by law, all rights are reserved and no part of this publication
covered by copyright may be reproduced or copied in any form or by any means except with the written
permission of CSIRO.

Important disclaimer
CSIRO advises that the information contained in this publication comprises general statements based on
scientific research. The reader is advised and needs to be aware that such information may be incomplete
or unable to be used in any specific situation. No reliance or actions must therefore be made on that
information without seeking prior expert professional, scientific and technical advice. To the extent
permitted by law, CSIRO (including its employees and consultants) excludes all liability to any person for
any consequences, including but not limited to all losses, damages, costs, expenses and any other
compensation, arising directly or indirectly from using this publication (in part or in whole) and any
information or material contained in it.

1
Acknowledgements

Time Animal Health (Australia) for provided training in the handling of sheep dipping chemical and
equipment. The biodegradability studies were conducted by RCC Ltd (Switzerland). Field trial samples were
analysed by Gribbles Analytic Laboratory (Australia). Ecotoxicology and metabolite data for LandguardTM,
diazinon and the diazinon metabolites were provided by the National Research Centre for Environmental
Toxicology (EnTox; Australia).

2
Summary

This report provides details of the LandguardTM A900 product developed by the Commonwealth Sientific
and Industrial Research Organization (CSIRO).

• LandguardTM is a suite of enzyme-based technology for the rapid detoxification of pesticides

• LandguardTM OP-A and LandguardTM A900 have been developed for the remediation of
organophosphate insecticide (OPs) residues
• Both LandguardTM OP-A and LandguardTM A900 are based on the bacterial phosphotriesterase
OpdA. LandguardTM OP-A is unmodified and LandguardTM A900 contains five amino acid
substitutions that improve its performance against some OPs
• The product is a freeze dried homogenate of a bacterial fermentation; there is no DNA in the
product
• LandguardTM has no observable toxic effects, although it can cause minor skin irritation
• LandguardTM is biodegradable and non-persistent in the environment
• The products of OP degradation by LandguardTM A900 are > 10,000-fold less toxic than the OPs
• When refrigerated, freeze-dried LandguardTM retains >90% of its activity for the first 4 months,
packaging will account for this rate of decay and provide a shelf-life of 6 months
• Field trials of LandguardTM A900 have demonstrated its efficacy in animal dips
• A dose rate of 1 g / 100 L is recommended to enable >99% reduction in OP concentration in 12
hours.

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Contents

Acknowledgements 2

Summary 3

1. Introduction 5

2. Bacterial phosphotriesterase 6

3. Production of LandguardTM 12

4. Biodegradability and toxicity of LandguardTM 13

5. Metabolite toxicity 16

6. Stability of Freeze-Dried LandguardTM 17

6. Efficacy of LandguardTM 19

References 21

Appendix 1: Related patents 24

Appendix 2: Product label 25

Appendix 3: OpdA (wild-type) protein sequence 26

Appendix 4: OpdA (A900) protein sequence 28

Appendix 5: Methyl parathion assay 30

4
1 Introduction

LandguardTM A900 is an enzyme-based technology developed by the CSIRO for the rapid remediation of
organophosphate insecticide residues from water and wetable surfaces [1], and is covered by a number of
patents (Appendix 1). Enzymes that degrade pesticides to compounds of lower toxicity are sourced from
nature (insects, bacteria etc)[2,3] and produced in a suitable fermentation organism (E. coli). Currently
LandguardTM A900 is produced as a freeze-dried homogenate of the production organism that has been
treated to remove the organisms DNA (Appendix 2 contains the label for this product).

A number of enzymes that target different pesiticide chemistries have been isolated and developed as
bioremediants for the LandguardTM range; the most advanced of these are the enzymes that target
organophosphate insecticides (LandgardTM OP and LandguardTM A900), synthetic pyrethroid insecticides
(LandguardTM SP) and triazine herbicides (LandguardTM TRZ).

The technical performance,[1,4,5] toxicity [1] and environmental fates [1] of LandguardTM have been
assessed, demonstrating that the product is both effective and suitable for use in environmental ground
water, such as irrigation tail water.

The product formulation (i.e. freeze-dried, wetable powder) is effective and convenient for use in static
water applications, such as animal dips. In these applications a dose rate of 1 g/100 L is recommended to
achieve > 99% reduction in contamination in the first 12 hours.

The CSIRO are currently developing time release formulae for dosing LandguardTM into moving water.

5
2 Bacterial phosphotriesterase

The LandguardTM A900 technology is based on a bacterial phosphotriesterase (PTE; EC 3.1.8.1), OpdA,
which is remarkably well suited for use as a free-enzyme bioremediant [3,6,7,8].

The gene encoding OpdA was isolated from an Agrobacterium species [9], as a chromosomally located
open-reading frame associated with transposable elements [10]. The encoded enzyme is a ca. 30 kDa
metal-dependent α8/β8 hydrolase [11,12,13], most closely related to another bacterial PTE, OPH, isolated
from Pseudomonas dimuta [14] and Flavobacterium sp. [15]. The activities and rates of OPH and OpdA are
similar, although OpdA has a ca. 10-fold higher activity than OPH against OPs with dimethyl side chains
[9,16]. Phosphotriesterases have been shown to be active against a wide range of OP insecticides,
including the high use insecticides parathion, chlorpyrifos, chlorfenvinphos and diazinon (Table 1).

OpdA hydrolyses one of the three phosphoester bonds of the phosphotriesters (Fig. 1) by nucleophilic
substitution (an SN2 mechanism) of the phosphoester group by a metal activated water [13,17,18,19]. This
reaction can approach diffusion limited rates of 107 sec-1.M-1 [20].

Figure 1. Hydrolysis of an organophosphorous insecticide (diazinon) by the phosphotriesterases

OpdA and OPH. The products are diethyl thiophosphoric acid and 2-isopropyl-4 methyl-pyrimidin-6-
ol.

Hydrolysis of a single phosphoester group yields a phosphodiester of considerably lower toxicity than the
parent phosphotriester. The efficiency of the wild-type enzyme varies depending on the
organophosphorous substrate, from 9.6 sec-1.M-1 for the Z-isomer of chlorfenvinphos up to greater than
1 x 107 sec-1.M-1 for paraoxon, methyl paraoxon, methyl parathion and methyl chlropyrifos oxon (Table 1).

6
Its broad substrate range, rapid rate of hydrolysis and cofactor-free reaction mechanism make OpdA an
ideal candidate as a bioremediant of OP insecticides [3,6,21,22,23].

Table 1. Insecticide and nerve agent phosphotriesters degraded by the wild-type

phosphotriesterases OpdA and OPH. Second order rate constants (k c a t /K M ) are shown.

Phosphotriester Structure kcat/KM Sources

-1 -1
(sec .M )*

S
O
P
S
HN 1
Acephate 1.8 x 10 [24,25]
O

S
O
P
O
O
O O 6
Coumaphos 1.5 x 10 [24,25,26]
Cl

O
O
P
O
O
Cl
Chlorfenvinphos (E- Cl 2
8.4 x 10 [27]
isomer)

Cl

O
O
P
O
O Cl
Cl
Chlorfenvinphos (Z- 0
9.6 x 10 [27]
isomer)

Cl

O S
P
O
O
N 5
Chlorpyrifos Cl Cl 2.8 x 10 [28]

Cl

O
O
P
O
S 3
Demeton-S S 1.6 x 10 [24,25,29]

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 S
O
P
O
O
N 5
Diazinon 1.5 x 10 [25]
N

O S
P
O
O 5
Dichlorvos Cl 8.1 x 10 [27]
Cl

O O
P
O
O
Diethyl 4-chlorophenyl 2
2.2 x 10 [17]
phosphate
Cl

O S
P
O
O
Diethyl 4-chlorophenyl 3
4.4 x 10 [17]
thiophosphate
Cl

O O
Diisopropyl P 4
O 7.8 x 10 [26,30,31]
F
fluorophosphate

S
O
P
O
S 3
Dimethoate NH 9.0 x 10 [32]
O

S
O
P
O
O

5
Fensulfothion 1.5 x 10 [33]
S O

O S
O
O P
O 1
Malathion O S 4.8 x 10 [24]
O

8
 O O
P
O
O
N 7
Methyl chlorpyrifos oxon Cl Cl 1.3 x 10 [32]

Cl

O S
P
O
O
N 4
Methyl chlorpyrifos thion Cl Cl 1.8 x 10 [32]

Cl

O
O
P
O O
7
Methyl paraoxon 1.1 x 10 [30,32]
NO2

S
O
P
O O
7 [13,24,30,34
Methyl parathion 1.2 x 10
]
NO2

O
O
P
O O
7 [26,29,30,31
Paraoxon 5.5 x 10
,35,36,37]
NO 2

S
O
P
O O
6
Parathion 2.7 x 10 [17,30,34]

NO 2

S
O P
O
S
3
Phosalone N 2.4 x 10 [25]
O
Cl O

S
O P O
O N 5 CS
Quinalphos 9.8 x 10
Unpublished
N

9
 O
O
P
O
O Cl

Tetrachlorvinphos ND [38]
Cl Cl

Cl

Enzyme engineering and in vitro evolution have been used extensively with the bacterial
phosphotriesterases to produce variant enzymes with significantly improved kinetic properties. This has
been facilitated by the wealth of structural and mechanistic data that is available for these enzymes
[12,13,16,17,18,20,27,39,40,41,42,43,44,45].

The value of being able to improve the properties of OpdA, and indeed any free-enzyme bioremediant, are
exemplified by the composition of LandguardTM A900. LandguardTM A900 is identical to the wild-type
enzyme at all amino acid positions except for five (Ala52Val, Ser64Ala, Lys157Arg, Asp204Glu and
Asn237Asp; Appendix 3 and Appendix 4). A900 possess higher kcat/Km values than the wild-type OpdA
against commercially important OP insecticides chlorpyrifos methyl, diazinon and parathion ethyl (Table 2).
The cofactor requirements, catalytic mechanism and hydrolysis products are identical to the wild-type
enzyme. A provisional patent covering the A900 variant of OpdA has been lodged.

Table 2. Comparison of the second order rate constants (k c a t /K M )* of OPH, OpdA and the rationally
designed OpdA variant A900 against commercially important OP insecticides. k c a t /K M values were
obtained for enzymes purified by the methods described in jackson et al, 2006 [12]. Hydrolysis rates
were determined at 25°C in MOPS buffer (pH8.0) using UV-vis spectroscopy at 250 nm (diazinon),
330 nm (chlorpyrifos ethyl and methyl) and 405 nm (parathion ethyl and methyl).

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 Insecticide Structure kcat/KM (sec-1.M-1)
OpdA A900
S CH3
O P O
H3C O N Cl
Chlorpyrifos ethyl 2.8 x 106 2.2 x 106
Cl
Cl

S CH3
O P O
H3C O N Cl
Chlorpyrifos methyl 1.2 x 104 3.7 x 105
Cl
Cl
S CH3
O P O
H3C O CH3

Diazinon N N 1.9 x 106 3.9 x 106

H3C CH3

S CH3
O P O
Parathion H3C O
1.7 x 107 9.4 x 107
ethyl + OH
N
O

S CH3
O P O
H3C O
Parathion methyl 5.0 x 106 3.6 x 106
+ OH
N
O

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3 Production of LandguardTM A900

LandguardTM A900 is produced by fermentation in Escherichia coli BL21 λDE3. E. coli BL21 λDE3 are
transformed with the OpdA A900 expression vector (pETCC2:A900), and a seed culture prepared by growth
over night in Erlenmeyer flasks.

Seed cultures are used to seed fermentation in a fed-batch fermenter in a defined medium, with
expression of the phosphotriesterase induced by the addition of isopropyl-β-D-thio-galactoside.

At the completion of the fermentation process the culture is homogenized, DNA removed by precipitation
with polyethyleneimine followed by clarification and freeze drying and packaging.

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4 Biodegradability and toxicity of LandguardTM A900

The safety of LandguardTM A900 to mammals was established in formal toxicity studies with Wistar rats
(Table 3). LandguardTM was either fed or applied dermally to the animals (Table 3) at dose rates of 50, 200
and 1000 mg/kg per day for 28 days (oral) or at 4 mL/kg of a 500 mg/mL solution per day for 15 days
(dermal). Biopsies revealed no noticeable detrimental effects of oral LandguardTM application in the rats,
whilst the dermal application produced a mild skin irritation with no clinical effects.

To test for any environmental toxicity of LandguardTM, it was applied at 1g/L to activated sludge, and at
100 mg/L to the alga Scenedesmus subspicatus, the arthropod Daphnia magna, and the fish species
Brachydanio rerio, with no adverse affects found in any of these indicator species. The dose rates used in
these toxicity and ecotoxicity studies were higher than in many of the successful field trials (see below),
demonstrating that this bioremediant could be safely used in environmental applications.

The ecotoxicology studies of LandguardTM were also complemented with a study of its biodegradability
(Figure 2). In this study the rate of digestion of LandguardTM was assessed in a manometric respirometry
test, whereby the amount of LandguardTM digested was calculated based on the biological oxygen demand
of activated sludge fed with LandguardTM as carbon source. An equivalent amount of carbon, provided as
the readily digestible compound sodium benzoate, was used as a positive control. The result of this test
was that > 95% of both the LandguardTM and the sodium benzoate were digested over a 15 day period,
showing that LandguardTM is readily degraded. Furthermore, there was no reduction in the biological
oxygen demand when sodium benzoate and LandguardTM were added simultaneously, suggesting that
LandguardTM has no inhibitory affect upon bacterial growth when it is not the sole carbon source.

The stability of LandguardTM in natural water was also monitored, using the hydrolysis of methyl parathion
as an indication of the relative amount of LandguardTM remaining in the sample (Table 3). In this
experiment the half-life of LandguardTM was seventy nine hours, with less than 1 percent of the original
activity remaining after seven days. These data suggest that LandguardTM is biodegradable, and has a
relatively short half-life in natural systems.

Table 3. Ecotoxicity and mammalian toxicity of Landguard TM

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 Test Monitoring Dose rate Effect of Landguard
time
Scenedesmus subspicatus 72 hour 100 mg/L No growth effects observed
Activated sludge 3 hour 1000 mg/L No inhibitory effect on bacterial
respiration
Brachydanio rerio 96 hour 100 mg/L No mortality or visible
abnormalities observed
Daphnia magna 48 hour 100 mg/L No mortality (immobilisation of
cells) observed
Wistar rat (oral) 28 day 50, 200 and All test animals survived with no
(n = 30, 5 male and 5 1000 apparent signs of toxicity. No
female at each dose rate) mg/kg/day adverse responses were observed.
Rats (Dermal) 15 days 4 mL/kg of a Slight general erythema observed
(n= 10, 5 male and 5 0.5 g/mL in test animals. No clinical effects
female) solution noted.

Figure 2. Biodegradability of Landguard TM (diamonds), sodium benzoate (triangles). Inhibition of the

biodegradation of sodium benzoate by Landguard T M was also assessed (squares).

Table 4. Stability of LandguardTM in natural water. The activity (U/L) of Landguard TM was measured
using methyl parathion as substrate. One unit of enzyme activity (U) is equivalent to the amount of
enzyme required to hydrolyse 1 µmol of methyl parathion per minute.

14
 Time (hours) Activity (U/L)* Activity (% initial)
0 2133 100%
17 2306 108%
24 2341 110%
48 1894 89%
96 624.4 29%
168 19.55 0.92%

Table 5. Toxicity of diazinon to Ceriodaphnia dubia before and after treatment with Landguard T M
dosed at 0.05g/L. EC50, effective concentration for 50% survival; LOEC, Lowest concentration at
which an effect is observed; NOEC: Highest concentration at with no effect is observed
Untreated Landguard TM OP Fold improved survival
diazinon treated diazinon*
24 48 24 48 24 hours 48
hours hours hours hours hours
EC 50 (µg/L) 0.389 0.195 73,000 46,100 1.9 x 105 1.9 x 105
LOEC (µg/L) 0.55 0.275 100,000 100,000
NOEC (µg/L) 0.275 0.138 30,000 30,000

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5 Metabolite toxicity

Detoxification of the pesticide was established by exposing an indicator arthropod species, Ceriodaphnia
dubia, to either untreated diazinon (50 mg/L) or diazinon (50 mg/L) that had been pre-treated with
LandguardTM (Table 5). The survival rate of the Ceriodaphnia was monitored after twenty four and forty
eight hours of exposure. At both time points the EC50 (effective concentration for 50% survival) for
Ceriodaphnia exposed to the LandguardTM treated diazinon was nearly 200,000 times greater than for
exposure to the untreated pesticide. Similarly impressive increases in the LOEC (Lowest concentration at
which an effect is observed; Table 5) and NOEC (Highest concentration at with no effect is observed; Table
5) were also observed. The products of diazinon hydrolysis by LandguardTM were confirmed by mass
spectroscopy to be diethyl thiophosphoric acid and 2-isopropyl-4 methyl-pyrimidin-6-ol, consistent with
the characterised mechanism of phosphotriesterases (Fig. 1). These data clearly demonstrate that
LandguardTM performs in a predictable manner and that its action significantly detoxifies the
phosphotriester insecticide.

Extensive ecotoxicological data are also available for another OP insecticide, chlorpyriphos, and its
hydrolysis products (diethyl thiophosphoric acid and TCP; 3,5,6 trichloropyridin-2-ol) [46]. Both products
are considerably less toxic than the parent compound. For example, chlorpyrifos has an LD50 of 4.2 µg/L for
Bluegill, compared with 12,500 µg/L for TCP (ca. 3,000 times less toxic) [47]. TCP does not cause
cholinesterase enzyme inhibition and is of low to moderate toxicity to aquatic and terrestrial biota [46].
The hydrolysis of insecticidal phosphotriesters catalysed by OpdA therefore represents a significant
reduction in their mammalian toxicities and ecotoxicities.

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6 Stability of Freeze-Dried LandguardTM

Early stability testing of freeze-dried LandguardTM A900 by Orica Australia Pty Ltd indicated that, when
stored at less than 5°C, LandguardTM retained more than 80 % of its activity after 6 months (Figure 3). In
these assessments LandguardTM that had been stored at 5°C was tested for activity using the standard
assay for methyl parathion activity (Appendix 5). CSIRO has conducted similar testing and found that the
Orica data were reproducible, and that >90% of the initial activity of the LandguardTM A900 is retained for
the first 4 months. The activity decay in the CSIRO experiments was nearly identical to that found by Orica.

Similar experiments were conducted by both CSIRO and Orica in which Landguard was stored at 25°C
(Figure 4). In these experiments >70% of activity was retained after 4 months, with both sets of data
showing similar rates of decay.

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 Stability of refrigerated LandguardTM A900 produced
by Orica Australia Pty Ltd and CSIRO
120

100

Residual activity (%) 80

60 4C (CSIRO)
5C (ORICA)
40

20

0
0 2 4 6 8
Time (months)

Figure 3. Stability of Landguard TM A900 over 6 months at <5°C.

Stability of unrefrigerated LandguardTM A900

produced by Orica Australia Pty Ltd and CSIRO
120

100
Residual activity (%)

80

60
25C (CSIRO)

40 25C (ORICA)

20

0
0 2 4 6 8
Time (months)

Figure 4. Stability of Landguard TM A900 over 6 months at 25°C.

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7 Efficacy of LandguardTM

The efficacy of the LanguardTM propduct has been investigated for a number of potential applications and
field trials have been conducted for the treatment of dip liquor, irrigation tail water, and soil and
commodity treatments. The details of these trials (below) indicate that LandguardTM is highly effective in
the treatment of aqueous contamination and the treatment of soils and commodities has a great deal of
early promise.

Parasite control often involves plunge-dipping livestock into quite concentrated OP insecticide solutions,
leaving pesticide-containing spent dip liquor that requires appropriate disposal. Free-enzyme
bioremediation provides a solution for the rapid clean up of these liquors, allowing them to be disposed of
safely. The OP-degrading LandguardTM was used in five trials in Australia to remediate diazinon from spent
liquors. The volumes of the dipping tanks varied from 3,000 – 8,000 litres, with diazinon concentrations
ranging from 0.91 – 75 mg/L (Table 6). The diazinon used was formulated by either Coopers (North Ryde,
NSW, Australia) or Vibrac Animal Health (Milperra, NSW, Australia). Enzyme amounts added varied
between 103 and 5150 U/L (where a unit, U, is defined as the amount of enzyme that is required to
degrade 1 µmol of methyl parathion in one minute), and treatment times ranged between 10 and 60
minutes.

The degree of diazinon degradation achieved varied in a predictable manner according to the amounts of
enzyme and substrate involved and the treatment time. However there were clearly a range of conditions
under which better than 99% degradation was consistently achieved. Notably also, the time periods tested
in these field trials was much shorter than a farmer may need (often overnight treatment will be quite
adequate), so enzyme dose rates can be reduced significantly, whilst still achieving the same levels of
decontamination. In fact dose rates of 1 g/100 L (equivalent of 20 U/L) are currently recommended for a
three hour treatment in order that a 99.99 % reduction in OP concentration can be achieved.

19
Table 6. Australian field trials of Landguard TM in spent diazinon-based sheep dip liquors. Trials
occurred in Victoria (Darlingtion and Lake Bolac), New South Wales (Gundagai), South Australia
(Loxton) and Tasmania (Flinder’s Island). One unit of enzyme activity (U) is equivalent to the
amount of enzyme required to hydrolyse 1 µmol of methyl parathion per minute.
Date Location* Formulation [Enzyme] (U/L) Time (min) [Diazinon] (mg/L) % hydrolysis
2004 Darlington Coopers 0 0 0.91 0
625 10 0.11 87.91
625 20 0.01 99.23

2004 Lake Bolac Virbac 0 0 34.7 0

1030 30 <0.01 >99.97
1030 60 <0.01 >99.97
257 30 <0.01 >99.97
515 30 <0.01 >99.97
1030 30 <0.01 >99.97

2005 Gundagai Coopers 0 0 59.3 0

515 30 9.6 83.81
515 60 2 96.63
103 30 47 20.74
257 30 33 44.35
515 30 12.3 79.26
2060 30 0.76 98.72

2005 Loxton Coopers 0 0 75 0

2575 30 0.41 99.45
2575 60 0.079 99.89
515 30 5.9 92.13
1030 30 2.3 96.93
2060 30 1.6 97.87
2575 30 0.71 99.05
3090 30 0.58 99.23
5150 30 0.21 99.72

2005 Flinders Island Coopers 0 0 39.4 0

724 20 <0.05 >99.87
724 30 <0.05 >99.87
724 30 <0.05 >99.87
0 0 30.5 0
839 20 <0.05 >99.84
839 30 <0.05 >99.84
839 30 <0.05 >99.84

20
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23
Appendix 1: Related Patents

TW7023

TW8498

TW7146

TW8047

TW8167

TW8198

24
Appendix 2: Product label

25
Appendix 3: OpdA (wild-type) protein sequence

Met Pro Ile Gly Thr Gly Asp Leu Ile Asn Thr Val Arg Gly Pro Ile
1 5 10 15

Pro Val Ser Glu Ala Gly Phe Thr Leu Thr His Glu His Ile Cys Gly
20 25 30

Ser Ser Ala Gly Phe Leu Arg Ala Trp Pro Glu Phe Phe Gly Ser Arg
35 40 45

Lys Ala Leu Ala Glu Lys Ala Val Arg Gly Leu Arg His Ala Arg Ser
50 55 60

Ala Gly Val Gln Thr Ile Val Asp Val Ser Thr Phe Asp Ile Gly Arg
65 70 75 80

Asp Val Arg Leu Leu Ala Glu Val Ser Arg Ala Ala Asp Val His Ile
85 90 95

Val Ala Ala Thr Gly Leu Trp Phe Asp Pro Pro Leu Ser Met Arg Met
100 105 110

Arg Ser Val Glu Glu Leu Thr Gln Phe Phe Leu Arg Glu Ile Gln His
115 120 125

Gly Ile Glu Asp Thr Gly Ile Arg Ala Gly Ile Ile Lys Val Ala Thr
130 135 140

Thr Gly Lys Ala Thr Pro Phe Gln Glu Leu Val Leu Lys Ala Ala Ala
145 150 155 160

Arg Ala Ser Leu Ala Thr Gly Val Pro Val Thr Thr His Thr Ser Ala
165 170 175

Ser Gln Arg Asp Gly Glu Gln Gln Ala Ala Ile Phe Glu Ser Glu Gly
180 185 190

Leu Ser Pro Ser Arg Val Cys Ile Gly His Ser Asp Asp Thr Asp Asp
195 200 205

Leu Ser Tyr Leu Thr Gly Leu Ala Ala Arg Gly Tyr Leu Val Gly Leu
210 215 220

Asp Arg Met Pro Tyr Ser Ala Ile Gly Leu Glu Gly Asn Ala Ser Ala
225 230 235 240

Leu Ala Leu Phe Gly Thr Arg Ser Trp Gln Thr Arg Ala Leu Leu Ile
245 250 255

Lys Ala Leu Ile Asp Arg Gly Tyr Lys Asp Arg Ile Leu Val Ser His
260 265 270

Asp Trp Leu Phe Gly Phe Ser Ser Tyr Val Thr Asn Ile Met Asp Val
275 280 285

Met Asp Arg Ile Asn Pro Asp Gly Met Ala Phe Val Pro Leu Arg Val
290 295 300

Ile Pro Phe Leu Arg Glu Lys Gly Val Pro Pro Glu Thr Leu Ala Gly
305 310 315 320

Val Thr Val Ala Asn Pro Ala Arg Phe Leu Ser Pro Thr Val Arg Ala
325 330 335

Val Val Thr Arg Ser Glu Thr Ser Arg Pro Ala Ala Pro Ile Pro Arg
340 345 350

26
Gln Asp Thr Glu Arg
355

27
Appendix 4: OpdA (A900) protein sequence

Met Pro Ile Gly Thr Gly Asp Leu Ile Asn Thr Val Arg Gly Pro Ile
1 5 10 15
Pro Val Ser Glu Ala Gly Phe Thr Leu Thr His Glu His Ile Cys Gly
20 25 30

Ser Ser Ala Gly Phe Leu Arg Ala Trp Pro Glu Phe Phe Gly Ser Arg
35 40 45

Lys Ala Leu Val Glu Lys Ala Val Arg Gly Leu Arg His Ala Arg Ala
50 55 60

Ala Gly Val Gln Thr Ile Val Asp Val Ser Thr Phe Asp Ile Gly Arg
65 70 75 80
Asp Val Arg Leu Leu Ala Glu Val Ser Arg Ala Ala Asp Val His Ile
85 90 95
Val Ala Ala Thr Gly Leu Trp Phe Asp Pro Pro Leu Ser Met Arg Met
100 105 110

Arg Ser Val Glu Glu Leu Thr Gln Phe Phe Leu Arg Glu Ile Gln His
115 120 125

Gly Ile Glu Asp Thr Gly Ile Arg Ala Gly Ile Ile Lys Val Ala Thr
130 135 140
Thr Gly Lys Ala Thr Pro Phe Gln Glu Leu Val Leu Arg Ala Ala Ala
145 150 155 160
Arg Ala Ser Leu Ala Thr Gly Val Pro Val Thr Thr His Thr Ser Ala
165 170 175
Ser Gln Arg Asp Gly Glu Gln Gln Ala Ala Ile Phe Glu Ser Glu Gly
180 185 190
Leu Ser Pro Ser Arg Val Cys Ile Gly His Ser Glu Asp Thr Asp Asp
195 200 205
Leu Ser Tyr Leu Thr Gly Leu Ala Ala Arg Gly Tyr Leu Val Gly Leu
210 215 220
Asp Arg Met Pro Tyr Ser Ala Ile Gly Leu Glu Gly Asp Ala Ser Ala
225 230 235 240
Leu Ala Leu Phe Gly Thr Arg Ser Trp Gln Thr Arg Ala Leu Leu Ile
245 250 255
Lys Ala Leu Ile Asp Arg Gly Tyr Lys Asp Arg Ile Leu Val Ser His
260 265 270

Asp Trp Leu Phe Gly Phe Ser Ser Tyr Val Thr Asn Ile Met Asp Val
275 280 285
Met Asp Arg Ile Asn Pro Asp Gly Met Ala Phe Val Pro Leu Arg Val
290 295 300

Ile Pro Phe Leu Arg Glu Lys Gly Val Pro Pro Glu Thr Leu Ala Gly
305 310 315 320

Val Thr Val Ala Asn Pro Ala Arg Phe Leu Ser Pro Thr Val Arg Ala
325 330 335

Val Val Thr Arg Ser Glu Thr Ser Arg Pro Ala Ala Pro Ile Pro Arg
340 345 350
28
Gln Asp Thr Glu Arg
355

29
Appendix 5: Methyl parathion assay

The activity of LandguardTM A900 was determined spectrophotometrically. A 10-fold dilution series of
LandguardTM A900 was produced in 10 mM Tris-HCl (pH 8.0) from a 0.1 g.mL-1 stock of LandguardTM A900,
covering a range of 10-1,000,000 fold dilution. Duplicate samples of 90 µL of each dilution was transferred
to a 96-well microtitre plate and 10 µL of a 2 mM solution of methyl parathion (in 100 % methanol) was
added to each well using an 8-channel pipette. The formation of product, paranitrophenol (PNP), was
monitored at 405 nM using a SpectraMax M5 spectrophotometer (Molecular Devices). A reading for each
sample was taken once every 10 seconds for 30 minutes. A standard curve of PNP (0-2 mM) was produced,
and used to determine the rate of hydrolysis in µMol per minute.

1 U of A900 activity is defined as the amount of enzyme required to hydrolyse 1 µMol of methyl parathion
in 1 minute.

30