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US5169554

The document describes an enzyme detergent formulation capable of detoxifying toxic organophosphorus acid compounds. The formulation contains a detergent and an enzymatic cell free extract from E. coli bacteria. The formulation is effective at hydrolyzing and detoxifying compounds like soman under mild conditions, preserving the functionality of treated items.
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0% found this document useful (0 votes)
21 views10 pages

US5169554

The document describes an enzyme detergent formulation capable of detoxifying toxic organophosphorus acid compounds. The formulation contains a detergent and an enzymatic cell free extract from E. coli bacteria. The formulation is effective at hydrolyzing and detoxifying compounds like soman under mild conditions, preserving the functionality of treated items.
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
Available Formats
Download as PDF, TXT or read online on Scribd
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|H|||||||||IIII USOO569554A

United States Patent (19) 11) Patent Number: 5,169,554


Akkara et al. 45) Date of Patent: Dec. 8, 1992
(54 ENZYME DETERGENT FORMULATION phates by o-Iodosobenzoate Functionalized Surfactant, J.
AND METHODS OF DETOXFYING TOXC American Chemical Society, 1986, 108,788-793.
ORGANOPHOSPHOROUS ACD "Soman Hydrolysis and Detoxication by a Thermo
COMPOUNDS philic Bacterial Enzyme", F. Hoskin et al., Enzymes
Hydrolysing Organophosphorus Compounds, pp.
75 Inventors: Joseph A. Akkara, Holliston; David 53-64, (1989).
L. Kaplan, Stow; Arthur M. Kaplan, "Soman-Hydrolyzing and -Detoxifying Properties of
Newton, all of Mass. an Enzyme from a Thermophilic Bacterium", G. Chet
73) Assignee: The United States of America as tur et al., Fundamental and Applied Technology, II, pp.
represented by the Secretary of the 373-380, (1988).
Army, Washington, D.C. Benschop et al., Fundamental and Applied Toxicology,
vol. 1, pp. 177-182 (1981).
21) Appl. No.: 417,614 Benschop et al., Journal of American Chemical Society,
vol. 103, pp. 4260-4262 (1981).
22 Filed: Oct. 4, 1989 Primary Examiner-A. Lionel Clingman
51) Int. Cl........................ C11D 17/00; C11D 7/06; Assistant Examiner-William S. Parks
CD 7/12 Attorney, Agent, or Firm-Richard J. Donahue,
52 U.S. Cl. ............................... 252/174.12; 252/156; Lawrence E. Labadini
252/173; 252/174.23; 252/174.24; 252/DIG. 57 ABSTRACT
12; 252/DIG. 14; 435/264
58) Field of Search ................... 252/156, 173, 174.12, An enzyme detergent composition capable of detoxify
252/174.23, 174.24, DIG. 12, DIG. 14; ing G-agents and other OPA chemicals comprises a
435/264 laundry detergent and an enzymatic cell free extract
from E. coli, the composition having a pH of about 6.5
(56) References Cited to 10 in water.
U.S. PATENT DOCUMENTS A method of hydrolyzing a hydrolyzable compound
3,649,454 3/972 Masao Isono et al. ........ 252/74.2 comprises contacting compound with an amount of an
4,865,983 9/1989 Durham ............. ... 252/742 aqueous suspension of the enzyme detergent composi
4,883,757 1/1989 Gutnick et al. ...................., 252/.351 tion of the invention under conditions affective to attain
the desired affect.
FOREIGN PATENT DOCUMENTS
A method of cleaning and detoxifying a product com
0273125 6/1988 European Pat. Off. . prising a hydrolyzable compound comprises contacting
OTHER PUBLICATIONS the product with an amount of an aqueous suspension of
the enzyme detergent composition of the invention
Hoskin et al., Two Enzymes for the Detotification of Or under conditions capable of attaining the desired affect,
ganophosphorous Compounds, Fundamentals of Applied and rinsing the product.
Toxicology, 4(2, Pt. 2), 165-172.
Moss et al., Efficient Catalytic Cleavage of Reactive Phos 6 Claims, 2 Drawing Sheets
U.S. Patent Dec. 8, 1992 Sheet 1 of 2 5,169,554

O--e NITIAL ENZYME ACTIVITY


O- - -o ACTIVITY AFTER 3 MN.

TEMPERATURE (C)

FIGURE 1. TEMP. PROFILE OF E, COL ENZYME


U.S. Patent Dec. 8, 1992 Sheet 2 of 2 5,169,554

O8

FIGURE 2, pH OPTIMUM OF E. COLT ENZYME ACTIVITY


5,169,554
1. 2
ENZYME DETERGENT FORMULATION AND BRIEF DESCRIPTION OF DRAWINGS
METHODS OF DETOXFYING TOXC FIG. 1 depicts the variation of the E. coli enzyme
ORGANOPHOSPHOROUS ACID COMPOUNDS activity with temperature both at an initial time and
after three minutes of contact.
The invention described herein may be manufac FIG. 2 depicts the variation of the E. coli enzyme
tured, used and licensed by or for the government for activity with the pH of the aqueous medium.
government purposes without the payment of any roy Other objects, advantages and features of the present
alty thereon. invention will become apparent to those skilled in the
10 art from the following discussion.
TECHNICAL FIELD
BEST MODE FOR CARRYING OUT THE
This invention relates to an enzyme detergent formu INVENTION
lation which is capable of cleaning and decontaminating The present invention arose from a desire by the
toxic organophosphorus acid compounds and their salts 15 inventors to improve on prior art detergent formula
from clothing and other articles. This is done under tions to provide an enzyme detergent preparation capa
mild conditions of pH and temperature which will not ble of rapid detoxification of clothing and other articles
adversely effect the functional properties of clothing contaminated with toxic organophosphorus acid com
and other articles. pounds and their salts under mild conditions which
BACKGROUND ART 20 would not adversely effect the functional properties of
contaminated clothing and other articles.
Many laundry detergents available in the market Toxic organophosphorus acids and their salts (OPA)
contain enzymes to facilitate the removal of protein, include organoflurophosphonates (warfare nerve agent,
carbohydrate and lipid based stains and soils from cloth called G-agents such as sonan), organophosphonates,
ing under mild laundering conditions. 25 organophosphinates and organophosphates (pesticides
Toxic organophosphorus acid compounds and other such as parathion, paroxon and methyl parathion).
salts can be encountered in industrial, agricultural and An enzyme detergent formulation has been devel
military operations. Such toxic materials can be re oped comprising commercially available detergent and
moved and detoxified by the application of harsh chem an enzyme from the bacterium Escherichia coli. The
icals or elevated temperatures, either of which may 30 hydrolysis of G-agents such as soman was increased up
degrade the articles being detoxified. to about 16-fold and more with the present enzyme
Thus, a need exists for a detergent formulation capa detergent formulation over that observed with the de
ble of rapid detoxification of toxic organophosphorus tergent formulation alone at a pH 9.5.
acid compounds and their salts under mild conditions Soman, hydrolyzed at pH 9.5 and 40° C., was detoxi
which will not have a harmful effect on articles so 35 fied up to 91% in 10 minutes in the presence of the
treated. enzyme detergent formulation of the invention. The
cleaning and decontaminating conditions required for
DISCLOSURE OF THE INVENTION this enzyme detergent formulation are very mild, which
This invention relates to an enzyme detergent compo preserve the functional finishes on the clothing on other
sition capable of detoxifying toxic organophosphorus articles being cleaned and decontaminated.
Moreover, this formulation is compatible with cur
acid compounds and salts thereof comprising rent Army field laundry equipment. This enzyme deter
a detergent formulation; and gent formulation can be used to decontaminate OPA
an enzymatic cell free extract from E. coli, the con chemicals, such as G-agents, in laundry applications in
position having a pH of about 5 to 9.5 in water. 45 place of super Tropical Bleach presently recommended
This invention also relates to a method of hydrolyz by the Army Field Manual.
ing and detoxifying toxic compounds of organophos A purpose of this invention is to provide a detergent
phorus acids and their salts comprising contacting said formulation that not only launders or cleans, but also
compounds with an amount of an aqueous suspension of decontaminates soiled clothing or other articles con
the enzyme detergent composition described above 50 taminated with, e.g., G-agents. Under normal laundry
under conditions effective to attain said hydrolysis and conditions G-agents are hydrolyzed slowly. The rate of
detoxification. hydrolysis or half-life of soman (pinacolyl methylphos
Also part of this invention is a method of cleaning and phonofloridate) at 25 C. and at pH 10 is 60 hours.
detoxifying a product contaminated with at least one 55 It is critical to catalytically destroy the agent at a
toxic compound selected from organophosphorus acids faster rate, while at the same time cleaning soiled cloth
and their salts, comprising ing and equipment under mild conditions and using
contacting the product with an amount of the aque equipment
enzyme
available in the field. The decontaminating
formulation provided therein extends the life
ous suspension of the enzyme detergent composi cycle of both field clothing and equipment, and reduces
tion of this invention under conditions capable of 60 logistic burdens.
attaining the desired affect; and The activities of decontaminating enzyme detergent
rinsing the product. formulations against OPA chemicals such as G-agents
A more complete appreciation of the invention and and G-agent surrogates have been determined at differ
many of the attendant advantages thereof will be ent pHs, temperatures, and with different substrates and
readily perceived as the same becomes better under 65 inhibitors. The activity of the enzyme itself against
stood by reference to the following detailed description G-agent and G-agent surrogates has also been studied in
when considered in connection with the accompanying the presence of different detergent components. Live
figures. agent testing with soman using enzyme detergent for
5,169,554
3 4
mulations and a non-enzymatic catalyst containing de known in the art and need not be described herein in
tergent formulations involved both the hydrolysis and further detail.
the detoxification of soman. Another particularly preferred embodiment of the
This invention provides an enzyme detergent compo composition further comprises a manganese compound
sition which is capable of detoxifying G-agents, the such as manganese chloride. However, other manga
composition comprising nese salts may also be utilized. Typically, the manganese
a laundry detergent; and compound is present in an amount of about 0.001 to 10
an enzymatic cell free extract from E. coli, the com wt %, and more preferably about 0.1 to 5 wt % of the
position having a pH of about 5 to 9.5 in water. composition.
A variety of laundry detergents may be utilized in the 10 Also part of this invention is the method of hydrolyz
present composition. Examples of laundry detergents ing an OPA chemical comprising contacting said con
are ionic and non-ionic types of detergents such as alkali pound with an amount of an aqueous suspension of the
detergents, type II laundry detergents, linear alkyl sul enzyme detergent composition described herein under
fonate, Tide "New Liquid" and type I non-ionic deter conditions effective to attain the desired effect.
15 Typically, the method may be conducted at a temper
gents. However, other detergents can also be utilized.
Particularly preferred are laundry detergents such as ature of about 20° to 50° C., and more preferably about
alkali detergent and laundry detergent type II. The 30 to 45° C. However, other temperature ranges within
amount of laundry detergent present in the composition the broader range may also be utilized.
is typically about 0.01 wt % to 10.0 wt.%, and more 20
The above method may be preferably practiced pre
preferably about 0.01 wt % to 50 wt.%. However, serving the pH at about 6.5 to 10.0, and more preferably
other amounts are also possible. about 7 to 9.5.
The enzymatic cell free extract from E. coli is typi Also part of this invention is a method of cleaning and
cally present in an amount of about 1.0 to 20 of the detoxifying a product contaminated with an OPA
composition, and more preferably 2.0 to 10 volume 25
chemical, comprises
percent of the composition. However, other amounts contacting the product with an amount of an aqueous
may also be utilized. suspension of the enzyme detergent formulation
The enzyme detergent formulation of this invention described herein under conditions capable of at
may also comprise a non-enzymatic catalyst, such as taining the desired effect; and
iodosobenzoate. Typically, this catalyst compound is 30
rinsing the product.
present in an amount of about 0.001 to 10 wt %, and Typically, the contacting step is conducted at a tem
more preferably about 0.001 to 5 wt % of the composi perature of about 20 to 50° C., and more preferably
tion. However, other amounts are also suitable about 25 to 45° C. The pH utilized for the practice of
The cell free extract may be obtained by sonicating E. the contacting step is preferably about 6.5 to 8, and
coli cells and then centrifuging the disrupted cells thus 35 more preferably about 7 to 7.5. Under these conditions
obtained. These are technologies which are standard in OPA chemicals may be detoxified while the product is
the art. cleaned.
In a particularly preferred embodiment of the enzyme Having now generally described this invention, the
detergent formulation described herein the enzymatic same will be better understood by reference to certain
cell free extract is obtained from E. coli having an 40 specific examples, which are included herein for pur
ATCC Accession No. 25922. A still more preferred poses of illustration only and are not intended to be
embodiment is that comprising the purified enzyme limiting of the invention or any embodiment thereof,
obtained from this microorganism. unless so specified.
The enzyme can be obtained from the E. coli microor EXAMPLES
ganism as follows. Escherichia coli (ATCC 25922 may 45
be grown in, e.g., trypticase soy broth (about 3%) and EXAMPLE 1.
incubated for about 18-40 hours at about 37 C. The Culture of Escherichia coli
cells may then be separated from the medium, washed E. coli ATCC No. 25922 was grown in trypticase soy
once in a buffer containing, e.g., about 400 mM sodium
chloride, about 40 mM potassium chloride and about 5 50 broth (3%) (Difco Laboratories, Detroit, Mich.) and
incubated for about 18 to 40 hours at 372 C. The cells
mM bistris at a pH about 7.2. The washed cells may were harvested, washed once with buffer containing
then be resuspended in the same buffer to give an about 400 nM sodium chloride, 40 mM potassium chloride and
10% (w/v) suspension. These bacterial cells may be 5 mM bis tris at pH 7.2.
broken up by ultrasonic oscillator at about 4 C., for, The bacteria were resuspended in the above buffer to
e.g., about 5 minutes. The output from the sonifier may 55
give 10% (w/v) suspension.
be pulsed and the output interrupted to maintain the
temperature of the cell suspension at about 4 C. The EXAMPLE 2
disrupted cell suspension may be centrifuged for about Preparation of Cell free Extract
30 minutes at about 4' C. at 30,000x g, and the superna
tant used as the source of E. coli enzyme (Akkara, J. A., The bacterial cell suspension prepared above was
Kaplan, D. L., and Kaplan, A. M., "Enzyme Formula ultrasonically disrupted by a Branson sonifier. Rat 4 C.,
tion for Laundering and Decontaminating Chemically for 5 minutes, using a microtip and with a pulsed input
Contained Clothing', Technical Report, Natick TR at 40% duty cycle and output control setting at 4
88/014L, U.S. Army Natick Research, Development (Model #350, Branson Sonic Power Co., Danbury,
and Engineering Center, Natick, MA (1988)). 65 Conn.).
In another preferred embodiment the enzymatic cell The pulsed input from the sonifier was for 30 seconds
free extract is provided in freeze dried form. The man followed by one minute cooling in between the sonifica
ner in which the freeze dried product is processed is tion. This cycle was repeated until the total sonification
5,169,554
5 6
time was 5 minutes. A cell free extract was prepared and iodosobenzoate) containing known amounts of fluo
from the disrupted cell suspension, after centrifugation ride before the catalytic activity was determined.
at 30,000x g for 30 minutes at 4 C. The activity was measured at room temperature un
The cell free crude extract was used for the determi less otherwise indicated.
nation of the enzyme activity, including G-agent hydro- 5 EXAMPLE 9
lysis and detoxification studies.
EXAMPLE 3
Agent Testing
Chemicals Soman testing with enzyme/iodosobenzoate deter
gent formulations was carried out. The agent testing
The following chemicals were purchased from com- 10 included both the hydrolysis and the detoxification of
mercial sources: diisopropyl fluorophosphonate (DFP), the agent.
bis para-nitrophenyl phosphate (bis pNPP), para The hydrolysis of soman was followed by measuring
nitrophenyl acetate, parathion, methyl parathion, pa fluoride released by the fluoride specific electrode. The
raoxon, 4 nitrophenyl ethyl (phenyl) phosphinate and 15 detoxification of soman by the enzyme/iodosobenzoate
iodosobenoic acid (IB). Soman (GD) used in the agent detergent formulations was calculated from the second
testing with detergent formulation was supplied by the order inhibition rate constant by the Ellman color reac
Illinois Institute of Technology Research Institute (II tion using eel acetylcholinesterase enzyme, with acetyl
TRI) thiocholine bromide as the substrate and dithiobisni
EXAMPLE 4 20 trobenzoic acid as the indicator (Ellman, G. L., Court
ney, K. D., Andres, V.Jr., and Featherstone, R. M., "A
Detergents New Rapid Determination of Acetylcholinesterase Ac
Alkali (CID-A-A-876), (U.S. Government Printing tivity" Biochem. Pharmacol. 7:88-95 (1961)).
Office, Customer Item Description, Alkali, Laundry EXAMPLE 10
CID-A-A-876, General Services Administration, Wash- 25
ington, D.C. (1980)), Laundry Detergent, Type II (P- Activity of E. coli Enzyme
D-245-E), non-ionic Detergent Type I (MIL-D-16791 FIG. 1 shows variation of the activity of the E. coli
F), Tide "New Liquid' (Procter and Gamble, Cincin enzyme with temperature, when bis para-nitrophenyl
nati, Ohio) and Linear Alkyl Sulfonate (Soap and De phosphate (bis pNPP) was used as the substrate. The
tergent Association, N.Y., N.Y.) were obtained from 30 activity of the enzyme increased with the increase in
commercial sources. incubation temperature up to 45° C., above which the
EXAMPLE 5 enzyme was deactivated very rapidly. The activity
Enzymes
reported is the net activity due to the enzyme after
subtracting the activity without the enzyme.
Commercial hydrolytic enzymes neutrase 1.5 G, 35 The second plot in FIG. 1 gives the rate of the reac
esperase 4.0 T, alcalase 2.0 T, savinase 8.0 SL, and Nue tion after three minutes into the reaction. The three
# , 6.0 S were obtained from Novo Laboratories, Wil minute plot clearly indicates that the rate of the reaction
ton, Conn., and stored at 4 C. These enzymes were in dropped to about 75% of the initial rate at 45° C. The
slurry (SL), granulated (G,T),;or powder (S) form. 40 pH optimum of the E. coli enzyme is given in FIG. 2,
EXAMPLE 6 when bis pNPP was used as the substrate. It appears
from the curve that the enzyme may have two pH op
Protein Determination tima, one at 7 to 7.5, and the second one near pH 8.5.
The protein concentration of the cell free extract of EXAMPLE 11
E. coli was determined by Bio Rad Protein Assay Kit 45 Substrate Specificity of E. coli Enzyme
(Bio Rad, Richmond, Cal.) using Coomassie Brilliant
Blue G-250 dye. The substrate specificity of the E. coli enzyme was
EXAMPLE 7 determined with different phosphate esters. Para
nitrophenyl acetate and bis pNPP had comparable ac
Fluoride Determination SO tivities. However, with other substrates such as: para
A fluoride specific electrode (Orion Research, Cam tivitythion, methyl parathion and paraoxon, the enzyme ac
bridge, Mass.) was connected with a Fisher Titrator was much lower as can be seen in Table i.
(Model #750, Medford, Mass.) with strip chart (Hitachi TABLE 1.
Recorder #056-1001) to determine the rate of hydroly Substrate Specificity of E. coli Enzyme
sis of DFP and soman by determining the fluoride that 55 Substrate Specific Activity
is released. bis para Nitrophenyl phosphate 0.128
EXAMPLE 8 para-Nitrophenyi acetate 0.30
Parathion O,042
Enzyme Assay 60 Methyl parathion 0,035
Paraoxon 0.02
A colorimetric method for the determination of the The activity of enzyme was measured at 37 C. at pH 7.2. The units of activity are
enzyme activity involved the measurement of para noles of paranitrophenol formed/min./mg protein.
nitrophenol formed at 405 nm in the presence of manga
nese chloride. Table 2 below shows the rate of hydrolysis of DFPat
DFP and sonan hydrolysis by the catalysts was fol 65 room temperature in the presence of different detergent
lowed by the measurement of the fluoride released formulations or detergent components. The net hydro
using the fluoride specific electrode. The electrode was lysis was much higher at pH 9.5 and in presence of
calibrated with detergent solutions (without enzyme detergent.
5,169,554
8
TABLE 2 EXAMPLE 3
Hydrolysis of DFP with E. coli Extract. Hydrolysis of Soman with Iodosobenzoate and
Hydrolyzing pH Detergent Type II and Alkali or Non-ionic Detergent
Mediurn 7.5 S.S 9.5 5 Type I
Alkali 0.02 0.05 0.18 Table 4 below presents the results of the hydrolysis of
Tided 0.008 0.0 O.O soman with iodosobenzoate, at 30° C. and 40 C., and in
Nonionic Detergent 0.0 0.005 0.03 the presence of various detergents.
bis Tris Propane 0.04 0.01 0.04
Glycine-NaOH ind? nd? 0.05 O With iodosobenzoate, the rate of hydrolysis of soman
Hydrolysis of DFP is followed by measuring the fluoride released with a fluoride
was higher at 40 C. than at 30° C. at pH 9.5 with either
specific electrode. The enzyme activity was measured as a moles of fluoride relea alkali or laundry detergent Type II. The hydrolysis of
sed/minute/mL of E. coli extract, soman was lower in the presence of non-ionic deter
One milliliter of E. coli extract contained 14.4 mg protein,
Alkali laundry CID-A-A-876. General Services Administration, Washington,
gent. Each formulation contains 0.075 wt/volume of
D.C. (1980). 15
iodosobenzoate.
Tide "New Liquid" (Procter and Gamble, Ohio). TABLE 4
Non-ionic Detergent Type I. Military Specification. Detergent. General Purpose
(Liquid, Nonionic), MIL-D-6791F. Hydrolysis of Soman with Iodosobenzoate?
No data. Hydrolyzing pH
Medium 7.5 8.S 9.5
20 I, 30 C.
EXAMPLE 12
Alkali 1.24 1.08 .78
Further Addition of Iodosobenzoate Detergent 1.25 S6 .64
Nonionic Detergent 0.97 0.74 0.73
Moss et al have shown that iodosobenzoic acid cata E. 40 C.
lytically hydrolyzed phospho-esters (Moss, R. A., Kim, 25 Alkali
Detergent
2.36
2.13
1.89
2.45
3.63
2.9
K. Y., and Swarup, S., "Efficient Catalytic Cleavage of Nonionic Detergent 145 20 1.9
Reactive Phosphates by an O-Iodosobenzoate Func Hydrolysis of Soman is followed by measuring the fluoride released with a fluoride
tionalized Surfactant' J. Amer. Chem. Soc., specific electrode. The activity was measured as u moles of fluoride released/.
minute/ng of iodsobenzoate.
108:788-783 (1986); Moss, R. A., and Swarup, S., "Sur Alkali Laundry CID-A-A-876, General Services Administration, Washington,
30 D.C.
face Specific Phosphate Cleavage of a Substrate Func (1980).
laundry Detergent Type II. Federal Specification Detergent. Laundry and Hand
tionalized Vesicular Surfactant' J. Amer, Chem. Soc., Washing (Granular), P-D-245.
108:5341-5342 (1986); Moss, R. A., Hendrickson, T. F., Non-ionic Detergent Type I. Military Specification, Detergent, General Purpose
(Liquid, Nonionic), MIL-D-6791F.
and Bizziggotti, G. O., "The Esterolytic Chemistry of a
Vesicular Thiocholine Surfactant' J. Amer. Chen. 35
Soc., 108:5520-5527 (1986)). The catalytic effects of EXAMPLE 14
iodosobenzoate in the presence of detergent formula Hydrolysis Detoxification of Soman with
tions or detergent components were determined and the Iodosobenzoate and Detergent Type II and Alkali
results are shown in Table 3 below. The formulation Table 5 below gives the data on the hydrolysis and
40
contains 0.0075 wt/volume of the iodosobenzoate. detoxification of soman by iodosobenzoate in presence
TABLE 3 of alkali and laundry detergent type II at 40° C. and at
Hydrolysis of DFP with iodosobenzoate
pH 9.5.
TABLE 5
-P
Hydrolyzing 7.5 8.5 9.5 45 Hydrolysis and Detoxification of Soman by Iodosobenzoate
Percent Percent
Alkali 0.06 0.16 0.3 Hydrolysis Detoxification
Nonionic Detergent 0.08 0.006 0.08
"Sea water synthetic? ind? 0.02 Ind I. Bufferb 20.3 18.3
Alkali 22.2 33.4
Sea water nd 0.03 d 50 Alkali -- Iodosobenzoate 41. 58.3
bis Tris Propane 0.03 0.05 0.04 II. Bufferb 2.9 S.4
Glycine-NaOH nd nd 0.05 Detergent 12.9 2.
he hydrolysis of DFP was followed by measuring the fluoride released with a Detergent + Iodosobensoate 36.1 37.4
fluoride specific electrode. The activity was measured as a moles of fluoride Hydrolysis of sonan is followed by measuring the fluoride released with the ion
released/minute/mg of iodsobenzoate. specific electrode. Detoxification of soman was determined by measuring the
'Aikali Laundry CID-A-A-876, General Services Administration, Washington, 55 inhibition of acetylcholinesterase enzyne.
D.C. (1980). Buffer: 0.05M glycine-NaOH pH 9.5.
Non-ionic Detergent Type I. Military Specification, Detergent. General Purpose Alkali, Laundry CID-A-A-876.
(Liquid, Non-ionic), MIL-D-16791F. Laundry detergent type II. Federal Specification. Detergent, Laundry and Hand
instant Ocean, Aquarium System, 814) Tyler Blvd., Mentor, OH. Washing (Granular), P-D-245E.
Collected from Narragansett Bay, RI.
No data.
60 EXAMPLE 1.5
DFP was hydrolyzed at about the same rate at pHs Detoxification Studies
8.5 and 9.5 in the presence of alkali. The hydrolysis of The detoxification studies were carried out by deter
DFP by iodosobenzoate in the presence of "Synthetic mining the inhibition of eel acetyl cholinesterase en
Sea Water' (Instant Ocean Aquarium System, Mention, 65 zyme (Ellman, G. L., Courtney, K. D., Andres, J. Jr.,
Oh.) or sea water (collected from Naragansett Bay, and Featherstone, R.M., "A New Rapid Determination
R.I.) at pH 8.5 showed lower activities. All formula of Acetylcohlinesterase Activity " Biochem. Phrama
tions contain 0.0075 wt/volume of iodosobenzoate. col. 7:88-95 (1961)). The data indicate that there was
5,169,554
10
significant hydrolysis and detoxification of soman in TABLE 8-continued
presence of alkali. Both hydrolysis and detoxification Hydrolysis of Soman with E. coli Enzyme at 30 C.
were increased by the presence of iodosobenzoate in the Hydrolyzing pH
detergent formulations. Detoxification was higher than Medium 7.5 8.5 9.5
hydrolysis in the presence of alkali and alkali in the Non-ionic Detergent 1.50 1.49 1.63
presence of iodosobenzoate.
Hydrolysis of Soman is followed by measuring the fluoride released with the ion
EXAMPLE 6 specific electrode. The activity of the enzyme was expressed as a moles of fluoride
released/minute/n of enzyme.
Hydrolysis with Commercially Available Enzymes. Alkali, Laundry CID-A-A-876.
Laundry Detergent Type II. Federal Specification. Detergent, Laundry and Hand
10 Washing
Commercially available hydrolytic enzymes were (Granular), P-D-245E.
Non-ionic Detergent Type I. Military Specification, detergent, General Purpose
also evaluated for hydrolysis of bis pNPP and DFP. (Liquid, Nonionic). MIL-D-6791F.
Table 6 below gives the results obtained with a number
of these enzymes purchased;from Novo Laboratories, The rates given in Table 8 above are the net activities
Conn. 15 due to the enzyme.
TABLE 6 The hydrolysis reaction was followed for 10 minutes
Phosphodiesterase Activity with Commercial Enzymes after the electrode was stabilized with the detergent
Enzyme Enzyme Activity formulation (without the catalyst).
Neutrase 1.5 Gb 3.0 EXAMPLE 1.8
Esperase 4.0 T. 0.09 20
Alcalase 2.0 Ta 0.0 Hydrolysis of Soman with the E. coli
Savinase 8.0 S. 0.07 Enzyme-Detergent at Different pHs
Nue #1, 6.0 Sf 0.09
The enzyme activity is measured by the hydrolysis of bis para-nitrophenyl phos Table 9 below shows data obtained for the hydrolysis
phate at 405 nm and at room temperature. The activity of the enzyme was neasured of soman with E. coli enzyme detergent formulation at
as H. moles of para-nitrophenol formed/minute/ing enzyme x 10. 25 40° C. incubation and at different pHs.
Batch #Pw 20508. Novo Laboratories, CT.
Batch if PE 80280, Novo Laboratories, CT. TABLE 9
Batch #PM 81601, Novo Laboratories, CT.
Batch iPB 60004, Novo Laboratories, CT. Hydrolysis of Soman with E. coli Enzyme at 40 C.
Batch #PN 20761. Novo Laboratories, CT. Hydrolyzing pH
Mediurn 7.5 8.5 9.5
Neutrase 1.5 G was the most active with bis pNPP as Alkali 3.58 3.92 2.44
the substrate. Neutrase .5 G was further evaluated Detergent 3.41 3.54 428
with DFP as the substrate at different pHs and in the Non-ionic Detergent 2.76 2.92 3.8
presence of Alkali as shown in Table 7 below. Hydrolysis of Sonan is followed by measuring the fluoride released with the ion
TABLE 7 35 specific electrode. The activity was measured as a moles of fluoride released/-
minute/mg of enzyme,
Hydrowsis of DFP with Neutrase Enzyme Alkali, Laundry CID-A-A-876, General Services Administration. Washington,
D.C. (1980).
Hydrolyzing pH laundry Detergent Type II. Federal Specification, detergent. Laundry and Hand
Medium 7.5 8.5 9.5 Washing (Granular). P-D-245E.
Non-ionic Detergent Type I. Military Specification, detergent, General Purpose
Alkali O.O O.O O. (Liquid. Nonionic), MIL-D-16791F.
bis Tris Propane O.O O.O 0.03 40
Hydrolysis of DFP is followed by measuring the fluoride released with a fluoride Other conditions of the experiments were the same as
specific electrode. The activity was measured as moles of fuoride released/. those given above. The enzyme activity with Alkali or
minute/mg of enzyme.
Neutrase 1.5 GBatch #Pw 20508, Novo Laboratories. CT.
Alkali. Laundry CID-A-A-876.
with the laundry detergent type II was increased two
45 fold when the temperature of the reaction mixture was
The hydrolytic activity of this enzyme was 0.1 umole increased from 30 C, to 40 C. at pHs 8.5 and 9.5.
per minute per milligram of Neutrase 1.5 G at pH 9.5. EXAMPLE 9
The hydrolysis and detoxification studies of soman by Detoxification of Soman with E. coli
neutrase in the presence of alkali and laundry detergent Enzyme-Detergent at Different pHs
type II also indicates that there was substantially no SO
catalytic breakdown of soman by this enzyme at pH 9.5 Table 10 below gives the results obtained for the
and at 40 C. detoxification of soman using the E. coli enzyme extract
with the detergent formulations at pH 7.0 and pH 9.5 at
EXAMPLE 17 40 C. incubation for 10 minutes (Ellman, G. L., Court
Hydrolysis of Soman by E. coli Enzyme in the presence 55 ney, K. D., Andres, V. Jr., and Featherstone, R. M., "A
of Alkali, Laundry Detergent Type II or Non-Ionic New Rapid Determination of Acetylcholinesterase Ac
Detergent at different pHs, tivity" Biochem. Pharmacol. 7:88-95 (1961)).
The reaction mixture was diluted after the 10 minutes
Table 8 below gives the results of the agent testing of incubation and the toxic fraction of soman that had
carried out using the E. coli extract in presence of alkali, 60 not been hydrolyzed was quantitated by the Ellman
laundry detergent type II, or non-ionic detergent at 30 reaction using eel acetyl cholinesterase enzyme inhibi
C. and at different pHs. t1O.
TABLE 8 The fluoride released from the agent by hydrolysis
Hydrolysis of Soman with E. coli Enzyme at 30" C." was monitored with the fluoride specific electrode dur
Hydrolyzing pH 65 ing the incubation of the enzyme detergent formulation
Mediurn 7.5 8.5 9.5 with the agent and also during the quantitation of toxic
Alkali 1.98 2.0 2.00 fractions of soman by acetylcholinesterase enzyme inhi
Detergent 1.88 2.03 1.90 bition. The detoxification of soman was increased by
5,169,554
11 12
the increase in pH from 7.0 to 9.5. Also the detoxifica number of bacterial cell free extracts (including from E.
tion is increased by the presence of manganese salt in coli ATCC 25922) and acetone powder preparations
the reaction mixture. This data are provided in Table 10 (Attaway, H., Nelson, J. O., Baya, A. M., Voll, M. A.,
below. White, W. E., Grimes, D. J., and Colwell, R. R., "Bac
TABLE 10 terial Detoxification of Diisopropyl Fluorophosphate"
Hydrolysis and Detoxification of Soman by E. coli Extract? Appl. and Environ. Microbiol. 53:1685-1689 (1987)).
Percent Percent The DFPase activities were determined in a buffer
Hydrolysis Detoxification medium and the activity reported as comparable to the
I. pH 7.0 activity reported here with a buffer system and given in
Detergent 2.6 3.3
10 Table 2 above. However, the hydrolytic activity of the
Detergent + Mnt 4.0 8.7 E. coli enzyme extract against DFP was significantly
Detergent -- Enz. Ext.
Detergent -- Enz. Ext. -- Mnt
25.3
500
16.7
22.
higher in the detergent formulations at pH 9.5 (see,
II. H9,5 Table 2).
Alkali 22.2 33.4 15
Earlier studies reported by Hoskin et al. showed that
Alkali -- Mint 1.6 34.0 the E. coli (ATCC 25922) enzyme hydrolyzed soman
Aikali -- Enz. Ext.
Alkali + Enz. Ext. -- Mnt
22.
48.2
28.
40.0
but did not detoxify the agent (Hoskin, F. C. G., Chet
Detergent 12.9 21. tur, G., Gallo, B.J., Robbins, F. M., and Walker, J. E.,
Detergent -- Mint 17.5 21,i "Hydrolysis and Detoxification of Soman and Dimebu
Detergent -- Enz. Ext. 3.2 35.4
20
by Microbial and Squid DFP-ases' Proceedings of the
Detergent -- Enz. Ext. -- Mn 44.7 40.8 1986 U.S. Army Chemical Research, Development and
Hydrolysis of Soman is followed by measuring the fluoride released with the ion
specific electrode at 40' C. Detoxification of Soman was determined by measuring
Engineering Center Scientific Conference on Chemical
the inhibition of eel acetylcholinesterase enzyme. Defense, pp. 283-288 (1987)).
Alkali.
0.3 mill E. coli enzyme extract.
Hoskin et al used partially purified enzyme (using
Laundry Detergent, Type II. 25 cold ethanol precipitation) followed by immobilization
of the enzyme on agarose resin. This enzyme-agarose
complex was used for detoxification studies. The results
EXAMPLE 2.0 of the present studies (see, Table 10) using crude E. coli
Enzyme Activity enzyme extract in the presence of laundry detergent
The temperature optimum for the E. coli enzyme was 30 type II show that detoxification was 45% of the total
45' C., above which the enzyme was rapidly inacti soman being hydrolyzed at pH 7.0, while detoxification
vated. It is, therefore important that the enzyme deter was 91% of the total soman hydrolyzed at pH 9.5.
gent formulation be used at 45° C. or below for activity. Hydrolysis and detoxification of soman by the E. coli
However, the incorporation of similar enzyme extracts enzyme in the presence of alkali were comparable to the
from thermophilous organisms such as Bacillus Stero 35 results obtained with laundry detergent type II. It is
thermophilus may provide higher temperature stability. interesting to note that Soman was detoxified at a
The net hydrolytic activity of the E. coli enzyme in higher rate than hydrolysis (i.e., fluorine release) in the
creased with an increase in pH up to 9.5, with both DFP presence of alkali.
and soman as the substrates, indicating the beneficial These hydrolysis and detoxification studies clearly
effect of the enzyme from E. coli in the detergent formu indicate the synergistic effect of soman detoxification in
lation. The pH optimum studies using bis pNPP (see, the presence of alkali. It is possible that soman is detoxi
FIG. 2) also indicated a second pH optimum for this fied in the presence of alkali by a mechanism other than
enzyme on the alkaline side. by fluorine removal, such as the cleavage of pinacolyl
The studies demonstrated an increase in catalytic alcohol (1,2,2-trimethylpropyl alcohol) from soman.
activity of the enzyme in the presence of alkali and 45 One of the end products of this reaction would be me
laundry detergent type II as pHs 8.5 and 9.5. These pHs thylfluorophosphonate, and this compound is not de
values not only increase the catalytic hydrolysis of the tected by the fluoride specific electrode. Moreover,
agent, but also increase the detergent of the laundry there is no information in the literature to indicate that
formulation in removing soil from clothing and other methylfluorophosphonate is inhibitory to the eel acetyl
articles. 50 cholinesterase enzyme used for the detoxification stud
Moreover, pH 9.5 represents fairly mild conditions ies of soman.
compared with standard chemical decontaminating Enzymes isolated from non-sporulating rod like obli
solutions currently used and thus could be used on a gate thermophiles (JD 100 and JD 300) have been
number of surfaces or conditions for decontamination shown to have catalytic activities against soman, both
along with cleaning. The E. coli enzyme is quite stable 55 hydrolysis and detoxification. The enzymes from these
when stored at 4 C. and retains its full catalytic activity thermophiles which have a high temperature optimum
for more than one year. The enzyme solution could also (stability and activity) against G-agents can also be
be freeze dried without any loss of activity. incorporated into the enzyme detergent formulation
A detergent formulation based on alkali, linear alkyl described herein and the laundry operation can be car
sulfonate, citric acid and freeze dried E. coli extract had ried out at high temperatures for complete detoxifica
the same DFPase activity as the enzyme extract with tion of G-agents (Hoskin, F. C. G., Chettur, G., Gallo,
detergent formulation at pH 9.5. The neutrase enzyme B. J., Robbins, F. M., and Walker, J. E., "Hydrolysis
from Novo Laboratories looks good for use in place of and Detoxification of Soman and Dimebu by Microbial
E. coli enzyme, even though the hydrolysis and detoxifi and Squid DFP-ases" Proceedings of the 1986 U.S.
cation of soman was not significant with the conditions 65 Army Chemical Research, Development and Engineer
used in this study. ing Center Scientific Conference on Chemical Defense,
A recent report by Attaway et al has demonstrated pp. 283-288 (1987); DeFrank, J. J., "Thermostable Bac
DFP hydrolyzing and detoxifying activities with a terial Enzymer for Organophosphorus Agent Detec
5,169,554
13 14
tion/Decontamination” Proceedings of the 1986 U.S. TABLE 2
Army Chemical Research, Development and Engineer Rate of Hydrolysis of Soman at
ing Center Scientific Conference on Chemical Defense, 40 C. in Trailer Mounted Laundry
pp. 635-641 (1987)). SOMAN
HYDROLYZED
EXAMPLE 2 OPERATION In MOLE 2
Addition of Iodosobenzoate SUDS 18 2.48
y 59 10.4
Iodosobenzoate was evaluated in this study for chem it 59 0.74
TOTAL 42.42
ical decontamination of OPA chemicals in order to 10 PERCENTAGE OF 6.4% 10 g/m2
generate comparative data for the non-enzymatic ap SOMAN HYDROLYZED 82.0% 2 g/m
proach to decontamination. Iodosobenzoate was tested
against DFP and soman and was shown to be catalyti This does not consider the fact that with purification
cally active against the agents in a detergent formula of the enzyme, increased concentration of the enzyme
tion. 15 in the detergent formulation and/or with increased
Iodosobenzoate was more active at pH 9.5 than at laundry time, this hydrolysis and detoxification can be
lower pHs. Hydrolysis and detoxification studies of increased significantly. The data contained in these two
soman by iodosobenzoate in the presence of alkali Tables are based on extrapolations from laboratory
clearly indicate that there was almost 100% detoxifica experiments.
tion of total soman hydrolysed. Iodosobenzoate was 20
EXAMPLE 23
also active against DFP in the presence of sea water.
The catalytic degradation of soman by iodosobenzo Conclusions
ate in the presence of sea water has to be studied at The catalytic activity against soman and other OPA
different temperatures and conditions before its poten 25
chemicals is up to about 16-fold and greater when the
tial use for decontamination of G-agents can be fully enzyme detergent formulation of the invention is uti
assessed, including possible Nave application in the lized over that observed with the detergent formulation
present of sea water. alone at pH 9.5. Soman hydrolyzed at pH 9.5 (at 40° C.)
in the enzyme detergent formulation is almost com
EXAMPLE 22 pletely (91%) detoxified.
Chemical Decontaminating Laundry Detergent 30 The present source of soman detoxifying enzyme,
Formulation
squid enzyme, is very expensive and the source is lim
ited. The E. coli enzyme can be produced in large quan
Enzymes are routinely incorporated in commercial tities and be more cost effective than the squid enzyme.
laundry detergent formulations. The primary purpose Another non-enzyme agent for the catalytic breakdown
of this study is to improve cleaning under mild laundry 35 of soman (both hydrolysis and detoxification) is iodoso
conditions by the use of proteases or lipases for the benzoic acid, which is shown in this study to be catalyti
removal of protein/lipid bound stains and/or soils. We cally active against soman and other agents in a deter
have demonstrated that an OPA chemical degrading gent formulation and sea water.
enzyme from E. coli can be incorporated into a deter The invention now being fully described, it will be
apparent to one of ordinary skill in the art that many
gent formulation with successful decontaminating ac changes and modifications can be made thereto without
tivity. This decontaminating activity was demonstrated departing from the spirit or scope of the invention as
at neutral to alkaline pHs. The incorporation of a laun said firth herein.
dry detergent type II and E. coli enzyme into a formula We claim:
tion at the concentrations evaluated in this study will 1. An enzyme detergent composition capable of de
produce the hydrolysis and detoxification of soman as 45 toxifying warfare nerve agents and other organophos
follows. phorus acid chemicals, comprising
If soman were present in contaminated clothing at 2 g a laundry detergent; and
per square meter (g/m2) then 82% would be hydro an enzymatic cell free extract from E. coli containing
organophosphorous acid anhydrolase the composi
lyzed and 74.6% detoxified in 33 minutes (see, Tables 11 50 tion having a pH of about 6.5 to 10 in water.
and 12 below). 2. The composition of claim 1, further comprising
If soman were present at 10 g/m2 of clothing, then iodosobenzoate.
16.4% would be hydrolyzed and 14.95 would be detoxi 3. The composition of claim 1, wherein the pH is
fied in 33 minutes. about 7.0 to 9.5.
TABLE 1.1 4. The enzyme detergent composition of claim 1,
55 wherein
Trailer Mounted Laundry for Overgarment 84 the enzymatic cell free extract is from E. coli having
OPERA- WATER LEVEL TIME TEMP. SUPPLIES an ATCC Accession No. 25922.
TION (inch) (min.) (C.) (per 13.6 kg) 5. The composition of claim 1, further comprising a
SUDS 1. 5 49 oz.
4 oz. 60 manganese compound.
6. A method of cleaning and detoxifying a product
f t 4 oz. contaminated with organophosphorous chemicals,
RNSE 2 28.5 comprising
P ya P contacting the product with an amount of an aqueous
suspension of the enzyme detergent composition of
3.6 kg (30 lbs) of Overgarment 84 were exposed with 10 g of soman per square 65 claim 1 at a temperature of about 20 to 50 degrees
meter, total Soman in the wash load was 258.6 g. The volune of water used per centigrade; and
cycle was 83 liters. The volume of clothing was 38 liters. Laundry Detergent Type
II was used at pH 9.5 with E. coli enzyme extract. rinsing the product.
sk k s: k

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