U.S. patent application number 11/001600 was filed with the patent office on 2009-12-31 for passivation of nerve agents by surface modified enzymes stabilized by non-covalent immobilization on robust, stable particles.
Invention is credited to Michael A. Markowitz, J. Matthew Mauro, Mehran Pazirandeh, Paul E. Schoen, Alok Singh.
Application Number | 20090325261 11/001600 |
Document ID | / |
Family ID | 29401827 |
Filed Date | 2009-12-31 |
United States Patent
Application |
20090325261 |
Kind Code |
A1 |
Singh; Alok ; et
al. |
December 31, 2009 |
Passivation of nerve agents by surface modified enzymes stabilized
by non-covalent immobilization on robust, stable particles
Abstract
Enzymes are modified by incorporating anchor sites for linking
the enzymes to a target surface without destroying the catalytic
activity of the enzymes. A stable carrier to accommodate and bind
the selected enzyme is constructed, and the enzyme is
non-covalently liked to the carrier, generally through metal salts
of iminodiacetate
Inventors: |
Singh; Alok; (Springfield,
VA) ; Pazirandeh; Mehran; (Silver Spring, MD)
; Schoen; Paul E.; (Alexandria, VA) ; Markowitz;
Michael A.; (Burke, VA) ; Mauro; J. Matthew;
(Silver Spring, MD) |
Correspondence
Address: |
NAVAL RESEARCH LABORATORY;ASSOCIATE COUNSEL (PATENTS)
CODE 1008.2, 4555 OVERLOOK AVENUE, S.W.
WASHINGTON
DC
20375-5320
US
|
Family ID: |
29401827 |
Appl. No.: |
11/001600 |
Filed: |
November 29, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09725309 |
Nov 29, 2000 |
6869784 |
|
|
11001600 |
|
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Current U.S.
Class: |
435/176 |
Current CPC
Class: |
C12N 11/02 20130101;
C12N 11/14 20130101; C12N 9/96 20130101; C12N 9/16 20130101 |
Class at
Publication: |
435/176 |
International
Class: |
C12N 11/14 20060101
C12N011/14 |
Claims
1-8. (canceled)
9. A method of reducing the presence of a contaminant comprising:
providing a genetically engineered thioesterase capable of reacting
with the contaminant to include one or more terminal histidine
residues; attaching the genetically engineered thioesterase to salt
groups selected from the group consisting of metal salts of
iminodiacetic acid, metal salts of nitrilotriacetic acid, and
mixtures thereof on the surface of a particulate inorganic carrier;
and contacting the attached thioesterase with a sample suspected of
containing the contaminant.
10. The method according to claim 9 wherein the metal salts are
selected from the group consisting of copper, nickel, cobalt, and
zinc salts.
11. The method according to claim 9 wherein the carrier is a metal
oxide ceramic particles that can be formed in the Stober process
starting with a metal alkoxide precursor.
12. The method according to claim 11 wherein the metal oxide
particles are selected from the group consisting of silica,
alumina, baria, titania, and zirconia.
13. The method according to claim 9 wherein the salt groups are
metal salts of iminodiacetic acid.
14. The method according to claim 9 wherein the salt groups are
metal salts of nitrilotriacetic acid.
15. The method of claim 9, wherein the thioesterase includes a
terminal polyhistidine chain.
16. The method of claim 9, wherein the attached thioesterase is
capable of detoxifying a nerve agent.
17. The method of claim 9, wherein the attached thioesterase is
catalytically active.
18-19. (canceled)
Description
FIELD OF THE INVENTION
[0001] The present invention relates to a
detoxification/decontamination system for nerve agents which has
long term stability over a wide temperature range.
BACKGROUND OF THE INVENTION
[0002] Nerve agents pose a growing threat to society whether they
are released accidentally or deliberately. Current means to counter
threats from nerve agents, although temporarily effective, are not
adequate. Currently, activated charcoal is used to filter nerve
agents from air and water; bleach solution or jet fuel is used for
decontaminating protective gear. However, these methods use
compositions which have undesirable properties including
corrosiveness, flammability, and toxicity. Moreover, these methods
can only be used on a small scale, and they are not effective over
an extended period of time.
[0003] Delivery of active enzyme systems to counter and detoxify
chemical and biological warfare agents is a promising and active
area of research. While some enzymes in their native form have
exhibited effectiveness against nerve agents, there are still many
challenges in developing effective detoxification systems,
including preservation of high catalytic activity in real
conditions, stability of the enzyme system after prolonged storage,
suitable means of delivery, and accessability of enzymes to threat
agents.
[0004] "Detoxifying Nerve Agents", C&E News Sep. 15, 1997, page
26 reports the current state of the art for detoxification of nerve
agents, with special reference to efforts on the part of the U.S.
Army. A class of enzymes that is known to catalyze the hydrolysis
of organophosphate compounds has been investigated for potential
decontamination. The organophosphate anhydrolases (OPAA: EC3.1.8.2)
catalyze the hydrolysis of many G-type chemical warfare nerve
agents. Specifically, these enzymes have activity against compounds
such as sarin, soman, and GF (O-cyclohexyl methylphosphono
fluoridate). Covalently linking enzymes to solid substrates and
embedding enzymes in polymer matrices are the two most common means
for enzyme immobilization. However, the covalent chemistry required
for linking an enzyme to a substrate often adversely affects the
enzyme's activity. Enzymes embedded in polymer matrices are not
accessible freely to the agents present in the surrounding
medium.
[0005] Branner-Jorgensen, in U.S. Pat. No. 4,266,029, disclose
immobilizing enzymes on a mineral oxide which has been coated with
gelatin and glutaraldehyde. However, these enzymes are used in
fluidized bed operations, and there is no indication that these
enzymes can be used to detoxify nerve agents.
[0006] Doctor et al., in U.S. Pat. No. 5,366,881, disclose mutant
cholinesterase which can be used for detoxifying organophosphates.
However, to maintain the activity of the cholinesterases, oximes
are added.
[0007] Tschang et al., in U.S. Pat. No. 4,461,832, disclose enzymes
embedded in silica gel in order to suspend the enzyme. There is no
indication that these enzymes retain their activity, or that these
enzymes can be used to detoxify nerve agents.
[0008] Recently, LeJeune and coworkers reported immobilizing
phosphotriesterases onto polyurethane polymers for decontamination
purposes (LeJeune et al., Biotechnology and Bioengineering 54:
105-114, 1997. However, there are several drawbacks to using
polyurethane for immobilizing phosphotriesterases. In addition to
being an environmentally unfriendly polymer, polyurethane may not
afford the maximal protein stability that can be achieved in the
protein's native environment. In addition, the enzymes used in
these studies have not been selected for use under field
conditions, and suffer many drawbacks, including inhibition by
substrate, low turnover, and low stability. Watkins et al.,
Biological Chemistry 272: 25596-25601, 1997) have demonstrated
enhanced rate of hydrolysis of phosphorus-fluorine bonds by
phosphotriesterases using engineered enzymes.
SUMMARY OF THE INVENTION
[0009] It is an object of the present invention to overcome the
deficiencies of the aforementioned prior art.
[0010] It is another object of the present invention to provide a
mutagenesis and selection/screening method to obtain enzymes with
the desired catalytic and stability properties.
[0011] It is a further object of the present invention to modify
the enzymes obtained for non-covalent immobilization on the surface
of polymerized vesicles.
[0012] It is another object of the present invention to modify the
enzymes obtained for non-covalent immobilization on the surface of
silica particles.
[0013] The following method is used to produce effective agents for
detoxifying/decommissioning nerve agents:
[0014] (1) Select a suitable enzyme.
[0015] (2) Modify the enzyme by incorporating anchor sites for
linking it to a target surface without destroying the catalytic
activity of the enzyme.
[0016] (3) Construct a stable carrier to accommodate and bind the
selected enzyme.
[0017] (4) Non-covalently link the enzyme to the colloids of
surface metal iminodiacetate groups and/or nitrilotriacetic acid
groups.
[0018] Once an enzyme has been selected for its catalytic and
stability properties, the enzyme is further modified for
non-covalent immobilization on the surface of polymerized vesicles.
The immobilization technique is the subject of U.S. Pat. No.
5,663,387, the entire contents of which are hereby incorporated by
reference. Polymerized liposomes are a prime substrate for
immobilizing active enzymes because they retain their structural
integrity in adverse chemical and physical environments, provide a
native environment for enzymes to sustain their activity, and
provide higher surface area to facilitate easy access of medium to
enzymes.
[0019] Silica particles can also be used as substrates for
non-covalent enzyme immobilization, because these particles have
high surface area and retain their structural integrity in adverse
chemical and physical environments. Silica particles with surface
IDA groups can be formed in at least one of two ways:
[0020] (1) Silica particle precursors, such as TEOS or TMOS are to
be co-hydrolyzed with IDA-modified alkoxysilantes using the Stober
process (Stober et al., Journal of Colloid Interface Science 26:
62, 1968); or
[0021] (2) IDA alkoxysilanes are grafted to the surface of silica
particles using well established procedures, Bradley et al.,
Langmuir 6: 792, 1990; Van Blaaderen et al., Langmuir 8:2921,
1992). After forming a metal IDA salt or a metal NTA salt,
non-covalent enzyme immobilization can proceed as previously
described. Since enzymes immobilized onto silica or other inorganic
particles can be packed into a variety of chromatographic columns
(liquid HPLC), they readily lend themselves to simultaneous
continuous-flow catalytic processing of multiple toxic agents. This
appears to be the first time that surface modified enzymes have
been immobilized on silica particles.
DETAILED DESCRIPTION OF THE INVENTION
[0022] According to the present invention, using either a liposome
or silica particle, customized immobilization protocols can be
developed and optimized for storing the enzyme systems under
otherwise adverse conditions.
[0023] Examples of enzymes which are useful in detoxifying nerve
agents are thioesterases, although the process of the present
invention can be used with any type of enzyme useful for destroying
waste materials. One example of this is lipases, which are used for
digesting waste onboard ships. The enzymes are genetically
engineered to include a poly-His tail as well as other stabilizing
amino acid substitutions. Non-covalent enzyme immobilization on
polymerized liposomes was effected by co-polymerizing amphiphiles
containing metal salts of iminodiacetic acid or nitrilotriacetic
acid with other polymerizable amphiphiles and then binding the
enzyme to the iminodiacetic acid-metal salts or NTA-metal salts on
the outer surfaces of the vesicles. This technique relies on the
strong binding affinity between iminodiacetate salts or NTA salts
and polyhistidine, which has been made available on the surface of
the enzyme selected for immobilization through genetic engineering.
The enzymes that can be used for this technique are those enzymes
that have appropriately reactive surface available histidines or
which have a histidine tag that can be added through site specific
metagenesis. This includes, of course, polyhistidine. Histidine
forms a strong bond with iminodiacetate salts, such as copper,
zinc, cobalt, and nickel iminodiacetate salts, and nitrilotriacetic
acid salts, such as copper, zinc, cobalt, and nickel salts. The
main criterion for this process to be effective is that the binding
site on the enzyme be far away from or innocuous to the function of
the enzyme's catalytic site. While silica is the preferred
inorganic surface because it is relatively inexpensive and its
properties are well understood, any type of metal oxide ceramic
particles that can be formed similar to the Stober process starting
with a metal alkoxide precursor can be used. Other types of
inorganic surfaces that can be used in the process of the present
invention include alumina, baria, titania, and gircinia.
[0024] Bachmair et al., in U.S. Pat. Nos. 5,646,017; 5,496,721;
5,196,321; 5,132,213; and 5,093,242, the entire contents of which
are hereby incorporated by reference, disclose methods for
designing or modifying protein structure at the protein or genetic
level to produce proteins having specified amino-termini in vivo or
in vitro. These methods can be used to produce proteins having
amino-termini on enzymes wherein genes encoding the enzymes can be
made to encode an amino acid of the desired class at the
amino-terminus so that the expressed enzyme exhibits a
predetermined amino-terminal structure which renders is
metabolically stable and able to bind to metal salts of
iminodiacetic acid which are copolymerized with amphiphiles.
Preferably, the amino-terminal structure is histidine, although
C-terminal or internal polyhis sequences will usually be
satisfactory as well.
[0025] A DNA sequence containing nucleotides coding for the enzyme
of interest, as well as nucleotides which code for an amino acid
sequence at the N-terminus or C-terminus of the enzyme such as
histidine which strongly bind to metal iminodiacetate or
nitrilotriacetic acid salts are operably linked to a promoter that
will permit expression of the enzyme in the cells of interest for
production thereon. This enzyme cassette is introduced into cells
for production of the stabilized enzyme, after which the stabilized
enzymes are recovered therefrom by conventional means.
[0026] The enzymes useful in detoxifying nerve agents are attached
to iminodiacetate salt groups on the surface of silica particles
formed by co-hydrolyzing TMOS with an IDA-alkoxysilane derivative.
The IDA-alkoxysilane accounted for 5 weight percent of the total
silica content. After particles were synthesized using the Stober
procedure, the copper salt of the surface IDA groups was formed by
adding an aliquot of 20% aqueous CuSO.sub.4 solution (wt/wt) to the
dry particles, and then suspending the particles using mild
sonication or vortex mixing. The suspension was centrifuged and the
supernatant was removed. This procedure was repeated, and the
resulting blue silica particles were washed with water by adding
the water to the particles, suspending the particles in solution,
and then centrifuging the suspension and removing the supernatant.
This procedure was repeated three times. Then, an aliquot of the
thioesterase in 0.05 M phosphate buffer, pH 7.2., was added to a
suspension of the particles in the same buffer. The suspension was
incubated at 4.degree. C. for three hours. The particles were then
centrifuged and the supernatant was removed. The particles were
then washed using the phosphate buffer described above. All
operations involving the enzyme were performed at 4.degree. C.
After the final washing, the particles were resuspended in the
buffer and stored for future use. The activity of the immobilized
enzyme was confirmed using standard procedures.
Examples
1. Cloning and Modifying Enzyme
[0027] The gene for thioesterase-1 (TE-1) of E. coli strain JM109
was cloned using a modification of the procedure published in
Escherichia coli: thioesterase I. Molecular cloning and sequencing
the structural gene and identification of a periplastic enzyme,
Hyeson Cho, John L. Carona (1993) Journal or Biological Chemistry
26:9238-9245. Briefly, amplified DNA encoding the TE-1 protein and
appropriate flanking nucleotide sequences was ligated into the DNA
vector PCR 2.1 (Invitrogen). After preparing of 140 micrograms of
the PCR2.1-TE1 vector DNA from 100 ml overnight culture, the
engineered TE-1 fragment was liberated from the intermediate vector
by digestion of 10 micrograms of this DNA with 20 units each of the
restriction endonucleases NdeI and XhoI at 37.degree. C. overnight.
The liberated TE-1 coding fragment was purified electrophoretically
on a 2% agarose gel. The stained gene fragment was excised from the
gel and subsequently obtained free of agarose using commercial
products (Qiagen).
[0028] The gene for N-terminal polyhistidine-modified TE-I was
prepared by enzymatically ligating approximately 300 mg of the gene
fragment described above with about 100 ng of pProEx-1 vector DNA
(Life Technologies) previously digested with NdeI and XhoI enzymes
and dephosphorylated with calf intestinal alkaline phosphatase.
Transformed E. coli DH5.alpha.F'LacI.sup.q cells (Life
Technologies) were screened for the presence of the TE-1 inserted
gene by electrophoretic analysis of differential whole-cell protein
profiles of cells taken from small scale cultures grown plus and
minus 1 mM isopropylthiogalactopyranoside (IPTG) chemical
inducer.
[0029] TE-1 was purified from 100 ml cell culture (LB/50
micrograms/ml carbenicillin) induced at 30.degree. C. with 1 mM for
about two hours (OD.sub.600 at induction .about.0.6). Cell
resuspension, sonic lysis, and chromatographic purification were
carried out according to published procedures published in Protein
Biotechnology (1993) Felix Franks, Human Press, Totwa, N.J., and
references cited therein. The final eluted TE-1 product, 14 ml, was
dialyzed for three days against 3 L 50 mM potassium phosphate
buffer, pH 7.2. The dialyzed product was concentrated in two stages
to 0.65 ml using Centriprep-10 and Centricon-10 centrifugal
concentrators at 4.degree. C. The final protein concentration of
0.35 mg/ml was evaluated against bovine serum albumin standard
protein using a Bio-Rad (Bradford method) assay kit.
2. Assay of Enzymatic Activity Immobilized TE-1
[0030] Samples of TE-1 immobilized on IDA silica were assayed for
their ability to hydrolyze p-nitrophenyl propionate (SIGMA)
according to published procedures. In a typical assay, equivalent
amounts of silica/enzyme slurry, or appropriate control samples, in
10 to 20 microliters were added to a 1.5 ml polypropylene conical
microcentrifuge tube that contained 0.97 ml physiologically
buffered saline (PBS) at pH 7.2, 3% v/v acetone, and 0.370 mM
p-nitrophenyl propionate. Each tube was capped, oriented on its
side, and shaken at 225 RPM at 30 C for 30 minutes. After 30
minutes, each sample was immediately centrifuged at room
temperature for exactly one minute. Then, 0.90 ml of each sample
was removed and immediately assayed spectrophotometrically at 346
nm. In one such assay, the background corrected results were as
follows:
TABLE-US-00001 Sample Activity (OD.sub.346 units/min .times.
10.sup.3 Cu.sup.2+ + IDA silica + TE-1 5.12 Cu.sup.2+ + IDA silica
0.47 IDA silica 0.67
3. Formulation and Catalytic Activity of Cu.sup.2+-IDA Silica
Particles
[0031] The silica particles were formed by co-hydrolyzing TMOS with
an IDA-alkoxysilane. The IDA-alkoxysilane accounted for 5 weight %
of the total silica content. After particle synthesis using the
Stober procedure, the copper salt of the surface IDA groups was
formed by adding an aliquot of aqueous 20% CuSO.sub.4 solution,
w/w, to the dry particles, and then suspending the particles using
mild sonication and vortex mixing. The suspension was centrifuged
and the supernatant was removed. This procedure was repeated, and
then the resulting blue silica particles were washed with water by
adding the water to the particles, suspending the particles in
solution, and then centrifuging the suspensions and removing the
supernatant. This procedure was repeated three times. A small
portion of these particles was further washed with an aqueous
saturated EDTA tetrasodium salt solution in a similar manner. Upon
adding the EDTA solution, the supernatant turned from clear to blue
and the particles turned from blue to white, demonstrating that
copper ions had been bound to the IDA groups on the surface of the
particles.
[0032] Then, the Cu.sup.2+-IDA particles were suspended by mild
sonication and vortex mixing in 1 ml of 0.005 M aqueous phosphate
buffer, pH 7.2. Then, 40 .mu.L of this suspension was added to a
test tube. 160 .mu.L of the buffer was added, and the resulting
suspension was cooled to 4.degree. C. After three hours at
4.degree. C., the catalytic activity of the particles was tested
using a thioesterase assay. The particles exhibited catalytic
activity as follows:
[0033] Cu.sup.2++IDA silica particles, 0.47 OD.sub.346
units/min.times.10.sup.3
[0034] IDA silica particles, 0.67 OD.sub.346
units/min.times.10.sup.3
[0035] This example demonstrates that the Cu.sup.2++IDA particles
have no catalytic activity in the absence of bound
thioesterase.
4. Binding and Catalytic Activity of Thioesterase on Cu.sup.2+-IDA
Silica Particles
[0036] Polyhistidine tagged thioesterase was noncovalently attached
to copper-IDA groups on the surface of silica particles made as in
Example 1 in the following manner: 40 .mu.L of the suspension of
the Cu.sup.2++IDA silica particles in 1 ml of 0.05M aqueous
phosphate buffer, pH 7.2., suspension was added to a test tube. 160
.mu.L of the buffer was added, and the resulting suspension was
cooled to 4.degree. C. Then, 10 .mu.L of the thioesterase in the
phosphate buffer was added to this suspension, which was then
incubated at 4.degree. C. for three hours. The particles were
centrifuged and the supernatant was removed, making sure that the
silica did not go dry. The particles were washed using the
phosphate buffer as described above. Eight mL of the buffer was
added to the particles, which were then suspended with mild
sonication, centrifuged, and the supernatant removed. This washing
procedure was repeated six times. All operations involving the
enzyme were performed at 4.degree. C. After the final washing, the
particles were resuspended in 1 mL of the buffer and stored for
future use. The activity of the immobilized enzyme was confirmed
using standard procedures. This sample, Cu.sup.2++IDA silica+TE-1
showed an activity of 5.12 OD.sub.346 units/min.times.10.sup.3.
This example demonstrates the sustained activity of polyhistidine
modified thioesterase bound to the Cu.sup.2+-IDA groups on the
silica particles.
5. Binding and Catalytic Activity of thioesterase on DA with Silica
Particles
[0037] The Cu.sup.2+-IDA silica particles that had been washed with
saturated aqueous tetrasodium EDTA solution were resuspended in 1
mL of 0.05 M aqueous phosphate buffer at pH 7.2. 40 .mu.L of the
suspension of this suspension was added to a test tube. 160 .mu.L
of the phosphate buffer was added, and the resulting suspension was
cooled to 4.degree. C. Then, 10 .mu.L of thioesterase in phosphate
buffer was added to this suspension, which was then incubated at
4.degree. C. for three hours. The particles were centrifuged and
the supernatant was removed, making sure that the silica did not go
dry. The particles were washed using the phosphate buffer as
described above. Eight mL of the buffer was added to the particles,
which were then suspended with mild sonication, centrifuged, and
the supernatant removed. This washing procedure was repeated six
times. All operations involving the enzyme were performed at
4.degree. C. After the final washing, the particles were
resuspended in 1 mL of the buffer and stored for future use. The
catalytic activity of these particles, as determined by the
thioesterase assay, was significantly less than the activity of the
enzyme bound to the Cu.sup.2+-IDA particles. This example
demonstrates that binding of the enzyme to the Cu.sup.2+-IDA groups
on the silica particles is required for optimal catalytic
activity.
[0038] The method of the present invention provides means for
stabilizing enzymes in such a fashion that the enzymes, by virtue
of their non-covalent bonding to the liposomes or silica, are
readily available to act on their substrates. The present invention
provides an effective system that uses the efficiency and
selectivity of enzymes in catalysis and utility of surfaces to
provide stability to sophisticated enzyme architecture.
[0039] The foregoing description of the specific embodiments will
so fully reveal the general nature of the invention that others
can, by applying current knowledge, readily modify and/or adapt for
various applications such specific embodiments without departing
from the generic concept, and, therefore, such adaptions and
modifications should and are intended to be comprehended within the
meaning and range of equivalents of the disclosed embodiments. It
is to be understood that the phraseology or terminology employed
herein is for the purpose of description and not of limitation.
* * * * *