U.S. patent application number 13/729639 was filed with the patent office on 2013-05-09 for methods, compositions and devices utilizing structurally stable cyanuric acid hydrolase.
This patent application is currently assigned to REGENTS OF THE UNIVERSITY OF MINNESOTA. The applicant listed for this patent is REGENTS OF THE UNIVERSITY OF MINNESOTA. Invention is credited to Michael J. Sadowsky, Jennifer L. Seffernick, Lawrence P. Wackett.
Application Number | 20130112608 13/729639 |
Document ID | / |
Family ID | 44068037 |
Filed Date | 2013-05-09 |
United States Patent
Application |
20130112608 |
Kind Code |
A1 |
Sadowsky; Michael J. ; et
al. |
May 9, 2013 |
METHODS, COMPOSITIONS AND DEVICES UTILIZING STRUCTURALLY STABLE
CYANURIC ACID HYDROLASE
Abstract
The present invention relates to stable cyanuric acid hydrolase
enzymes, compositions, and devices for use in the treatment of a
liquid, such as water. The present invention also relates to
methods of using these enzymes, compositions and devices for the
treatment of a liquid, such as water.
Inventors: |
Sadowsky; Michael J.; (St.
Paul, MN) ; Seffernick; Jennifer L.; (St. Paul,
MN) ; Wackett; Lawrence P.; (St. Paul, MN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
REGENTS OF THE UNIVERSITY OF MINNESOTA; |
St. Paul |
MN |
US |
|
|
Assignee: |
REGENTS OF THE UNIVERSITY OF
MINNESOTA
St. Paul
MN
|
Family ID: |
44068037 |
Appl. No.: |
13/729639 |
Filed: |
December 28, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
12879903 |
Sep 10, 2010 |
8367389 |
|
|
13729639 |
|
|
|
|
61241797 |
Sep 11, 2009 |
|
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Current U.S.
Class: |
210/287 ;
435/231 |
Current CPC
Class: |
C12N 9/86 20130101; A62D
3/02 20130101; A62D 2101/45 20130101; C02F 3/342 20130101 |
Class at
Publication: |
210/287 ;
435/231 |
International
Class: |
C02F 3/34 20060101
C02F003/34; C12N 9/86 20060101 C12N009/86 |
Claims
1. An isolated or purified structurally stable cyanuric acid
hydrolase (CAH) enzyme.
2. The CAH of claim 1 having a K.sub.m value for cyanuric acid of
25-150 .mu.M.
3. The CAH of claim 1 having a k.sub.cat value for cyanuric acid of
4.8-76 s.sup.-1.
4. The CAH of claim 1, wherein the enzyme is thermostable.
5. The CAH of claim 1, wherein the enzyme retains at least 30%
enzymatic activity at a temperature above 25.degree. C.
6. The CAH of claim 1, wherein the enzyme comprises amino acid
sequence TEGNG(C/G/L)(V/M/A)ND(Y/F)(T/S)R (SEQ ID NO:15).
7. The CAH of claim 1, wherein the enzyme comprises amino acid
sequence (M/F/L/I)(V/I/M)(M/F/W)SGG
(D/E/G/P)G(V/I/L/G/A)(L/I/M/A)(S/T/A/C)PHX(T/I/L/S)(V/I/L)(F/I/V)
(SEQ ID NO:17), wherein X is any amino acid.
8. The CAH of claim 1, wherein the enzyme comprises amino acid
sequence TEGNGCVNDFTR (SEQ ID NO:16) and amino acid sequence
FIMSGGEGVMTPHTVF (SEQ ID NO:18).
9. The CAH of claim 1, wherein the enzyme is 350-380 amino acids in
length.
10. The CAH of claim 1, wherein the enzyme has a pI of about
5-6.
11. The CAH of claim 1, wherein the enzyme is from Moorella
thermoacetica.
12. The CAH of claim 11, wherein the enzyme is from Moorella
thermoacetica ATCC 39073.
13. The CAH of claim 1, wherein the enzyme has a specific activity
of 12-18 .mu.mmol/min/mg with cyanuric acid as substrate and less
than 2 .mu.mol/min/mg with barbituric acid as substrate.
14. A composition for remediation of a liquid comprising the CAH of
claim 1 and polyethylene glycol (PEG) and/or KCl.
15. The composition of claim 14, wherein the PEG is PEG4000.
16. The composition of claim 14, wherein the PEG is present at a
concentration of 5-50% by weight.
17. The composition of claim 14, wherein KCl is present at a
concentration of 50-500 mM.
18. The composition of claim 14, wherein the liquid is water.
19. A device for remediation of a liquid comprising a matrix and
the stable cyanuric acid hydrolase of claim 1.
20. The device of claim 19, wherein the device further comprises a
casing or housing for the matrix, wherein water flows through the
at least one casing and contacts the enzyme.
21. The device of claim 19, further comprising a permeable layer.
Description
REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority under 35 U.S.C. 119(e) from
U.S. application Ser. No. 12/879,903, filed Sep. 10, 2010, which
claims priority under 35 U.S.C. 119(e) from U.S. Provisional
Application Ser. No. 61/241,797 filed Sep. 11, 2009, which is
incorporated herein by reference.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which
has been submitted in ASCII format via EFS-Web and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on Jan. 19, 2011, is named 09531291.txt and is 13,619 bytes in
size.
BACKGROUND OF THE INVENTION
[0003] s-Triazine compounds have diverse applications as
herbicides, resins, and disinfectants. The s-triazine herbicides,
such as atrazine, help promote high-yields and sustainability in
agricultural crops.
[0004] Melamine, or triamino-s-triazine, is a high volume
industrial chemical. Melamine-based polymers have outstanding
thermosetting properties, are ideal for their use in kitchen
utensils and plates, and as high-pressure laminates such as
Formica, and as whiteboards. Di- and tri-chloroinated isocyanuric
acids find widespread application as disinfectants, algicides, and
bactericides. The chlorinated isocyanuric acids are used in water
treatment, in the textile industry as bleaching compounds, and in
preventing and curing diseases in husbandry and fisheries. A major
use of these compounds is for swimming pool chlorination. They have
outstanding performance for maintaining an elevated, stable,
chlorine content by dissolving slowly in water, allowing a
continuous metered dosing of chlorine.
[0005] Degradation of these and other s-triazine compounds results
in the production of cyanuric acid (FIG. 1). Cyanuric acid has come
under increased scrutiny because of its potential involvement in
co-mediating toxicity resulting from the ingestion of melamine
(Puschner, B., et al., 2007. J Vet Diagn Invest 19:616-24).
Recently, melamine has been found in adulterated pet food and baby
formula. Melamine and its metabolite cyanuric acid co-crystallize
at low concentrations and are implicated in acute renal failure in
cats that have consumed adulterated food products (Puschner, B., et
al., 2007. J Vet Diagn Invest 19:616-24). Cyanuric acid degradation
is also of interest from the perspective of environmental
remediation. The use of di- or tri-chloroisocyanuric acid in pool
water results in spontaneous chemical dechlorination that
disinfects the water, but also produces as a by-product large
amounts of cyanuric acid. High levels of cyanuric acid perturb the
equilibrium of dissolution of chloro-cyanuric acids preventing
dechlorination by additional chlorinated isocyanuric acid. If this
happens, disinfection is not achieved. As a result, swimming pools
must be emptied and refilled, using water and causing discharge
issues. It would be desirable to remediate pool water in situ,
maintaining disinfection ability, conserving water, saving money
and extending pool water use.
SUMMARY OF THE INVENTION
[0006] The present invention provides an isolated or purified
structurally stable cyanuric acid hydrolase (CAH) enzyme. In
certain embodiments, the CAH has a K.sub.m value for cyanuric acid
of 25-150 .mu.M. In certain embodiments, the CAH has a K.sub.m
value for cyanuric acid of 100-130 .mu.M. In certain embodiments,
the CAH has a k.sub.cat value for cyanuric acid of 4.8-76 s.sup.-1.
In certain embodiments, the enzyme is thermostable. In certain
embodiments, the CAH is thermostable such that the enzyme retains
at least 30% (e.g., at least 40%, at least 50%, at least 95%, or
any other value between 30% and 100%) enzymatic activity at a
temperature above 25.degree. C., and has activity up to 70.degree.
C. In certain embodiments, the enzyme is stable when stored at
between room temperature and -80.degree. C. (e.g., between
20.degree. C. and -80.degree. C., or any other value in between)
for at least 8 weeks. In certain embodiments, the enzyme is stable
for at least 1 year. In certain embodiments, the enzyme retains
enzymatic activity at a pH of from 5.5 to 10.5. In certain
embodiments, the enzyme is from Moorella thermoacetica, such as
from Moorella thermoacetica ATCC 39073.
[0007] In certain embodiments, the CAH enzyme contains at least 19
Lys residues. In certain embodiments, the CAH enzyme contains at
least 40 Arg and/or Lys residues. In certain embodiments, the CAH
enzyme contains at least 50 Asp and/or Glu residues.
[0008] In certain embodiments, the CAH enzyme comprises amino acid
sequence TEGNG(C/G/L)(V/M/A)ND(Y/F)(T/S)R (SEQ ID NO:15), such as
sequence TEGNGCVNDFTR (SEQ ID NO:16). In certain embodiments, the
CAH enzyme comprises amino acid sequence (M/F/L/I)(V/I/M)(M/F/W)SGG
(D/E/G/P)G(V/I/L/G/A)(L/I/M/A)(S/T/A/C)PHX(T/I/L/S)(V/I/L)(F/UV)
(SEQ ID NO:17), wherein X is any amino acid, such as sequence
FIMSGGEGVMTPHTVF (SEQ ID NO:18). In certain embodiments, the enzyme
comprises amino acid sequence TEGNG(C/G/L)(V/M/A)ND(Y/F)(T/S)R (SEQ
ID NO:15) and amino acid sequence (M/F/L/I)(V/I/M)(M/F/W)SGG
(D/E/G/P)G(V/I/L/G/A)(L/I/M/A)(S/T/A/C)PHX(T/I/L/S)(V/I/L)(F/I/V)
(SEQ ID NO:17), wherein X is any amino acid. In certain
embodiments, the enzyme comprises amino acid sequence TEGNGCVNDFTR
(SEQ ID NO:16) and amino acid sequence FIMSGGEGVMTPHTVF (SEQ ID
NO:18).
[0009] In certain embodiments, the enzyme is 350-380 amino acids in
length. In certain embodiments, the enzyme has a pI of about
5-6.
[0010] In certain embodiments, the CAH is SEQ ID NO: 3:
TABLE-US-00001 MQKVEVFRIPTASPDDISGLATLIDSGKINPAEIVAILGKTEGNGCVND
FTRGFATQSLAMYLAEKLGISREEVVKKVAFIMSGGTEGVMTPHITVFV
RKDVQEPAKPGKRLAVGVAFTRDFLPEELGRMEQVNEVARAVKEAMKDA
QIDDPRDVHFVQIKCPLLTAERIEDAKRRGKDVVVNDTYKSMAYSRGAS
ALGVALALGEISADKISNEAICHDWNLYSSVASTSAGVELLNDEIIVVG
NSTNSASDLVIGHSVMKDAIDADAVRAALKDAGLKFDCCPPAEELAKIV
NVLAKAEAASSGTVRGRRNTMLDDSDINHTRSARAVVNAVIASVVGDPM
VYVSGGAEHQGPDGGGPIAVIARV.
[0011] In certain embodiments, the CAH has an SDS-PAGE protein band
corresponding to about 40 kDa. In certain embodiments, the CAH has
a specific activity of 12-18 .mu.mmol/min/mg with cyanuric acid as
substrate and a specific activity of less than 2 mmol/min/mg with
barbituric acid as substrate. In certain embodiments, the CAH has a
specific activity of 15.7 .mu.mol/min/mg with cyanuric acid as
substrate, but does not show detectable activity with barbituric
acid. In certain embodiments, the CAH enzyme shows less that 10%
detectable activity with barbituric acid as compared to the
enzyme's activity with cyanuric acid.
[0012] In certain embodiments, the CAH enzyme contains at least 19
Lys residues. In certain embodiments, the CAH enzyme contains at
least 40 Arg and/or Lys residues. In certain embodiments, the CAH
enzyme contains at least 50 Asp and/or Glu residues.
[0013] The present invention provides an isolated or purified
nucleic acid molecule comprising SEQ ID NO: 4:
TABLE-US-00002 CTACACCCTGGCAATAACAGCAATTGGGCCACCGCCATCAGGCCCTTGA
TGCTCTGCACCACCGGAAACGTAGACCATAGGATCTCCTACCACGCTGG
CAATAACAGCATTTACTACTGCTCGCGCCGAGCGGGTATGATTGATATC
AGAGTCATCAAGCATCGTGTTACGCCTACCCCTTACTGTACCAGAAGAT
GCGGCCTCAGCCTTGGCCAGTACATTAACGATCTTAGCAAGCTCTTCTG
CTGGCGGGCAACAATCAAATTTTAAACCGGCATCTTTAAGGGCAGCACG
TACTGCATCAGCGTCAATGGCATCCTTCATAACAGAGTGGCCTATAACC
AAATCACTGGCACTATTGGTAGAGTTTCCTACTACGATAATTTCGTCAT
TAAGAAGTTCAACCCCCGCTGACGTCGAAGCCACACTAGAGTAGAGATT
CCAGTCATGACAAATTGCTTCGTTGCTAATCTTATCCGCAGATATCTCG
CCCAGTGCGAGGGCCACTCCGAGAGCTGAGGCGCCACGTGAGTAAGCCA
TTGATTTATAAGTGTCATTTACCACAACATCTTTCCCGCGTCGCTTGGC
ATCCTCAATTCTTTCAGCAGTCAAAAGCGGGCACTTTATCTGAACAAAG
TGAACGTCGCGGGGATCATCTATTTGGGCGTCTTTCATAGCCTCTTTTA
CAGCTCGAGCCACTTCGTTTACCTGTTCCATCCGGCCCAATTCTTCCGG
CAGAAAGTCCCGCGTAAAAGCTACGCCTACTGCCAAGCGCTTTCCTGGC
TTAGCTGGTTCCTGGACATCTTTTCGGACAAAGACAGTAATGTGCGGCG
TCATAACACCCTCAGTACCGCCTGACATTATAAACGCAACTTTTTTTAC
AACTTCTTCGCGGCTTATTCCCAATTTTTCTGCTAGATACATTGCTAGA
GATTGGGTAGCAAAACCGCGAGTAAAATCGTTAACACAACCATTACCTT
CCGTCTTGCCCAGAATAGCTACAATTTCAGCCGGATTAATCTTCCCTGA
GTCAATCAAAGTAGCCAACCCGCTGATATCATCAGGTGAGGCTGTTGGG
ATACGAAAGACTTCAACTTTTTGCAT.
[0014] The present invention provides a composition for remediation
of a liquid comprising the CAH as described above, along with
polyethylene glycol (PEG) and/or KCl. In certain embodiments, the
PEG is PEG4000. In certain embodiments, the PEG is present at a
concentration of 50-500 mM. In certain embodiments, the composition
comprises 50-500 mM KCl. In certain embodiments, the liquid is
water.
[0015] The present invention provides a device for remediation of a
liquid comprising a matrix and one or more structurally stable
cyanuric acid hydrolases or a composition for remediation of a
liquid as described above. In certain embodiments, the liquid is
water. In certain embodiments, the device further comprises a
casing or housing for the matrix. In certain embodiments, the
matrix is water-insoluble. In certain embodiments, the
water-insoluble matrix is granular and/or porous. In certain
embodiments, the water-insoluble matrix is an organic matrix or an
inorganic matrix. In certain embodiments, the matrix is an organic
matrix and the organic matrix is plastic, nylon, activated carbon,
cellulose, agarose, chitin, chitosan, collagen and/or polystyrene.
In certain embodiments, the matrix is an inorganic matrix and the
inorganic matrix is glass, zeolite, silica, alumina, titania,
zirconia, calcium alginate and/or celite. In certain embodiments,
the device further comprises a permeable layer. In certain
embodiments, the enzyme is imbedded in or on the permeable layer.
In certain embodiments, the device further comprises at least one
casing, wherein water flowing through the at least one casing
contacts the enzyme.
[0016] The present invention provides methods of remediating a
liquid comprising contacting the liquid from a circulating
reservoir with the enzyme, composition, or the device described
herein above to reduce the concentration of cyanuric acid in the
liquid. In certain embodiments, the enzyme is present in pellet
form. In certain embodiments, the liquid is contacted with the
device described hereinabove by passing the water over or through
the device. In certain embodiments, the circulating liquid
reservoir is a pool, a swimming pool, a spa, a hot tub, a whirlpool
bath, a fountain or a waterslide. In certain embodiments, the
concentration of cyanuric acid in the liquid subsequent to
treatment is less than 100 ppm. In certain embodiments, the
concentration of cyanuric acid in the liquid subsequent to
treatment ranges from 70 ppm to 30 ppm. In certain embodiments, the
enzyme is present at a concentration of at least 2.5 mg per one
cubic meter of the liquid. In certain embodiments, the liquid flows
through the device. In certain embodiments, the liquid treatment
comprises reducing a concentration of cyanuric acid in the liquid.
In certain embodiments, the liquid treatment is effected during a
time period of 20 hours or less. In certain embodiments, the
passing of the liquid through the device is effected at a flow rate
of at least 10 cubic meters per hour. In certain embodiments, the
liquid is water.
BRIEF DESCRIPTION OF DRAWINGS
[0017] FIG. 1. Atrazine, ametryn, trichloroisocyanuric acid and
melamine are all metabolized via cyanuric acid that is transformed
to biuret by the action of cyanuric acid hydrolases.
[0018] FIG. 2. Tree showing relatedness of cyanuric acid hydrolases
known or identified in different bacteria. The degree of
relatedness shown is based on amino acid sequence identity.
[0019] FIG. 3. Substrate specificity of the cyanuric acid hydrolase
from M. thermoacetica ATCC 39073. (A) Compounds that are
substrates. (B) Compounds that are not substrates.
[0020] FIG. 4. Stability of enzyme activity vs. temperature at pH
8.0 for purified cyanuric acid hydrolases from M. thermoacetica
ATCC 39073 ( ) compared with TrzD (.box-solid.) and AtzD
(.tangle-solidup.). Enzymes were maintained at the temperature
indicated for 30 minutes prior to assay under standard
conditions.
[0021] FIG. 5. Comparative reaction and metabolic pathways for
barbiturase (A) and cyanuric acid hydrolase (B) (Soong, C. L., J.
Ogawa, E. Sakuradani, and S. Shimizu. 2002. Barbiturase, a novel
zinc-containing amidohydrolase involved in oxidative pyrimidine
metabolism. J. Biol. Chem. 277:7051-7058).
DETAILED DESCRIPTION OF THE INVENTION
[0022] Hypochlorous acid (HOCl) is a common source of free
chlorine. Chemical compounds that release free chlorine are the
most commonly used sanitizers in swimming pools. Hypochlorous acid,
however, is highly unstable, and readily decomposes into inactive
breakdown products, such as hydrochloric acid, water and oxygen,
via UV radiation-driven photochemical reactions upon exposure to
direct sun light, and/or upon exposure to moderate and high
temperatures. On a typical summer day, up to 90% of the total
active chlorine species are lost within two to three hours. In
order to control these effects and preserve the effectiveness of
the chlorine, the chlorine-stabilizing agent cyanuric acid (also
called s-triazinetrione or isocyanuric acid) is often added to the
water. Cyanuric acid, as well as cyanurate salts and various
derivatives thereof are compounds which protect the chlorine from
the negative effects of UV and heat, and therefore practically
reduce the amount of chlorine which needs to be added to the water
in order to maintain safe conditions of disinfection. The
protection action of these compounds is achieved by the ability of
free chlorine (Cl.sup.+) to reversibly bind to the nitrogen atoms
in the cyanuric acid ring. With a correct dosing, cyanuric acid can
reduce the chlorine consumption. However, incorrect balance of
cyanuric acid can create an over-protective effect and hence
substantially decrease the effectiveness of chlorine as a
disinfectant.
[0023] Excessive amounts of cyanuric acid drive the equilibrium
towards the uptake of free chlorine. Hence, excessive amounts of
cyanuric acid cause the chlorine to become progressively
over-stabilized, reducing the availability of free chlorine and
interfering with its disinfection function. The phenomenon, known
as "chlorine-lock", takes place when the concentration of cyanuric
acid reaches over 100 ppm (0.77 mM). Chlorine-lock expresses itself
similarly to inadequately low chlorine level, in clouding of the
pool's water which, apart from an aesthetic nuisance, is a clear
indication that the water is no longer safe for use. Once added to
the pool, cyanuric acid does not dissipate or degrade
substantially. It is removed from the pool only by splash-out and
backwash waste procedures or dilution. Typically, cyanuric acid
level is lowered by draining part of the pool's water and diluting
the remaining water with fresh water. If the cyanuric acid level
exceeds 100 ppm considerably, the pool should be partially or
totally drained and have its inner-walls scrubbed (cyanuric acid
will sediment on the sides of the pool). This time-consuming and
water-wasteful process is extremely costly not only in terms of
water but also in loss of the pool's operational time, additional
stabilized chlorine added, and the so far unavoidable reiterative
nature of the overall process needed to maintain the balance
between the concentration of reactive chlorine species and the
concentration of cyanuric acid.
[0024] The present invention provides structurally stable cyanuric
acid hydrolase enzymes, compositions and devices for removing
excess cyanuric acid, e.g., from pool water, without the need to
drain the water and/or diluting the remaining water.
[0025] Structurally Stable Cyanuric Acid Hydrolase Enzymes
[0026] A cyanuric acid hydrolase is an enzyme that specifically
hydrolyzes cyanuric acid. As used herein "specifically hydrolyzes"
means that less than 1% of the enzyme's activity is on other
substrates besides cyanuric acid. As used herein, a "cyanuric acid
hydrolase" is an enzyme that hydrolytically catalyzes the
ring-opening reaction that converts cyanuric acid to biuret.
Cyanuric acid hydrolase enzymes are well known in the art and have
been isolated from various sources, some of which were
characterized by their amino acid sequence, K.sub.M (Michaelis
constant), Vmax, inhibitors thereof, and other biochemical
parameters. The Michaelis constant represents the dissociation
constant (affinity for substrate) of the enzyme-substrate complex.
Low values indicate that this complex is held together very tightly
and rarely dissociates without the substrate first reacting to form
the product. In order that an enzyme would be used effectively for
treating liquids (such as water) in large volumes and rate, the
enzyme needs to be an efficient catalyst; hence the biometric
parameters of cyanuric acid hydrolase are significant in the
context of the present invention. The catalysis parameters of
cyanuric acid hydrolase on cyanuric acid, namely K.sub.M values of
25-125 .mu.M as presented herein, signify that these enzymes can be
used effectively to reduce the concentration of cyanuric acid in
the liquid, such as water, so as to achieve a concentration lower
than the chlorine-lock concentration of 100 ppm (corresponding to
0.77 mM). Even at the highest allowable concentration of cyanuric
acid in such water, 0.62 mM, the enzyme is highly effective and can
produce the desired hydrolysis. The cyanuric acid hydrolase of the
present invention has k.sub.cat values for cyanuric acid of 4.8-76
s.sup.-1.
[0027] The present inventors discovered that the number of Lys
residues is considerably higher in Moorella as compared to other
enzymes with similar structure and function. AtzD had 7, TrzD had
12, but Moorella CAH had 21. Many of these are conservative
changes, replacing the lower numbers of Arg's, but some are not.
The inventors also looked at acidic and basic residues as a
potential metric for salt bridges. [0028] Arg+Lys=32 AtzD/36TrzD/41
Moorella (due mainly to the Lys as mentioned above) [0029]
Asp+Glu=43 atzd/50 trzd/50 Moorella
[0030] The global amino acid composition of these various proteins
was also examined. The CAH from Bradyrhizobium USDA 110 was also
compared as a control for random deviations. The relative stability
for these proteins is as follows: [0031] Bradyrhizobium CAH (loses
activity in weeks)<AtzD (stable for months at 4.degree. C., but
loses activity by 9 months)<TrzD (kept activity for 9 mo., but
cannot freeze without loss of activity)<Moorella
[0032] The following values are expressed in % amino acids instead
of absolute numbers of particular amino acids, as given above:
[0033] R+K=8.7 brady/8.8 atzd/9.7 trzd/11.1 Moorella [0034]
E+D=10.6 brady/11.9atzd/13.5trzd/13.7 Moorella
[0035] Sources of cyanuric acid hydrolases include man-made
biological sources such as native and/or genetically modified
microorganisms, plants and animals, which produce or over-produce
the enzyme. The present inventors discovered a class of cyanuric
acid hydrolases that are unusually stable at various temperatures.
These enzymes are "structurally stable" in that they retain their
catalytic activity at a broad temperature range, and can be stored
for long periods of time under a wide range of conditions with
minimal decrease in enzymatic activity. In certain embodiments, the
cyanuric acid hydrolase retains at least 30% enzymatic activity at
a temperature above 25.degree. C. In certain embodiments, the
cyanuric acid hydrolase is a thermostable enzyme. In general,
thermostable enzymes also have a greater overall stability under a
variety of conditions, such as to immobilization, to salt, to
solvents, to low osmotic strength, etc. Thermostable enzymes hold
their structural elements together more tightly, preventing the
protein from irreversibly denaturing.
[0036] Many s-triazine compounds degrade to the metabolic
intermediate cyanuric acid. Cyanuric acid can be further
metabolized to biuret by cyanuric acid hydrolase. Cyanuric acid
accumulates in swimming pools due the breakdown of the sanitizing
agents di- and tri-chloroisocyanuric acid. The present inventors
have discovered structurally stable cyanuric acid hydrolases that
can be used in pool water remediation. In one embodiment, cyanuric
acid hydrolase from the thermophile Moorella thermoacetica ATCC
39073 was cloned, expressed in Escherichia coli, and purified to
homogeneity. The recombinant enzyme was found to have a broader
temperature range and greater stability at both elevated and low
temperatures, in comparison to previously described cyanuric acid
hydrolases. The enzyme had a narrow substrate specificity acting
only on cyanuric acid and N-methylisocyanuric acid. The M.
thermoacetica enzyme did not require metals or other discernible
cofactors for activity.
[0037] It was unexpected that the M. thermoacetica cyanuric acid
amidohydrolase ATCC 39073 had enzymatic activity as a cyanuric acid
hydrolase. In earlier studies, the M. thermoacetica cyanuric acid
amidohydrolase from ATCC 39073 was indicated instead as a
barbiturase. Support for it being a barbiturase was based on the
fact that it has 48 percent amino acid-identity to barbiturase (48%
ID). For example, a barbiturase from Streptosporangium roseum DSM
43021 is 43 and 46% identical to AtzD and TrzD, even though AtzD
and TrzD are both cyanuric acid hydrolases. Thus, an enzyme's
percent-identity with other known enzymes of a certain specificity
does not guarantee that the enzyme of interest will also have the
same substrate specificity (i.e., it is isofunctional). Merely
observing the sequence of the putative gene in its genome context
is not indicative of its function.
[0038] Cyanuric acid amidohydrolases share the following sequence
consensus strings (where only one of the amino acids in parentheses
is present in that position):
TABLE-US-00003 SEQ ID Sequence NO (G/A)KTEGNG(C/G)VND(Y/F)(T/S)R 5
(V/I)MSGGTEG(V/A/G)(L/M)(S/A/T)PH 6 E(E/H/D/A)XG(R/T) 7
(D/Q)(L/V/A)H(F/Y/L)VQ(V/I)KCPLLT 8
SM(G/A/S)(Y/F/L)(S/N)R(G/A)A(S/A)ALG 9
(S/A/G)(A/T/C)S(S/A/G)G(I/V/G/S)ELX2(N/D/C/H)(V/E)X4G(M/N)(S/A)X2(S/A/W)
10 HX(V/E)MXD(A/G)(I/M)D 11 KAE 12
(R/D)(G/N)XR(H/N)(T/I)M(L/H/N)(S/T/D/E)D(S/T)D(I/V)(N/S/A)XTR(H/S)AR(A/G)
13 (V/I/L)(F/Y)VSGG(S/A/G)EHQGP(A/D/P)GGGP 14
[0039] "Naturally occurring" is used to describe a substance or
molecule that can be found in nature as distinct from being
artificially produced. For example, a protein sequence present in
an organism (including a virus), which can be isolated from a
source in nature and which has not been intentionally modified in
the laboratory, is naturally occurring.
[0040] A "variant" of an enzyme is a sequence that is substantially
similar to the sequence of the native enzyme. "Wild-type" or
"naturally occurring" or "native" refers to the normal gene, or
organism found in nature without any known mutation. Variant amino
acid sequences include synthetically derived amino acid sequences,
or recombinantly derived amino acid sequences. Generally, amino
acid sequence variants of the invention will have at least 40, 50,
60, to 70%, e.g., 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, to 79%,
generally at least 80%, e.g., 81%-84%, at least 85%, e.g., 86%,
87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, to 98%,
sequence identity to the native (endogenous) amino acid
sequence.
[0041] The present invention includes variants of
naturally-occurring cyanuric acid hydrolases. By "variant" an
enzyme is intended as an enzyme derived from the native enzyme by
deletion (so-called truncation) or addition of one or more amino
acids to the N-terminal and/or C-terminal end of the native enzyme;
deletion or addition of one or more amino acids at one or more
sites in the native enzyme; or substitution of one or more amino
acids at one or more sites in the native enzyme. The enzyme of the
invention may be altered in various ways including amino acid
substitutions, deletions, truncations, and insertions. Methods for
such manipulations are generally known in the art. For example,
amino acid sequence variants of the enzyme can be prepared by
mutations in the DNA. Methods for mutagenesis and nucleotide
sequence alterations are well known in the art. The substitution
may be a conserved substitution. A "conserved substitution" is a
substitution of an amino acid with another amino acid having a
similar side chain. A conserved substitution would be a
substitution with an amino acid that makes the smallest change
possible in the charge of the amino acid or size of the side chain
of the amino acid (alternatively, in the size, charge or kind of
chemical group within the side chain) such that the overall enzyme
retains its spatial conformation but has altered biological
activity. For example, common conserved changes might be Asp to
Glu, Asn or Gln; His to Lys, Arg or Phe; Asn to Gln, Asp or Glu and
Ser to Cys, Thr or Gly. Alanine is commonly used to substitute for
other amino acids. The 20 essential amino acids can be grouped as
follows: alanine, valine, leucine, isoleucine, proline,
phenylalanine, tryptophan and methionine having nonpolar side
chains; glycine, serine, threonine, cystine, tyrosine, asparagine
and glutamine having uncharged polar side chains; aspartate and
glutamate having acidic side chains; and lysine, arginine, and
histidine having basic side chains.
[0042] As used herein, "sequence identity" or "identity" in the
context of two nucleic acid or polypeptide sequences makes
reference to a specified percentage of residues in the two
sequences that are the same when aligned for maximum correspondence
over a specified comparison window, as measured by sequence
comparison algorithms or by visual inspection. When percentage of
sequence identity is used in reference to proteins it is recognized
that residue positions which are not identical often differ by
conservative amino acid substitutions, where amino acid residues
are substituted for other amino acid residues with similar chemical
properties (e.g., charge or hydrophobicity) and therefore do not
change the functional properties of the molecule. When sequences
differ in conservative substitutions, the percent sequence identity
may be adjusted upwards to correct for the conservative nature of
the substitution. Sequences that differ by such conservative
substitutions are said to have "sequence similarity" or
"similarity." Means for making this adjustment are well known to
those of skill in the art. Typically this involves scoring a
conservative substitution as a partial rather than a full mismatch,
thereby increasing the percentage sequence identity. Thus, for
example, where an identical amino acid is given a score of 1 and a
non-conservative substitution is given a score of zero, a
conservative substitution is given a score between zero and 1. The
scoring of conservative substitutions is calculated, e.g., as
implemented in the program PC/GENE (Intelligenetics, Mountain View,
Calif.).
[0043] As used herein, "comparison window" makes reference to a
contiguous and specified segment of an amino acid or polynucleotide
sequence, wherein the sequence in the comparison window may
comprise additions or deletions (i.e., gaps) compared to the
reference sequence (which does not comprise additions or deletions)
for optimal alignment of the two sequences. Generally, the
comparison window is at least 20 contiguous amino acid residues or
nucleotides in length, and optionally can be 30, 40, 50, 100, or
longer.
[0044] As used herein, "percentage of sequence identity" means the
value determined by comparing two optimally aligned sequences over
a comparison window, wherein the portion of the polypeptide or
polynucleotide sequence in the comparison window may comprise
additions or deletions (i.e., gaps) as compared to the reference
sequence (which does not comprise additions or deletions) for
optimal alignment of the two sequences. The percentage is
calculated by determining the number of positions at which the
identical nucleic acid base or amino acid residue occurs in both
sequences to yield the number of matched positions, dividing the
number of matched positions by the total number of positions in the
window of comparison, and multiplying the result by 100 to yield
the percentage of sequence identity.
[0045] The term "substantial identity" of polynucleotide sequences
means that a polynucleotide comprises a sequence that has at least
70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, or 79%, at least 80%,
81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, at least 90%, 91%,
92%, 93%, or 94%, and at least 95%, 96%, 97%, 98%, or 99% sequence
identity, compared to a reference sequence using one of the
alignment programs described using standard parameters. One of
skill in the art will recognize that these values can be
appropriately adjusted to determine corresponding identity of
proteins encoded by two nucleotide sequences by taking into account
codon degeneracy, amino acid similarity, reading frame positioning,
and the like. Substantial identity of amino acid sequences for
these purposes normally means sequence identity of at least 70%, at
least 80%, 90%, or at least 95%.
[0046] The term "substantial identity" in the context of a peptide
indicates that a peptide comprises a sequence with at least 70%,
71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, or 79%, 80%, 81%, 82%, 83%,
84%, 85%, 86%, 87%, 88%, or 89%, at least 90%, 91%, 92%, 93%, or
94%, or 95%, 96%, 97%, 98% or 99%, sequence identity to the
reference sequence over a specified comparison window. An
indication that two peptide sequences are substantially identical
is that one peptide is immunologically reactive with antibodies
raised against the second peptide. Thus, a peptide is substantially
identical to a second peptide, for example, where the two peptides
differ only by a conservative substitution.
[0047] For sequence comparison, typically one sequence acts as a
reference sequence to which test sequences are compared. When using
a sequence comparison algorithm, test and reference sequences are
input into a computer, subsequence coordinates are designated if
necessary, and sequence algorithm program parameters are
designated. The sequence comparison algorithm then calculates the
percent sequence identity for the test sequence(s) relative to the
reference sequence, based on the designated program parameters.
[0048] The term "amino acid" includes the residues of the natural
amino acids (e.g., Ala, Arg, Asn, Asp, Cys, Glu, Gln, Gly, His,
Hyl, Hyp, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and
Val) in D or L form, as well as unnatural amino acids (e.g.,
phosphoserine, phosphothreonine, phosphotyrosine, hydroxyproline,
gamma-carboxyglutamate; hippuric acid, octahydroindole-2-carboxylic
acid, statine, 1,2,3,4,-tetrahydroisoquinoline-3-carboxylic acid,
penicillamine, ornithine, citruline, .alpha.-methyl-alanine,
para-benzoylphenylalanine, phenylglycine, propargylglycine,
sarcosine, and tert-butylglycine). The term also comprises natural
and unnatural amino acids bearing a conventional amino protecting
group (e.g., acetyl or benzyloxycarbonyl), as well as natural and
unnatural amino acids protected at the carboxy terminus (e.g., as a
(C.sub.1-C.sub.6)alkyl, phenyl or benzyl ester or amide; or as an
.alpha.-methylbenzyl amide). Other suitable amino and carboxy
protecting groups are known to those skilled in the art (See for
example, T. W. Greene, Protecting Groups In Organic Synthesis;
Wiley: New York, 1981, and references cited therein).
[0049] The invention encompasses isolated or substantially purified
protein compositions. In the context of the present invention, an
"isolated" or "purified" polypeptide is a polypeptide that exists
apart from its native environment and is therefore not a product of
nature. The terms "polypeptide" and "protein" are used
interchangeably herein. An isolated protein molecule may exist in a
purified form or may exist in a non-native environment such as, for
example, a transgenic host cell or bacteriophage. For example, an
"isolated" or "purified" protein, or biologically active portion
thereof, is substantially free of other cellular material, or
culture medium when produced by recombinant techniques, or
substantially free of chemical precursors or other chemicals when
chemically synthesized. A protein that is substantially free of
cellular material includes preparations of protein or polypeptide
having less than about 30%, 20%, 10%, 5%, (by dry weight) of
contaminating protein. When the protein of the invention, or
biologically active portion thereof, is recombinantly produced,
preferably culture medium represents less than about 30%, 20%, 10%,
or 5% (by dry weight) of chemical precursors or
non-protein-of-interest chemicals. Fragments and variants of the
disclosed proteins or partial-length proteins encoded thereby are
also encompassed by the present invention. By "fragment" or
"portion" is meant a full length or less than full length of the
amino acid sequence of a protein.
[0050] Thus, the genes and nucleotide sequences of the invention
include both the naturally occurring sequences as well as mutant
forms. Likewise, the polypeptides of the invention encompass
naturally occurring proteins as well as variations and modified
forms thereof. Such variants will continue to possess the desired
activity. The deletions, insertions, and substitutions of the
polypeptide sequence encompassed herein are not expected to produce
radical changes in the characteristics of the polypeptide. However,
when it is difficult to predict the exact effect of the
substitution, deletion, or insertion in advance of doing so, one
skilled in the art will appreciate that the effect will be
evaluated by routine screening assays.
[0051] Individual substitutions deletions or additions that alter,
add or delete a single amino acid or a small percentage of amino
acids (typically less than 5%, more typically less than 1%) in an
encoded sequence are "conservatively modified variations," where
the alterations result in the substitution of an amino acid with a
chemically similar amino acid. Conservative substitution tables
providing functionally similar amino acids are well known in the
art. The following five groups each contain amino acids that are
conservative substitutions for one another: Aliphatic: Glycine (G),
Alanine (A), Valine (V), Leucine (L), Isoleucine (I); Aromatic:
Phenylalanine (F), Tyrosine (Y), Tryptophan (W); Sulfur-containing:
Methionine (M), Cysteine (C); Basic: Arginine (R), Lysine (K),
Histidine (H); Acidic: Aspartic acid (D), Glutamic acid (E),
Asparagine (N), Glutamine (Q). In addition, individual
substitutions, deletions or additions which alter, add or delete a
single amino acid or a small percentage of amino acids in an
encoded sequence are also "conservatively modified variations."
[0052] By "portion" or "fragment," as it relates to a nucleic acid
molecule, sequence or segment of the invention, when it is linked
to other sequences for expression, is meant a sequence having at
least 80 nucleotides, more preferably at least 150 nucleotides, and
still more preferably at least 400 nucleotides. If not employed for
expressing, a "portion" or "fragment" means at least 9, preferably
12, more preferably 15, even more preferably at least 20,
consecutive nucleotides, e.g., probes and primers
(oligonucleotides), corresponding to the nucleotide sequence of the
nucleic acid molecules of the invention.
[0053] Nucleic Acids Encoding Structurally Stable Cyanuric Acid
Hydrolases
[0054] The present invention includes isolated nucleic acids and
vectors that encode the structurally stable cyanuric acid
hydrolases described above.
[0055] The term "nucleic acid" refers to deoxyribonucleotides or
ribonucleotides and polymers thereof in either single- or
double-stranded form, composed of monomers (nucleotides) containing
a sugar, phosphate and a base which is either a purine or
pyrimidine. Unless specifically limited, the term encompasses
nucleic acids containing known analogs of natural nucleotides that
have similar binding properties as the reference nucleic acid and
are metabolized in a manner similar to naturally occurring
nucleotides. Unless otherwise indicated, a particular nucleic acid
sequence also implicitly encompasses conservatively modified
variants thereof (e.g., degenerate codon substitutions) and
complementary sequences as well as the sequence explicitly
indicated. Specifically, degenerate codon substitutions may be
achieved by generating sequences in which the third position of one
or more selected (or all) codons is substituted with mixed-base
and/or deoxyinosine residues. A "nucleic acid fragment" is a
fraction of a given nucleic acid molecule. Deoxyribonucleic acid
(DNA) in the majority of organisms is the genetic material while
ribonucleic acid (RNA) is involved in the transfer of information
contained within DNA into proteins. The term "nucleotide sequence"
refers to a polymer of DNA or RNA that can be single- or
double-stranded, optionally containing synthetic, non-natural or
altered nucleotide bases capable of incorporation into DNA or RNA
polymers. The terms "nucleic acid," "nucleic acid molecule,"
"nucleic acid fragment," "nucleic acid sequence or segment," or
"polynucleotide" may also be used interchangeably with gene, cDNA,
DNA and RNA encoded by a gene.
[0056] The invention encompasses isolated or substantially purified
nucleic acid compositions. In the context of the present invention,
an "isolated" or "purified" DNA molecule or an "isolated" or
"purified" polypeptide is a DNA molecule that exists apart from its
native environment and is therefore not a product of nature. An
isolated DNA molecule may exist in a purified form or may exist in
a non-native environment such as, for example, a transgenic host
cell or bacteriophage. For example, an "isolated" or "purified"
nucleic acid molecule, or biologically active portion thereof, is
substantially free of other cellular material, or culture medium
when produced by recombinant techniques, or substantially free of
chemical precursors or other chemicals when chemically synthesized.
In one embodiment, an "isolated" nucleic acid is free of sequences
that naturally flank the nucleic acid (i.e., sequences located at
the 5' and 3' ends of the nucleic acid) in the genomic DNA of the
organism from which the nucleic acid is derived. For example, in
various embodiments, the isolated nucleic acid molecule can contain
less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of
nucleotide sequences that naturally flank the nucleic acid molecule
in genomic DNA of the cell from which the nucleic acid is derived.
Fragments and variants of the disclosed nucleotide sequences
encoded thereby are also encompassed by the present invention. By
"fragment" or "portion" is meant a full length or less than full
length of the nucleotide sequence encoding the amino acid sequence
of a protein.
[0057] The term "gene" is used broadly to refer to any segment of
nucleic acid associated with a biological function. Thus, genes
include coding sequences and/or the regulatory sequences required
for their expression. For example, gene refers to a nucleic acid
fragment that expresses mRNA, functional RNA, or specific protein,
including regulatory sequences. Genes also include nonexpressed DNA
segments that, for example, form recognition sequences for other
proteins. Genes can be obtained from a variety of sources,
including cloning from a source of interest or synthesizing from
known or predicted sequence information, and may include sequences
designed to have desired parameters.
[0058] A "variant" of a molecule is a sequence that is
substantially similar to the sequence of the native molecule. For
nucleotide sequences, variants include those sequences that,
because of the degeneracy of the genetic code, encode the identical
amino acid sequence of the native protein. Naturally occurring
allelic variants such as these can be identified with the use of
well-known molecular biology techniques, as, for example, with
polymerase chain reaction (PCR) and hybridization techniques.
Variant nucleotide sequences also include synthetically derived
nucleotide sequences, such as those generated, for example, by
using site-directed mutagenesis that encode the native protein, as
well as those that encode a polypeptide having amino acid
substitutions. Generally, nucleotide sequence variants of the
invention will have at least 40, 50, 60, to 70%, e.g., preferably
71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, to 79%, generally at least
80%, e.g., 81%-84%, at least 85%, e.g., 86%, 87%, 88%, 89%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, to 98%, sequence identity to the
native (endogenous) nucleotide sequence.
[0059] "Conservatively modified variations" of a particular nucleic
acid sequence refers to those nucleic acid sequences that encode
identical or essentially identical amino acid sequences, or where
the nucleic acid sequence does not encode an amino acid sequence,
to essentially identical sequences. Because of the degeneracy of
the genetic code, a large number of functionally identical nucleic
acids encode any given polypeptide. For instance the codons CGT,
CGC, CGA, CGG, AGA, and AGG all encode the amino acid arginine.
Thus, at every position where an arginine is specified by a codon,
the codon can be altered to any of the corresponding codons
described without altering the encoded protein. Such nucleic acid
variations are "silent variations" which are one species of
"conservatively modified variations." Every nucleic acid sequence
described herein which encodes a polypeptide also describes every
possible silent variation, except where otherwise noted. One of
skill will recognize that each codon in a nucleic acid (except ATG,
which is ordinarily the only codon for methionine) can be modified
to yield a functionally identical molecule by standard techniques.
Accordingly, each "silent variation" of a nucleic acid which
encodes a polypeptide is implicit in each described sequence.
[0060] A "vector" is defined to include, inter alia, any plasmid,
cosmid, phage or binary vector in double or single stranded linear
or circular form which may or may not be self transmissible or
mobilizable, and which can transform prokaryotic or eukaryotic host
either by integration into the cellular genome or exist
extrachromosomally (e.g., autonomous replicating plasmid with an
origin of replication).
[0061] "Cloning vectors" typically contain one or a small number of
restriction endonuclease recognition sites at which foreign DNA
sequences can be inserted in a determinable fashion without loss of
essential biological function of the vector, as well as a marker
gene that is suitable for use in the identification and selection
of cells transformed with the cloning vector. Marker genes
typically include genes that provide tetracycline resistance,
hygromycin resistance or ampicillin resistance.
[0062] "Expression cassette" as used herein means a DNA sequence
capable of directing expression of a particular nucleotide sequence
in an appropriate host cell, comprising a promoter operably linked
to the nucleotide sequence of interest which is operably linked to
termination signals. It also typically comprises sequences required
for proper translation of the nucleotide sequence. The coding
region usually codes for a protein of interest but may also code
for a functional RNA of interest, for example antisense RNA or a
nontranslated RNA, in the sense or antisense direction. The
expression cassette comprising the nucleotide sequence of interest
may be chimeric, meaning that at least one of its components is
heterologous with respect to at least one of its other components.
The expression cassette may also be one that is naturally occurring
but has been obtained in a recombinant form useful for heterologous
expression. The expression of the nucleotide sequence in the
expression cassette may be under the control of a constitutive
promoter or of an inducible promoter that initiates transcription
only when the host cell is exposed to some particular external
stimulus. In the case of a multicellular organism, the promoter can
also be specific to a particular tissue or organ or stage of
development.
[0063] Such expression cassettes will comprise the transcriptional
initiation region of the invention linked to a nucleotide sequence
of interest. Such an expression cassette is provided with a
plurality of restriction sites for insertion of the gene of
interest to be under the transcriptional regulation of the
regulatory regions. The expression cassette may additionally
contain selectable marker genes.
[0064] "Coding sequence" refers to a DNA or RNA sequence that codes
for a specific amino acid sequence and excludes the non-coding
sequences. It may constitute an "uninterrupted coding sequence",
i.e., lacking an intron, such as in a cDNA or it may include one or
more introns bounded by appropriate splice junctions. An "intron"
is a sequence of RNA which is contained in the primary transcript
but which is removed through cleavage and re-ligation of the RNA
within the cell to create the mature mRNA that can be translated
into a protein.
[0065] The terms "open reading frame" and "ORF" refer to the amino
acid sequence encoded between translation initiation and
termination codons of a coding sequence. The terms "initiation
codon" and "termination codon" refer to a unit of three adjacent
nucleotides (`codon`) in a coding sequence that specifies
initiation and chain termination, respectively, of protein
synthesis (mRNA translation).
[0066] "Operably-linked" refers to the association of nucleic acid
sequences on single nucleic acid fragment so that the function of
one is affected by the other. For example, a regulatory DNA
sequence is said to be "operably linked to" or "associated with" a
DNA sequence that codes for an RNA or a polypeptide if the two
sequences are situated such that the regulatory DNA sequence
affects expression of the coding DNA sequence (i.e., that the
coding sequence or functional RNA is under the transcriptional
control of the promoter). Coding sequences can be operably-linked
to regulatory sequences in sense or antisense orientation.
[0067] Compositions for Remediation of Liquid
[0068] Certain embodiments of the present invention provide
compositions for use in the remediation of a liquid, such as water,
for example swimming pool water. The compositions include one or
more structurally stable cyanuric acid hydrolases described above.
In certain embodiments, the composition further comprises
polyethylene glycol and/or KCl. The compositions can be used for
treating a liquid in order to maintain a chemical balance in the
liquid by reducing the level of cyanuric acid in the liquid.
[0069] Devices for Remediation of Liquid
[0070] Certain embodiments of the present invention provide devices
for use in the remediation of a liquid, such as water, for example
swimming pool water. The devices include a matrix and one or more
structurally stable cyanuric acid hydrolases described above, where
the enzymes are incorporated in, into, or on the matrix. In certain
embodiments, the enzymes are incorporated in or on a
water-insoluble matrix, which serves as a solid support for the
enzyme, namely, it provides a stationary object with respect to the
water and the various chemicals dissolved in it. The
water-insoluble matrix allows performing a continuous and/or
repetitive contact of the treated water with the enzyme, as well as
maintaining the enzyme affixed, thus eliminating loss of the enzyme
due to leaching out.
[0071] Many commercially available solid-phase synthesis columns,
purification and ion-exchange columns are packed with granular
and/or porous water-insoluble and water-permeable matrices that are
suitable for protein immobilization applications, or can readily be
modified so as to be suitable for protein immobilization, and
therefore are suitable for use as the water-insoluble matrix
according to the present invention. Such granular and/or porous
water-insoluble matrices are well known in the art and are used in
various applications such as filtration and chromatography.
Representative examples include, without limitation, organic
substances such as nylons, polystyrenes, polyurethanes and other
synthetic polymers and co-polymers, activated carbon, cellulose,
agarose, chitin, chitosan and collagen, and inorganic substances
such as beads, filters, cloth, glass, plastic, zeolite, silica,
alumina, titania, zirconia, calcium alginate and celite.
[0072] Other forms of organic polymers, copolymers and cross-linked
derivatives thereof, and inorganic materials such as diatomaceous
earths and other types of molecular sieves, typically used in
various water filtrations, can be used as a granular and/or porous
water-insoluble matrix, according to the present invention, on or
in which an enzyme can be incorporated.
[0073] The term "incorporated," as used herein, refers to any mode
of contact between the water-insoluble matrix and the enzyme which
achieves immobilization of the enzyme with respect to the matrix,
thus rendering a biochemically active enzyme insoluble, or in other
words immobilized, and in some cases more protected, than the
soluble enzyme.
[0074] Incorporation of an enzyme into or on the matrix can be
effected by attachment via any type of chemical bonding, including
covalent bonds, ionic (electrostatic) bonds, hydrogen bonding,
hydrophobic interactions, metal-mediated complexation,
affinity-pair bonding and the like, and/or by attachment via any
type of physical interaction such as magnetic interaction, surface
adsorption, encapsulation, entrapment, entanglement and the like.
The enzyme(s) can be incorporated in and/or on physical structural
elements of a water-insoluble matrix. In cases where the structural
elements of the matrix are granular but not porous, such as, for
example, in cases where the matrix is made of solid glass beads or
particles, or solid plastic beads or particles, the enzyme(s) is
incorporated on the surface of the beads or particles, and the
water that flows in the channels between the beads or particles
comes in contact with the enzyme(s), thus allowing the
amide-containing compounds dissolved in the water to be
enzymatically degraded.
[0075] In cases where the structural element of the matrix is
porous but not granular, such as, for example, in cases where the
matrix is extruded zeolite blocks, carbonaceous blocks or solid
plastic foam blocks, the enzyme(s) is incorporated in the cavities,
on the inner surface of the innate inter-connected pores and
channels which are characteristic to such matrices, as well as on
the outer surface of the block, and the water that flows in the
inter-connected pores and channels comes in contact with the
enzyme(s). In cases where the structural elements of the matrix are
granular and porous, such as, for example, in cases where the
matrix is zeolite granules or molecular sieves pellets, the
enzyme(s) is incorporated on the surface of the granules or pellets
and in the inner surface of the pores and channels of these
matrices, and the water that flows between the granules or pellets
as well as through them comes in contact with the enzyme(s), thus
allowing the amide-containing compounds dissolved in the water to
be enzymatically degraded.
[0076] In certain embodiments, the incorporation of the enzyme to
the water-insoluble matrix is effected by a combination of chemical
and physical attachments such as covalent bonding and
entanglement.
[0077] In certain embodiments of the present invention, the
incorporation of the enzyme to the water-insoluble matrix is
effected by covalently attaching the enzyme to the water-insoluble
matrix (the solid support) by conventional methods known in the art
for enzyme immobilization.
[0078] Exemplary immobilization techniques are described for
example in U.S. Pat. Nos. 4,071,409, 4,090,919, 4,258,133,
4,888,285, 5,177,013, 5,310,469, 5,998,183, 6,905,733, and
6,987,079, U.S. Patent Application Publication No. 2003/0096383,
and in Yan-A-X. et al, 2002, Applied Biochemistry and
Biotechnology, Vol. 101(2), pp. 113-130(18); and Ye, Yun-hua et al,
2004, Peptide Science, Vol. 41, pp 613-616, which are incorporated
herein by reference. Briefly, protein immobilization by covalent
bonding to a solid matrix, according to certain embodiments of the
present invention, is based on coupling two functional groups, as
these are defined hereinbelow, one within the matrix (e.g., on its
surface) and the other within the enzyme (e.g., on its surface),
either directly or via a spacer. The spacer can be, for example, a
bifunctional moiety, namely, a compound having at least two
functional groups which are capable of forming covalent bonds with
functional groups of both the matrix and the enzyme. As used
herein, the phrase "functional group" describes a chemical group
that has certain functionality and therefore can participate in
chemical reactions with other components which lead to chemical
interactions as described hereinabove (e.g., a bond formation). The
phrase "cross-linking agent," as used herein, refers to a
bifunctional compound that can promote or regulate intermolecular
interactions between polymer chains, linking them together to
create a more rigid structure. Cross-links are bonds linking
functional groups of polymers and/or other substances, so as to
form intermolecular interactions therebetween and, as a result, a
three-dimensional network interconnecting these substances.
Cross-linking can be effected via covalent bonds, metal
complexation, hydrogen bonding, ionic bonds and the like.
[0079] Water-treatment devices that are suitable for use in the
context of the present invention are described, for example, in
U.S. Pat. Nos. 4,532,040, 4,935,116, 5,055,183, 5,478,467,
5,855,777, 5,980,761, 6,257,242 and 6,325,929, which are
incorporated by reference.
[0080] Water treatment devices utilized in circulating reservoirs
typically form a part of a larger system, which is typically
referred to as a water plant. Typical water treatment devices used
in water plants of circulating reservoirs exert their designated
treatment action when water flows therethrough, either by means of
a pump or by gravity. The water flows into the system, enters the
device, and passes through a water-permeable and water-insoluble
matrix within the device, which effects the designated treatment
action, typically filtration of insoluble particulates and objects,
chemical exchange of solutes and ions and dissolution and addition
of chemicals into the water.
[0081] The device containing the composition described herein can
therefore be any device, or part of a device through which water
flows during the process of treating the water. Such a device can
be, for example, one or more of a filter, a filter cartridge, an
ion-exchanger, an erosion feeder and the likes, as is exemplified
hereinbelow. The device may be a removable device such as a
removable filter cartridge. Such a removable device can be
manufactured and sold separately as a "replacement" cartridge.
[0082] Thus, according to certain embodiments, the composition of
the present invention can be added to a water-treatment device
having a water-treatment substance embedded therein which effects
the originally designated treatment action of these devices, or
replace that substance altogether.
[0083] The device, according to the present embodiments, can form a
part of a comprehensive water treatment system, which exerts other
water treatment actions, such as filtration of solid particulates
and addition of chemicals. Water that flows through such a
water-treatment system also flows through the device presented
herein. The system can be design such that all its water capacity
flows through the device, or such that only a part of its water
capacity flows therethrough.
[0084] Typically, the flow rate can be adjusted per device for the
optimal function of the system and every device in it. For an
efficient function of the present device, which includes an
immobilized active enzyme, the amount of enzyme, amount of
water-insoluble matrix, overall shape of the device and flow-rate
need to be designed to as to suit the system's layout, water
capacity (power) and the expected rate at which the concentration
of an amide-containing compound such as, for example, cyanuric
acid, is required to be reduced. The rate of an amide-containing
compound reduction depends on the enzymatically catalyzed reaction
condition, e.g., temperature, pH, ionic strength and, in relevance
to this case, water flow. All the abovementioned parameters are
considered while designing the device.
[0085] The incorporation of enzymes to water-insoluble matrices is
typically measured in international units of activity. An
international unit (IU) of an enzyme is defined as the amount of
enzyme that produces one micromole of a reaction product in one
minute under defined reaction conditions. The amount of IU which
can be incorporated to a matrix depends on the type of matrix and
incorporation technique, surface area of the matrix, the
availability and chemical reactivity of functional groups suitable
for conjugation in both the enzyme and the matrix, and on the
residual enzymatic activity subsequent to the incorporation
process. Typical enzyme load ranges from a few IU to hundreds of IU
of an enzyme per cm.sup.3 of matrix material. An optimal load,
namely, the optimal amount of enzyme to be incorporated per a unit
volume of water-insoluble matrix material, is an example of one
parameter that is considered while designing the device.
[0086] The water-treatment device presented herein is shaped and
sized, and its through-flow is designed, so as to achieve optimized
efficacy in reducing the concentration of the desired
amide-containing compound (e.g., cyanuric acid). For example, using
the enzymatic catalysis parameters presented hereinabove for
structurally stable cyanuric acid hydrolase, one can calculate that
for a water quantum of 100 cubic meters, 250 mg of cyanuric acid
hydrolase is capable of treating this water quantum by decreasing
the cyanuric acid concentration from 100 ppm to 50 ppm within a
time period of 20 hours. Considering typical water pumps used in
water treatment systems of pools, which can transfer an average of
11 cubic meters per hour, this water quantum will be treated by 250
mg of cyanuric acid amidohydrolase once in 9.09 hours and more than
twice in 20 hours, which is an acceptable rate of cyanuric acid
degradation.
[0087] A reduction of 50 ppm in cyanuric acid concentration
translates to approximately 50 grams of cyanyric acid (about 0.4
moles) per cubic meter of water at chlorine-lock conditions.
Therefore, about 280 IU of cyanuric acid amidohydrolase are
required in order to reduce the concentration of cyanuric acid in
one cubic meter of water within a time period of 24 hours.
[0088] As used herein, the term "about" means.+-.10%.
[0089] Thus, according to certain embodiments of the present
invention the amount of cyanuric acid hydrolase required to treat
one cubic meter of water within a time period of 24 hours ranges
from 0.5 mg to 10 mg per, preferably 1 to 5, and more preferably
the amount of cyanuric acid amidohydrolase is at least 2.5 mg per
one cubic meter of treated water.
[0090] As mentioned hereinabove, in the water-treatment device
described herein, the composition presented herein is embedded in a
casing.
[0091] The casing may be used so as to avoid sweeping of the
composition-of-matter by the water passing through the device.
Another purpose of a casing is to form the desired shape and
cross-section of the device, which will optimize its function and
maintain a continuous, void-free bed of the composition-of-matter
presented herein. The casing material is preferably selected
suitable for water high-pressure, and is typically water-insoluble
and water-tight. Furthermore, the casing material is preferably
selected inactive and stable with respect to water and the
chemicals that are typically found in circulating reservoirs.
Examples for suitable casing materials include, without limitation,
plastic, galvanized metal and glass. In preferred embodiments, the
device for water treatment of the present invention includes a
casing with two parallel perforated faces, constituting a
semi-closed compartment, whereby the composition presented herein
fills, or partially fills the compartment. The casing thus has one
perforated face for a water inlet, and the other perforated face
for a water outlet. The water to be treated (containing the
amide-containing compound(s)) enters the inlet, pass through the
compartment containing the composition, and come in contact with
the permeable and water-insoluble matrix having the enzyme(s)
incorporated therein or thereon.
[0092] In certain embodiments, the device may include an
immobilizing matrix that has a permeable layer. Such an
enzyme-containing matrix could serve as a stationary phase for the
reservoir's water.
[0093] Other exemplary devices for water treatment according to
certain embodiments of the present invention may be a filter
cartridge, similar to that disclosed, for example, in U.S. Pat. No.
6,325,929, and containing, as the composition, an extruded solid,
water-permeable carbonaceous material block as a water-insoluble
matrix and one or more amidohydrolase enzyme(s) incorporated in and
on the carbonaceous block.
[0094] Methods of Remediating Water
[0095] Certain embodiments of the present invention provide devices
for use in the remediation of water, such as swimming pool water.
In certain embodiments, the method involves the treatment of water
with the enzymes, compositions or devices described above. In
certain embodiments, in order for the treatment to be effective, it
is desirable that the water flow at a certain rate so as to come in
contact with an effective amount of the hydrolase for a certain
period of time.
[0096] In certain embodiments, the method involves adding the CAH
enzyme to a liquid, such as water, in the form of a free enzyme, or
can be present as part of a device or part of a device through
which water flows through or over during the process of treating
the water.
[0097] In certain embodiments of the invention, the water treatment
is effected by reducing a concentration of cyanuric acid in the
water. In methods of the present invention, the water is contacted
with the device described hereinabove, such as by passing the water
through the device.
[0098] The phrase "circulating reservoir," as used herein, refers
to a structure for holding a relatively large amount of water. The
relatively large amount of water means that the water is not
replaced after every use, or rarely replaced in general for a long
period of time in terms of months and hence maintaining the water
is typically achieved by a circulating procedure. In order to
maintain the water, it at least partially pumped or otherwise
transferred out of the structure and then back into the reservoir
by means of a water transferring device such as, for example, a
pump, while being passed via a water treatment plant. Typical,
presently used, water treatment plants include water treatment
devices, such as, for example, sensors, detectors, heaters,
coolers, chemical feeders, chemical exchangers and filters of
various purposes and designs.
[0099] In certain embodiments the circulating reservoirs are public
and/or private reservoirs that are used by humans for hygiene,
sports, professional training, recreation, amusement, therapeutic
and general bathing and for ceremonial and aesthetic purposes, and
include, without limitation, pools, artificial ponds and lakes,
swimming pools, spas, hot tubs, whirlpool baths, fountains and
waterslides. The water treatment system that houses the device and
effect water flow through the device by means of, for example,
water pumps, distribution manifolds, hoses and pipes, spigots and
valves.
[0100] As discussed above, chlorine-lock occurs when the
concentration of cyanuric acid reaches 100 ppm, rendering the
quality of the water in the circulating reservoir unacceptable. In
general, water needed to be treated is generally at 50-200 ppm, and
the goal is to get it below 30 ppm. Thus, in certain embodiments,
the method acts to reduce the concentration of cyanuric acid in the
water, subsequent to the treatment, to less than 100 ppm. In
certain embodiments, the concentration of cyanuric acid in the
water, subsequent to the treatment, ranges from 70 ppm to 30
ppm.
[0101] In certain embodiments, the amount of the structurally
stabile cyanuric acid hydrolase is at least 2.5 mg per one cubic
meter of the water.
[0102] In addition to treating the water of circulating reservoirs
in order to reach the desired concentration of cyanuric acid in the
water, in certain embodiments the desired effect of water treatment
would be achieved within a relatively short period of time. The
time period should be minimized so as to avoid loss of operational
time of circulating reservoir, and avoid the risk of reaching
chlorine-lock due to the continuous addition of stabilized
sanitizers. The length of the time period within which the
treatment takes place depends on the amount of water to be treated,
the capacity of the water-treatment system and the amount and the
catalytic efficiency of the enzyme, as discussed hereinabove.
[0103] To demonstrate an exemplary implementation of the method of
treating water according to the present invention, one can consider
an exemplary circulating reservoir such as an Olympic swimming
pool. An Olympic swimming pool that meets international standards
as defined by The International Swimming Federation, must be 50
meters in length by 25 meters wide by at least 2 meters in depth.
Among other standards, the water must be kept at 25-28.degree. C.
and the lighting level at greater than 1500 lux. There are thus at
least 2500 cubic meters of water (660,430 U.S. liquid gallons)
which must be treated in a standard Olympic pool.
[0104] Using the calculation for the sufficient amount of cyanuric
acid hydrolase needed to treat 100 cubic-meters of water, i.e., to
reduce the cyanuric acid concentration from 100 ppm to 50 ppm
within about 20 hours, as presented hereinabove, the water of an
Olympic pool in a state of chlorine-lock should be passed twice
through one or more devices, as presented herein, and be brought in
contact with a composition-of-matter comprising cyanuric acid
hydrolase, according to the present invention. The molecular weight
of cyanuric acid is 129.07, so 50 ppm of a 100,000 L pool has 38.7
moles of cyanuric acid. Assuming the enzyme had 10 .mu.mol/min/mg
specific activity and contact time was 1200 minutes, then 3.2 g of
enzyme would be required. This assumes that the enzyme had infinite
number of turnovers and that it retained its original specific
activity. This would be static contact time. With water flowing
through the filter, the contact time would really be less than
this. Basically, it would be 3870 divided by the number of minutes
the water was in contact with the enzyme, in order to give the
number of grams of enzyme needed. If one assumes that the water is
pumped through the filter two times, then 1935 divided by the
number of minutes the water was in contact with the filter on each
pass would give the number of grams of enzyme needed. For
hypothetical sake, if one had a filter with a 5 minute contact time
on each pass, then for two passes, one would need 397 grams of
enzyme with a specific activity of 10 .mu.mol/min/mg. If it was a
one minute contact time then for the same two passes, one would
need 1935 grams of enzyme.
GENERAL TERMINOLOGY
[0105] "Synthetic" nucleic acids are those prepared by chemical
synthesis. The nucleic acids may also be produced by recombinant
nucleic acid methods. "Recombinant nucleic acid molecule" is a
combination of nucleic acid sequences that are joined together
using recombinant nucleic acid technology and procedures used to
join together nucleic acid sequences as described, for example, in
Sambrook and Russell (2001).
[0106] As used herein, the term "nucleic acid" and "polynucleotide"
refers to deoxyribonucleotides or ribonucleotides and polymers
thereof in either single- or double-stranded form, composed of
monomers (nucleotides) containing a sugar, phosphate and a base
that is either a purine or pyrimidine. Unless specifically limited,
the term encompasses nucleic acids containing known analogs of
natural nucleotides which have similar binding properties as the
reference nucleic acid and are metabolized in a manner similar to
naturally occurring nucleotides. Unless otherwise indicated, a
particular nucleic acid sequence also implicitly encompasses
conservatively modified variants thereof (e.g., degenerate codon
substitutions) and complementary sequences as well as the sequence
explicitly indicated. Specifically, degenerate codon substitutions
may be achieved by generating sequences in which the third position
of one or more selected (or all) codons is substituted with
mixed-base and/or deoxyinosine residues.
[0107] A "nucleic acid fragment" is a portion of a given nucleic
acid molecule. Deoxyribonucleic acid (DNA) in the majority of
organisms is the genetic material while ribonucleic acid (RNA) is
involved in the transfer of information contained within DNA into
proteins. The term "nucleotide sequence" refers to a polymer of DNA
or RNA which can be single- or double-stranded, optionally
containing synthetic, non-natural or altered nucleotide bases
capable of incorporation into DNA or RNA polymers.
[0108] The terms "nucleic acid," "nucleic acid molecule," "nucleic
acid fragment," "nucleic acid sequence or segment," or
"polynucleotide" may also be used interchangeably with gene, cDNA,
DNA and RNA encoded by a gene, e.g., genomic DNA, and even
synthetic DNA sequences. The term also includes sequences that
include any of the known base analogs of DNA and RNA.
[0109] A "variant" of a molecule is a sequence that is
substantially similar to the sequence of the native molecule. For
nucleotide sequences, variants include those sequences that,
because of the degeneracy of the genetic code, encode the identical
amino acid sequence of the native protein. Naturally occurring
allelic variants such as these can be identified with the use of
well-known molecular biology techniques, as, for example, with
polymerase chain reaction (PCR) and hybridization techniques.
Variant nucleotide sequences also include synthetically derived
nucleotide sequences, such as those generated, for example, by
using site-directed mutagenesis that encode the native protein, as
well as those that encode a polypeptide having amino acid
substitutions. Generally, nucleotide sequence variants of the
invention will have in at least one embodiment 40%, 50%, 60%, to
70%, e.g., 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, to 79%,
generally at least 80%, e.g., 81%-84%, at least 85%, e.g., 86%,
87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, to 98%,
sequence identity to the native (endogenous) nucleotide
sequence.
[0110] The term "gene" is used broadly to refer to any segment of
nucleic acid associated with a biological function. Genes include
coding sequences and/or the regulatory sequences required for their
expression. For example, gene refers to a nucleic acid fragment
that expresses mRNA, functional RNA, or a specific protein,
including its regulatory sequences. Genes also include nonexpressed
DNA segments that, for example, form recognition sequences for
other proteins. Genes can be obtained from a variety of sources,
including cloning from a source of interest or synthesizing from
known or predicted sequence information, and may include sequences
designed to have desired parameters. In addition, a "gene" or a
"recombinant gene" refers to a nucleic acid molecule comprising an
open reading frame and including at least one exon and (optionally)
an intron sequence. The term "intron" refers to a DNA sequence
present in a given gene which is not translated into protein and is
generally found between exons.
[0111] "Naturally occurring," "native" or "wild type" is used to
describe an object that can be found in nature as distinct from
being artificially produced. For example, a nucleotide sequence
present in an organism (including a virus), which can be isolated
from a source in nature and which has not been intentionally
modified in the laboratory, is naturally occurring. Furthermore,
"wild-type" refers to the normal gene, or organism found in nature
without any known mutation.
[0112] "Homology" refers to the percent identity between two
polynucleotides or two polypeptide sequences. Two DNA or
polypeptide sequences are "homologous" to each other when the
sequences exhibit at least about 75% to 85% (including 75%, 76%,
77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, and 85%), at least about
90%, or at least about 95% to 99% (including 95%, 96%, 97%, 98%,
99%) contiguous sequence identity over a defined length of the
sequences.
[0113] "Operably-linked" nucleic acids refers to the association of
nucleic acid sequences on single nucleic acid fragment so that the
function of one is affected by the other, e.g., an arrangement of
elements wherein the components so described are configured so as
to perform their usual function. For example, a regulatory DNA
sequence is said to be "operably linked to" or "associated with" a
DNA sequence that codes for an RNA or a polypeptide if the two
sequences are situated such that the regulatory DNA sequence
affects expression of the coding DNA sequence (i.e., that the
coding sequence or functional RNA is under the transcriptional
control of the promoter). Coding sequences can be operably-linked
to regulatory sequences in sense or antisense orientation. Control
elements operably linked to a coding sequence are capable of
effecting the expression of the coding sequence. The control
elements need not be contiguous with the coding sequence, so long
as they function to direct the expression thereof. Thus, for
example, intervening untranslated yet transcribed sequences can be
present between a promoter and the coding sequence and the promoter
can still be considered "operably linked" to the coding
sequence.
[0114] As discussed above, the terms "isolated and/or purified"
refer to in vitro isolation of a nucleic acid, e.g., a DNA or RNA
molecule from its natural cellular environment, and from
association with other components of the cell, such as nucleic acid
or polypeptide, so that it can be sequenced, replicated, and/or
expressed. For example, "isolated nucleic acid" may be a DNA
molecule that is complementary or hybridizes to a sequence in a
gene of interest and remains stably bound under stringent
conditions (as defined by methods well known in the art). Thus, the
RNA or DNA is "isolated" in that it is free from at least one
contaminating nucleic acid with which it is normally associated in
the natural source of the RNA or DNA and in one embodiment of the
invention is substantially free of any other mammalian RNA or DNA.
The phrase "free from at least one contaminating source nucleic
acid with which it is normally associated" includes the case where
the nucleic acid is reintroduced into the source or natural cell
but is in a different chromosomal location or is otherwise flanked
by nucleic acid sequences not normally found in the source cell,
e.g., in a vector or plasmid.
[0115] As used herein, the term "recombinant nucleic acid," e.g.,
"recombinant DNA sequence or segment" refers to a nucleic acid,
e.g., to DNA, that has been derived or isolated from any
appropriate cellular source, that may be subsequently chemically
altered in vitro, so that its sequence is not naturally occurring,
or corresponds to naturally occurring sequences that are not
positioned as they would be positioned in a genome that has not
been transformed with exogenous DNA. An example of preselected DNA
"derived" from a source would be a DNA sequence that is identified
as a useful fragment within a given organism, and which is then
chemically synthesized in essentially pure form. An example of such
DNA "isolated" from a source would be a useful DNA sequence that is
excised or removed from said source by chemical means, e.g., by the
use of restriction endonucleases, so that it can be further
manipulated, e.g., amplified, for use in the invention, by the
methodology of genetic engineering.
[0116] Thus, recovery or isolation of a given fragment of DNA from
a restriction digest can employ separation of the digest on
polyacrylamide or agarose gel by electrophoresis, identification of
the fragment of interest by comparison of its mobility versus that
of marker DNA fragments of known molecular weight, removal of the
gel section containing the desired fragment, and separation of the
gel from DNA. Therefore, "recombinant DNA" includes completely
synthetic DNA sequences, semi-synthetic DNA sequences, DNA
sequences isolated from biological sources, and DNA sequences
derived from RNA, as well as mixtures thereof.
[0117] Nucleic acid molecules having base substitutions (i.e.,
variants) are prepared by a variety of methods known in the art.
These methods include, but are not limited to, isolation from a
natural source (in the case of naturally occurring sequence
variants) or preparation by oligonucleotide-mediated (or
site-directed) mutagenesis, PCR mutagenesis, and cassette
mutagenesis of an earlier prepared variant or a non-variant version
of the nucleic acid molecule.
[0118] The invention will now be illustrated by the following
non-limiting Examples.
Example 1
Thermostable Cyanuric Acid Hydrolase
[0119] Microbial enzymatic degradation of cyanuric acid has been
studied previously (Devers, M., et al., 2007. FEMS Microbiol Lett.
273:78-86; Eaton, R. W., and J. S. Karns. 1991. J. Bacteriol.
173:1363-1366; Fruchey, I., N. et al., 2003. Appl Environ
Microbiol. 69:3653-7; Smith, D., S. Alvey, and D. E. Crowley. 2005.
FEMS Microbiol Ecol. 53:265-73). Two distinct but homologous
enzymes, AtzD from Pseudomonas sp. strain ADP (Fruchey, I., N. et
al., 2003. Appl Environ Microbiol. 69:3653-7) and TrzD from
Pseudomonas sp. strain NRRLB-12227 (Smith, D., S. Alvey, and D. E.
Crowley. 2005. FEMS Microbiol Ecol. 53:265-73), have been studied
in detail. These enzymes, known as cyanuric acid hydrolases,
catalyze the conversion of cyanuric acid to biuret (FIG. 1). Biuret
is not considered toxic to humans and degrades more readily than
cyanuric acid.
[0120] Barbiturase is the only protein known to be homologous to
cyanuric acid hydrolase that has a defined and different
physiological function. Barbiturase catalyzes the conversion of
barbituric acid to ureidomalonic acid in organisms that catabolize
pyrimidines by the oxidative pathway. Barbiturase is unstable at
4.degree. C. in the absence of ethylene glycol and dithiothrietol
(DTT). Furthermore, activity is completely lost when the protein is
maintained at 55.degree. C. for 30 minutes (Soong, C. L., et al.,
2002. J Biol Chem. 277:7051-8). AtzD and TrzD are relatively stable
at 4.degree. C., but they lose activity when frozen. Moreover, the
thermostability properties of AtzD and TrzD are not well studied,
but these enzymes are derived from mesophilic bacteria. In this
context, the inventors initiated a search to identify a stable
cyanuric acid hydrolase. Enzymes that are more stable to
temperature changes are more stable to many environmental factors.
Thus, a thermostable enzyme would be most applicable to pool water
and other remediation efforts.
[0121] The inventors identified a cyanuric acid hydrolase homolog
in Moorella thermoacetica ATCC 39073, an anaerobic, acetogenic
bacterium that is able to grow at 65.degree. C. The gene was cloned
into E. coli, the protein was expressed at high levels, the
recombinant E. coli degraded cyanuric acid, and the enzyme was
obtained in homogeneous form by a convenient one-step purification.
The enzyme's function as a cyanuric acid hydrolase was confirmed,
and it was shown to be significantly more stable than other known
members of the cyanuric acid protein family.
[0122] Materials and Methods
[0123] Chemicals.
[0124] 5-nitrobarbituric acid, 5-methylbarbituric acid,
2-methylamino-4,6-dihydroxy-s-triazine,
1,3,5-trimethoxy-s-triazine, methyl isocyanurate,
2-(3-methoxypropylamino)-4,6-dihydroxy-s-triazine,
triphenoxy-s-triazine, 2-secbutyl-4,6-dihydroxy-s-triazine,
N-phenyl ammelide, 2-ethylamino-4,6-dihydroxy-s-triazine,
triisopropoxy-s-triazine, trimethyl isocyanurate, and 1,3-dimethyl
isocyanurate were prepared as previously described (Smolin, E. M.,
and L. Rapoport. 1959. s-Triazines and Derivatives, p. 269-308. In
A. Weissberger (ed.), The chemistry of heterocyclic compounds.
Interscience Publishers Inc., New York). Hexazinone,
4,6-dihydroxy-2-mecapto pyrimidine and
4,6-dihydroxy-5-nitro-pyrimidine were purchased from
Sigma-Aldrich.
[0125] Bacterial Strains and Growth Conditions.
[0126] M. thermoacetica ATCC 39073, formerly known as Clostridium
thermoacticum, was obtained from the ATCC and grown anaerobically
at 55.degree. C. in serum bottles (125 ml) or in rubber-stoppered,
screw-capped infusion flasks (1200 ml; Muller-Krempel, Bulach,
Switzerland. The bacterium was grown in an anaerobic medium
containing the following per liter: 5 g yeast extract, 5 g
tryptone, 6.4 g Na.sub.2HPO.sub.4, 6.1 g NaH.sub.2PO.sub.4, 0.4 g
NH.sub.4Cl, 0.3 g MgCl.sub.2.6H.sub.2O, 0.05 g
CaCl.sub.2.2H.sub.2O, 0.04 g Fe(NH.sub.4)(SO4).sub.2.6H.sub.2O, and
10 ml of a mineral mix which was previously described (Marsili, E.,
et al., 2008. Appl Environ Microbiol. 74:7329-37). After
autoclaving, 18 g glucose and 0.2 mg of biotin, cyanocobalamin,
flavin mononucletide, folic acid, nicotinic acid, p-aminobenzoic
acid and thiamine pyrophosphate were added individually.
Immediately before inoculation, 10 ml of a reducing solution,
containing 0.36 g of Na.sub.2S.9H.sub.2O and 0.36 g of cysteine
HCl, were added. The pH of all media was adjusted to 6.6 prior to
the addition of 2 g/l NaHCO3. The media was flushed with
oxygen-free N.sub.2--CO.sub.2 (80:20 [vol/vol]) for 30 min prior to
sealing with butyl rubber stoppers.
[0127] Protein Identification and Comparisons.
[0128] The BLAST algorithm was used to identify homologs of AtzD,
TrzD, and barbiturase. Sequence alignments were done with Clustal W
(Thompson, J. D., D. G. Higgins, and T. J. Gibson. 1994. Nucleic
Acids Res. 22:4673-4680.), and protein trees were constructed with
the maximum likelihood algorithm in PHYLIP (Felsenstein, J. 1989.
Cladistics 5:164-166). The protein sequences (GenBank accession
numbers) used are as follows: Pseudomonas sp. strain ADP AtzD
(AAK50331), Pseudomonas sp. strain NRRLB-12227 (now called
Acidovorax avenae subsp. citrulli) TrzD (AAC61577), Rhodococcus
erythropolis barbiturase (CAC86669), Chelatobacter heintzii
(AAK52819), Moorella thermoacetica ATCC 39073 (YP.sub.--430955),
Bradyrhizobium japonicum USDA 110 (BAC52546), and Arthrobacter sp.
strain AD25 (ABK41866).
[0129] Cloning and Expression.
[0130] Total genomic DNA was extracted from cell pellets of M.
thermoacetica ATCC 39073, Pseudomonas sp. strain NRRLB-12227 and
Pseudomonas sp. strain ADP, as previously described (Sambrook, J.,
E. F. Fritsch, and T. Maniatis. 1989. Molecular Cloning: A
Laboratory Manual, 2nd ed. Cold Spring Harbor Press, Cold Spring
Harbor, N.Y.; van der Maarel, et al., 1996. Appl Environ Microbiol.
62:3978-84). ORF Moth.sub.--2120 from M. thermoacetica ATCC 39073
was PCR amplified, using primers
5'-GCGAATTCCATATGCAAAAAGTTGAAGTCTT-3' (SEQ ID NO:1) and
5'-GCCAAGCTTCTACACCCTGGCAATAACAG-3' (SEQ ID NO:2) (NdeI and HindIII
restriction enzyme sites underlined, respectively). The gene was
cloned into a pET28b+ vector (Novagene, Madison, Wis.). The
resulting vector, pET28b+::Moth.sub.--2120, was transformed into E.
coli BRL21(DE3) pLysS, and grown at 37.degree. C. in Luria-Bertani
medium (Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989.
Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor
Press, Cold Spring Harbor, N.Y.) containing 50 .mu.g kanamycin and
25 .mu.g chloramphenicol per ml. When the culture reached an
optical density at 600 nm of 0.5, 1 mM
isopropyl-D-thiogalactopyranoside (IPTG) was added, and the induced
cells were grown overnight at 30.degree. C. A similar cloning,
expression, and purification system was used to clone and His-tag
the trzD gene from P. sp strain NRRLB-12227 and atzD from P. sp
strain ADP to obtain these enzymes for comparison.
[0131] Enzyme Purification.
[0132] For enzyme purification, 2 L of E. coli BRL21(DE3)
(pET28b+::Moth.sub.--2120) cells were grown, as described above.
The culture was centrifuged at 10,000.times.g for 20 min at
4.degree. C., and the pellet was resuspended (1 ml per gram cell)
in 100 mM potassium phosphate buffer, pH 7.0. The cell suspension
was passed three times through a chilled Amicon French pressure
cell, operated at 140 MPa, and the crude cell lysate was
centrifuged at 18,000.times.g for 90 min at 4.degree. C. The enzyme
was purified using a 5 ml HiTrap chelating HP column (Amersham
Pharmacia Biotech AB, Uppsala, Sweden) and a Pharmacia FPLC LKB
system (Amersham Pharmacia Biotech AB, Uppsala, Sweden). The column
was prepared as per manufacturers instructions and equilibrated
with 60 ml of 100 mM potassium phosphate buffer, pH 7.0. After
loading the protein, the column was washed with a series of step
gradients consisting of 0.05 M, 0.1 M, 0.25 M, and 0.5 M imidazole
in 100 mM potassium phosphate buffer (pH 7.0). Purified protein was
analyzed by sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) for purity and subunit molecular weight
determination by comparison to broad range standards (Bio-Rad
Laboratories, Hercules, Calif.). The purified protein was dialyzed
at 4.degree. C. against 100 mM potassium phosphate buffer (pH 7.0)
to remove imidazole.
[0133] Enzyme Assay and Kinetic Constant Determinations.
[0134] Enzyme activity was monitored on a Beckman DU 640
spectrophotometer (Beckman Coulter, Fullerton, Calif.). Cyanuric
acid and barbituric acid concentrations were measured at 214 nm
(extinction coefficient=9200 cm.sup.-1M.sup.-1) and 256 nm,
respectively. Reactions were carried out in 0.5 ml 25 mM Tris-HCl
buffer, pH 8.0 with 100 .mu.M cyanuric acid or barbituric acid at
30.degree. C. Reactions were initiated by the addition of the
enzyme. Kinetic parameters were determined by obtaining rates at
cyanuric acid concentrations ranging from 10 to 110 .mu.M. The data
was plotted using Lineweaver-Burke plots.
[0135] Conversion of cyanuric acid to biuret was confirmed using
high-pressure liquid chromatography (HPLC). Control samples,
standards, and enzymatic reactions were set up in 5 mM phosphate
buffer (pH 7.0). Samples were analyzed by HPLC, using a
Hewlett-Packard HP 1100 system equipped with a photodiode array
detector interfaced to an HP Chemstation. A mixed-mode
C.sub.8/anion 7.mu. column (250 by 4.6 mm) (Alltech, Deerfield,
Ill.) was used with an isocratic mobile phase consisting of 95%
methanol and 5% 5 mM phosphate buffer (pH 7.0).
[0136] Substrate Specificity Analysis.
[0137] Seventeen compounds structurally analogous to cyanuric acid
were tested as substrates: 5-nitrobarbituric acid,
5-methylbarbituric acid, 2-methylamino-4,6-dihydroxy-s-triazine,
1,3,5-trimethoxy-s-triazine, 2-(3-methoxy
propylamino)-4,6-dihydroxy-s-triazine, triphenoxy-s-triazine,
2-secbutyl-4,6-dihydroxy-s-triazine, N-phenyl ammelide,
2-ethylamino-4,6-dihydroxy-s-triazine, triisopropoxy-s-triazine,
trimethyl isocyanurate, 1,3-dimethyl isocyanurate, hexazinone,
4,6-dihydroxy-2-mecapto pyrimidine,
4,6-dihydroxy-5-nitro-pyrimidine, barbituric acid and methyl
isocyanurate. The compounds (100 .mu.M) were incubated with 50
.mu.g of purified enzyme in 25 mM Tris-HCl buffer (pH 8.0) for 30
min. Changes in the UV spectra were recorded. In cases where
changes in the spectra were detected, an appropriate wavelength
with absorbance differences between the substrate and product was
chosen for further kinetic study. For N-methylisocyanurate,
absorbance was measured at 214 nm (extinction coefficient=9500
cm.sup.-1M.sup.-1).
[0138] Effect of pH and Temperature on Enzymatic Activity.
[0139] The optimum pH of the enzyme was determined at 30.degree. C.
with the following buffers: 25 mM NaHCO.sub.3/Na.sub.2CO.sub.3, pH
5-7; 25 mM Tris-HCl, pH 7-9; NaHCO.sub.3/Na.sub.2CO.sub.3, pH
9.5-10.5. The temperature optimum was determined by assaying the
enzyme activity in 25 mM Tris-HCl, pH 8.0 at temperatures ranging
from 25.degree. C. to 70.degree. C.
[0140] Temperature Stability of the Enzyme.
[0141] The thermal stability of the enzyme was determined by
incubating the enzyme at various temperatures for 30 min prior to
activity determinations. Studies of the stability during storage
were performed at 4.degree. C. or -80.degree. C. with or without
additives. The additives were 0.2 mM of dithiothreitol (DTT), 10%
ethylene glycol, 25% glycerol and 25% polyethylene glycol (PEG)
4000. The activity was checked every two weeks, as described
above.
[0142] Metal Chelator Effects.
[0143] Purified protein was incubated at room temperature with 5 mM
1,10-phenanthroline, 8-hydroxyquinoline-5-sulfonic acid, or
ethylenediaminetetraacetic acid (EDTA) for 24 hours. PD-10
desalting columns (GE Healthcare) were used, as per the
manufacturer's instructions, to remove the chelator from the
enzyme. Enzymatic activity was measured with cyanuric acid as
substrate, as described above.
[0144] Effects of Metals.
[0145] Enzyme was incubated with 0.2 mM of each metal salt for 60
min at 4.degree. C. For each treatment, specific activity was
determined, as described above. The final metal concentration in
the assay buffer was 0.1 mM. The metals tested were CoCl.sub.2,
MnSO.sub.4, NiCl.sub.2, CuCl.sub.2, ZnSO.sub.4, FeCl.sub.3, and
FeSO.sub.4. The following salts were also tested at the final
concentrations indicated: 1 mM CaCl.sub.2 and 2 mM MgCl.sub.2.
[0146] Metal Analysis.
[0147] Enzyme was hydrolyzed with 6M hydrochloric acid at
110.degree. C. for 24 hrs under vacuum. Metal content and protein
concentrations were determined as previously described (de Souza,
M. L., et al., 1996. J. Bacteriol. 178:4894-4900).
[0148] Results
[0149] Identification of ORF Moth.sub.--2120.
[0150] The BLAST algorithm was used to search GenBank for amino
acid sequences homologous to AtzD, TrzD, and barbiturase. A
subgroup of the sequences identified were found to cluster most
closely to the cyanuric acid hydrolases (FIG. 2). The protein from
Chelatobacter heintzii was identical to TrzD, and the protein from
Arthrobacter AD25 was 99% identical to AtzD. The protein from
Bradyrhizobium japonicum USDA 110 was 56%, 66%, and 44% identical
to TrzD, AtzD, and barbiturase, respectively. A predicted protein
sequence derived from the genome sequence of Moorella thermoacetica
ATCC 39073 (gi83590946; locus tag Moth 2120) was 64%, 57%, and 48%
identical to TrzD, AtzD, and barbiturase, respectively. This
revealed two divergent homologs to known cyanuric acid hydrolases,
but the known ability of M. thermoacetica to grow at elevated
temperatures made this protein more attractive for further
study.
[0151] Protein Characterization and Determination of Catalytic
Activity.
[0152] The putative cyanuric acid hydrolase homolog encoded by ORF
Moth.sub.--2120 from M. thermoacetica ATCC 39073 was cloned and
expressed in E. coli as described above. The resultant E. coli
strain, unlike the wild-type strain, showed clearing zones on agar
plates containing a suspension of 130 mM cyanuric acid. The
recombinant protein was expressed with an N-terminal His-tag that
allowed its purification to homogeneity in a single step. SDS-PAGE
showed a single protein band corresponding to 40 kDa. This agrees
with the calculated molecular weight of 38.9 kDa for a polypeptide
encoded by this ORF.
[0153] The purified enzyme in vitro hydrolyzed cyanuric acid with a
specific activity of 15.7 .mu.mol/min/mg but did not show
detectable activity with barbituric acid, even with the use of 50
.mu.g of protein in a single assay. These results indicate that ORF
Moth 2120 is a cyanuric acid hydrolase. Consistent with this view,
the enzyme showed a physiologically reasonable K.sub.m value of 110
.mu.M with cyanuric acid as substrate. The k.sub.cat was determined
to be 10.6 s.sup.-1 with a k.sub.cat/K.sub.m of 1.0 10.sup.5
M.sup.-1s.sup.-1. This compares with the published values for
K.sub.m and k.sub.cat of 57 .mu.M and 6.8 s.sup.-1 for AtzD (7),
and 50 .mu.M and 250 s.sup.-1 for TrzD (Smith, D., S. Alvey, and D.
E. Crowley. 2005. FEMS Microbiol Ecol. 53:265-73).
[0154] Substrate Specificity.
[0155] Seventeen compounds that are structurally analogous to
cyanuric acid were tested as substrates for hydrolysis by the
Moorella cyanuric acid hydrolase (FIG. 3). Of these, only methyl
isocyanurate was a substrate, with a rate of 0.13 .mu.mol/min/mg,
slightly less than 1% of the rate of the preferred substrate
cyanuric acid. Based on these analyses, the substrates for this
enzyme appear to require three nitrogen atoms in a six member ring
as typical of s-triazine compounds, three carbonyl oxygen atoms on
the carbons of the ring, and at least two of the ring nitrogens
having hydrogens, while the other nitrogen atom can be bonded to a
hydrogen or a methyl group.
[0156] Optimum pH and Temperature of the Enzyme.
[0157] The activity for cyanuric acid hydrolase from M.
thermoacetica ATCC 39073 was determined at pH values ranging from
5.5 to 10.5.
[0158] Maximum activity was achieved at pH 8.0. This pH optimum
agrees with the other characterized cyanuric acid hydrolases
(Fruchey, I., N. et al., 2003. Appl Environ Microbiol. 69:3653-7;
Smith, D., S. Alvey, and D. E. Crowley. 2005. FEMS Microbiol Ecol.
53:265-73). The effects of temperature on enzyme activity were also
determined in the range of 25-70.degree. C. The activity of the
enzyme steadily increased with temperature, reaching a maximum at
70.degree. C. of 24 .mu.mol per min per mg protein. Temperatures
greater than 70.degree. C. could not be assayed due to the
limitations of our equipment. The optimum temperature over this
range was 70.degree. C., which is much higher than the
45-50.degree. C. range reported for TrzD (Smith, D., S. Alvey, and
D. E. Crowley. 2005. FEMS Microbiol Ecol. 53:265-73).
[0159] Temperature Stability of the Enzyme.
[0160] The thermostability of the enzyme at pH 8.0 was also tested
by incubating the enzyme for 30 min prior to determining the
activity (FIG. 4). At 50.degree. C., 95% of initial activity
remained with the Moorella enzyme, while at 70.degree. C., 30% of
the initial activity remained. This contrasts with other members of
this family of proteins. AtzD had no loss of activity at 40.degree.
C., but all activity was lost when incubated at 50.degree. C.
Likewise, TrzD lost most of its activity when the temperature was
increased to 50.degree. C. (FIG. 4). Barbiturase is also thermally
sensitive and reported to have no residual activity after a 30 min
incubation at 55.degree. C. (Soong, C. L., et al., 2002. J Biol
Chem. 277:7051-8) (18). These results suggest that the Moorella
cyanuric acid hydrolase is structurally and catalytically more
stable than other members of this family of enzymes.
[0161] Storage stability of the enzyme at 4.degree. C. and
-80.degree. C. in the presence and absence of various additives was
also examined (Table 1).
TABLE-US-00004 TABLE 1 Storage stability of purified cyanuric acid
hydrolase from M. thermoacetica ATCC 39073 at pH 8.0 as a function
of time and additives.sup.a. Temperature Activity (.mu.mol per min
per mg protein).sup.b (.degree. C.) Additive 0 week 2 week 4 week 6
week 8 week 4.degree. C. enzyme only 11.9 .+-. 0.23 12.5 .+-. 0.2
15.9 .+-. 0.7 16.6 .+-. 0.9 16.9 .+-. 1 10% ethylene glycol 12.3
.+-. 1.2 13.8 .+-. 0.3 11.3 .+-. 0.3 10.5 .+-. 0.9 11.4 .+-. 0.6
0.2 mM of DTT 13.4 .+-. 0.9 15.3 .+-. 0.5 17.7 .+-. 0.7 14.8 .+-.
0.3 18.3 .+-. 0.6 25% glycerol 14.3 .+-. 1.9 13.4 .+-. 0.1 12.2
.+-. 0.5 9.8 .+-. 0.5 12.0 .+-. 1.3 25% PEG 4000 31.8 .+-. 1.8 20.0
.+-. 0.2 31.0 .+-. 0.5 22.2 .+-. 0.6 18.7 .+-. 0.1 -80.degree. C.
enzyme only 11.9 .+-. 0.2 14.9 .+-. 0.1 14.3 .+-. 0.3 12.3 .+-. 0.4
11.7 .+-. 1.1 10% ethylene glycol 12.3 .+-. 1.2 13.3 .+-. 0.3 14.1
.+-. 0.3 13.6 .+-. 0.8 13.0 .+-. 0.2 0.2 mM of DTT 13.4 .+-. 0.9
14.7 .+-. 0.2 14.3 .+-. 0.3 12.2 .+-. 0.3 11.8 .+-. 0.3 25%
glycerol 14.3 .+-. 1.9 14.0 .+-. 0.2 14.0 .+-. 0.9 10.7 .+-. 1.0
11.0 .+-. 1.2 25% PEG 4000 31.8 .+-. 1.8 21.4 .+-. 0.7 22.0 .+-.
0.4 21.0 .+-. 0.3 17.1 .+-. 0.4 .sup.aValues are the mean .+-.
standard error of three replicates. .sup.bActivity was measured
using the standard assay at 25.degree. C.
[0162] Among the additives were 10% ethylene glycol, 0.2 mM DTT,
25% glycerol, and 25% PEG4000. The enzymatic activity was monitored
every 2 weeks for a total of 8 weeks. The enzyme without additives
showed no loss of activity after 8 weeks at both storage
temperatures. This suggested that the enzyme was stable at low
temperatures. Though the enzymatic activity was unchanged in the
frozen sample after 8 weeks, the specific activity of the sample
stored at 4.degree. C. increased to 142% and 136% of initial
specific activity compared to the sample without additives and 0.2
mM DTT, respectively. The addition of 25% PEG 4000 had a
stimulatory affect upon activity prior to storage, increasing
activity by 270%. However, activity for the PEG-stimulated enzyme
subsequently decreased to around 1.4-1.6 times initial activity
without additives over the 8 week time course at both temperatures.
All other additives, including 10% ethylene glycol and 25%
glycerol, had little or no positive effect on sustaining
activity.
[0163] Effect of Metal Ions and Chelators.
[0164] The affects of divalent metals upon native enzyme activity
were monitored (Table 2).
TABLE-US-00005 TABLE 2 Effect of metal ions on the activity of
purified cyanuric acid hydrolase from M. thermoacetica.sup.a Metal
Concentration (mM) Relative activity CoCl.sub.2 0.2 92 .+-. 5
MnSO.sub.4 0.2 96 .+-. 3 ZnSO.sub.4 0.2 51 .+-. 4 CuCl.sub.2 0.2 83
.+-. 3 FeCl.sub.3 0.2 105 .+-. 4 NiCl.sub.2 0.2 79 .+-. 3
CaCl.sub.2 1.0 121 .+-. 7 MgCl.sub.2 2.0 119 .+-. 2 .sup.aValues
are the mean .+-. standard error of three replicates.
[0165] Zn(II) had the greatest inhibitory affect, with slight
decreases in activity observed in the presence of Cu(II) and
Ni(II). At higher concentrations, Ca(II) and Mg(II) caused slight
increases to 121% and 119% of native activity, respectively.
Chelators, including EDTA, o-phenanthroline, and
8-hydroxyquinoline-5-sulfonic acid, even with 24 h incubations,
failed to alter activity. These data suggested that either a metal
could not be removed or that metals were not necessary for
catalytic activity.
[0166] Quantitation of Bound Metal.
[0167] The metal content of the enzyme was analyzed by ICP. The
only metals detected above background were zinc and nickel which
were present at 0.08 and 0.05 molar metal to subunit
stoichiometries, respectively. These results indicated that no
catalytically-significant metals were present in the enzyme
preparation having a k.sub.cat/K.sub.m of 1.0 10.sup.5
M.sup.-1s.sup.-1, a significant catalytic rate. Similar results
were obtained with AtzD (Fruchey, I., et al., 2003. Appl Environ
Microbiol. 69:3653-7).
[0168] Discussion
[0169] The cyanuric acid hydrolase enzymes AtzD from Pseudomonas
sp. strain ADP (Fruchey, I., et al., 2003. Appl Environ Microbiol.
69:3653-7) and TrzD from Pseudomonas sp. strain NRRLB-12227 (Smith,
D., S. Alvey, and D. E. Crowley. 2005. FEMS Microbiol Ecol.
53:265-73) have been purified and characterized. These enzymes were
identified in organisms isolated for their ability to degrade
atrazine and melamine, respectively. Both organisms mineralize the
compounds via metabolic pathways that proceed through cyanuric acid
as an intermediate. Cyanuric acid also forms abiotically via the
spontaneous decomposition of chlorinated isocyanuric acids (Cant ,
R., et al., 2000. Anal. Chem. 72:5820-8). The breakdown is an
intended process as it serves to disinfect pools by slowly
generating hypochlorite. After a time, however, cyanuric acid
accumulates, rendering further chlorination ineffective. Various
methods to remove cyanuric acid chemically have been tried, but
none have proven commercially successful to date. To deal with this
issue, enzymatic treatment of cyanuric acid in pool water has been
considered. One impediment to this application has been the
relative instability of the cyanuric acid hydrolases known to
date.
[0170] The known cyanuric acid hydrolases, AtzD and TrzD, have been
observed to lose activity during freezing, a typical method used to
maintain enzyme stability and deter microbial and proteolytic
breakdown of proteins. The goal of this study was to identify a
more stable cyanuric acid hydrolase enzyme for use in
bioremediation applications. In this context, the inventors
identified an AtzD/TrzD homolog from M. thermoacetica ATCC 39073.
This is the first cyanuric acid hydrolase purified from an organism
not previously shown to degrade s-triazine compounds. Genome
context gives little indication of a native function for this gene.
Upstream of Moth.sub.--2120 are genes that encode for a CdaR
transcriptional regulator, two hypothetical proteins, an FdrA
protein implicated in regulating diverse cellular processes through
FtsH, and another hypothetical protein. Downstream there is a
hypothetical protein, uracil-xanthine permease, carbamate kinase,
and methyltransferase genes.
[0171] The AtzD gene homolog from M. thermoacetica was cloned, the
recombinant enzyme was purified. Characteristics of the enzyme were
studied with respect to catalysis and stability. Table 3 compares
the properties of this new enzyme with those of AtzD and TrzD.
TABLE-US-00006 TABLE 3 Comparison of Cyanuric Acid Hydrolases
Properties Moorella TrzD AtzD Mol. wt. (calc.) (kDa) 38.9 39.4 38.2
pH optimum 8.0 8.0-8.5(8) 8.2(7) Temp. optimum (.degree. C.) 70
45-50(8) 30(6) Thermostability.sup.a (.degree. C.) 20-65 20-45
20-40 k.sub.cat (s.sup.-1) 10.6 250(8) 6.8 .+-. 0.7(7) K.sub.M
(.mu.M) 110 50(8) 57 .+-. 10(7) k.sub.cat/K.sub.M
(s.sup.-1M.sup.-1) 1.0 10 .sup.5 5 10 .sup.6 1.2 10 .sup.5 Known
substrates Cyanuric acid, N- Cyanuric acid(8) Cyanuric acid, N-
methylisocyanuric methylisocyanuric acid acid (7) Metal content
None n.d. None(7) Metal affects on Cu(II), Zn(I), Ni(II) Mg(II),
Mn(II) no affect No stimulatory activity inhibitory at 0.2 mM; 1
mM; Co(II), Cu(II), affects with Zn(II), Ca(II), Mg(II) slight
Fe(II) slight inhibitory Cu(II), Fe(II), Co(II), increase at 1.0
and 1 mM; Zn(II) 1 mM or Ni(II) (7) 2.0 mM, respectively greatly
inhibitory(8) Influence of chelators No affect EDTA, o- No affect
EDTA(8) No affect EDTA and on activity phenanthroline, or 8-
o-phenanthroline hydroxyquinoline-5- sulfonic acid
.sup.aTemperature where activity above 50% after a 30 min
incubation. n.d. = not determined
[0172] The cyanuric acid hydrolase from M. thermoacetica ATCC 39073
was shown to have cyanuric acid hydrolase activity, with K.sub.m
and k.sub.cat values for cyanuric acid of 110 .mu.M and 10.6
s.sup.-1, respectively. The enzyme displayed a high degree of
substrate discrimination, only catalyzing reactions with cyanuric
acid and its close structural analog N-methylisocyanuric acid, but
with no other analogous structure. This mirrors the restricted
reactivity found with AtzD (Fruchey, I., et al., 2003. Appl Environ
Microbiol. 69:3653-7) and indicates that, although the sequences of
the cyanuric acid hydrolases are only 57-64% identical, the
proteins share a similar substrate range. The key difference
between the M. thermoacetica cyanuric acid hydrolase and AtzD/TrzD
is in the greater stability of the former enzyme. The new enzyme
had its highest catalytic activity at 70.degree. C. (the highest
temperature that our assays were able to maintain), was stable when
stored for 30 min at 50.degree. C., and was able to be stored
frozen for long periods of time. In combination, these properties
make this a superior enzyme for cyanuric acid remediation.
[0173] Barbiturase, a cyanuric acid homolog, has been proposed to
be a member of the amidohydrolase superfamily (Soong, C. L., et
al., 2002. J Biol. Chem. 277:7051-8). In general, amidohydrolase
superfamily members contain one or two metal atoms per subunit,
with zinc being the most commonly identified metal. The enzymes
AtzA, AtzB, AtzC and TrzN, which are in the pathway of atrazine
degradation, are all metalloenzymes and members of this superfamily
(Seffernick, J. L., et al., 2007. J. Bacteriol. 189:6989-97;
Seffernick, J. L., et al., 2002. Biochemistry 41:14430-7; Shapir,
N., et al., 2002. J. Bacteriol. 184:5376-84; Shapir, N., et al.,
2006. J. Bacteriol. 188:5859-64). However, sequence analysis of the
AtzD/TrzD/barbiturase family of proteins revealed that they are not
members of this common superfamily. Instead, these enzymes are very
distant from any other proteins of known function, and cluster as
an isolated island in sequence space. Because of the lack of
sequence and evolutionary links to other well-characterized amidase
enzymes, this family must be considered independently. Consistent
with this, a metal was not necessary for catalysis by the M.
thermoaceticum cyanuric acid hydrolase. Previously, active AtzD was
found not to contain significant levels of metals (Fruchey, I., N.
et al., 2003. Appl Environ Microbiol. 69:3653-7).
[0174] In conclusion, the cyanuric acid hydrolase from M.
thermoacetica ATCC 39073 is not a metalloenzyme. Furthermore, it is
an outstanding thermophilic enzyme that is stable at high or low
temperatures.
Example 2
Defining the Cyanuric acid hydrolase/Barbiturase Protein Family
[0175] As sequence databases grown and alignment methods become
more sophisticated, many protein superfamilies have swelled to
include tens or hundreds of thousands of members. Dozens of those
members typically have available X-ray structures. This provides
structure and function information that newly discovered members of
the superfamily can be anchored to and thus provide immediate clues
as its structure. A notable exception to this paradigm is the small
cluster of related sequences that belong to proteins known to be,
or annotated as, cyanuric or barbituric acid hydrolases. These
related proteins catalyze the opening of structurally analogous
nitrogen heterocyclic rings (FIG. 1). The barbituric acid
hydrolases, also known as barbiturases, react with a pyrimidine
ring and carry out recycling of cytosine and uracil. The cyanuric
acid hydrolases have been largely identified in bacteria that
catabolize anthropogenic s-triazine ring compounds such as the
herbicide atrazine, or the monomer melamine. The barbiturases and
cyanuric acid hydrolases characterized to date do not show
cross-reactivity with each other substrates; they are each quite
specific.
[0176] In pairwise comparisons, the barbiturases and cyanuric acid
hydrolases generally show 40-60% relatedness to each other. There
are annotated proteins that show approximately 50% sequence
relatedness to each of a known cyanuric acid hydrolase and a known
barbiturase. Thus, it can be difficult to discern from sequence
alone if a protein is a cyanuric acid hydrolase or a barbiturase.
It also is possible that some of the annotated proteins catalyze a
different reaction altogether.
[0177] In light of these issues, the current study sought to
further define the cyanuric acid/barbiturase protein family. The
total membership in this family to-date was defined. Bioinformatics
initially delineated each protein as a cyanuric acid hydrolase,
barbiturase, or an unknown function protein. Several proteins from
diverse were purified to homogeneity and most were shown to have
cyanuric acid hydrolase activity and were not active with
barbituric acid. One protein was a barbiturase. Another protein was
observed to be unreactive with any substrate tested. A previous
study had suggested that barbiturase was a member of the
amidohydrolase superfamily and thus was a metalloenzyme. The
present study finds no sequence relatedness to the amidohydrolase
superfamily and no evidence for the participation of metals in
catalysis by any member of the cyanuric acid/barbiturase family of
proteins.
MATERIALS AND METHODS
[0178] Spectrophotometric Assay for Cyanuric Acid Degradation.
[0179] The rate of cyanuric acid hydrolysis catalyzed by pure or
partially purified enzyme preparation was determined by monitoring
the disappearance of cyanuric at 220 nm over time. The
spectrophotometer was blanked with 1 ml of 25 mM Tris-HCl, pH 8.0.
Enzyme was added to create an absorbance of 0.05 to 0.4, and
reblanked. To this solution, 1 .mu.l of 0.15 M cyanuric acid was
added. The observed extinction coefficient for cyanuric acid in
this range is 5.654 A220 nm/mM. When 25 mM Tris-HCl, pH 8.25 was
used, the extinction coefficient for cyanuric acid was 5.848 A220
nm/mM. The absorbance of cyanuric acid was monitored over time and
used to calculate the activity of the enzyme.
[0180] Preparation of Soluble Crude Extract.
[0181] E. coli was grown overnight at 30.degree. C. with shaking at
225 rpm in 1 L LB broth containing 50 .mu.g/ml ampicillin.
Isopropyl-1-thio-B-D-galactoside (IPTG) was added to a final
concentration of 1 mM when the culture reached OD600 nm 0.5. Cells
were harvested by centrifugation at 5000.times.g for 15 minutes at
4.degree. C., washed with 500 ml sterile PBS, and resuspended in 40
ml 25 mM MOPS, pH 7.0. The cells were lysed by one passage through
a cold French pressure cell at 18,000 lb/in2. The extract was
clarified initially by centrifugation at 13,000.times.g for 15
minutes at 4.degree. C., followed by centrifugation at
25,000.times.g for 90 minutes at 4.degree. C. This supernatant was
designated the crude, cell-free soluble enzyme fraction and was
used for further enzyme purification.
[0182] Results
[0183] The subunit sizes of the different cyanuric acid/barbiturase
homologs were typically 350-380 amino acids with a molecular weight
of approximately 40,000. The polypeptides are generally acidic with
pI values in the range of 5-6.
[0184] The k.sub.cat and K.sub.m values for the cyanuric acid
hydrolases are within an order of magnitude of each other.
K.sub.cat ranges from 5-73 s.sup.-1 and the K.sub.m from 19-58
.mu.M. These give k.sub.cat/K.sub.m in the range of 10.sup.5 to
10.sup.6. These values suggest that cyanuric acid hydrolysis is the
true physiological function of these enzymes.
TABLE-US-00007 TABLE 4 Kinetic Properties of Cyanuric Acid
Hydrolases Organism k.sub.cat (s.sup.-1) K.sub.m (.mu.M)
k.sub.cat/K.sub.m (s.sup.-1 M.sup.-1) TrzD (literature) 250 50 5 10
.sup.6 TrzD (current study) 14.2 .+-. 0.4 58 .+-. 7 2.5 10 .sup.5
Pseudomonas sp. ADP-AtzD 6.8 .+-. 0.7 57 .+-. 10 1.2 10 .sup.5
(literature) Pseudomonas sp. ADP-AtzD 73 .+-. 6 23 .+-. 7 3.2
10.sup.6 (current study) Moorella thermoacetica ATCC 10.6 110 1.0
10 .sup.5 39073 (literature) Bradyrhizobium japonicum 9.3 .+-. 0.7
50 .+-. 10 1.9 10 .sup.5 USDA 110 Rhizobium leguminosarum 5 .+-. 1
130 .+-. 60 3.8 10 .sup.4 bv. viciae 3841 Methylobacterium sp. 4-46
17 .+-. 2 69 .+-. 16 2.5 10 .sup.5 Note: All enzymes in the current
study were stored at 4.degree. C. and never frozen. Stability was
monitored to ensure no deactivation occurred throughout the
study.
[0185] Although the foregoing specification and examples fully
disclose and enable the present invention, they are not intended to
limit the scope of the invention, which is defined by the claims
appended hereto.
[0186] All publications, patents and patent applications are
incorporated herein by reference. While in the foregoing
specification this invention has been described in relation to
certain embodiments thereof, and many details have been set forth
for purposes of illustration, it will be apparent to those skilled
in the art that the invention is susceptible to additional
embodiments and that certain of the details described herein may be
varied considerably without departing from the basic principles of
the invention.
[0187] The use of the terms "a" and "an" and "the" and similar
referents in the context of describing the invention are to be
construed to cover both the singular and the plural, unless
otherwise indicated herein or clearly contradicted by context. The
terms "comprising," "having," "including," and "containing" are to
be construed as open-ended terms (i.e., meaning "including, but not
limited to") unless otherwise noted. Recitation of ranges of values
herein are merely intended to serve as a shorthand method of
referring individually to each separate value falling within the
range, unless otherwise indicated herein, and each separate value
is incorporated into the specification as if it were individually
recited herein. All methods described herein can be performed in
any suitable order unless otherwise indicated herein or otherwise
clearly contradicted by context. The use of any and all examples,
or exemplary language (e.g., "such as") provided herein, is
intended merely to better illuminate the invention and does not
pose a limitation on the scope of the invention unless otherwise
claimed. No language in the specification should be construed as
indicating any non-claimed element as essential to the practice of
the invention.
[0188] Embodiments of this invention are described herein,
including the best mode known to the inventors for carrying out the
invention. Variations of those embodiments may become apparent to
those of ordinary skill in the art upon reading the foregoing
description. The inventors expect skilled artisans to employ such
variations as appropriate, and the inventors intend for the
invention to be practiced otherwise than as specifically described
herein. Accordingly, this invention includes all modifications and
equivalents of the subject matter recited in the claims appended
hereto as permitted by applicable law. Moreover, any combination of
the above-described elements in all possible variations thereof is
encompassed by the invention unless otherwise indicated herein or
otherwise clearly contradicted by context.
Sequence CWU 1
1
18131DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 1gcgaattcca tatgcaaaaa gttgaagtct t
31229DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 2gccaagcttc tacaccctgg caataacag 293367PRTMoorella
thermoacetica 3Met Gln Lys Val Glu Val Phe Arg Ile Pro Thr Ala Ser
Pro Asp Asp 1 5 10 15 Ile Ser Gly Leu Ala Thr Leu Ile Asp Ser Gly
Lys Ile Asn Pro Ala 20 25 30 Glu Ile Val Ala Ile Leu Gly Lys Thr
Glu Gly Asn Gly Cys Val Asn 35 40 45 Asp Phe Thr Arg Gly Phe Ala
Thr Gln Ser Leu Ala Met Tyr Leu Ala 50 55 60 Glu Lys Leu Gly Ile
Ser Arg Glu Glu Val Val Lys Lys Val Ala Phe 65 70 75 80 Ile Met Ser
Gly Gly Thr Glu Gly Val Met Thr Pro His Ile Thr Val 85 90 95 Phe
Val Arg Lys Asp Val Gln Glu Pro Ala Lys Pro Gly Lys Arg Leu 100 105
110 Ala Val Gly Val Ala Phe Thr Arg Asp Phe Leu Pro Glu Glu Leu Gly
115 120 125 Arg Met Glu Gln Val Asn Glu Val Ala Arg Ala Val Lys Glu
Ala Met 130 135 140 Lys Asp Ala Gln Ile Asp Asp Pro Arg Asp Val His
Phe Val Gln Ile 145 150 155 160 Lys Cys Pro Leu Leu Thr Ala Glu Arg
Ile Glu Asp Ala Lys Arg Arg 165 170 175 Gly Lys Asp Val Val Val Asn
Asp Thr Tyr Lys Ser Met Ala Tyr Ser 180 185 190 Arg Gly Ala Ser Ala
Leu Gly Val Ala Leu Ala Leu Gly Glu Ile Ser 195 200 205 Ala Asp Lys
Ile Ser Asn Glu Ala Ile Cys His Asp Trp Asn Leu Tyr 210 215 220 Ser
Ser Val Ala Ser Thr Ser Ala Gly Val Glu Leu Leu Asn Asp Glu 225 230
235 240 Ile Ile Val Val Gly Asn Ser Thr Asn Ser Ala Ser Asp Leu Val
Ile 245 250 255 Gly His Ser Val Met Lys Asp Ala Ile Asp Ala Asp Ala
Val Arg Ala 260 265 270 Ala Leu Lys Asp Ala Gly Leu Lys Phe Asp Cys
Cys Pro Pro Ala Glu 275 280 285 Glu Leu Ala Lys Ile Val Asn Val Leu
Ala Lys Ala Glu Ala Ala Ser 290 295 300 Ser Gly Thr Val Arg Gly Arg
Arg Asn Thr Met Leu Asp Asp Ser Asp 305 310 315 320 Ile Asn His Thr
Arg Ser Ala Arg Ala Val Val Asn Ala Val Ile Ala 325 330 335 Ser Val
Val Gly Asp Pro Met Val Tyr Val Ser Gly Gly Ala Glu His 340 345 350
Gln Gly Pro Asp Gly Gly Gly Pro Ile Ala Val Ile Ala Arg Val 355 360
365 41104DNAMoorella thermoacetica 4ctacaccctg gcaataacag
caattgggcc accgccatca ggcccttgat gctctgcacc 60accggaaacg tagaccatag
gatctcctac cacgctggca ataacagcat ttactactgc 120tcgcgccgag
cgggtatgat tgatatcaga gtcatcaagc atcgtgttac gcctacccct
180tactgtacca gaagatgcgg cctcagcctt ggccagtaca ttaacgatct
tagcaagctc 240ttctgctggc gggcaacaat caaattttaa accggcatct
ttaagggcag cacgtactgc 300atcagcgtca atggcatcct tcataacaga
gtggcctata accaaatcac tggcactatt 360ggtagagttt cctactacga
taatttcgtc attaagaagt tcaacccccg ctgacgtcga 420agccacacta
gagtagagat tccagtcatg acaaattgct tcgttgctaa tcttatccgc
480agatatctcg cccagtgcga gggccactcc gagagctgag gcgccacgtg
agtaagccat 540tgatttataa gtgtcattta ccacaacatc tttcccgcgt
cgcttggcat cctcaattct 600ttcagcagtc aaaagcgggc actttatctg
aacaaagtga acgtcgcggg gatcatctat 660ttgggcgtct ttcatagcct
cttttacagc tcgagccact tcgtttacct gttccatccg 720gcccaattct
tccggcagaa agtcccgcgt aaaagctacg cctactgcca agcgctttcc
780tggcttagct ggttcctgga catcttttcg gacaaagaca gtaatgtgcg
gcgtcataac 840accctcagta ccgcctgaca ttataaacgc aacttttttt
acaacttctt cgcggcttat 900tcccaatttt tctgctagat acattgctag
agattgggta gcaaaaccgc gagtaaaatc 960gttaacacaa ccattacctt
ccgtcttgcc cagaatagct acaatttcag ccggattaat 1020cttccctgag
tcaatcaaag tagccaaccc gctgatatca tcaggtgagg ctgttgggat
1080acgaaagact tcaacttttt gcat 1104514PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 5Xaa
Lys Thr Glu Gly Asn Gly Xaa Val Asn Asp Xaa Xaa Arg 1 5 10
613PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 6Xaa Met Ser Gly Gly Thr Glu Gly Xaa Xaa Xaa Pro
His 1 5 10 75PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 7Glu Xaa Xaa Gly Xaa 1 5
813PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 8Xaa Xaa His Xaa Val Gln Xaa Lys Cys Pro Leu Leu
Thr 1 5 10 912PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 9Ser Met Xaa Xaa Xaa Arg Xaa Ala Xaa Ala
Leu Gly 1 5 10 1022PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 10Xaa Xaa Ser Xaa Gly Xaa Glu Leu Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Gly Xaa Xaa Xaa Xaa Xaa 20
119PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 11His Xaa Xaa Met Xaa Asp Xaa Xaa Asp 1 5
123PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 12Lys Ala Glu 1 1321PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 13Xaa
Xaa Xaa Arg Xaa Xaa Met Xaa Xaa Asp Xaa Asp Xaa Xaa Xaa Thr 1 5 10
15 Arg Xaa Ala Arg Xaa 20 1417PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 14Xaa Xaa Val Ser Gly Gly Xaa
Glu His Gln Gly Pro Xaa Gly Gly Gly 1 5 10 15 Pro 1512PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 15Thr
Glu Gly Asn Gly Xaa Xaa Asn Asp Xaa Xaa Arg 1 5 10
1612PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 16Thr Glu Gly Asn Gly Cys Val Asn Asp Phe Thr Arg
1 5 10 1717PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 17Xaa Xaa Xaa Ser Gly Gly Xaa Gly Xaa Xaa Xaa Pro
His Xaa Xaa Xaa 1 5 10 15 Xaa 1816PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 18Phe Ile Met Ser Gly Gly
Glu Gly Val Met Thr Pro His Thr Val Phe 1 5 10 15
* * * * *