Note: Descriptions are shown in the official language in which they were submitted.
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PCT/LS2013/021133
COMPOSITIONS USEFUL
FOR THE HYDROLYSIS OF GUAR IN HIGH pH ENVIRONMENTS AND
METHODS RELATED THERETO
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BACKGROUND OF THE INVENTION
Field of the Invention
[0001] The invention relates to gelled fracturing fluids used in well bore
operations. More
specifically, the present invention relates to methods of hydrolyzing gelled
fracturing fluids
using enzymes incorporated in the gelled fracturing fluids, particularly in
environments having
elevated pH values.
Description of the Related Art
[0002] Hydraulic fracturing is used to create subterranean fractures that
extend from the
borehole into rock formation in order to increase the rate at which fluids can
be produced by the
formation. Generally, a high viscosity fracturing fluid is pumped into the
well at sufficient
pressure to fracture the subterranean formation. In order to maintain the
increased exposure to
the formation, a solid proppant is added to the fracturing fluid which is
carried into the fracture
by the high pressure applied to the fluid.
[0003] Some conventional fracturing fluids include guar gum (galactomannans)
or guar gum
derivatives, such as hydroxypropyl guar (HPG), carboxymethyl guar (CMG), or
carboxymethylhydroxypropyl guar (CMHPG). These polymers can be crosslinked
together in
order to increase their viscosities and increase their capabilities of
proppant transport.
[0004] Once the formation is adequately fractured and the proppant is in
place, the fracturing
fluid is recovered typically through the use of breakers. Breakers generally
reduce the fluid's
viscosity to a low enough value that allows the proppant to settle into the
fracture and thereby
increase the exposure of the formation to the well. Breakers work by reducing
the molecular
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weight of the polymers, which "breaks" the polymer. The fracture then becomes
a high
permeability conduit for fluids and gas to be produced back to the well.
[0005] Besides providing a breaking mechanism for the gelled fluid to
facilitate recovery of the
fluid, breakers can also be used to control the timing of the breaking of the
fracturing fluids,
which is important. Gels that break prematurely can cause suspended proppant
material to settle
out of the gel before being introduced a sufficient distance into the produced
fracture. Premature
breaking can also result in a premature reduction in the fluid viscosity
resulting in a less than
desirable fracture length in the fracture being created.
[0006] On the other hand, gelled fluids that break too slowly can cause slow
recovery of the
fracturing fluid and a delay in resuming the production of formation fluids.
Additional problems
can result, such as the tendency of proppant to become dislodged from the
fracture, resulting in a
less than desirable closing and decreased efficiency of the fracturing
operation.
[0007] For purposes of the present application, premature breaking will be
understood to mean
that the gel viscosity becomes diminished to an undesirable extent before all
of the fluid is
introduced into the formation to be fractured.
[0008] Optimally, the fracturing gel will begin to break when the pumping
operations are
concluded. For practical purposes, the gel should be completely broken within
a specific period
of time after completion of the fracturing period. At higher temperatures, for
example, about 24
hours is sufficient. A completely broken gel will be taken to mean one that
can be flushed from
the formation by the flowing formation fluids or that can be recovered by a
swabbing operation.
In the laboratory setting, a completely broken, non-crosslinked gel is one
whose viscosity is
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either about 10 centipoises or less as measured on a Model 50 Fann viscometer
RUB! at 300 rpm
or less than 100 centipoises by Brookfield viscomctcr spindle #1 at 0.3 rpm.
[00091 By way of comparison, certain gels, such as those based upon guar
polymers, undergo a
natural break without the intervention of chemical additives. The break time
can be excessively
long, however. Accordingly, to decrease the break time of gels used in
fracturing, chemical
agents are incorporated into the gel and become a part of the gel itself
Typically, these agents
are either oxidants or enzymes that operate to degrade the polymeric gel
structure.
[0010] However, obtaining controlled breaks using various chemical agents,
such as oxidants or
enzymes, has proved difficult. Common oxidants are ineffective at low
temperature ranges from
ambient temperature to 130 F. The common oxidants require either higher
temperatures to
cause homolytic cleavage of the peroxide linkage or a coreactant to initiate
cleavage. Common
oxidants do not break the polysaccharide backbone into monosaccharide units.
The breaks are
nonspecific, creating a mixture of macromolecules. Further, common oxidants
are difficult to
control because they not only attack the polymer, but they also react with any
other molecule
that is prone to oxidation. Oxidants can react, for example, with the tubing
and linings used in
the oil industry, as well as, resins on resin coated proppants.
[0011] Enzymes, on the other hand, are catalytic and substrate specific and
will catalyze the
hydrolysis of specific bonds on the polymer. Using enzymes for controlled
breaks circumvents
the oxidant temperature problems, as the enzymes are effective at the lower
temperatures. An
enzyme will degrade many polymer bonds in the course of its useful lifetime.
Unfortunately,
enzymes operate under a narrow pH range and their functional states are often
inactivated at high
pH values. Conventional enzymes used to degrade galactomannans have maximum
catalytic
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activities under mildly acidic to neutral conditions (pH 5 to 7). Activity
profiles have indicated
that the enzyme retains little to no activity past this point. Enzymatic
activity rapidly declines
after exceeding pH 8.0 and denatures above pH 9Ø In the case of borate
crosslinked guar gels,
the gels are also pH dependant requiring pH in excess of 8.0 to initiate
gellation. As the pH
increases, the resulting gel becomes stronger. Normally, when enzymes are used
with borate
crosslinked fluids these gels are buffered to maintain a pH range of 8.2 to
8.5 to ensure both
gellation and enzymatic degradation. This technique requires high
concentrations of both borate
and enzyme. Unfortunately, while ensuring good breaks, the initial gel
stability and proppant
transport capability is weakened. The determination of the optimum enzyme
concentration is a
compromise between initial gel stability and an adequate break.
[0012] Because most guar polymers are crosslinked at pH values between 9.5 and
11.0 for
fracturing applications, a need exists for a breaker that can degrade guar-
based fracturing fluids
within that range, such as at pH ranges > 10.5. A need also exists for a gel
system for a well
fracturing operation that can break the gel polymers within a wide range of pH
values at low to
moderate temperatures without interfering with the crosslinking chemistry. It
would be
advantageous to provide an enzyme breaker system for a gelled fracturing fluid
that produces a
controlled break over a wide pH range and at low temperatures and that
decreases the amount
and size of residue left in the formation after recovery of the fluid from the
formation.
SUMMARY OF THE INVENTION
[0013] In view of the foregoing, methods and compositions for fracturing
subterranean
formations are provided that effectively hydrolyze fracturing fluids,
particularly at elevated pH
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values. The methods and compositions for fracturing subterranean formations
use enzyme
breakers that arc effective at elevated pH values.
[0014] As an embodiment of the present invention, a method of fracturing a
subterranean
formation that is penetrated by a well bore is provided. In this embodiment, a
crosslinked
polymer gel is provided that includes an aqueous fluid, a hydratable polymer,
a crosslinking
agent capable of crosslinking the hydratable polymer, and a glycoside
hydrolase enzyme breaker.
Glycoside hydrolases hydrolyze the glycosidic bond between two or more
carbohydrates or
between a carbohydrate and a non-carbohydrate moiety. The crosslinkcd polymer
gel is then
pumped to a desired location within the well bore under sufficient pressure to
fracture the
surrounding subterranean formation. Once the fracturing is complete, the
enzyme breaker is
allowed to degrade the crosslinked polymer gel so that it can be recovered or
removed from the
subterranean formation. The enzyme breaker is catalytically active and
temperature stable in a
temperature range of about 60 F to about 225 F.
[0015] Preferred glycoside hydrolase enzyme breakers are those selected from
the group
consisting of glycoside hydrolase family 5 and glycoside hydrolase family 26
as well as mixtures
thereof
[0016] In another embodiment, the preferred enzyme breaker is an alkaline P-
mannanase.
Especially preferred alkaline I3-mannanase breakers are those derived from a
gene having
engineered restriction endonuclease sites, such as Xhol and Bell, flanking the
5' and the 3' ends
of the gene coding for the alkaline 13-mannanase enzyme. In an aspect, the
gene is codon
optimized for expression in E. coli. In another aspect, the gene produces a
GST-mannanase
fusion protein.
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[0017] Another embodiment relates to a method of fracturing a subterranean
formation that
surrounds a well bore is provided as another embodiment of the present
invention. In this
embodiment, a crosslinked polymer gel is formed that includes an aqueous
fluid, a hydratable
polymer, a crosslinking agent capable of crosslinking the hydratable polymer
and an enzyme
breaker selected from the group consisting of glycoside hydrolase family 5 and
glycoside
hydrolase family 26 to produce a crosslinked polymer as well as those alkaline
I3-mannanases
derived from a gene having engineered restriction endonuclease sites, such as
Xhol and Bell,
flanking the 5' and the 3' ends of the gene coding for the alkaline P-
mannanase enzyme. In an
aspect, the gene is codon optimized for expression in E. co/i. In another
aspect, the gene
produces a GST-mannanase fusion protein. Once the crosslinked polymer gel is
formed, it is
pumped to a desired location within the well bore under sufficient pressure to
fracture the
surrounding subterranean formation. The enzyme breaker is allowed to degrade
the crosslinked
polymer gel so that it can be recovered or removed from the subterranean
formation.
The enzyme breaker is catalytically active and temperature stable in a
temperature range of about
60 F to about 225 F and in a pH range of about 7 to about 12, with the
maximum catalytic
activity at pH 10.5 ¨ 11.5.
[0018] Besides the method embodiments, compositions are also provided as
embodiments of the
present invention. As another embodiment of the present invention, a
fracturing fluid
composition is provided. The fracturing fluid comprises an aqueous fluid, a
hydratable polymer,
a crosslinking agent capable of crosslinking the hydratable polymer, and an
enzyme breaker such
as those selected from the group consisting of glycoside hydrolase family 5
and glycoside
hydrolase family 26 as well as alkaline P-mannanase such as those designated
above. As with
the other embodiments, the enzyme breaker is catalytically active and
temperature stable in a
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temperature range of about 60 F to about 225 F and in a pH range of about 7
to about 12,
with the maximum catalytic activity at pH 10.5 - 11.5.
100191 In an embodiment of the present invention, the enzyme breaker used in
the methods
and compositions described herein may be derived from a gene having engineered
restriction
endonuclease sites, such as Xhol and Bell, flanking the 5' and the 3' ends of
the gene coding
for the alkaline 13-mannanase enzyme. In an aspect, the gene is codon
optimized for
expression in E. coli. In another aspect, the gene produces a GST-mannanase
fusion protein.
[0019a] Accordingly, in one aspect of the present invention there is provided
A method of
fracturing a subterranean formation that surrounds a well bore, the method
comprising the
steps of: a) combining an aqueous fluid, a hydratable polymer, a crosslinking
agent, and an
enzyme breaker comprising an alkaliphile derived enzyme breaker selected from
the group
consisting of glycoside hydrolase 'family 5, glycoside hydrolase family 26 and
mixtures
thereof wherein the enzyme breaker of glycoside hydrolase family 5 is selected
from the
group consisting of endoglucanase, exo-1,3-glucanase, endo-1,6-glucanase,
xylanase,
endoglycoceramidase, chitosanase, 13-mannosidase, cellulose, glucan P-1,3-
glucosidase,
licheninase, glucan endo-1,6-13-glucosidase, mannan endo-i3-1,4-mannosidase,
endo-p-1,4-
xylanase, cellulose 13-1,4-cellobiosidase, xyloglucan-specific endo-13-1,4-
glucanase, mannan
transglycosylase, endo-13-1,6-galactanase, endoglycoceramidase and mixtures
thereof; b)
injecting the crosslinked polymer gel into the well bore and into contact with
the formation
under sufficient pressure to fracture the surrounding subterranean formation;
and c) allowing
the enzyme breaker to degrade the crosslinked polymer gel so that it can be
removed from the
subterranean formation, the enzyme breaker being catalytically active and
temperature stable
in a temperature range of about 60 F to about 225 F and in a pH range of
about 7 to about
12, wherein: i) the enzyme breaker is stored at or below refrigeration
temperature prior to
being combined with the aqueous fluid, hydratable polymer and crosslinking
agent, or ii) the
enzyme breaker is frozen, or iii) prior to combining the enzyme breaker with
the aqueous
fluid, the hydratable polymer and the crosslinking agent: (A) an alkaliphile
from which the
enzyme of the enzyme breaker is derived is frozen, (B) the product of step (A)
is thawed, and
(C) the enzyme breaker is derived from the alkaliphile.
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BRIEF DESCRIPTION OF THE DRAWINGS
[0020] Figure IA is the sequence of the gene that codes for an enzyme breaker
made in
accordance with embodiments of the present invention;
[0021] Figure 1B is a comparison of the gene sequence of Figure 1A with the
gene sequence
of a gene coded for a prior art enzyme;
[0022] Figure 2 is a schematic illustrating the creation of the plasmids pGS-
21a-hp/3 and
pUC57-hpfl which may be used in the enzyme breaker in accordance with
embodiments of
the present invention;
[0023] Figure 3 is a graph that illustrates the degradation in the viscosity
of 18 ppt
crosslinked guar GW-3 after 1 hour and 18 hours at pH values 11.0, 12.0, and
13.0 by an
enzyme breaker made in accordance with embodiments of the present invention;
[0024] Figure 4 is a bar graph that illustrates the degradation in the
viscosity of 18 ppt
crosslinked guar GW-3 after 1 hour and 18 hours at pH values 1 1.0, 12.0, and
13.0
comparing
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the crosslinked guar GW-3 having no enzyme breaker and the crosslinked guar GW-
3 having an
enzyme breaker made in accordance with embodiments of the present invention;
[0025] Figure 5 is a graph that illustrates the viscosity reduction in 50 ppt
non-crosslinked guar
GW-3 after 18 hours using an enzyme breaker made in accordance with
embodiments of the
present invention;
[0026] Figure 6 is a graph that illustrates the effect on the viscosity of 30
ppt crosslinked guar
GW-3 at a pH of 10.5 using different loadings of an enzyme breaker made in
accordance with
embodiments of the present invention;
[0027] Figure 7 is a graph that illustrates the effect on the viscosity of 30
ppt crosslinked guar
GW-3 at a pH of 10.5 of adding different types of divalent cations to an
enzyme breaker made in
accordance with embodiments of the present invention; and
[0028] Figure 8 is a graph that illustrates the shelf-life of an enzyme
breaker made in accordance
with the embodiments of the present invention, wherein the enzyme breaker was
stored at
various concentrations and temperatures and its activity reported with respect
to time.
[0029]
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DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
[0030] Illustrative embodiments of the invention are described below as they
might be employed
in the operation and in the treatment of oilfield applications. In the
interest of clarity, not all
features of an actual implementation are described in this specification. It
will of course be
appreciated that in the development of any such actual embodiment, numerous
implementation-
specific decisions must be made to achieve the developers' specific goals,
which will vary from
one implementation to another. Moreover, it will be appreciated that such a
development effort
might be complex and time-consuming, but would nevertheless be a routine
undertaking for
those of ordinary skill in the art having the benefit of this disclosure.
Further aspects and
advantages of the various embodiments of the invention will become apparent
from
consideration of the following description.
[0031] As an embodiment of the present invention, a method of fracturing a
subterranean
formation that surrounds a well bore is provided. In this embodiment, a
crosslinked polymer gel
is provided that includes an aqueous fluid, a hydratable polymer, a
crosslinking agent capable of
crosslinking the hydratable polymer and a glycoside hydrolase enzyme breaker
to produce a
crosslinked polymer. The erosslinked polymer gel is then injected to a desired
location within
the well bore and into contact with the formation under sufficient pressure to
fracture the
surrounding subterranean formation. Once the fracturing is complete, the
enzyme breaker is
allowed to degrade the crosslinked polymer gel so that it can be recovered or
removed from the
subterranean formation. The enzyme breaker is catalytically active and
temperature stable in a
temperature range of about 60 F to about 225 F.
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[0032] Another method of fracturing a subterranean formation that surrounds a
well bore is
provided as another embodiment of the present invention. In this embodiment, a
gelled fluid is
formed by combining an aqueous fluid, a hydratable polymer, and a glycoside
hydrolase enzyme
breaker. A crosslinking agent capable of crosslinking the gelled fluid is then
added to form a
crosslinked polymer gel having sufficient viscosity to facilitate fracturing
of the formation.
Once the crosslinked polymer gel is formed, it is injected to a desired
location within the well
bore and into contact with the formation under sufficient pressure to fracture
the surrounding
subterranean formation. The enzyme breaker is allowed to degrade the
crosslinked polymer gel
so that it can be recovered or removed from the subterranean formation. The
enzyme breaker is
catalytically active and temperature stable in a temperature range of about 60
F to about 225 F
and in a pH range of about 7 to about 12, with the maximum catalytic activity
at pH 10.5 ¨ 11.5.
[0033] Besides the method embodiments, compositions are also provided as
embodiments of the
present invention. As another embodiment of the present invention, a
fracturing fluid
composition is provided. The fracturing fluid comprises an aqueous fluid, a
hydratable polymer,
a crosslinking agent capable of crosslinking the hydratable polymer, and a
glycoside hydrolase
enzyme breaker. As with the other embodiments, the enzyme breaker is
catalytically active and
temperature stable in a temperature range of about 60 F to about 225 F and
in a pH range of
about 7 to about 12, with the maximum catalytic activity at pH 10.5 ¨ 11.5.
[0034] The enzyme breaker of the present invention preferably comprises
glycoside hydrolases
of Family 5 or Family 26 of the Carbohydrate-Active enZYmes (CAZy), as updated
on January
9, 2012, developed by the Glycogenomics group at AFMB in Marseille, France.
Glycoside
hydrolase family 5 (GH5) are retaining enzymes with several known activities;
endoglucanase
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(EC:3.2.1.4); beta-mannanase (EC:3.2.1.78); exo-1,3-glucanase (EC:3.2.1.58);
endo-1,6-
glucanasc (EC:3.2.1.75); xylanasc (EC:3.2.1.8); cndoglycoceramidase
(EC:3.2.1.123). Glycoside
hydrolases of Family 5 include chitosanase (EC 3.2.1.132); P-mannosidase (EC
3.2.1.25);
cellulase (EC 3.2.1.4); glucan13-1,3-glucosidase (EC 3.2.1.58); licheninase
(EC 3.2.1.73); glucan
endo-1,6-13-glucosidase (EC 3.2.1.75); mannan endo-13-1,4-mannosidase (EC
3.2.1.78); endo-13-
1,4-xylanase (EC 3.2.1.8); cellulose 13-1,4-cellobiosidase (EC 3.2.1.91); 13-
1,3-mannanase (EC
3.2.1.-); xyloglucan-specific endo-3-1,4-glucanase (EC 3.2.1.151); mannan
transglycosylase (EC
2.4.1.-); endo-P-1,6-galaetanase (EC 3.2.1.164); and endoglycoceramidase (EC
3.2.1.123).
[0035] In a preferred embodiment, enzymes of Subfamily 8 of Glycoside
hydrolase Family 5
may be used. Such retaining enzymes include the enzyme breaker derived from a
gene of the
alkaliphilic Bacillus sp. N16-5. Such enzymes exhibit a pH optimum of
enzymatic activity at
about 9.5 and fold into a (Pict)(8)-barrel fold with two active site glutamic
acids being
approximately 200 residues apart in sequence and located at the C-terminal
ends of 13-strands 4
(acid/base) and 7 (nueleophile).
[0036] In another preferred embodiment, the enzyme breaker is an alkaline 13-
mannanase such as
the alkaline P-mannanase derived from a gene having engineered restriction
endonuclease sites,
such as Xhol and Bell, flanking the 5' and the 3' ends of the gene coding for
the alkaline [3-
mannanase enzyme. In an aspect, the gene is codon optimized for expression in
E. coll. In
another aspect, the gene produces a GST-mannanase fusion protein.
[0037] The enzyme breaker of the present invention can be prepared in
accordance with the
methods described in Example 1 of this specification. The enzyme breaker of
the present
invention catalyzes the random hydrolysis of 3-(1,4) mannosidic linkages and
can be used to
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break the polymer backbone of galactomannan polymers. Unlike the conventional
enzyme prior
art products, the enzyme breaker of the present invention does not require the
action of an
associated a-galactosidase in order to function.
[0038] In embodiments of the present invention, the enzyme breaker is derived
from a gene
having engineered restriction endonuclease sites, such as Xhol and Bell,
flanking the 5' and the
3' ends of the gene coding for the I3-mannanase enzyme. In an aspect, the gene
is codon
optimized for expression in E. coll. In another aspect, the gene codes for an
expressed N-
terminal GST fusion protein.
[0039] In addition, enzymes within Glycoside Hydrolases of Family 26 (GH26)
are preferred.
GH26 encompasses 13-mannanase (EC 3.2.1.78, mainly mannan endo-1,4-beta-
mannosidases
which randomly hydrolyze 1,4-beta-D-linkages in mannans, galactomannans,
glucomannans and
galactoglucomannans, as well as 3-1,3-xylanase (EC 3.2.1.32). The glycoside
hydrolases of GH
26 display little, if any, activity towards other plant cell wall
polysaccharides.
[0040] The enzyme breaker may be stored prior to being combined into the
aqueous fluid. In an
embodiment, for instance, the enzyme breaker may be refrigerated prior to
being combined with
the aqueous fluid. In another embodiment, the alkaliphile from which the
enzyme breaker is
derived may be refrigerated prior to being combined with the aqueous fluid.
The enzyme, upon
being removed from storage, may then be derived from the alkaliphile. The
enzyme breaker is
typically brought to room temperature prior to being combined with the aqueous
fluid.
[0041] In another embodiment, the enzyme breaker or the alkaliphile from which
the enzyme is
derived may be stored in a frozen state. In this embodiment, the enzyme
breaker or the
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alkaliphile may be combined with a winterizing agent, such as a glycerol,
during storage. Upon
being thawed, the enzyme breaker may be derived from the alkaliphilc and
preferably brought to
room temperature prior to being added to the aqueous fluid. Where the enzyme
is stored in a
frozen state, the enzyme is thawed and preferably brought to room temperature
prior to being
introduced to the aqueous fluid.
[0042] The enzyme can be diluted in various concentrations that are effective
and convenient for
use in fracturing jobs. In an aspect, the enzyme breaker of the present
invention is diluted to a
concentration of about 1:24 and is present in the crosslinkcd polymer gel in a
range of about 0.25
gpt to about 4 gpt; alternatively, in a range of about 0.5 gpt to about 2.5
gpt; alternatively, in a
range of about 0.5 gpt to about 1 gpt; or alternatively, in a range of about 1
gpt to about 2 gpt.
Other suitable dilution concentrations and amounts of enzyme breaker will be
apparent to those
of skill in the art and are to be considered within the scope of the present
invention. In an aspect,
the total protein concentration of the stock enzyme breaker from which the
dilutions are made is
greater than 1 mg/mL.
[0043] Conventional enzymes used to degrade galactomannans work well in
environments
having a pH value between 4.0 and 8Ø At elevated pH ranges (> pH 10.0) these
enzymes
quickly denature and lose activity. The typical 0-mannanase enzyme has a pH
optimum of ¨5Ø
This would suggest that the enzyme loses >90% of its activity at pH values
greater than 8Ø
Since most guar polymers are crosslinked at pH values between 9.5 and 11.0 for
fracturing
applications, it is beneficial to have an enzyme that can degrade guar under
elevated pH
conditions without an additional step to reduce the pH first.
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[0044] As indicated herein, the enzyme breaker of the present invention can be
used in a wide
range of temperatures and pH values. In an aspect, the enzyme breaker of the
present invention
can be used in applications having a temperature that ranges from about 60 F
to about 225 F;
or alternatively, in a range from about 120 F to about 225 F. In yet another
aspect, the enzyme
breaker can be catalytically active and temperature stable in a pH range of
about 7 to about 12;
alternatively, in a range of about 9.5 to about 11.5; or alternatively, in a
range from about 10.5 to
about 11.
[0045] In an aspect, the enzyme breaker of the present invention can include
an alkaline enzyme.
As used herein, the term "alkaline enzyme" generally refers to enzymes that
display their
maximum catalytic activity somewhere within a pH range of about 8.0 to about
14Ø In an
aspect, the maximum catalytic activity of the alkaline enzyme can be at pH
values above 9Ø In
an aspect, the alkaline enzyme is derived from an alkaliphilic organism. As
used herein, the term
"alkaliphilic organism" generally refers to extremophilic organisms that
thrive in alkaline
conditions somewhere in the pH range of about 8.0 to about 14Ø
[0046] The methods and compositions described herein can be used with a
variety of hydratable
polymers. In an aspect, the hydratable polymer has repeating units of mannose
linked by 3-(1,4)
mannosidic linkages. In another aspect, the hydratable polymer comprises guar,
guar
derivatives, cellulose derivatives, water soluble biopolymers, or combinations
thereof. Other
suitable types of hydratable polymers that can be used in the methods and
compositions
described herein will be apparent to those of skill in the art and are to be
considered within the
scope of the present invention.
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[00471 Because the enzyme breaker of the present invention has a maximum
activity under
alkaline pH ranges, it can be combined with other breakers that operate in
different pH ranges to
allow for better control of hydrolysis of fracturing fluids over a much
greater pH range. In an
aspect, the crosslinked polymer gel can further include a second enzyme
breaker that is
catalytically active and temperature stable in a pH range of about 4 to about
8. Suitable enzymes
that can be used include those described in U.S. Patent No. 5,201,370.
[0048] Divalent cations can affect the activity of the enzyme breaker of the
present invention, as
shown and described in Example 5. In an aspect, the erosslinked polymer gel
can further include
a divalent cation. Suitable divalent cations can include Mg', Co, or Me2-.
Other suitable
divalent cations that can be used in the present invention will be apparent to
those of skill in the
art and are to be considered within the scope of the present invention,
[0049] The methods and compositions described herein can be used with a wide
variety of
crosslinking agents. A suitable crosslinking agent can be any compound that
increases the
viscosity of the hydratable polymer by chemical crosslinking, physical
crosslinking, or any other
mechanisms. For example, the gellation of the hydratable polymer can be
achieved by
crosslinking the hydratable polymer with metal ions including borate
compounds, zirconium
compounds, titanium compounds, aluminum compounds, antimony compounds,
chromium
compounds, iron compounds, copper compounds, zinc compounds, or mixtures
thereof. One
class of suitable crosslinking agents is zirconium-based crosslinking agents.
Suitable
crosslinking agents can include zirconium oxychloride, zirconium acetate,
zirconium lactate,
zirconium malate, zirconium glycolate, zirconium lactate triethanolamine,
zirconium citrate, a
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zirconate-based compound, zirconium triethanolamine, an organozirconate, or
combinations
thereof. XLW-14 is a
particularly suitable zirconate-based crosslinking agent that is
commercially available from Baker Hughes Incorporated and described in U.S.
Patent No.
4,534,870. Suitable
borate- containing
crosslinking agents can include, for example, alkaline earth metal borates,
alkali metal-alkaline
earth borates, probertite, ulexite, nobleite, frolovite, colemanite, calcined
colernanite, priceite,
pateroniate, hydroboractie, kaliborite, or combinations thereof. Suitable
titanium-containing
crosslinking agents can include, for example, titanium lactate, titanium
Imitate, titanium citrate,
titanium ammonium lactate, titanium triethanolamine, titanium acetylacetonate,
or combinations
thereof. Suitable aluminum-containing crosslinking agents can include, for
example, aluminum
lactate, aluminum citrate, or combinations thereof. Other suitable
crosslinking agents that are
compatible with the compositions and methods described herein will be apparent
to those of skill
in the art and are to be considered within the scope of the present invention.
[0050] Besides the polymers, crosslinking agents, and enzyme breakers
described herein,
various additives can be useful in the present invention. Additives used in
the oil and gas
industry and known in the art, including but not limited to, corrosion
inhibitors, non-emulsifiers,
iron control agents, delay additives, silt suspenders, flowback additives,
proppants, and gel
breakers, can also be used in embodiments of the present invention. Other
suitable additives
useful in the present invention will be apparent to those of skill in the art
and arc to be
considered within the scope of the present invention.
[0051] The amount of crosslinking agent and other additives used in the
present invention can
vary depending upon the desired effect of the additives. For example, the
crosslinking agent can
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be present in the crosslinked polymer gel in an amount sufficient to provide
the desired degree of
crosslinking between molecules within the hydratablc polymer. The amounts of
additives that
can be used in the present invention will be apparent to those of skill in the
art and are to be
considered within the scope of the present invention.
[0052] As an advantage of the present invention, less enzyme breaker of the
present invention
can be used, when compared with conventional prior art enzymes. The reduction
in the amount
of enzyme breaker needed results in a cost savings in terms of enzyme
production, shipping, and
storage.
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EXAMPLES
[0053] The following examples are included to demonstrate the use of
compositions in
accordance with embodiments of the present invention. It should be appreciated
by those of skill
in the art that the techniques disclosed in the examples that follow represent
techniques
discovered by the inventors to function well in the practice of the invention.
However, those of
skill in the art should, in light of the present disclosure, appreciate that
many changes can be
made in the specific embodiments that are disclosed and still obtain a like or
similar result
without departing from the scope of the invention.
Example 1
[0054] A novel 13-mannanase enzyme was first isolated in the alkaliphilic
extremophile, Bacillus
sp. N16-5 (see Ma et al. (2004) Characterization and Gene Cloning of a Novel
13-mannanase
from Alkaliphilic Bacillus sp. N16-5, Extrentophiles 8, 447-454). The gene
coding this 13-
mannanase was sequenced and the sequence data deposited in the NCBI (National
Center for
Biotechnology Information) PubMed database under the accession number
AY534912. This
gene structure was shown to code for a 50.7 kDa protein with a 32 amino acid
signal sequence
that was post-tranlationaly processed. The smaller, mature form of the enzyme
was found to be
secreted from the microorganism into the extracellular environment.
[0055] To prepare the enzyme breaker of the present invention, the gene
structure coding for the
13-mannanase enzyme isolated by Ma et al. was reengineered to remove the
portion coding for
the protein signal sequence in an attempt to produce a gene product with more
stability, activity
and yield than its wild type precursor isolated by Ma et al. Additionally, the
gene sequence was
codon optimized for expression in E. coli using GenScript's Codon Optimization
algorithm to
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increase the efficiency of its expression in E. coll. Finally, Xho 1 and Bell
restriction
endonuclease sites were engineered into the 5' and 3' ends of the gene,
respectively. The gene,
N16-5, used in the enzyme breaker of the present invention has a 75% identical
sequence with
the wild type p-mannanase gene, as shown in Figures lA and 1B. In Figure 1B,
the top line of
the gene sequence is that of a prior art gene made in accordance with methods
taught by Ma et al
and the second line of the gene sequence is that of the enzyme breaker of the
present invention.
The gene sequences for the wild-type gene and the optimized gene were aligned
with the
ClustalW sequence alignment algorithm so that a comparison could be made on a
line-by-line
basis of the two genes. The gene of the present invention, designated hp/3,
was cloned into the
expression vector pGS-21a and the cloning vector pUC57. This created two new
plasmids, pGS-
21- hpfi and pUC57- hp13, which are shown in Figure 2. Ampr codes for a 13-
lactamase,
rep(pMB1) and fl on represent the origin of replication in their respective
vectors, and MCS
represents the Multiple Cloning Site. The coding region for the GST fusion
site is represented
by the gene gst. The pGS-21a expression vector contains a region coding for
glutathione S-
transferase (GST) protein that can be used to help purify the P-mannanase. The
resultant gene
product is a GST-mannanase fusion protein.
[0056] The plasmids pGS-21a-hpfi and pUC57-hpfl were transformed into
competent BL21
(DE3) E. coil and cultured in 5 mL LB-Miller nutrient media at 98.6 F at 200
RPM for 16
hours. The culture broth was supplemented with 100 ug/mL ampicillan that was
used as an
inoculum for a 100 mL culture of E. coli harboring the plasmids pGS-21a-hpfl
and pUC57- hpfl.
The cultures were grown at 104 F and 200 RPM. After 4 hours, isopropyl-P-D-
thiogalactopyranoside (IPTG) was added to the culture to a final concentration
of 0.1 m1\4. After
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3 hours of incubation in the presence of IPTG, the cells were chilled to 39 F
and harvested by
centrifugation. The culture medium was then discarded and the cells stored at -
4 F until use.
[0057] Cells were thawed and resuspended in 5 mLs chilled 50 mM sodium
phosphate buffer.
Lysozyme was added to a final concentration of 1 mg/mL and the culture was
incubated at room
temperature for 30 minutes. Nucleic acids were disrupted by brief pulses of
sonication. The
resultant cell free extract (CFX) was then passed through a GST high-affinity
resin (Genscript)
following manufacturer's directions. Eluted fractions were pooled,
concentrated, and dialyzed
into 20 mM 2-(n-cyclohexylamino)ethane sulfonic acid (CHES) buffer, pH 9Ø
Enzyme purity
was estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis
(SDS-PAGE) and
found to be > 95% pure.
[0058] The plasmid pUC57-hp13 was transformed into competent DH5aE. co/i and
cultured in
TYE nutrient media at 30 C at 200 RPM for 40 hours. The culture broth was
supplemented with
50 ug/mL ampicillan and 4% glycerol. After 16 hours, fresh ampicillan was
added to the culture
broth. After 40 hours of incubation, the cells were chilled to 4 C and
harvested by centrifugation
at 3,000 rpm for 20 minutes. The culture medium was then discarded and the
cells stored at ¨20
C until use.
[0059] Cells were resuspended in 4.5 times (w/v) chilled 50 mM sodium
phosphate buffer, pH
8.0 and sonicated on ice for three minutes. Cell debris was removed by
centrifugation and the
supernatant was retained for tests.
[0060] In a comparative study, cells were sonicated as above and the sonicate
(cell extract) was
used as the enzyme sample in Examples 2 and 3.
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Example 2
[0061] In this Example, the ability of the enzyme breaker of the present
invention to hydrolyze
the polymannan backbone of the guar polymer was examined. As shown in Figures
2, 3, and 4,
the enzyme breaker of the present invention, Enzyme Hp-13, comprising the
alkaline 13-
mannanasc effectively hydrolyzes the guar polymer at elevated pH ranges.
Enzyme Hp-13 can be
used as a standalone product to degrade high pH guar gels or in combination
with the existing
conventional enzyme products to degrade guar gels over a much broader pH
range.
[0062] The Hp-1.3 enzyme was tested against crosslinkcd guar polymer gels at
pH 11.0, 12.0 and
13.0 using 18 ppt (pounds per thousand pounds fluid) guar polymer GW-3 that is
commercially
available from Baker Hughes Incorporated. The viscosity of each of these
polymer gels was
measured at 1 hour and at 18 hours to observe degradation in the viscosity. As
can be seen in
Figures 3 and 4, the alkaline 13-mannanase of the present invention provides
almost complete
reduction in the viscosity of the guar after 18 hours across all pH ranges
tested. Without
enzyme, the fluid does not break across all pH ranges tested. The control (no
enzyme) at pH
12.0 is included for comparison purposes (Figure 4).
Example 3
[0063] In this Example, the activity of the enzyme breaker of the present
invention Enzyme Hp-
13 from Example 1 was evaluated. The enzyme breaker from Example 1 was added
to 50 ppt
GW-3 polymer. The reduction in viscosity was measured for the GW-3 polymer in
a pH range
of about 4 to about 13, as shown in Figure 5. The total reduction across the
pH values is
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normalized to itself. As can be seen in Figure 5, Enzyme Hp-I3 appears to show
the greatest
activity at a pH value of about 11.
Example 4
[0064] This Example indicates that the enzyme breaker of the present invention
(Enzyme Hp-13)
maintains its effectiveness in a wide range of temperatures, even up to about
225 F, and in a
wide range of loadings. To demonstrate the effectiveness of the enzyme breaker
of the present
invention at 225 F, five samples of crosslinked GW-3 polymer were prepared,
each having a
different loading of the enzyme breaker of the present invention contained
therein. Sample A
represents a crosslinked polymer having no enzyme breaker. Sample B represents
a crosslinked
polymer having 0.5 gpt of the diluted enzyme breaker of the present invention.
Sample C
represents a crosslinked polymer having 1 gpt of the diluted enzyme breaker of
the present
invention. Sample D represents a crosslinked polymer having 2 gpt of the
diluted enzyme
breaker of the present invention. Sample E represents a crosslinked polymer
having 4 gpt of the
diluted enzyme breaker of the present invention. As shown in Figure 6, a
loading of 0.5 gpt
enzyme (Sample B) is sufficient to reduce the viscosity of the fracturing
fluid to below 200 cps
after approximately 3 hours. Additionally, there is no re-healing of the
polymer once it cools to
room temperature (data not shown). Higher loadings of enzyme have shown to be
too aggressive
in the degradation of the polymer leading to a rapid decrease in the viscosity
of the fluid.
[0065] As shown in Figure 6, Enzyme Hp-I3 is voracious and quickly reduces the
viscosity of the
fracturing fluid. In the event that an operator wishes to maintain a high
viscosity fluid for a
longer period of time, a way to slow the activity of the enzyme would be
beneficial. It would be
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important to slow the activity of the enzyme and not hinder or abolish any of
the catalytic
parameters lest re-healing of the cooled polymer result.
Example 5
[0066] Divalent cations can have beneficial or detrimental effects on the
activity of the enzyme
breakers of the present invention. As previously reported in Ma et al. (2004)
Characterization
and Gene Cloning of a Novel (3-mannanase from Alkaliphilic Bacillus sp. N16-5,
Extremophiles
8, 447 - 454, 1.0 mM Mg2-' has the effect of increasing Enzyme Hp-13's
activity while the
presence of 1.0 mM Co2-' decreases enzyme activity. Enzyme Hp-I3 was incubated
in the
presence of 1.0 mM of each of the divalent cations and the activity of the
samples was measured
against crosslinked 30 ppt GW-3, pH 10.5. As shown in Figure 7, Enzyme Hp-I3
has a dramatic
increase in activity when incubated in the presence of 1.0 Mg2-'. While 1.0 mM
Co2-' does
appear to have a slight decrease in the activity of the enzyme, when compared
to the activity of
the enzyme without the divalent cation, this effect does not appear to be very
significant.
Additional tests are required to confirm or deny this result. There are also
additional metal ions
that can be employed to reduce the activity of the enzyme. Currently, the use
of divalent cations
to increase or reduce the rate of catalysis appears promising.
Example 6
[0067] To compare the stability of the enzyme breaker of the present
invention, several enzyme
samples were prepared and compared. The results are shown in Figure 8. Sample
A represents
data for 1.0 mg/mL Enzyme Hp-13 samples stored at 40 F and 72 F, and 5.0
mg/mL Enzyme
Hp-I3 samples stored at 40 F, 72 F, and 120 F. In all cases the data was the
same. Sample B
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represents data for 1.0 mg/mL Enzyme Hp-I3 samples stored at 120 F. To
measure enzyme
activity, incubated enzyme samples were diluted so that the final, working
concentration of
Enzyme Hp-I3 samples was 0.4 ng mL-1 (nanograms/milliliter). Enzymes were
incubated at 102
F in the presence of crosslinked 20 ppt GW-3, pH 10.5, for 16 hours. After 16
hours, sample
viscosities were measured on a Fann 35 after solutions were allowed to cool to
room temperature
for at least 60 minutes to allow rehealing of the crosslinked polymer.
[0068] Typically, the more concentrated an enzyme solution is, the longer it
maintains its
conformational stability and, thus, its activity. Enzyme stocks of varying
concentrations were
prepared and stored at various temperatures as described in this Example and
shown in Figure 8.
Highly concentrated Enzyme Hp-I3 (Sample A) showed remarkable stability, even
after storage
of 2 weeks at 120 F as evidenced by no observable decrease in activity. Even
the 1 mg/mL
stock of Enzyme Hp-I3 (Sample B) was stable over the course of two weeks.
However, there was
an observable decrease in activity of the sample stored at 120 F. This result
was not unexpected
as an increase in temperature leads to a larger increase in the conformational
entropy of the
enzyme in dilute solutions as compared to those in concentrated solutions.
Over time, this leads
to larger populations of unfolded (and inactive) states in the enzyme sample.
[0069] The enzyme breaker of the present invention, Enzyme Hp-3, appears to be
the most
stable when stored as a highly concentrated stock solution (> 5 mg/mL). To
increase longevity
of the enzyme in transit and/or storage, the concentration of the stock
solution of Enzyme Hp-I3
should be > 5.0 mg/mL.
[0070] All of the compositions and/or methods disclosed and claimed herein can
be made and
executed without undue experimentation in light of the present disclosure.
While the
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compositions and methods of this invention have been described in terms of
preferred
embodiments, it will be apparent to those of skill in the art that variations
can be applied to the
compositions and/or methods and in the steps or in the sequence of steps of
the methods
described herein without departing from the concept, spirit and scope of the
invention. More
specifically, it will be apparent that certain agents that are chemically
related can be substituted
for the agents described herein while the same or similar results would be
achieved. All such
similar substitutes and modifications apparent to those skilled in the art are
deemed to be within
the scope and concept of the invention.
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