Classifications

• • C—CHEMISTRY; METALLURGY
  • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
  • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
  • C09K8/00—Compositions for drilling of boreholes or wells; Compositions for treating boreholes or wells, e.g. for completion or for remedial operations
  • C09K8/60—Compositions for stimulating production by acting on the underground formation
  • C09K8/62—Compositions for forming crevices or fractures
  • C09K8/66—Compositions based on water or polar solvents
  • C09K8/68—Compositions based on water or polar solvents containing organic compounds
  • C09K8/685—Compositions based on water or polar solvents containing organic compounds containing cross-linking agents
• • C—CHEMISTRY; METALLURGY
  • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
  • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
  • C09K8/00—Compositions for drilling of boreholes or wells; Compositions for treating boreholes or wells, e.g. for completion or for remedial operations
  • C09K8/60—Compositions for stimulating production by acting on the underground formation
  • C09K8/84—Compositions based on water or polar solvents
  • C09K8/86—Compositions based on water or polar solvents containing organic compounds
  • C09K8/88—Compositions based on water or polar solvents containing organic compounds macromolecular compounds
  • C09K8/887—Compositions based on water or polar solvents containing organic compounds macromolecular compounds containing cross-linking agents
• • C—CHEMISTRY; METALLURGY
  • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
  • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
  • C09K8/00—Compositions for drilling of boreholes or wells; Compositions for treating boreholes or wells, e.g. for completion or for remedial operations
  • C09K8/60—Compositions for stimulating production by acting on the underground formation
  • C09K8/84—Compositions based on water or polar solvents
  • C09K8/86—Compositions based on water or polar solvents containing organic compounds
  • C09K8/88—Compositions based on water or polar solvents containing organic compounds macromolecular compounds
  • C09K8/90—Compositions based on water or polar solvents containing organic compounds macromolecular compounds of natural origin, e.g. polysaccharides, cellulose
• • C—CHEMISTRY; METALLURGY
  • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
  • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
  • C09K2208/00—Aspects relating to compositions of drilling or well treatment fluids
  • C09K2208/24—Bacteria or enzyme containing gel breakers

Definitions

• 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.
• 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.
• a high viscosity fracturing fluid is pumped into the well at sufficient pressure to fracture the subterranean formation.
• a solid proppant is added to the fracturing fluid which is carried into the fracture by the high pressure applied to the fluid.
• 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.
• guar gum galactomannans
• HPG hydroxypropyl guar
• CMG carboxymethyl guar
• CMHPG carboxymethylhydroxypropyl guar
• 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 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.
• 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.
• 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.
• the fracturing gel will begin to break when the pumping operations are concluded.
• 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.
• a completely broken, non-crosslinked gel is one whose viscosity is either about 10 centipoises or less as measured on a Model 50 Fann viscometer Rl/Bl at 300 rpm or less than 100 centipoises by Brookfield viscometer spindle #1 at 0.3 rpm.
• Enzymes 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 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 9.0.
• 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.
• methods and compositions for fracturing subterranean formations that effectively hydrolyze fracturing fluids, particularly at elevated pH values.
• the methods and compositions for fracturing subterranean formations use enzyme breakers that are effective at elevated pH values.
• a method of fracturing a subterranean formation that is penetrated by a well bore is provided.
• 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 crosslinked polymer gel is then 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.
• 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.
• the preferred enzyme breaker is an alkaline ⁇ -mannanase.
• Especially preferred alkaline ⁇ -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 ⁇ -mannanase enzyme.
• the gene is codon optimized for expression in E. coli.
• the gene produces a GST-mannanase fusion protein.
• 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.
• 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 ⁇ -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 ⁇ -mannanase enzyme.
• the gene is codon optimized for expression in E. coli.
• the gene produces a GST-mannanase fusion protein.
• 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 - 1 1.5.
• compositions are also provided as embodiments 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 ⁇ -mannanase such as those designated above.
• 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.
• the enzyme breaker used in the methods and compositions described herein may be derived from a gene having engineered restriction endonuc lease sites, such as Xhol and Bell, flanking the 5' and the 3' ends of the gene coding for the alkaline ⁇ -mannanase enzyme.
• the gene is codon optimized for expression in E. coli.
• the gene produces a GST-mannanase fusion protein.
• Figure 1A is the sequence of the gene that codes for an enzyme breaker made in accordance with embodiments of the present invention.
• Figure IB is a comparison of the gene sequence of Figure 1A with the gene sequence of a gene coded for a prior art enzyme
• Figure 2 is a schematic illustrating the creation of the plasmids pGS-21a-/z/? ? and pUC57- ⁇ which may used in the enzyme breaker in accordance with embodiments of the present invention
• 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
• 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 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;
• 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
• 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
• 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.
• 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.
• a method of fracturing a subterranean formation that surrounds a well bore is provided.
• 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 crosslinked 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.
• 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.
• 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 - 1 1.5.
• compositions are also provided as embodiments 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.
• 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 - 1 1.5.
• 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 (EC:3.2.1.4); beta-mannanase (EC:3.2.1.78); exo-l ,3-glucanase (EC:3.2.1.58); endo-1 ,6- glucanase (EC:3.2.1.75); xylanase (EC:3.2.1.8); endoglycoceramidase (EC:3.2.1.123).
• Glycoside hydrolases of Family 5 include chitosanase (EC 3.2.1.132); ⁇ -mannosidase (EC 3.2.1.25); cellulase (EC 3.2.1.4); glucan -l ,3-glucosidase (EC 3.2.1.58); licheninase (EC 3.2.1.73); glucan endo- l ,6" -glucosidase (EC 3.2.1.75); mannan endo ⁇ -l ,4-mannosidase (EC 3.2.1.78); endo- ⁇ - 1 ,4-xylanase (EC 3.2.1.8); cellulose -l ,4-cellobiosidase (EC 3.2.1.91); ⁇ -1 -mannanase (EC 3.2.1.-); xyloglucan-specific endo ⁇ - l ,4-glucanase (EC 3.2.1.151); mannan transglycosylase (EC 2.4.1.-); endo- "l
• 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 ( /a)(8)-barrel fold with two active site glutamic acids being approximately 200 residues apart in sequence and located at the C-terminal ends of ⁇ -strands 4 (acid/base) and 7 (nucleophile).
• the enzyme breaker is an alkaline ⁇ -mannanase such as the alkaline ⁇ -mannanase derived from a gene having engineered restriction endonuclease sites, such as Xho l and Bell , flanking the 5 ' and the 3' ends of the gene coding for the alkaline ⁇ - mannanase enzyme.
• the gene is codon optimized for expression in E. coli.
• the gene produces a GST-mannanase fusion protein.
• 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 ⁇ -(1 ,4) mannosidic linkages and can be used to break the polymer backbone of galactomannan polymers.
• the enzyme breaker of the present invention does not require the action of an associated a-galactosidase in order to function.
• the enzyme breaker is derived from a gene having engineered restriction endo nuclease sites, such as Xho l and Bell , flanking the 5' and the 3' ends of the gene coding for the ⁇ -mannanase enzyme.
• the gene is codon optimized for expression in E. coli.
• the gene codes for an expressed N- terminal GST fusion protein.
• GH26 Glycoside Hydrolases of Family 26
• GH26 encompasses ⁇ -mannanase (EC 3.2.1.78, mainly mannan endo- 1 ,4-beta-mannosidases which randomly hydro lyze 1 ,4-beta-D-linkages in mannans, galactomannans, glucomannans and galactoglucomannans, as well as -l ,3-xylanase (EC 3.2.1.32).
• the glycoside hydrolases of GH 26 display little, if any, activity towards other plant cell wall polysaccharides.
• the enzyme breaker may be stored prior to being combined into the aqueous fluid.
• the enzyme breaker may be refrigerated prior to being combined with the aqueous fluid.
• 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.
• the enzyme breaker or the alkaliphile from which the enzyme is derived may be stored in a frozen state.
• the enzyme breaker or the alkaliphile may be combined with a winterizing agent, such as a glycerol, during storage.
• a winterizing agent such as a glycerol
• the enzyme breaker may be derived from the alkaliphile and preferably brought to room temperature prior to being added to the aqueous fluid.
• 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.
• the enzyme can be diluted in various concentrations that are effective and convenient for use in fracturing jobs.
• the enzyme breaker of the present invention is diluted to a concentration of about 1 :24 and is present in the crosslinked 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.
• the total protein concentration of the stock enzyme breaker from which the dilutions are made is greater than 1 mg/mL.
• 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.
• 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 1 1.5; or alternatively, in a range from about 10.5 to about 1 1.
• the enzyme breaker of the present invention can include an alkaline enzyme.
• alkaline enzyme generally refers to enzymes that display their maximum catalytic activity somewhere within a pH range of about 8.0 to about 14.0.
• the maximum catalytic activity of the alkaline enzyme can be at pH values above 9.0.
• the alkaline enzyme is derived from an alkaliphilic organism.
• alkaliphilic organism generally refers to extremophilic organisms that thrive in alkaline conditions somewhere in the pH range of about 8.0 to about 14.0.
• the methods and compositions described herein can be used with a variety of hydratable polymers.
• the hydratable polymer has repeating units of mannose linked by ⁇ -(1 ,4) mannosidic linkages.
• 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.
• 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.
• 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, which is hereby incorporated by reference in its entirety
• Divalent cations can affect the activity of the enzyme breaker of the present invention, as shown and described in Example 5.
• the crosslinked polymer gel can further include a divalent cation.
• Suitable divalent cations can include Mg 2+ , Co 2+ , or Me 2+ .
• 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.
• 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.
• 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.
• 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 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, which is incorporated by reference in its entirety.
• Suitable borate-containing crosslinking agents can include, for example, alkaline earth metal borates, alkali metal- alkaline earth borates, probertite, ulexite, nobleite, frolovite, colemanite, calcined colemanite, priceite, pateroniate, hydroboractie, kaliborite, or combinations thereof.
• Suitable titanium-containing crosslinking agents can include, for example, titanium lactate, titanium malate, 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.
• 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.
• corrosion inhibitors non-emulsifiers
• iron control agents including but not limited to, corrosion inhibitors, non-emulsifiers, iron control agents, delay additives, silt suspenders, flowback additives, proppants, and gel breakers
• delay additives including but not limited to, delay additives, silt suspenders, flowback additives, proppants, and gel breakers
• Other suitable additives useful 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.
• the amount of crosslinking agent and other additives used in the present invention can vary depending upon the desired effect of the additives.
• the crosslinking agent can be present in the crosslinked polymer gel in an amount sufficient to provide the desired degree of crosslinking between molecules within the hydratable 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.
• a novel ⁇ -mannanase enzyme was first isolated in the alkaliphilic extremophile, Bacillus sp. 16-5 (see Ma et al. (2004) Characterization and Gene Cloning of a Novel ⁇ -mannanase from Alkaliphilic Bacillus sp. 16-5, Extremophiles 8, 447-454).
• the gene coding this ⁇ - 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.
• the gene structure coding for the ⁇ -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 increase the efficiency of its expression in E. coli. Finally, Xho l 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 ⁇ -mannanase gene, as shown in Figures 1A and IB.
• 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 ⁇ was cloned into the expression vector pGS-21a and the cloning vector pUC57.
• the plasmids pGS-21 a-/z/? ? and pXJC57-hpfi were transformed into competent BL21 (DE3) E. coli 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-/z/? ? and pUC57- ⁇ .
• the cultures were grown at 104 °F and 200 RPM.
• IPTG isopropyl ⁇ -D- thiogalactopyranoside
• the plasmid pUC57- z/? ? was transformed into competent OH5 E. coli 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.
• the ability of the enzyme breaker of the present invention to hydrolyze the polymannan backbone of the guar polymer was examined.
• the enzyme breaker of the present invention Enzyme ⁇ - ⁇ , comprising the alkaline ⁇ - mannanase effectively hydrolyzes the guar polymer at elevated pH ranges.
• Enzyme ⁇ - ⁇ 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.
• the ⁇ - ⁇ enzyme was tested against crosslinked 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.
• the alkaline ⁇ -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 2 the activity of the enzyme breaker of the present invention Enzyme Hp- ⁇ 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 normalized to itself.
• Enzyme ⁇ - ⁇ appears to show the greatest activity at a pH value of about 11.
• This Example indicates that the enzyme breaker of the present invention (Enzyme ⁇ - ⁇ ) maintains its effectiveness in a wide range of temperatures, even up to about 225 °F, and in a wide range of loadings.
• enzyme breaker of the present invention Enzyme ⁇ - ⁇
• 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.
• 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.
• Enzyme ⁇ - ⁇ is voracious and quickly reduces the viscosity of the fracturing fluid.
• a way to slow the activity of the enzyme would be beneficial. It would be 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.
• Divalent cations can have beneficial or detrimental effects on the activity of the enzyme breakers of the present invention.
• 1.0 mM Mg 2+ has the effect of increasing Enzyme ⁇ - ⁇ ' ⁇ activity while the presence of 1.0 mM Co 2+ decreases enzyme activity.
• Enzyme ⁇ - ⁇ 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.
• Enzyme ⁇ - ⁇ has a dramatic increase in activity when incubated in the presence of 1.0 Mg 2+ . While 1.0 mM Co 2+ 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.
• Sample A represents data for 1.0 mg/mL Enzyme ⁇ - ⁇ samples stored at 40 °F and 72°F, and 5.0 mg/mL Enzyme ⁇ - ⁇ samples stored at 40 °F, 72 °F, and 120°F. In all cases the data was the same.
• Sample B represents data for 1.0 mg/mL Enzyme ⁇ - ⁇ samples stored at 120 °F. To measure enzyme activity, incubated enzyme samples were diluted so that the final, working concentration of Enzyme ⁇ - ⁇ 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.
• 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 ⁇ - ⁇ (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 ⁇ - ⁇ (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.
• the enzyme breaker of the present invention Enzyme ⁇ - ⁇ , 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 ⁇ - ⁇ should be > 5.0 mg/mL.
• compositions and/or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the 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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