U.S. patent application number 09/742737 was filed with the patent office on 2001-06-14 for compositions for fracturing subterranean formations.
Invention is credited to Kelly, Robert M., Khan, Saad A., Leduc, Pascal, Prud'homme, Robert K., Tayal, Akash.
Application Number | 20010003315 09/742737 |
Document ID | / |
Family ID | 22779797 |
Filed Date | 2001-06-14 |
United States Patent
Application |
20010003315 |
Kind Code |
A1 |
Kelly, Robert M. ; et
al. |
June 14, 2001 |
Compositions for fracturing subterranean formations
Abstract
A method of fracturing a subterranean formation which surrounds
a well bore comprises the steps of providing a fracturing fluid,
and injecting the fracturing fluid into the well bore at a pressure
sufficient to form fractures in the subterranean formation which
surrounds the well bore. The pressure is then released from the
fracturing fluid, after which the fluid may be removed from the
well and the well placed into production. The fracturing fluid
comprises an aqueous liquid, a polysaccharide soluble or
dispersible in the aqueous liquid in an amount sufficient to
increase the viscosity of the aqueous liquid, an enzyme breaker
which degrades said polysaccharide at a temperature above
180.degree. F. Fracturing fluid compositions and enzyme breaker
systems useful for carrying out the invention are also
disclosed.
Inventors: |
Kelly, Robert M.; (Cary,
NC) ; Khan, Saad A.; (Raleigh, NC) ; Leduc,
Pascal; (Raleigh, NC) ; Tayal, Akash;
(Raleigh, NC) ; Prud'homme, Robert K.; (Princeton
Junction, NJ) |
Correspondence
Address: |
MYERS BIGEL SIBLEY & SAJOVEC
PO BOX 37428
RALEIGH
NC
27627
US
|
Family ID: |
22779797 |
Appl. No.: |
09/742737 |
Filed: |
December 21, 2000 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
09742737 |
Dec 21, 2000 |
|
|
|
09185120 |
Nov 3, 1998 |
|
|
|
6197730 |
|
|
|
|
09185120 |
Nov 3, 1998 |
|
|
|
08403078 |
Mar 13, 1995 |
|
|
|
5869435 |
|
|
|
|
08403078 |
Mar 13, 1995 |
|
|
|
08209679 |
Mar 10, 1994 |
|
|
|
5421412 |
|
|
|
|
Current U.S.
Class: |
166/300 ;
166/308.3; 507/201 |
Current CPC
Class: |
Y10S 507/923 20130101;
C09K 8/685 20130101; Y10S 507/921 20130101; C09K 8/62 20130101;
Y10S 507/922 20130101; E21B 43/267 20130101 |
Class at
Publication: |
166/300 ;
166/308; 507/201 |
International
Class: |
E21B 043/26 |
Goverment Interests
[0001] This invention was made with Government support under grant
number BCS-93-10964 from the National Science Foundation. The
Government has certain rights to this invention.
Claims
That which is claimed is:
1. A method of fracturing a subterranean formation which surrounds
a well bore, comprising the steps of: (a) providing a fracturing
fluid comprising: (i) an aqueous liquid; (ii) a polysaccharide
soluble or dispersible in said aqueous liquid in an amount
sufficient to increase the viscosity of said aqueous liquid; and
(iii) an enzyme breaker which degrades said polysaccharide at a
temperature above 180.degree. F.; then (b) injecting said
fracturing fluid into said well bore at a pressure sufficient to
form fractures in the subterranean formation which surrounds said
well bore; and then (c) releasing the pressure from said fracturing
fluid.
2. A method according to claim 1, wherein said fracturing fluid
further comprises proppant particles.
3. A method according to claim 1, wherein said fracturing fluid
further comprises a crosslinking agent for crosslinking said
polysaccharide.
4. A method according to claim 1, wherein said enzyme breaker
comprises a mannanase which degrades said polysaccharide at a
temperature above 180.degree. F.
5. A method according to claim 4, wherein said enzyme breaker
further comprises an .alpha.-galactosidase which degrades said
polysaccharide at a temperature above 180.degree. F.
6. A method according to claim 1, wherein said enzyme breaker
degrades said polysaccharide at a temperature of from 180.degree.
F. to 212.degree. F.
7. A method according to claim 1, wherein said enzyme breaker is
essentially incapable of degrading said polysaccharide at a
temperature of 100.degree. F. or less.
8. A method according to claim 1, wherein said providing step is
carried out at a temperature of 100.degree. F. or less.
9. A method according to claim 1, wherein: said subterranean
formation surrounding said well bore has a temperature greater than
180.degree. F.; said enzyme breaker comprises at least one enzyme
which is (a) essentially incapable of degrading said polysaccharide
at a temperature of 100.degree. or less, and (b) degrades said
polysaccharide at a temperature of at least 180.degree. F. to
212.degree. F.; and said fracturing fluid is maintained at a
temperature of 100.degree. F. or less prior to said injecting
step.
10. A hydraulic fracturing fluid useful for fracturing a
subterranean formation which surrounds a well bore, said fracturing
fluid comprising: (a) an aqueous liquid; (b) a polysaccharide
soluble or dispersible in said aqueous liquid in an amount
sufficient to increase the viscosity of said aqueous liquid; and
(c) an enzyme breaker which degrades said polysaccharide at a
temperature between 180.degree. F. and 280.degree. F., said enzyme
breaker included in an amount effective to degrade said
polysaccharide at said temperature.
11. A fracturing fluid according to claim 10, wherein said
polysaccharide is guar gum.
12. A fracturing fluid according to claim 10, said fracturing fluid
further comprising a crosslinking agent for crosslinking said
polysaccharide.
13. A fracturing fluid according to claim 10, further comprising
proppant particles.
14. A fracturing fluid according to claim 10, wherein said enzyme
breaker comprises a mannanase which degrades said polysaccharide at
a temperature above 180.degree. F.
15. A fracturing fluid according to claim 14, wherein said enzyme
breaker further comprises an .alpha. -galactosidase which degrades
said polysaccharide at a temperature above 180.degree. F.
16. A breaker composition useful for preparing hydraulic fracturing
fluids for fracturing a subterranean formation which surrounds a
well bore, said enzyme breaker comprising, in combination, a
mannanase which degrades polysaccharide at a temperature above
180.degree. F. and an .alpha. -galactosidase which degrades
polysaccharide at a temperature above 180.degree. F.
17. A breaker composition according to claim 16, wherein said
composition is an aqueous composition.
18. A breaker composition according to claim 16, wherein said
.alpha.-galactosidase: (a) hydrolyzes .alpha.-1,6 hemicellulolytic
linkages in galactomannans; (b) is isolated from a
hyperthermophilic organism; (c) is active at a temperature above
180.degree. F.; and (d) is essentially inactive at a temperature of
100.degree. F. or less.
Description
FIELD OF THE INVENTION
[0002] This invention concerns thermostable enzyme breakers for the
hydrolysis of galactomannans in hydraulic fracturing fluids.
BACKGROUND OF THE INVENTION
[0003] When the pressure of oil or gas in a reservoir declines as
oil or gas is taken from that reservoir, production from a well in
that reservoir declines and the economic viability of the well
declines until it is no longer profitable to operate (even though
it continues to produce gas or oil). Production can be increased
from such wells through oil well stimulation. In addition, where
forming a bore hole into a reservoir is very expensive, such as in
offshore drilling, it is desirable to stimulate production from a
single well.
[0004] Oil well stimulation typically involves injecting a
fracturing fluid into the well bore at extremely high pressures to
create fractures in the rock formation surrounding the bore. The
fractures radiate outwardly from the well bore, typically from 100
to 1000 meters, and extend the surface area from which oil or gas
drains into the well. The fracturing fluid typically carries a
propping agent, or "proppant", such as sand, so that the fractures
are propped open when the pressure on the fracturing fluid is
released, and the fracture closes around the propping agent. This
leaves a zone of high permeability (the propping agent trapped and
compacted in the fracture in the subterranean formation.
[0005] The fracturing fluid typically contains a water soluble
polymer, such a guar gum or a derivative thereof, which provides
appropriate flow characteristics to the fluid and suspends the
proppant particles therein. When pressure on the fracturing fluid
is released and the fracture closes around the propping agent,
water is forced therefrom and the water-soluble polymer forms a
compacted cake. This compacted cake can prevent oil or gas flow if
not removed. To solve this problem, "breakers" are included in the
fracturing fluid.
[0006] Currently, breakers are either enzymatic breakers or
oxidative breakers. The enzyme breakers are preferable, because (a)
they are true "catalysts", (b) they are relatively high in
molecular weight and do not leak off into the surrounding
formation, and (c) they are less susceptible to dramatic changes in
activity by trace contaminants. Oxidative breakers, on the other
hand, are low in molecular weight and leak off into the formation,
and they are active only over a very narrow temperature range.
Enzyme breakers, however, are inactive at higher temperatures,
limiting their use to shallow wells. It would accordingly be highly
desirable to have enzyme breakers that operate at higher
temperatures to enable fracturing of deep wells. See generally J.
Gulbis, Fracturing Fluid Chemistry, in RESERVOIR STIMULATION, Chap.
4 (J. J. Economides and K. G. Nolte, Eds., 2d Ed. 1989).
[0007] U.S. Pat. No. 4,996,153 to Cadmus and Slodki discloses a
heat-stable enzyme breaker which may be used as a viscosity breaker
in oil recovery, but this breaker is a xanthanase for degrading
zanthan-based rather than guar-based fracturing fluids, and is only
said to be active at 55.degree. C. (156.6.degree. F.).
[0008] U.S. Pat. No. 5,201,370 to Tjon-Joe-Pin discloses enzyme
breakers for galactomannan-based fracturing fluids, which enzyme
breakers are galactomannases that hydrolyze the
1,6-.alpha.-D-galactomannosidic and the 1,4-.beta.-D-mannosidic
linkages in the guar polymer, but these are said to only be active
at low to moderate temperatures of about 50.degree. F. to
180.degree. F.
[0009] U.S. Pat. No. 4,250,044 to Hinkel concerns a tertiary
amine/persulfate breaker system, and not an enzyme system.
[0010] In view of the foregoing, there is a continued need for
thermostable enzyme breakers useful for fracturing subterranean
formations in the course of oil and gas well stimulation.
SUMMARY OF THE INVENTION
[0011] A first aspect of the present invention is a method of
fracturing a subterranean formation which surrounds a well bore.
The method comprises the steps of providing a fracturing fluid, and
injecting the fracturing fluid into the well bore at a pressure
sufficient to form fractures in the subterranean formation which
surrounds the well bore. The pressure is then released from the
fracturing fluid, after which the fluid may be removed from the
well and the well placed into production. The fracturing fluid
comprises an aqueous liquid, a polysaccharide soluble or
dispersible in the aqueous liquid in an amount sufficient to
increase the viscosity of the aqueous liquid, an enzyme breaker
which degrades said polysaccharide at a temperature above
180.degree. F.
[0012] A second aspect of the present invention is a hydraulic
fracturing fluid useful for fracturing a subterranean formation
which surrounds a well bore. The fracturing fluid comprises an
aqueous liquid; a polysaccharide soluble or dispersible in the
aqueous liquid in an amount sufficient to increase the viscosity of
said aqueous liquid (said polysaccharide typically included in said
aqueous liquid in an amount of from about 0.1 to 1 percent by
weight); and an enzyme breaker which degrades said polysaccharide
at a temperature between 180.degree. F. and 280.degree. F., the
enzyme breaker included in an amount effective to degrade the
polysaccharide at that temperature.
[0013] A third aspect of the present invention is an enzyme breaker
useful for preparing hydraulic fracturing fluids for fracturing a
subterranean formation which surrounds a well bore. The enzyme
breaker comprises in combination, a mannanase which degrades
polysaccharide at a temperature above 180.degree. F. and an
.alpha.-galactosidase which degrades polysaccharide at a
temperature above 180.degree. F.
[0014] A fourth aspect of the present invention is a heat-stable
.alpha.-galactosidase composition which hydrolyzes .alpha. -1,6
hemicellulolytic linkages in galactomannans, is isolated from
hyperthermophilic organisms (e.g., as a cell-free extract thereof),
is active at a temperature above 180.degree. F., and is essentially
inactive at a temperature of 100.degree. F. or less.
[0015] In addition to making available enzyme breakers for higher
temperature wells, a still further advantage or the present
invention is that, by employing enzyme breakers which are
essentially inactive at the temperature at which the fracturing
fluid is initially provided, the problem of premature breaking of
the fracturing fluid is inhibited or reduced. As discussed in U.S.
Pat. No. 3,922,173 to Misak at columns 1-2, premature breaking of
the gelled fracturing fluid (or "sanding out") can cause suspended
proppant particles to settle out of the fracturing fluid before
being introduced a sufficient distance into the fractures. This
causes blockage of the fracture and/or an undesirable diminution of
potential fracture width. The problem of premature breaking is
reduced or inhibited in the present invention because the enzyme
breaker is essentially inactive at the temperature at which the
fracturing fluid is initially provided (e.g., usually ambient
temperature, and not more than 90 or 100.degree. F.). The enzyme
becomes active at the elevated temperatures encountered in the
subterranean formation surrounding the well bore, which is
precisely the point at which enzyme activity leading to a breaking
of the fracturing fluid viscosity is desired. Thus, the present
invention advantageously provides a temperature control means for
controlling the timing of activation of the enzyme breaker.
[0016] E. Luthi et al., Appl. Environ. Microbiol. 57, 694 (1991),
and M. Gibbs et al., Appl. Environ. Microbiol. 58, 3864 (1992),
both concern a heat-stable .beta.-mannanase, but do not disclose a
heat stable a-galactosidase, do not suggest their use in oil well
fracture fluids, and do not address the problem of premature
breaking.
[0017] The foregoing and other objects and advantages of the
present invention are explained in detail in the drawings herein
and the specification set forth below.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] FIG. 1 shows the effect of temperature (given in degrees
centigrade) on relative activity of the galactosidase activity and
mannanase activity found in an enzyme breaker composition prepared
from Thermotoga neopolitana 5068.
[0019] FIG. 2 shows the effect of the enzyme breaker composition of
FIG. 1 on the viscosity of a 0.7% guar solution after 6 hours at
85.degree. C. Viscosity, .eta. (poise) is given on the vertical
axis from 0.5 to 10.0, and shear rate, .gamma. (sec.sup.-1) is
given on the horizontal axis from 1.0 to 100.0. Open circles
represent values from a control solution with no enzyme added, and
filled circles represent values from the solution with enzyme
added.
[0020] FIG. 3 is essentially the same as FIG. 2, except the guar
solution was held for 6 hours at 98.degree. C.
[0021] FIG. 4 is essentially the same at FIGS. 2 and 3, except the
guar solution was held for 9 hours at 98.degree. C., and the enzyme
breaker was prepared from Pyrococcus furlosus.
DETAILED DESCRIPTION OF THE INVENTION
[0022] Fracturing fluids used to carry out the present invention
are, in general, prepared from an aqueous base fluid such as water,
brine, aqueous foams or water-alcohol mixtures. Any suitable mixing
apparatus may be used to provide the fracturing fluid. The pH of
the fluid is typically from about 2 to 11 or 12. The fracturing
fluid includes a polysaccharide as a gelling agent, as discussed
below, and typically includes other ingredients such as proppant
particles and crosslinking agents to crosslink the polysaccharide
gelling agent, also discussed below.
[0023] Polysaccharides soluble or dispersible in an aqueous liquid
include industrial gums such as those generally classified as
exudate gums, seaweed gums, seed gums, microbial polysaccharides,
and hemicelluloses (cell wall polysaccharides found in land plants)
other than cellulose and pectins. Examples include xylan, mannan,
galactan, L-arabino-xylans, L-arabino-D-glucurono-D-xylans,
D-gluco-D-mannans, D-Galacto-D-mannans, arabino-D-galactans, algins
such as sodium alginate, carrageenin, fucordan, laminarin, agar,
gum arabic, gum ghatti, karaya gum, tamarind gum, tragacanth gum,
locust bean gum, cellulose derivative such as hydroxyethylcellulose
or hydroxypropylcellulose, and the like. Particularly preferred are
the hydratable polysaccharides having galactose and/or mannose
monosaccharide components, examples of which include the
galactomannan gums, guar gum and derivatized guar gum. Examples of
particularly preferred thickening agents are guar gum,
hydroxypropyl guar, and carboxymethyl hydroxypropyl guar.
[0024] The amount of polysaccharide included in the fracturing
fluid is not particularly critical, so long as the viscosity of the
fluid is sufficiently high to keep the proppant particles suspended
therein during the injecting step. Thus, depending upon the
application, the polysaccharide is included in the fracturing fluid
in an amount of from about 10 to 150 pounds of polysaccharide per
1000 gallons of aqueous liquid, and more preferably in an amount of
from about 20 to 100 pounds of polysaccharide per 1000 gallons of
aqueous solution (about 2.4 to 12 kg/m.sup.3).
[0025] Any crosslinking agent may be used to carry out the present
invention. Examples include metal ions including aluminum,
antimony, zirconium an titanium containing compounds including
organotitantates (see, e.g., U.S. Pat. No. 4,514,309). Borate
crosslinking agents or borate ion donating materials, are currently
preferred. Examples of these include the alkali metal and alkaline
earth metal borates and boric acid, such as sodium borate
decahydrate. The crosslinking agent is typically included in an
amount in the range of from about 0.0245 to 0.18% by weight of the
aqueous fluid or more.
[0026] Proppant particles or propping agents are typically added to
the base fluid prior to the addition of the crosslinking agent.
Propping agents include, for example, quart sand grains, glass and
ceramic beads, walnut shell fragments, alluminum pellets, nylon
pellets, and the like. The propping agents are typically included
in an amount of from 1 to 8 or even 18 pounds per gallon of
fracturing fluid composition. Particle size of the proppant
particles is typically in the range of about 200 to about 2 mesh on
the U.S. Sieve Series scale. The base fluid may also contain other
conventional fracturing fluid additives, such as buffers,
surfactants, antioxidants, corrosion inhibitors, bactericides,
etc.
[0027] Enzyme breaker compositions useful for carrying out the
present invention may be provided in any suitable physical form,
such as concentrated or dilute aqueous solutions, lyophylized
powders, etc. The compositions contain an enzyme effective for
degrading the particular crosslinking polysaccharide employed as
the gelling agent. The compositions typically include a
.beta.-Mannanase which degrades .beta.-1,4 hemicellulolytic
linkages in galactomannan compounds such as guar gums at a
temperature above 180.degree. F., and/or an .alpha.-galactosidase
which degrades .alpha.-1,6 hemicellulolytic linkages in
galactomannan compounds such as guar gums at a temperature above
180.degree. F. Typically, the enzyme breaker composition and one or
more of the enzymes therein are at least capable of degrading the
polysaccharide at temperatures of from 180.degree. F. to
212.degree. F. Preferably, as discussed above, the enzyme breaker
and one or more of the enzymes therein are also essentially
incapable of degrading the polysaccharide at a temperature of 90 or
100.degree. F. or less (e.g., have relative activity of 0.1 or even
0.05 of optimum activity (1.0) at these temperatures).
[0028] Enzymes useful for preparing enzyme breaker compositions
used in the present invention may be obtained from
hyperthermophilic microorganisms. Hyperthermophilic microorganisms
are microorganisms which grow at temperatures higher than
90.degree. C., and have an optimal growth temperature higher than
80.degree. C. Generally, hyperthermophilic organisms are either
hyperthermophilic bacteria or hyperthermophilic Archaea (as
categorized according to C. Woese et al., Proc. Natl. Acad. Sci.
USA 87, 4576-4579 (1990)). These organisms are found in, for
example, the Thermoproteales, Sulfolobales, Pyrodictiales,
Thermococcales, Archaeoglobales, Methanococcales,
Methanobacteriales, Methanopyrales, Thermotogales, and Aquificales
groups. Typically these organisms are found in the genera Aquifex,
Archaeoglobus, Thermotoga, Thermoproteus, Staphylothermus,
Desulfurococcus, Thermofilum, Pyrobaculum, Acidianus,
Desulfurolobus, Pyrodictium, Thermodiscus, Pyrococcus,
Thermococcus, Hyperthermus, Methanococcus, Methanothermus, and
Methanopyrus. See, e.g., Biocatalysis at Extreme Temperatures, pgs.
4-22 (M. Adams and R. Kelly Eds. 1992) (ACS Symposium Series 498).
The genera Thermotoga, Thermococcus, and Pyrococcus are
particularly convenient sources of organisms useful for carrying
out the present invention. Specific organisms from which enzymes
useful in carrying out the present invention may be obtained
include, but are not limited to, Pyrococcus furiosus, Pyrococcus
furiosus GBD, Thermotoga maritima, Thermus aquaticus, Thermus
thermophilous, Thermococcus litoralis, ES-1, ES-4, etc. The enzymes
are identified in bacteria supernatant or lysed cell extracts by
conventional techniques, such as by isolating and culturing the
organisms on media which contain the appropriate growth substrates
(e.g., for .alpha.-D-galactosidase activity on the substrate
p-Nitrophenyl-.alpha.-D-Galactopyranoside; for .beta.-D-mannanase
activity on the substrate p-Nitrophenyl-.beta.-D-Mannopyranoside),
DNA screening with consensus oligonucleotide probes, "shotgun"
cloning and screening of transformed host cells with antibodies
and/or gel plate assays (e.g., plates containing the growth
substrates given above), etc. The enzymes may be produced by any
suitable means, including either conventional fermentation in a
high temperature fermentor or by genetic engineering
techniques.
[0029] The present invention may be carried out on subterranean
formations which surround any type of well bore, including both oil
and gas well bores, with the fracturing fluid being provided and
injected and pressure released, etc., all in accordance with
procedures well known to those skilled in the art. As noted above,
the invention is particularly advantageously employed when the
subterranean formation surrounding the well bore to be fractured
has a temperature greater than 180.degree. F., up to 280.degree. F.
or more (it being understood that other subterranean formations
which surround the well bore, which may or may not be integral with
the subterranean formation having the aforesaid temperature may or
may not be fractured and may or may not have the aforesaid
temperature range).
[0030] In one embodiment of the invention, the step of providing
the fracturing fluid (e.g. preparing and mixing the fluid on-site)
is carried out at a temperature of 90 or 100.degree. F. or less,
the fracturing fluid thereby being maintained at a temperature of
90 to 100.degree. F. or less prior to the injecting step. This
serves to reduce premature breaking of the crosslinked
polysaccharide and sanding out of the fracturing fluid, as
discussed above.
[0031] The present invention is explained in greater detail in the
following non-limiting Examples, where "DSM" means Deutsche
Sammlung von Mikroorganimen (Braunsschweig, Federal Republic of
Germany), "M" means Molar, ".mu.M" means microMolar, ".mu.m" means
micrometer, "ml" means microliter, "L" means liter, "mg" means
microgram, "g" means gram, "rpm" means revolutions per minute,
"PSIG" means pounds per square inch gauge, and temperatures are
given in degrees Centigrade unless otherwise indicated.
EXAMPLE 1
Thermotoga neoplitana Culture Conditions
[0032] Thermotoga neopolitana DSM 5068 cells were cultured in an
artificial sea water (ASW) based media supplemented with 0.1% yeast
extract and 0.5% tryptone, with 0.5% lactose and 0.03% guar gum
added as inducers (the guar gum was obtained from Rhne-Poulenc).
The media composition per liter was: NaCl 15.0 g, Na.sub.2SO.sub.4
2.0 g, Mgcl.sub.2.6H.sub.2O 2.0 g, CaCl.sub.2.2H2O 0.50 g,
NaHCO.sub.3 0.25 g, K.sub.2HPO.sub.4 0.10 g, KBr 50 mg,
H.sub.3BO.sub.3 20 mg, KI 20 mg, Fe(NH.sub.4).sub.2(SO.sub.4).sub.2
15 mg, Na.sub.2WO.sub.4.2H.sub.2O 3 mg, and NiCl.sub.26H.sub.2O 2
mg. The lactose, guar gum, K.sub.2HPO.sub.4,
Fe(NH.sub.4).sub.2(SO.sub.4).sub.2 were added after sterilization.
However, K.sub.2HPO.sub.4 and Fe(NH.sub.4).sub.2(SO.sub.4)- .sub.2
were filtered through a 0.2 .mu.m filter prior to addition.
[0033] First, inocula were grown in closed bottles (125 ml) under
anaerobic conditions. The cultures were prepared by heating the
media-containing bottles to 98.degree. C. for 30 minutes, then
sparging with N.sub.2, and then adding Na.sub.2S.9H.sub.2O (0.5
g/L) from a 50 g/L stock solution. Prior to anaerobic inoculation,
cultures were cooled to 80.degree. C.
[0034] Large scale cultures were grown in a semi-batch fashion
using a 8-liter fermentor (Bioengineering Lab Fermenter type 1
1523). Oxygen was removed by continuous flow of nitrogen of
approximately 5 liter/minute. Temperature and agitation were
monitored using a conventional data acquisition system and were set
at 85.+-.2.degree. C. and 150 rpm respectively. Growth was
monitored by epifluorescent microscopy using acridine orange
stain.
[0035] Cells were harvested in late exponential growth phase (1.5
to 2.10.sup.8 cells/ml). 350 liters were harvested and concentrated
down to approximately 60 liters using a 0.45 .mu.m PELLICON.TM.
cross-flow filter from Millipore. The retentate was then
centrifuged at 8000 rpm for 30 minutes in 1-liter bottles. Cells
were frozen at -20.degree. C. until use.
EXAMPLE 2
Preparation of T. neopolitana Cell Extract
[0036] Frozen cells of T. neopolitana DSM 5068 prepared as
described in Example 1 above were resuspended in 430 ml of 0.1M
sodium phosphate buffer, pH 7.4 and disrupted by one passage
through a French pressure cell at 1100 PSIG. NaN.sub.3 (0.01%) was
added at this stage to prevent contamination. Cell debris was
removed by centrifugation at 10,000 g for 30 minutes, and the
soluble fraction was used as the crude enzyme preparation.
EXAMPLE 3
Detection of Enzyme Activity
[0037] .alpha.-Galactosidase and .beta.-mannanase activities were
determined by using p-nitrophenyl-.alpha.-D-galactopyranoside and
p-nitrophenyl-.beta.-D-mannopyranoside respectively as substrates.
Spectrophotometric readings were taken with a Lamda 3
spectrophotometer (The Perkin-Elmer Corp., Connecticut) with a
thermostated six cell transport. A liquid-circulating temperature
bath (VWR Scientific model 1130) containing a 1:1 mixture of
ethylene glycol and water was used to maintain the desired
temperature in the cell holder. This temperature was monitored with
a thermocouple mounted in a cuvette that was placed in the cell
transport. The six-cell transport was controlled and data were
collected and analyzed by Perkin-Elmer software run on a
microcomputer.
[0038] Routine enzymatic assays for .alpha.-galactosidase and
.beta.-mannanase were conducted as follows. For each assay, 1.1 ml
portions of substrate consisting 10 mM of substrate in 0.1M sodium
phosphate buffer (pH 7.4) were pipetted into quartz cuvettes. The
cuvettes were inserted into the cell holder, which was heated at
the desired temperature, and preincubated for at least 10 minutes
to allow the substrate to reach assay temperature. After the
preincubation, 0.1 ml of sample was added to the cuvettes and the
formation of the p-nitrophenyl (PNP) was followed by monitoring the
optical density at 405 nm. A blank containing the same amount of
sample in 0.1M sodium phosphate buffer (pH 7.4) was also monitored
to correct for interferences. At temperatures below 100.degree. C.
and for a short period of time (less than 15 minutes) no
nonenzymatic release of PNP was noticed. One unit of
.alpha.-galactosidase or .beta.-mannanase activity was defined as
the amount of enzyme releasing 1 nmol of PNP per minute under the
specified assay conditions.
[0039] Temperature optima were determined by performing the
appropriate assay at the temperature indicated.
[0040] FIG. 1 shows the results obtained at 75.degree. C.,
85.degree. C. and 96.degree. C. for the cell extract of the
large-scale culture prepared as described in Examples 1 and 2 above
for both .alpha.-galactosidase and .beta.-mannanase activities.
Assays were performed as described above.
[0041] Relative activity is defined as the measured activity
divided by the maximum activity (.alpha.-galactosidase activity at
96.degree. C.).
[0042] Both activities increase with temperature but none of them
reach an optimum in the considered temperature range. This result,
however, shows that the optimal temperature for both activities is
equal or greater than 96.degree. C.
EXAMPLE 4
Pyrococcus furiosus Culture Conditions and Preparation of Cell
Extract
[0043] Pyrococcus furiosus (DSM 3638) is grown in essentially the
same manner as described in F. Bryant and M. Adams, J. Biol. Chem.
264, 5070-5079 (1989). Cell extract from the P. furiosus cultures
is prepared in essentially the same manner as given in Example 2
above.
EXAMPLE 5
Rheological Testing Solutions
[0044] Standardized guar gum solutions were made for rheological
testing. The composition for a preferred solution is shown in Table
1. The guar solution is prepared by using a blender set at low
speed to provide a shallow vortex of water. The guar is sprinkled
slowly onto the free surface to produce a uniform dispersion. KCl,
glutaraldehyde and sodium thiosulfate are added quickly. The total
mixing time is about two minutes. The glutaraldehyde is used as a
bactericide, and the sodium thiosulfate serves as an antioxidant.
The samples are then mixed for 20 hours using a horizontal
shaker.
1TABLE 1 Rheological Test Solution MATERIAL AMOUNT Deionized water
100 g Guar gum (powder) 0.7 9 Potassium chloride 2 g 25%
glutaraldehyde solution in water 50 ml Sodium thiosulfate 0.5 g
EXAMPLE 6
Rheological Testing of Enzyme Preparations
[0045] Standard guar solutions for carrying out Theological tests
prepared as described in Example 4 above are mixed with enzyme
prepared as described in Examples 1 and 2 above and then incubated
at 85.degree. C./98.degree. C. using a shaking oil bath. Samples
are also incubated without any enzyme as a control.
[0046] Rheological measurements were performed using a Rheometrics
Mechanical Spectrometer (RMS 800) using a cone and plate geometry
with a diameter of 50 mm and a cone angle of 0.04 radians. The
sample is loaded onto the rheometer and steady shear tests are
performed (.gamma. range 1-100 sec.sup.-1) Steady shear viscosities
are obtained this way for the samples with and without the enzyme.
A lower viscosity curve for the sample with enzyme indicates
effectiveness of the enzyme in degrading the guar gum solution.
[0047] FIG. 2 plots the data for the samples incubated for 6 hours
at a temperature of 85.degree. C. with T. neopolitana cell extract.
Note that the samples used did not have an antioxidant and the
viscosity curves are thus shifted downward compared to the other
plots.
[0048] FIG. 3 shows data for samples incubated for 6 hours with T.
neopolitana cell extract at 98.degree. C.
[0049] FIG. 4 show data for samples incubated for 9 hours with P.
furiosus cell extract at 98.degree. C.
[0050] The low shear rate region in the foregoing figures has been
shown sensitive to polymer microstructure and the large drops in
viscosity at these shear rates (.sup.-1-100 sec.sup.-1) indicates
breakup of the microstructure.
[0051] The foregoing is illustrative of the present invention, and
not to be construed as limiting thereof. The invention is defined
by the following claims, with equivalents of the claims to be
included therein.
* * * * *