CA1279180C - Process utilizing a polysaccharide polymer - Google Patents
Process utilizing a polysaccharide polymerInfo
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- CA1279180C CA1279180C CA000615544A CA615544A CA1279180C CA 1279180 C CA1279180 C CA 1279180C CA 000615544 A CA000615544 A CA 000615544A CA 615544 A CA615544 A CA 615544A CA 1279180 C CA1279180 C CA 1279180C
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Abstract
A POLYSACCHARIDE POLYMER MADE BY XANTHOMONAS
ABSTRACT OF THE INVENTION
A polysaccharide polymer is disclosed which is a better viscosifier of water than xanthan gum. The polysaccharide polymer and its non-acetylated form, are comprised of glucose and mannose moieties in a ratio of about 2:1. The invention also discloses Xanthomonas mutants which produce the polysaccharide polymer but which do not produce xanthan gum. Methods of preparing the polysaccharide polymers and of their use are also described.
ABSTRACT OF THE INVENTION
A polysaccharide polymer is disclosed which is a better viscosifier of water than xanthan gum. The polysaccharide polymer and its non-acetylated form, are comprised of glucose and mannose moieties in a ratio of about 2:1. The invention also discloses Xanthomonas mutants which produce the polysaccharide polymer but which do not produce xanthan gum. Methods of preparing the polysaccharide polymers and of their use are also described.
Description
127~
A POLYSACCHARIDE POLYMER MADE BY XANTHOMONAS
This application is a division of our copending Canadian patent application Serial No. 577,352 filed on September 14, 1988.
BACKGROUND OF THE INVENTION
Xanthan gum is produced by bacteria of the genus Xanthomonas, such as the species campestris, albilineans, fra~ria, vesicatoria, and the like. Xanthan gu m is 8 widely used product due to its unusual physical properties: extremely high specific viscosity and pseudo-plasticity. It is ~ommonly used in foods as a thickening agent and in secondflry oil recovery as mobility control and profile modification agents and in petroleum drilling fluids.
Chemically, xanthan gum is an anionic heteropolysaccharide. The repeating unit of the polymer is a pentamer composed of five sugar moieties: two glucose, one glucuronic scid and two mannose moieties.
They are flrranged such that the glucose moieties form the backbone of the polymer chain, and side chains of mannose-glucuronic acid-mannose gene~ally extend from alternate glucose moieties. Often this bssic structure is specifically acetylated and/or pyruvylated. (Janson, P.E., Kenne, L., and Lindberg, B., Carbohydrate Research, 45, 2~5-282 (l975); Melton, L.D.~ Mindt, L., Rees, D.A., and Sanderson, G.R., Carboyhydrate Research, 46, 245-257 (1976).) The structure is depicted below:
L~ ~
to~ , ...~,~.~.
In spite of the broad utility of naturally occurring x~nthan gum, there are some situations where its physical properties become limiting.
In particular, in secondary oil recovery it ~3 not uncommon for the 5 temperature of the oil-bearing reservoir and salt concentrations in the reservoir brine to be higher than are optimal ~or xanthan solutions.
When these conditions oc~ur, xanthan can precipitate, flocculate and/or lose viscosity. Therefore there is a need for new viseosi~ying products which perform well at high temperature and high salt conditions.
10 ~ SUMMARY OF THE INVENTION
.
An object of the invention is to provide a process for recovering oil from an oil-bearing subterranean formation using novel viscosifying products.
- 3 ~
In accordance with another aspect of this invention claimed in our parent Canadian patent application Serial No. 513,834 which issued as Canadian Patent 1,247,033 on December 20, 1988, there is provided a composition comprising a polysaccharide polymer containing essentially no glucuronic acid moieties having a D-glucose:D-mannose ratio of about 2:1, wherein the D-glucose ~oieties are linked in a beta-[1,4] configuration to form the polymer backbone, and the D~mannose moieties are each linked in an alpha-[1,3] configuration generally to alternate glucose moieties. This composition can be used in the process of the present application.
The polysaccharide polymer which can be used in this invention can be made by blocking one of the steps in xanthan gum biosynthesis. Therefore, rather than having a three-sugar side-chain extending from the backbone of beta-[1,4]-D-glucose as in xanthan gum, the polysaccharide polymer of this invention has a single sugar moiety generally linked to alternate glucose moieties of the backbone. The polysaccharide polymer of this invention is herein termed "polytrimer" because it consists of a repeating trimer unit, glucose-glucose-mannose. Its structure is shown below, where n is the ~ number of repeating units in the polymer.
~;~;~
01~ OH
~Lo '~27918C) As shown by the above, the polytrimer consists of D-mannose linked alpha-[1,3] generally to alternate moieties of beta-~1,4] linked D~
glucose. As in xanthan gum, an acetic scid moiety can be, but is not always, esterified at the 6-O position of mannose, as described in Sutherland, I.W., Carbohydrate Polymers, ~ 107-115, (1981). Although the structure of the polysaccharide polymer is thought to be . as shown, it is possible that under certain conditions of synthesis, a mannose moiety may not always be linked at alternating glucose residues.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 depicts the assumed pathway of xanthan gum biosynthe-sis. It is bssed on the data of several laboratoriesO See, lelpi, L.7 Couso, R.O., and Dankert, :~I.A., Biochem. Biophy. Res. Comm., 102, 1400-1408 (1981), FEBS Letters, 130, 253-256 (1981), 8iochem. Intern~, 6, 323-333 (1983~; Osborn, M.J. and Weiner, I.M., J. Biol. Chem., 243, 2631-2639 (196~); Troy, F.A., Annual Reviews of Microbiology, 33J
519-560 (1979). Abbreviations used are: glu=glucose, gluA=glucuronic acid, man=mannose, glu-glu=cellobiose, P=phosphate, PP=pyrophosphQte, C55=isoprenoid lipid carrier, PEP=phosphoenolpyruvste, AcCoA=acetyl coenzyme A, I-V=glycosyltransferases, UDP=uridine 5'-diphosphate, GDP=guanosine 5'~iphosph~te.
Figure 2 shows the viscosities of solutions of polytrimer and xanthan gum, each at 1000 ppm in 10 weight percent NaCI brine, AS a function of shear rate.
Figure 3 shows the ratio of viscosities of solutions of 1,000 ppm polytrimer to xanthan gum as a function of brine salinity.
Figure 4 shows the ratio of viscosities of solutions of polytrimer to xanthan gum as a function of polymer concentration in 10 weight percent NaCl brine.
Figure 5 shows the ratio of viscosities of solutions of 1,000 ppm polytrimer to xanthan gum as a function of temperature in brines of various salinities.
DETAILED DESCRIPTION OF THE INVENTION
The polysaccharide polymer of tbis invention can be made with a cell-free enzyme system or can be made by growing cells of an appro-priate mutant strain. Other means of preparing the polysacchsride polymer are also described below.
The basic method relating to the use of ~ cell-free system to make xanthan gum is described in lelpi, L., Couso, R.O., and Dankert, M.A. (FEBS Letters, 130, 253-256, (1981)~ and c~n also be employed to make the polysaccharide polymer of this invention. For exsmple, wild~
type Xanthomonas campestris cells can be lysed by a freeze-thdw process and the substrates for polytrimer synthesis, UDP-glucose and GDP-mannose, with or without acetyl~oA, c~n be added to the lysate.
Alternate means of lysis mfly be used including but not limited to sonication, detergent treatment, enzyme treatment and combinations thereof. The lysate may be used in its crude ~orm, or purification of the enzymes may be employed. The enzymes of the xanthan gum biosynthetic pathway covalently join the glucose and mannose moieties as in the normal pathway. Since the enzymes have no UDP~lucuronic acid to add to the nascent chains, the pathway is blocke~ at reaction IV ~see pathway, Figure 1,) snd the intermediate isoprenoid lipi~ pyro~
phosphate-glucose-glucose-mannose accumuletes. Surprisingly, the xanthan polymerase which ordinarily ~cts on lipid-linked pentamer (glucose-glucose-mannose-glucuronic aci~mannose) is ~ble to polymerize lipid-linked trimer, (glucose~lucose-mannose.) Thus, the polytrimer of the present invention can be synthesized in vitro.
The cell-free synthesis of polytrimer described abave shows that Xanthomonas campestris cells hsve all the enzymes necessary to syn-thesize polytrimer. However, to use whole cells to synthesize polytrimer in vivo, Q means of blocking xanthan gum synthesis at resction IY (see Figure 1) is required. Mutagenesis can be employed 3 to block reaction IV.
9~8C3 Transposons, including but not limited to TnlO ~nd Tn903, can be used to mutagenize Xanthomonss. These transposons con~er re~istance to tetracycline and kanamycin, respectively. Transposons have the ability to insert themselYes into genes; when they do so, they cause mutations by interrupting the coding sequence, (Kleckner, N., Annual Reviews of Geneticst 15, 3~1 (1981).) The transposons can be introduced into Xanthomonas on a so-called ~uicide vector, such as pRK2013. This vector has the ability to tr~nsfer itself into non-enteric bacteria, such QS Xanthomonas, but cannot maintain itself (replicate) in that host, (Ditta, G., Corbin, D., Helinski, D.R., Proc.
Natl. Acad. Sci. USA, 77, 7347-7351 (1980). Thus, if the suicide vector is introduced into a population of Xanthomonas cells, and that population is subsequently challenged with either tetracycline or kanamycin, the individuals which survive are those in which one of the transposons has inserted itself into the genome of Xanthomonas. Survi-Yors of such a challenge can be screenea for those which haYe lost the ability to make xRnthan gum. Such mutants appear less mucoid than wild-type Xanthomonas.
In other embodiments of the invention, other means of mutagenesis can be employed to generate mutants which have lost the ability to make xanthan gum. Such me~ns will readily occur to one skilled in the art, and include, without limitation, irradiation, recombinant DNA technology, and chemical mutagen treatment (Miller, J.H., ExQeriments in Molecular Genetics (1972); Davis, R.W., 13otstein, D., and Roth, J.R., Advanced Bacterial Genetics (1980);
Maniatis, T., Fritsch, E.F., Sambrook, J., Molecular Clonin~ (1982), Cold Spring H~rbor).
Although mutsnts can first be chosen which appear less mucoid than wild-type, those desired retain the ability to make some polysaccharide. Cell-free extracts of each of the xanthan gum deficient mutants can be prepared and tested by adding different combinations of substrates and analyzing the products. For example, if 12791~0 UDP~glucose, GDP-mannose, and UDP-glucuronic scid Are added as substrates, the product should be Identical to that produced when UDP~lucose snd GDP-mannose are added. Alternstively, appropriate mutants can be detected by assaying the culture broth of each mut~nt for the presence of polytrimer. ThlLs xanthan gum deficient mutants can be found which appear to be blocked at reaction IV of the xanthan gum pathwsy. A mutsnt of this description has been placed on file at the American Type Culture Collection, Rockville, Maryland, as ATCC No. 53195. Such mutants can be used to synthesize polytrimer in vivo.
Although glycosyltransferase IV mutants have been employed in the examples to make the polytrimer of the present invention, other embodiments of the invention contemplste use of mutants in UDP-glucuronic acid metabolism. Such a mutant has been isolated and deposited at the American Type Culture Collection, Rockville, Maryland, under the ATCC No. 53196.
lt is not beyond the scope of the in~ention to employ an enzyme inhibitor of wild-type glycosyltransferase IV or of UDP-glucuronic acid biosynthesis to arrive at the same product. Still other alternatives for producing polytrimer are contemplated including enzymatic and chemical degrsdation of natural xanthan gum ss, for example, by removing the terminal mannose and glucuronic acid moieties from the side chsins o~ xanthan gum.
Using similar schemes to mutagenize strains of Xanthomonas, it is possible to obtain mutants which produce other new polysaccharide polymers. For exsmple, a mutation in the acetylase gene yields com-pletely non-acetylated xanthan gum. When an acetylase mutstion and a glycosyltransferase IV mutation ~re put in the same strain (a double mutant), a non-acetylaled polytrimer is produced. Other mutstions and combinations of mutations of the xsnthan pathway are possible to yield new products.
~27~80 - The mutants can be grown under conditions known in the art for growth of wild-type Xanthomonas. For example, they can be grown on suitable sssimilable carbon sources such as glueose, sucrose, maltose, starch, invert sugar, complex carbohydrates such QS molasses or corn syrup, various organic acids snd the like. Mixtures of carbon sources can also be employed. The concentration of carbon source supplied is often between about 10 and 60 grams per litetO Also necessary for growth are an assimilable source of organic or inorganic nitrogen, gen-erslly between about 0.1 and 1.0 grams per llter, and minerals, the choice of which are easily within the skill of the artO Examples of suitable nitrogen sources are ammonium salts, nitrate, urea, yeast extract, peptone, or other hydrolyzed proteinaceous materials or mix-tures thereof. Examples of suitable minerals include phosphorous, sulfur, potassium, sodium, iron, msgnesium; these are often added with a chelating sgent such as EDTA or citric acid.
Optimal temperatures for growth of Xsnthomonas generally are between about 18 and 35C, preferably between ~bout 28 and 32C.
anthomonas cells are grown aerobically by supplying air or oxygen so thst an adequate level of dissolved oxygen is maintained, for example, above about 10% of saturation. Preferably the level is kept above about 20%. The pH often is maintained at about 6.0 to 8.0, prefer-ably at about 6.5 to 7.5.
The polysaccharide polymer of the present invention can be recovered from fermentation broths by a suitable means. Precipitation with isopropanol, ethanol or other suitable alcohol readily yields the polytrimer gum. Generally, alcohols are added to a concentration of about 50 to 7596, on the basis of volume, preferably in the presence of potassium chloride, sodium chloride or other salt. Alternatively, the polymer can be recovered from the broth by ultrafiltration.
When chemical analyses are performed on polytrimer gum to determine the ratio of glucose:mannose, a variation from the theoret-ical value of 2:1 is found. The same type of variation is found when ~7~ O
g analyzing xanthan gum. Measured ranges of the ratio of glucose:
mannose will generslly be between about 1.4:1 and about 2.4:1. Pref-erably the ratio will be between 1.7:1 and ~.1:1.
Levels of acetylstion of the mannose residues of the polyssccharide polymer vary. In addition, it is not beyond the scope of the invention to employ a microorganism to m~ke the polysaccharide polymer which is incapable of acetylating the mannose residue~ such as acetylase-deficient mutants. In such a case there will be no acetylated mannose residues in the polyssccharide polymer.
Typically, concentrations o~ polytrimer in the fermentation broth are about 0.1% (w/w). Routine testing of fermentation condi tions And classical and recombinant DNA strain improvement techniques, all within the skill of the art, can be employed to improve the yield.
On a weight basis, polytrimer is superior to xanthan as a viscosifier of an aqueous medium. The viscosity of solutions of polytrimer is retained at conditions of high temperatures and/or high salinity. Such solutions can be prepaied at any desirable concen-trations, preferably between about 0.0196 and about 15%, by dissolving the polysaccharide polymer in an aqueous medium. The product of this invention is ideally suited for use in secondary oil recovery. The same techniques as sre used with xanthan gum in the art, and are well-known in secondary oil recovery, are appropriate with the polysaccharide polymer. See, for example, Lindblom, G.P., et al., U.S.
3,198,~68.
Mobility control solutions for use in enhanced oil recovery can be prepared from the polysaccharide polymer. Concentrations of from about 100 to about 3,000 ppm of the polys~ccharide polymer are appropriate for such mobility control solutions. Other known additiYes may also be used in, or in combinfltion with, these solutions to further enhance oil recovery. Such additives include, for example, surfactants and alkaline agents.
~2~3180 The polysacch~ride polymer, like x~nthan gum, can slso ~e used as a thickening agent in foods, cosmetics, medicinal form~ations, p~per sizings, drilling muds, printing inks, and the like. In addition it can be used to reduce frictional drag of fluid ilow in pipes.
The following examples are provided by way of exemplification and are not intended to limit the scope o~ the invention.
Example 1 This ex~mple shows how the produzt of the present invention can be prepared in vitro~ ~nd identiries it AS a truncated product of the ~canthan pathway.
Preparation of Lysates Xanthomonas campestris B1~59 S4-L w~s ob$sined from Northern Regional Research Laboratories of the U.S. Department of Agriculture.
Bacteria were grown in YM (yeast- nalt medium~ supplemented with 2%
(w/v) glucose as described by Jeanes, A., et 81. ~U.S. Department of Agriculture, ARS-NC-51, 14 pp (1976)). Cultures wer~ grown to l~te log phase at 30C at 300 rpm. The cells were titered on YM plus 2% (w/v) glucose plates at 30C. The cells were harvested by centrifugation and washed with cold Tris-HCl, 70mM, pH 8.2. Washed cells were resuspended in Tris-HCl, 70mM, p~I 5.2 with lOmM EDTA
and were freeze-thawed three times by a procedure similar to Garcia, R.C., et al. ~European Journal of Biochemistry 43, 93-105, (1974)).
This procedure ruptured the cells, as was eqidenced by the increased viscosity of the suspensions and the complete loss of cell viability (one in 106 survivors) after this treatment. The freeze-thawed lysates were frozen in aliquots at -80 C. Protein concentration was determined with BIO RAD assay (BIO RAD Laboratories, Richmond, California) and was found to be 5 to 7 mg cell protein per ml of lysate.
Biosynthetic Assay Procedure 3n As described in lelpi, L., Couso, R.O., and Dankert, M.A., FEBS
Letters, 130, 253-256 (1981), an aliquot of freeze-thawed lysate (equivalent to 300 to 400 ug protein), DNAase I (10 ug/ml), and MgCl2 ~2~ 80 (8 mM) were preincubated at 20(`' for twenty minutes. An equal volume of 70 mM Tris-HCI, pH 8.2, with the desired radiol~beled sugar nucleotides (UDP-glucose and GDP- mannose), with or without UDP-glucuronic acid, was added snd incubated at 20C. At various times~
S the reactions were stopped by the l~ddition of EDTA to 4m M. The samples were centrifuged; the pellets were washed two times with - buffer. The supernatants were combined, carrier ~anthan (100 ug) WAS
added, and the xanth~n plus synthe~ized polymer were precipitated with ethanol(~0%)-KCl(0.8%). The precipitated polymer was resuspended in water and reprecipitated two more times to remove unincorporated label. Radioactivity incorporated into the gum iraction was determined in a liquid scintillation counter, ~nd the data were processed to obtain incorporation in terms Or pmoles.
~L2~L130 TABI,E 1 lncorporation of l~beled sugars by freeze-thQw cell lysate o~ X. ~ampestris B1459 S4-L into gum Gum Fract i on ~ pmol ?
I ncuba t i on Mi x[ 3H]man E 1 4c] gl c gl c/man _________________~_~__ ___~____~___~____ .
+UDPG, GDPM 98 201 2.1 +UDPG, GDPM, UDP-GA1540 1562 1.0 dpm/pmol 3H = 442 14C = 37. 5 _~ _ , , .
UDPG = UDP-glucose glc = glucose GDPM = COP-mannose man = mannose - UDP-GA = UDP-glucuronic acid dpm = disintegrations per minute pmo 1 = p i como l e Cell Iysates oî B1459 S4-L were incubated at 20C for 30 minutes and processed to give the gum fractions as described in the text. The . .
molar ratio of glucose to mQnnose is the ratio of pmoles of incorpor-ated carbon-l~i to tritium labeled sugars in the gum ~ractions.
~79~
In the presence of all three sugar constituents, the ratio of glucose: msnnose was 1.0:1, as expected for xanthsn gum. When UDP-glucuronic acid was absent, the ratio was 2.1:1. See Table 1.
This ratio is consistent with the hypothesis that the polysaccharide polymer is formed of trimer units which are intermediates in the xanthsn gum biosynthetic pathway.
A pulse-chase in vitro experiment showed that lipid-linked cellobiose (a glucose dirner) W8S processed to lipid-linked trimer (glucose-glucose-mannose) and subsequently to polytrimer gum. A
freeze-thaw lysate of strain B1459 S4-L was prepared ss described above. UDP-[14C]glucose was sdded to the lysate, comprising the "pulse", and radiolabeled cellobiose accumulated on the lipid carrier during an incubstion of 13 minutes. The "chase" consisted of addition of 100-fold excess unlabeled UDP~lucose as well ss GDP{3H~mannose.
Aliquots of the incubation mixture of lysate snd sugar nucleotides were removed at various times and processed to produce fln organic extract (lipid carrier-linked fraction) and an aqueous fraction (containing gum).
The oligosaccharides o~ the organic extrsct were acid hydrolyzed from the lipid carrier, dephosphorylated~ separated by thin l~yer chromstography, removed from the chromstogr~ms and the radiolabel quantitated. The results are shown in Tsble 2.
~ ....... .
127~
. . .
Fate of UDP-[14C~ glucose in l)ulse-chase in vitro experiment with cell lysates of B145g S4-L
Pulse (12 min) 9 pmol Lipid-linked cellobiose Chase (4 min)1 pmol Lipid-linked cellobiose 10 pmol Li pi d-l i nked t r imer - Chase (16 min) 0.5 pmol Lipid-linked cellobiose 6 pmol Li pi d-l i nked tr imer 3 pmol Soluble polytrimer Chase ( 48 mi n ) O . 2 pmol Li pi d-l i nked cel lobi ose 0.4 pmol Lipid-linked trimer 10 pmol Soluble polytrimer The experimental conditions and the processing of the organic fraction and the soluble gum fraction sre described in the text of Example lo ~279~
The labeled glucose from ~JDP-[14C]glucose, as can be seen in Table 2, was immedistely incorporat,ed into llpid-linked cellobiose in the ~pulse". Upon addition of GDP-mannose and excess UDP~lucose (the chsse), the labeled cellobiose was conYerted rapidly to labeled lipid-linked trimer, which WQS lster detected as polytrimer gum in the aque-ous fraction, at about 16 minutes after the chase began. This demonstrates the precursor-product relationships o~ UDP-glucose, lipid-linked cellobiose, lipid-linked trimer, and polytrimer gum, and their relationships to the xanthan biosynthetic pathway.
Example 2 This example demonstrates the molar ratio of glucose to mQnnose in polytrimer gum synthesized in vitro by a glycosyltransferase IV-deficient mutant.
The method of preparing the lysate is described above in Exam-ple 1. The strain used to prepare the lysate was that designated Al`CC No. 53195. Added to the lysate were either 1, 2 or 3 nucleotide-charged sugars, consisting of UDP-[14C]glucose alone9 UDP-[14C]glucose and GDP-[3H]mannose, or UDP-[14C]g Icose, GDP-[3H]mannose and unlabeled UDP~lucuronic acid. At 30 minutes after addition of the sugar substrates, the aqueous fraction was processed and analyzed as described in Example 1. Results are shown in Table 3. When two sugQr substrates, UDP-glucose and GDP-mannose, were present in the incub~tion mixture the molar ratio of glucose to mannose found in the gum was 2.4:1. When all three sugar substrates were incubated together with the lysate, the resulting gum had a 2.3:1 molar ratio of glucose to mannose.
~Z7~
Incorporation of labeled s~gsrs by ~reeze thas~ cell lysate of ATCC Ns. 53195 into polytrfmer gum ~;urn Fract i on ( Dmol ) -Resction Mix t3H]m~n [14C]glc glc/m~n ____ ~____________ ___~_____~________________ ;
+ 2 UDPG, GDPM 71 17 4 2 . 4 + 3 U.DPG, GDPM, UDP -GA 6 5 1 5 2 2 . 3 dpm/pmol 3H = 340 14C = 40 __________~_ __~______ ~_______~________ ___ Abbrevistions are e~plained in legend to Table 1.
Cell Iysates of ATCC No. 53195 were incubsted st 20C for 30 minutes in the reaction mixes indicated and processed to give the gum fract30ns ~s described in Example 1. The molar ratio of glucose to mannose indicated is the ratio of pmoles of incorporated carbon-14 to tritium labeled sugars in the processed fract30ns.
~7~0 ., .
The presence of UDP~lucuronic acid has no effect on the ratio o glucose to mannose incorporated into a polysacch~ride polymer when the cell-free lysate used is from a glycosyltransferase IV~eficient mutant. The biochemical phenotype of the mutant lysate when incubated with all three sugars is analogous to that Or the wild-type lysate when incubated with only two sugar substrates, in that the in vitro produced gums both have a molar ratio of spproximately 2:1 of glucose to mannose moieties.
Example 3 This example demonstrates that the trimeric intermediate which is polymerized to form polytrimer gum has the ssme anomeric configu-ration of the sugars as in xanthan gum. In addition it demonstrates that the mannose of the trimer is attached to the non-reducing glucose of cellobiose in the lipid-linked intermediate.
Alpha-mannosidase (EC 3.2.1.24) 2nd beta-glucosidase (EC 3.2.1.21)-were used to singly or sequentislly treat the trimeric oligosaccharide which had been synthesized and double labeled in vitro as described in Example 1. Alpha-mannosidase will hydrolyze terminal, unsubstituted mannose residues attached through an alpha-1 linkage. Beta-glucosidase will hydrolyze terminal, unsubstituted D~lucosyl residues attached in a beta-1 linkage.
The trimer was removed from the lipid and dephosphorylated.
This was then deacetylated by base treatment, such as pH12 for 2 to 3 hours, becsuse alphs-mannosidase cannot recognize acetylated mannose 2 5 moieties.
The results were as follows. Treatment of trimeric oligosaccharide with beta~lucosidase left it unchanged. When slpha-mannosidase was used to treat the trimeric oligosacchsride, cellobiose and mannose were formed. When the trimeric oligomer was treated ~ with alpha-mannosid~se, first, and beta-glucosidase, second, glucose and mannose were formed. The results confirm that mannose is attached to the non--reducing glucose by an alpha-linkage in the trimeric 1279~30 intermediate, and that the glucose moleties are bets-linked. This confirms that trimer is an intermediate product of the normal xanthan enzyme pathway.
Example 4 This example shows the methods of mutagenesis and screening which were employed to generste the mutant strains which are xanthan gum deficient due to a lesion in th~e gene for glycosyltransferase IV.
Xflnthomonas campestris, genetically m~rked with 8 chromosomal resistance to streptomycin, W8S used QS a recipient in ~ conjugation with E. coli LE392 containing plasmid pRK2û13::TnlO. Plasmid pRK2013 contains Tn903 which encodes kanamycin resistance, ~Figurski, D.H., and Helinski, D.R., Proc. Natl. Acsd. Sci., U.S.A., 76-, 16~8-1652 (1979);) and the plasmid cannot replicate in Xanthomonas, (Ditta, G., et al., supra.) Transposon TnlO encodes r~sistance to tetracycline.
Transconjugants were selected which were resistant to streptomycin and kanamycin, or streptomycin and tetracycline. The former occurred at a frequency of about 4 X 10~6/recipient and presumably resulted from a transposition of Tn903. The latter occurred at a frequency of about 3 X 10~6/recipient ~nd presumably resulted from a transposition of TnlO into the genome of Xanthomonas campestris.
Auxotrophs were found among these transconjugants at 8 frequency of about 296; their needs were widely distributed among the various nutritional requirements. This indicates that these transposons do not have a particularly preferred locus for insertion in Xanthomonas. Prototrophic revertants of the auxotrophs were selected, and most were found to be drug-sensitive; this suggests that the auxotrophies were caused by transposon insertion.
To screen for xanthan gum deficient mutants among the doubly resistant transconjugants, Congo Red dye, which enhances the morphological distinction between xanthan gum producJng and non-producing colonies, WQS added to the solid media. Colonial morphology was examined after 7 to 12 days incubation at 30C. Xanthan gum deficient mutants were found at a frequency of approximately lO-~.
12~ 8~
. -- 19 -To identify a glycosyltransfersse IV mut~nt from among the xanthan gum deficient mutsnts, freez:e-thflw Iysates of each were pre-2ared. Radiolabeled UDP-glucose and GDP-mannose were added with or without UDP-glucuronic acid, The desired mutants made Q gum 5 having a glucose:m~nnose ratio of ~bout 2:1, irrespective of the presence of UDP~lucuronic acid. SeYeral mutants were fouhd of this description. They cont~in lesions due to TnlO insertion. Mutants induced by Tn903 were also found having this phenotype. In addition mutants have been isolated having this phenotype which were induced 10 by nitrosoguanidine.
Example 5 This example demonstrates the use of a glycosyltransferase IV
deficient mutant to produce polytrimer gum in vivo.
To obtain in v~vo synthesized gums, five liters each of wild-type 15 NRRL B-1459 S4-L ~nd the glycosyltransferase IY deficient mutant oî
Example 4 (ATCC No. 53195) were aerobically grown in a fermenter st 28C to 32C, with the pH controlled at pH 6.0 to 8Ø A minimal medium was used contsining 10 g/l potassium phosphate, 1.43 g/l ammonium sulfate, 2 g/l citric acid, 30 g/l glucose, ~nd trace ele-20 ments. After 145 hours, the gums were recovered and purified. The cells were removed by centrifugation and the gums precipitated from the broth by addition of isoprop~nol (55% v/v) and sodium chloride (0.5% w/v). The precipitates were collected by filtration and redissolved in water. The gums were reprecipitated with isopropsnol 25 (55q6 v/v) without salt and redissolved in water. The preparations were di~lyzed using 1~,000 MW cutoff membrane dialysis tubing against water for three days.
The glucose:mannose ratios were determined by complete acid hydrolysis of the polysaccharide polymers with subsequent analysis by 30 high performance liquid chromatography (HPLC), and found to conform to the ratios found for the in vitro synthesized polym ers. The glycosyltransferase IV deficient mutant designated ATCC No. 53l~5 12"~9 3L8V
made a gum with Q glucose to mannose ratio o~ &bout 2.15:1, whereas the wil~type made R gum of ratio ebout 0.96:1.
Other in v~vo produced sampl~s of polytrimer gum were assayed by HPLC or by enzymatic ~nelyses of the sugsrs after acid hydrolysis.
For the twenty-four enalyses performed, the molar ratios r~nge from 1.43:1 to 2.44:1 of glucose to mannose. The mean ratio v~as 1.90 ~
0.15:1 for polytrimer made by the glycosyltransfer~se IV deficient mutant strain.
Also shown by the HPLC analysis of the in vlvo produced polytrimer within detectsble limits were: 1-the absence of glucuronic acid; 2-the absence of pyruvate; 3-the presence of acetate; 4-the absence of sugars other than glucose and mannose.
Example 6 This example shows that polytrimer provides aqueous solutions which exhibit improved rheological properties compared to xanthan gum over ~ range of temperatures snd inorganic salt concentrations.
Solutions of polytrimer gum (synthesized in vivo in accordance with Example 5) and xanthan gum (purified Pfizer Flocon 48G0, were prepared ~t a concentration of 1,000 ppm in a water containing 10 weight percent sodium chloride. Polytrimer gum shows substantially greater viscosity than xanthan gum over e wide range of shear rates (Figure 2).
The ratio of polytrimer to xanthan viscosity at room temperature varies with water salinity and is between 2 and 2.5 over a salinity range of 0 to 20 weight percent sodium chloride, as shown in Figure 3. The ratio of polytrimer viscosity to xenthan viscosity also varies with polymer concentration (Figure 4). Finally, the improvement in polytrimer viscosity over xanthan viscosity increases with temper~ture over a range of 25 to 75C, ror w~ter sslinities of 0 to 20 weight 3 0 percent sodium chloride (Figure 5~.
Since vsriations of this invention will be apparent to those skilled in the srt, it is intended that this invention be limited only by the scope Or the claims.
A POLYSACCHARIDE POLYMER MADE BY XANTHOMONAS
This application is a division of our copending Canadian patent application Serial No. 577,352 filed on September 14, 1988.
BACKGROUND OF THE INVENTION
Xanthan gum is produced by bacteria of the genus Xanthomonas, such as the species campestris, albilineans, fra~ria, vesicatoria, and the like. Xanthan gu m is 8 widely used product due to its unusual physical properties: extremely high specific viscosity and pseudo-plasticity. It is ~ommonly used in foods as a thickening agent and in secondflry oil recovery as mobility control and profile modification agents and in petroleum drilling fluids.
Chemically, xanthan gum is an anionic heteropolysaccharide. The repeating unit of the polymer is a pentamer composed of five sugar moieties: two glucose, one glucuronic scid and two mannose moieties.
They are flrranged such that the glucose moieties form the backbone of the polymer chain, and side chains of mannose-glucuronic acid-mannose gene~ally extend from alternate glucose moieties. Often this bssic structure is specifically acetylated and/or pyruvylated. (Janson, P.E., Kenne, L., and Lindberg, B., Carbohydrate Research, 45, 2~5-282 (l975); Melton, L.D.~ Mindt, L., Rees, D.A., and Sanderson, G.R., Carboyhydrate Research, 46, 245-257 (1976).) The structure is depicted below:
L~ ~
to~ , ...~,~.~.
In spite of the broad utility of naturally occurring x~nthan gum, there are some situations where its physical properties become limiting.
In particular, in secondary oil recovery it ~3 not uncommon for the 5 temperature of the oil-bearing reservoir and salt concentrations in the reservoir brine to be higher than are optimal ~or xanthan solutions.
When these conditions oc~ur, xanthan can precipitate, flocculate and/or lose viscosity. Therefore there is a need for new viseosi~ying products which perform well at high temperature and high salt conditions.
10 ~ SUMMARY OF THE INVENTION
.
An object of the invention is to provide a process for recovering oil from an oil-bearing subterranean formation using novel viscosifying products.
- 3 ~
In accordance with another aspect of this invention claimed in our parent Canadian patent application Serial No. 513,834 which issued as Canadian Patent 1,247,033 on December 20, 1988, there is provided a composition comprising a polysaccharide polymer containing essentially no glucuronic acid moieties having a D-glucose:D-mannose ratio of about 2:1, wherein the D-glucose ~oieties are linked in a beta-[1,4] configuration to form the polymer backbone, and the D~mannose moieties are each linked in an alpha-[1,3] configuration generally to alternate glucose moieties. This composition can be used in the process of the present application.
The polysaccharide polymer which can be used in this invention can be made by blocking one of the steps in xanthan gum biosynthesis. Therefore, rather than having a three-sugar side-chain extending from the backbone of beta-[1,4]-D-glucose as in xanthan gum, the polysaccharide polymer of this invention has a single sugar moiety generally linked to alternate glucose moieties of the backbone. The polysaccharide polymer of this invention is herein termed "polytrimer" because it consists of a repeating trimer unit, glucose-glucose-mannose. Its structure is shown below, where n is the ~ number of repeating units in the polymer.
~;~;~
01~ OH
~Lo '~27918C) As shown by the above, the polytrimer consists of D-mannose linked alpha-[1,3] generally to alternate moieties of beta-~1,4] linked D~
glucose. As in xanthan gum, an acetic scid moiety can be, but is not always, esterified at the 6-O position of mannose, as described in Sutherland, I.W., Carbohydrate Polymers, ~ 107-115, (1981). Although the structure of the polysaccharide polymer is thought to be . as shown, it is possible that under certain conditions of synthesis, a mannose moiety may not always be linked at alternating glucose residues.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 depicts the assumed pathway of xanthan gum biosynthe-sis. It is bssed on the data of several laboratoriesO See, lelpi, L.7 Couso, R.O., and Dankert, :~I.A., Biochem. Biophy. Res. Comm., 102, 1400-1408 (1981), FEBS Letters, 130, 253-256 (1981), 8iochem. Intern~, 6, 323-333 (1983~; Osborn, M.J. and Weiner, I.M., J. Biol. Chem., 243, 2631-2639 (196~); Troy, F.A., Annual Reviews of Microbiology, 33J
519-560 (1979). Abbreviations used are: glu=glucose, gluA=glucuronic acid, man=mannose, glu-glu=cellobiose, P=phosphate, PP=pyrophosphQte, C55=isoprenoid lipid carrier, PEP=phosphoenolpyruvste, AcCoA=acetyl coenzyme A, I-V=glycosyltransferases, UDP=uridine 5'-diphosphate, GDP=guanosine 5'~iphosph~te.
Figure 2 shows the viscosities of solutions of polytrimer and xanthan gum, each at 1000 ppm in 10 weight percent NaCI brine, AS a function of shear rate.
Figure 3 shows the ratio of viscosities of solutions of 1,000 ppm polytrimer to xanthan gum as a function of brine salinity.
Figure 4 shows the ratio of viscosities of solutions of polytrimer to xanthan gum as a function of polymer concentration in 10 weight percent NaCl brine.
Figure 5 shows the ratio of viscosities of solutions of 1,000 ppm polytrimer to xanthan gum as a function of temperature in brines of various salinities.
DETAILED DESCRIPTION OF THE INVENTION
The polysaccharide polymer of tbis invention can be made with a cell-free enzyme system or can be made by growing cells of an appro-priate mutant strain. Other means of preparing the polysacchsride polymer are also described below.
The basic method relating to the use of ~ cell-free system to make xanthan gum is described in lelpi, L., Couso, R.O., and Dankert, M.A. (FEBS Letters, 130, 253-256, (1981)~ and c~n also be employed to make the polysaccharide polymer of this invention. For exsmple, wild~
type Xanthomonas campestris cells can be lysed by a freeze-thdw process and the substrates for polytrimer synthesis, UDP-glucose and GDP-mannose, with or without acetyl~oA, c~n be added to the lysate.
Alternate means of lysis mfly be used including but not limited to sonication, detergent treatment, enzyme treatment and combinations thereof. The lysate may be used in its crude ~orm, or purification of the enzymes may be employed. The enzymes of the xanthan gum biosynthetic pathway covalently join the glucose and mannose moieties as in the normal pathway. Since the enzymes have no UDP~lucuronic acid to add to the nascent chains, the pathway is blocke~ at reaction IV ~see pathway, Figure 1,) snd the intermediate isoprenoid lipi~ pyro~
phosphate-glucose-glucose-mannose accumuletes. Surprisingly, the xanthan polymerase which ordinarily ~cts on lipid-linked pentamer (glucose-glucose-mannose-glucuronic aci~mannose) is ~ble to polymerize lipid-linked trimer, (glucose~lucose-mannose.) Thus, the polytrimer of the present invention can be synthesized in vitro.
The cell-free synthesis of polytrimer described abave shows that Xanthomonas campestris cells hsve all the enzymes necessary to syn-thesize polytrimer. However, to use whole cells to synthesize polytrimer in vivo, Q means of blocking xanthan gum synthesis at resction IY (see Figure 1) is required. Mutagenesis can be employed 3 to block reaction IV.
9~8C3 Transposons, including but not limited to TnlO ~nd Tn903, can be used to mutagenize Xanthomonss. These transposons con~er re~istance to tetracycline and kanamycin, respectively. Transposons have the ability to insert themselYes into genes; when they do so, they cause mutations by interrupting the coding sequence, (Kleckner, N., Annual Reviews of Geneticst 15, 3~1 (1981).) The transposons can be introduced into Xanthomonas on a so-called ~uicide vector, such as pRK2013. This vector has the ability to tr~nsfer itself into non-enteric bacteria, such QS Xanthomonas, but cannot maintain itself (replicate) in that host, (Ditta, G., Corbin, D., Helinski, D.R., Proc.
Natl. Acad. Sci. USA, 77, 7347-7351 (1980). Thus, if the suicide vector is introduced into a population of Xanthomonas cells, and that population is subsequently challenged with either tetracycline or kanamycin, the individuals which survive are those in which one of the transposons has inserted itself into the genome of Xanthomonas. Survi-Yors of such a challenge can be screenea for those which haYe lost the ability to make xRnthan gum. Such mutants appear less mucoid than wild-type Xanthomonas.
In other embodiments of the invention, other means of mutagenesis can be employed to generate mutants which have lost the ability to make xanthan gum. Such me~ns will readily occur to one skilled in the art, and include, without limitation, irradiation, recombinant DNA technology, and chemical mutagen treatment (Miller, J.H., ExQeriments in Molecular Genetics (1972); Davis, R.W., 13otstein, D., and Roth, J.R., Advanced Bacterial Genetics (1980);
Maniatis, T., Fritsch, E.F., Sambrook, J., Molecular Clonin~ (1982), Cold Spring H~rbor).
Although mutsnts can first be chosen which appear less mucoid than wild-type, those desired retain the ability to make some polysaccharide. Cell-free extracts of each of the xanthan gum deficient mutants can be prepared and tested by adding different combinations of substrates and analyzing the products. For example, if 12791~0 UDP~glucose, GDP-mannose, and UDP-glucuronic scid Are added as substrates, the product should be Identical to that produced when UDP~lucose snd GDP-mannose are added. Alternstively, appropriate mutants can be detected by assaying the culture broth of each mut~nt for the presence of polytrimer. ThlLs xanthan gum deficient mutants can be found which appear to be blocked at reaction IV of the xanthan gum pathwsy. A mutsnt of this description has been placed on file at the American Type Culture Collection, Rockville, Maryland, as ATCC No. 53195. Such mutants can be used to synthesize polytrimer in vivo.
Although glycosyltransferase IV mutants have been employed in the examples to make the polytrimer of the present invention, other embodiments of the invention contemplste use of mutants in UDP-glucuronic acid metabolism. Such a mutant has been isolated and deposited at the American Type Culture Collection, Rockville, Maryland, under the ATCC No. 53196.
lt is not beyond the scope of the in~ention to employ an enzyme inhibitor of wild-type glycosyltransferase IV or of UDP-glucuronic acid biosynthesis to arrive at the same product. Still other alternatives for producing polytrimer are contemplated including enzymatic and chemical degrsdation of natural xanthan gum ss, for example, by removing the terminal mannose and glucuronic acid moieties from the side chsins o~ xanthan gum.
Using similar schemes to mutagenize strains of Xanthomonas, it is possible to obtain mutants which produce other new polysaccharide polymers. For exsmple, a mutation in the acetylase gene yields com-pletely non-acetylated xanthan gum. When an acetylase mutstion and a glycosyltransferase IV mutation ~re put in the same strain (a double mutant), a non-acetylaled polytrimer is produced. Other mutstions and combinations of mutations of the xsnthan pathway are possible to yield new products.
~27~80 - The mutants can be grown under conditions known in the art for growth of wild-type Xanthomonas. For example, they can be grown on suitable sssimilable carbon sources such as glueose, sucrose, maltose, starch, invert sugar, complex carbohydrates such QS molasses or corn syrup, various organic acids snd the like. Mixtures of carbon sources can also be employed. The concentration of carbon source supplied is often between about 10 and 60 grams per litetO Also necessary for growth are an assimilable source of organic or inorganic nitrogen, gen-erslly between about 0.1 and 1.0 grams per llter, and minerals, the choice of which are easily within the skill of the artO Examples of suitable nitrogen sources are ammonium salts, nitrate, urea, yeast extract, peptone, or other hydrolyzed proteinaceous materials or mix-tures thereof. Examples of suitable minerals include phosphorous, sulfur, potassium, sodium, iron, msgnesium; these are often added with a chelating sgent such as EDTA or citric acid.
Optimal temperatures for growth of Xsnthomonas generally are between about 18 and 35C, preferably between ~bout 28 and 32C.
anthomonas cells are grown aerobically by supplying air or oxygen so thst an adequate level of dissolved oxygen is maintained, for example, above about 10% of saturation. Preferably the level is kept above about 20%. The pH often is maintained at about 6.0 to 8.0, prefer-ably at about 6.5 to 7.5.
The polysaccharide polymer of the present invention can be recovered from fermentation broths by a suitable means. Precipitation with isopropanol, ethanol or other suitable alcohol readily yields the polytrimer gum. Generally, alcohols are added to a concentration of about 50 to 7596, on the basis of volume, preferably in the presence of potassium chloride, sodium chloride or other salt. Alternatively, the polymer can be recovered from the broth by ultrafiltration.
When chemical analyses are performed on polytrimer gum to determine the ratio of glucose:mannose, a variation from the theoret-ical value of 2:1 is found. The same type of variation is found when ~7~ O
g analyzing xanthan gum. Measured ranges of the ratio of glucose:
mannose will generslly be between about 1.4:1 and about 2.4:1. Pref-erably the ratio will be between 1.7:1 and ~.1:1.
Levels of acetylstion of the mannose residues of the polyssccharide polymer vary. In addition, it is not beyond the scope of the invention to employ a microorganism to m~ke the polysaccharide polymer which is incapable of acetylating the mannose residue~ such as acetylase-deficient mutants. In such a case there will be no acetylated mannose residues in the polyssccharide polymer.
Typically, concentrations o~ polytrimer in the fermentation broth are about 0.1% (w/w). Routine testing of fermentation condi tions And classical and recombinant DNA strain improvement techniques, all within the skill of the art, can be employed to improve the yield.
On a weight basis, polytrimer is superior to xanthan as a viscosifier of an aqueous medium. The viscosity of solutions of polytrimer is retained at conditions of high temperatures and/or high salinity. Such solutions can be prepaied at any desirable concen-trations, preferably between about 0.0196 and about 15%, by dissolving the polysaccharide polymer in an aqueous medium. The product of this invention is ideally suited for use in secondary oil recovery. The same techniques as sre used with xanthan gum in the art, and are well-known in secondary oil recovery, are appropriate with the polysaccharide polymer. See, for example, Lindblom, G.P., et al., U.S.
3,198,~68.
Mobility control solutions for use in enhanced oil recovery can be prepared from the polysaccharide polymer. Concentrations of from about 100 to about 3,000 ppm of the polys~ccharide polymer are appropriate for such mobility control solutions. Other known additiYes may also be used in, or in combinfltion with, these solutions to further enhance oil recovery. Such additives include, for example, surfactants and alkaline agents.
~2~3180 The polysacch~ride polymer, like x~nthan gum, can slso ~e used as a thickening agent in foods, cosmetics, medicinal form~ations, p~per sizings, drilling muds, printing inks, and the like. In addition it can be used to reduce frictional drag of fluid ilow in pipes.
The following examples are provided by way of exemplification and are not intended to limit the scope o~ the invention.
Example 1 This ex~mple shows how the produzt of the present invention can be prepared in vitro~ ~nd identiries it AS a truncated product of the ~canthan pathway.
Preparation of Lysates Xanthomonas campestris B1~59 S4-L w~s ob$sined from Northern Regional Research Laboratories of the U.S. Department of Agriculture.
Bacteria were grown in YM (yeast- nalt medium~ supplemented with 2%
(w/v) glucose as described by Jeanes, A., et 81. ~U.S. Department of Agriculture, ARS-NC-51, 14 pp (1976)). Cultures wer~ grown to l~te log phase at 30C at 300 rpm. The cells were titered on YM plus 2% (w/v) glucose plates at 30C. The cells were harvested by centrifugation and washed with cold Tris-HCl, 70mM, pH 8.2. Washed cells were resuspended in Tris-HCl, 70mM, p~I 5.2 with lOmM EDTA
and were freeze-thawed three times by a procedure similar to Garcia, R.C., et al. ~European Journal of Biochemistry 43, 93-105, (1974)).
This procedure ruptured the cells, as was eqidenced by the increased viscosity of the suspensions and the complete loss of cell viability (one in 106 survivors) after this treatment. The freeze-thawed lysates were frozen in aliquots at -80 C. Protein concentration was determined with BIO RAD assay (BIO RAD Laboratories, Richmond, California) and was found to be 5 to 7 mg cell protein per ml of lysate.
Biosynthetic Assay Procedure 3n As described in lelpi, L., Couso, R.O., and Dankert, M.A., FEBS
Letters, 130, 253-256 (1981), an aliquot of freeze-thawed lysate (equivalent to 300 to 400 ug protein), DNAase I (10 ug/ml), and MgCl2 ~2~ 80 (8 mM) were preincubated at 20(`' for twenty minutes. An equal volume of 70 mM Tris-HCI, pH 8.2, with the desired radiol~beled sugar nucleotides (UDP-glucose and GDP- mannose), with or without UDP-glucuronic acid, was added snd incubated at 20C. At various times~
S the reactions were stopped by the l~ddition of EDTA to 4m M. The samples were centrifuged; the pellets were washed two times with - buffer. The supernatants were combined, carrier ~anthan (100 ug) WAS
added, and the xanth~n plus synthe~ized polymer were precipitated with ethanol(~0%)-KCl(0.8%). The precipitated polymer was resuspended in water and reprecipitated two more times to remove unincorporated label. Radioactivity incorporated into the gum iraction was determined in a liquid scintillation counter, ~nd the data were processed to obtain incorporation in terms Or pmoles.
~L2~L130 TABI,E 1 lncorporation of l~beled sugars by freeze-thQw cell lysate o~ X. ~ampestris B1459 S4-L into gum Gum Fract i on ~ pmol ?
I ncuba t i on Mi x[ 3H]man E 1 4c] gl c gl c/man _________________~_~__ ___~____~___~____ .
+UDPG, GDPM 98 201 2.1 +UDPG, GDPM, UDP-GA1540 1562 1.0 dpm/pmol 3H = 442 14C = 37. 5 _~ _ , , .
UDPG = UDP-glucose glc = glucose GDPM = COP-mannose man = mannose - UDP-GA = UDP-glucuronic acid dpm = disintegrations per minute pmo 1 = p i como l e Cell Iysates oî B1459 S4-L were incubated at 20C for 30 minutes and processed to give the gum fractions as described in the text. The . .
molar ratio of glucose to mQnnose is the ratio of pmoles of incorpor-ated carbon-l~i to tritium labeled sugars in the gum ~ractions.
~79~
In the presence of all three sugar constituents, the ratio of glucose: msnnose was 1.0:1, as expected for xanthsn gum. When UDP-glucuronic acid was absent, the ratio was 2.1:1. See Table 1.
This ratio is consistent with the hypothesis that the polysaccharide polymer is formed of trimer units which are intermediates in the xanthsn gum biosynthetic pathway.
A pulse-chase in vitro experiment showed that lipid-linked cellobiose (a glucose dirner) W8S processed to lipid-linked trimer (glucose-glucose-mannose) and subsequently to polytrimer gum. A
freeze-thaw lysate of strain B1459 S4-L was prepared ss described above. UDP-[14C]glucose was sdded to the lysate, comprising the "pulse", and radiolabeled cellobiose accumulated on the lipid carrier during an incubstion of 13 minutes. The "chase" consisted of addition of 100-fold excess unlabeled UDP~lucose as well ss GDP{3H~mannose.
Aliquots of the incubation mixture of lysate snd sugar nucleotides were removed at various times and processed to produce fln organic extract (lipid carrier-linked fraction) and an aqueous fraction (containing gum).
The oligosaccharides o~ the organic extrsct were acid hydrolyzed from the lipid carrier, dephosphorylated~ separated by thin l~yer chromstography, removed from the chromstogr~ms and the radiolabel quantitated. The results are shown in Tsble 2.
~ ....... .
127~
. . .
Fate of UDP-[14C~ glucose in l)ulse-chase in vitro experiment with cell lysates of B145g S4-L
Pulse (12 min) 9 pmol Lipid-linked cellobiose Chase (4 min)1 pmol Lipid-linked cellobiose 10 pmol Li pi d-l i nked t r imer - Chase (16 min) 0.5 pmol Lipid-linked cellobiose 6 pmol Li pi d-l i nked tr imer 3 pmol Soluble polytrimer Chase ( 48 mi n ) O . 2 pmol Li pi d-l i nked cel lobi ose 0.4 pmol Lipid-linked trimer 10 pmol Soluble polytrimer The experimental conditions and the processing of the organic fraction and the soluble gum fraction sre described in the text of Example lo ~279~
The labeled glucose from ~JDP-[14C]glucose, as can be seen in Table 2, was immedistely incorporat,ed into llpid-linked cellobiose in the ~pulse". Upon addition of GDP-mannose and excess UDP~lucose (the chsse), the labeled cellobiose was conYerted rapidly to labeled lipid-linked trimer, which WQS lster detected as polytrimer gum in the aque-ous fraction, at about 16 minutes after the chase began. This demonstrates the precursor-product relationships o~ UDP-glucose, lipid-linked cellobiose, lipid-linked trimer, and polytrimer gum, and their relationships to the xanthan biosynthetic pathway.
Example 2 This example demonstrates the molar ratio of glucose to mQnnose in polytrimer gum synthesized in vitro by a glycosyltransferase IV-deficient mutant.
The method of preparing the lysate is described above in Exam-ple 1. The strain used to prepare the lysate was that designated Al`CC No. 53195. Added to the lysate were either 1, 2 or 3 nucleotide-charged sugars, consisting of UDP-[14C]glucose alone9 UDP-[14C]glucose and GDP-[3H]mannose, or UDP-[14C]g Icose, GDP-[3H]mannose and unlabeled UDP~lucuronic acid. At 30 minutes after addition of the sugar substrates, the aqueous fraction was processed and analyzed as described in Example 1. Results are shown in Table 3. When two sugQr substrates, UDP-glucose and GDP-mannose, were present in the incub~tion mixture the molar ratio of glucose to mannose found in the gum was 2.4:1. When all three sugar substrates were incubated together with the lysate, the resulting gum had a 2.3:1 molar ratio of glucose to mannose.
~Z7~
Incorporation of labeled s~gsrs by ~reeze thas~ cell lysate of ATCC Ns. 53195 into polytrfmer gum ~;urn Fract i on ( Dmol ) -Resction Mix t3H]m~n [14C]glc glc/m~n ____ ~____________ ___~_____~________________ ;
+ 2 UDPG, GDPM 71 17 4 2 . 4 + 3 U.DPG, GDPM, UDP -GA 6 5 1 5 2 2 . 3 dpm/pmol 3H = 340 14C = 40 __________~_ __~______ ~_______~________ ___ Abbrevistions are e~plained in legend to Table 1.
Cell Iysates of ATCC No. 53195 were incubsted st 20C for 30 minutes in the reaction mixes indicated and processed to give the gum fract30ns ~s described in Example 1. The molar ratio of glucose to mannose indicated is the ratio of pmoles of incorporated carbon-14 to tritium labeled sugars in the processed fract30ns.
~7~0 ., .
The presence of UDP~lucuronic acid has no effect on the ratio o glucose to mannose incorporated into a polysacch~ride polymer when the cell-free lysate used is from a glycosyltransferase IV~eficient mutant. The biochemical phenotype of the mutant lysate when incubated with all three sugars is analogous to that Or the wild-type lysate when incubated with only two sugar substrates, in that the in vitro produced gums both have a molar ratio of spproximately 2:1 of glucose to mannose moieties.
Example 3 This example demonstrates that the trimeric intermediate which is polymerized to form polytrimer gum has the ssme anomeric configu-ration of the sugars as in xanthan gum. In addition it demonstrates that the mannose of the trimer is attached to the non-reducing glucose of cellobiose in the lipid-linked intermediate.
Alpha-mannosidase (EC 3.2.1.24) 2nd beta-glucosidase (EC 3.2.1.21)-were used to singly or sequentislly treat the trimeric oligosaccharide which had been synthesized and double labeled in vitro as described in Example 1. Alpha-mannosidase will hydrolyze terminal, unsubstituted mannose residues attached through an alpha-1 linkage. Beta-glucosidase will hydrolyze terminal, unsubstituted D~lucosyl residues attached in a beta-1 linkage.
The trimer was removed from the lipid and dephosphorylated.
This was then deacetylated by base treatment, such as pH12 for 2 to 3 hours, becsuse alphs-mannosidase cannot recognize acetylated mannose 2 5 moieties.
The results were as follows. Treatment of trimeric oligosaccharide with beta~lucosidase left it unchanged. When slpha-mannosidase was used to treat the trimeric oligosacchsride, cellobiose and mannose were formed. When the trimeric oligomer was treated ~ with alpha-mannosid~se, first, and beta-glucosidase, second, glucose and mannose were formed. The results confirm that mannose is attached to the non--reducing glucose by an alpha-linkage in the trimeric 1279~30 intermediate, and that the glucose moleties are bets-linked. This confirms that trimer is an intermediate product of the normal xanthan enzyme pathway.
Example 4 This example shows the methods of mutagenesis and screening which were employed to generste the mutant strains which are xanthan gum deficient due to a lesion in th~e gene for glycosyltransferase IV.
Xflnthomonas campestris, genetically m~rked with 8 chromosomal resistance to streptomycin, W8S used QS a recipient in ~ conjugation with E. coli LE392 containing plasmid pRK2û13::TnlO. Plasmid pRK2013 contains Tn903 which encodes kanamycin resistance, ~Figurski, D.H., and Helinski, D.R., Proc. Natl. Acsd. Sci., U.S.A., 76-, 16~8-1652 (1979);) and the plasmid cannot replicate in Xanthomonas, (Ditta, G., et al., supra.) Transposon TnlO encodes r~sistance to tetracycline.
Transconjugants were selected which were resistant to streptomycin and kanamycin, or streptomycin and tetracycline. The former occurred at a frequency of about 4 X 10~6/recipient and presumably resulted from a transposition of Tn903. The latter occurred at a frequency of about 3 X 10~6/recipient ~nd presumably resulted from a transposition of TnlO into the genome of Xanthomonas campestris.
Auxotrophs were found among these transconjugants at 8 frequency of about 296; their needs were widely distributed among the various nutritional requirements. This indicates that these transposons do not have a particularly preferred locus for insertion in Xanthomonas. Prototrophic revertants of the auxotrophs were selected, and most were found to be drug-sensitive; this suggests that the auxotrophies were caused by transposon insertion.
To screen for xanthan gum deficient mutants among the doubly resistant transconjugants, Congo Red dye, which enhances the morphological distinction between xanthan gum producJng and non-producing colonies, WQS added to the solid media. Colonial morphology was examined after 7 to 12 days incubation at 30C. Xanthan gum deficient mutants were found at a frequency of approximately lO-~.
12~ 8~
. -- 19 -To identify a glycosyltransfersse IV mut~nt from among the xanthan gum deficient mutsnts, freez:e-thflw Iysates of each were pre-2ared. Radiolabeled UDP-glucose and GDP-mannose were added with or without UDP-glucuronic acid, The desired mutants made Q gum 5 having a glucose:m~nnose ratio of ~bout 2:1, irrespective of the presence of UDP~lucuronic acid. SeYeral mutants were fouhd of this description. They cont~in lesions due to TnlO insertion. Mutants induced by Tn903 were also found having this phenotype. In addition mutants have been isolated having this phenotype which were induced 10 by nitrosoguanidine.
Example 5 This example demonstrates the use of a glycosyltransferase IV
deficient mutant to produce polytrimer gum in vivo.
To obtain in v~vo synthesized gums, five liters each of wild-type 15 NRRL B-1459 S4-L ~nd the glycosyltransferase IY deficient mutant oî
Example 4 (ATCC No. 53195) were aerobically grown in a fermenter st 28C to 32C, with the pH controlled at pH 6.0 to 8Ø A minimal medium was used contsining 10 g/l potassium phosphate, 1.43 g/l ammonium sulfate, 2 g/l citric acid, 30 g/l glucose, ~nd trace ele-20 ments. After 145 hours, the gums were recovered and purified. The cells were removed by centrifugation and the gums precipitated from the broth by addition of isoprop~nol (55% v/v) and sodium chloride (0.5% w/v). The precipitates were collected by filtration and redissolved in water. The gums were reprecipitated with isopropsnol 25 (55q6 v/v) without salt and redissolved in water. The preparations were di~lyzed using 1~,000 MW cutoff membrane dialysis tubing against water for three days.
The glucose:mannose ratios were determined by complete acid hydrolysis of the polysaccharide polymers with subsequent analysis by 30 high performance liquid chromatography (HPLC), and found to conform to the ratios found for the in vitro synthesized polym ers. The glycosyltransferase IV deficient mutant designated ATCC No. 53l~5 12"~9 3L8V
made a gum with Q glucose to mannose ratio o~ &bout 2.15:1, whereas the wil~type made R gum of ratio ebout 0.96:1.
Other in v~vo produced sampl~s of polytrimer gum were assayed by HPLC or by enzymatic ~nelyses of the sugsrs after acid hydrolysis.
For the twenty-four enalyses performed, the molar ratios r~nge from 1.43:1 to 2.44:1 of glucose to mannose. The mean ratio v~as 1.90 ~
0.15:1 for polytrimer made by the glycosyltransfer~se IV deficient mutant strain.
Also shown by the HPLC analysis of the in vlvo produced polytrimer within detectsble limits were: 1-the absence of glucuronic acid; 2-the absence of pyruvate; 3-the presence of acetate; 4-the absence of sugars other than glucose and mannose.
Example 6 This example shows that polytrimer provides aqueous solutions which exhibit improved rheological properties compared to xanthan gum over ~ range of temperatures snd inorganic salt concentrations.
Solutions of polytrimer gum (synthesized in vivo in accordance with Example 5) and xanthan gum (purified Pfizer Flocon 48G0, were prepared ~t a concentration of 1,000 ppm in a water containing 10 weight percent sodium chloride. Polytrimer gum shows substantially greater viscosity than xanthan gum over e wide range of shear rates (Figure 2).
The ratio of polytrimer to xanthan viscosity at room temperature varies with water salinity and is between 2 and 2.5 over a salinity range of 0 to 20 weight percent sodium chloride, as shown in Figure 3. The ratio of polytrimer viscosity to xenthan viscosity also varies with polymer concentration (Figure 4). Finally, the improvement in polytrimer viscosity over xanthan viscosity increases with temper~ture over a range of 25 to 75C, ror w~ter sslinities of 0 to 20 weight 3 0 percent sodium chloride (Figure 5~.
Since vsriations of this invention will be apparent to those skilled in the srt, it is intended that this invention be limited only by the scope Or the claims.
Claims
CLAIM.:
1. A process for the recovery of oil from an oil-bearing subterranean formation comprising: injecting a solution containing a polysaccharide polymer having a glucose:mannose ratio of about 2:1 into a well to displace trapped oil from the porous rock, and collecting the displaced oil.
1. A process for the recovery of oil from an oil-bearing subterranean formation comprising: injecting a solution containing a polysaccharide polymer having a glucose:mannose ratio of about 2:1 into a well to displace trapped oil from the porous rock, and collecting the displaced oil.
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US762,878 | 1985-08-06 | ||
US06/762,878 US4713449A (en) | 1985-08-06 | 1985-08-06 | Polysaccharide polymer made by xanthomonas |
CA000513834A CA1247033A (en) | 1985-08-06 | 1986-07-15 | Polysaccharide polymer made by xanthomonas |
CA000577352A CA1264537A (en) | 1985-08-06 | 1988-09-14 | Process utilizing a polysaccharide polymer |
Related Parent Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CA000577352A Division CA1264537A (en) | 1985-08-06 | 1988-09-14 | Process utilizing a polysaccharide polymer |
Publications (1)
Publication Number | Publication Date |
---|---|
CA1279180C true CA1279180C (en) | 1991-01-22 |
Family
ID=27167616
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CA000615544A Expired - Fee Related CA1279180C (en) | 1985-08-06 | 1989-10-26 | Process utilizing a polysaccharide polymer |
Country Status (1)
Country | Link |
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CA (1) | CA1279180C (en) |
-
1989
- 1989-10-26 CA CA000615544A patent/CA1279180C/en not_active Expired - Fee Related
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