Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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:
-- 2 --
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.
9180
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
-- 10 -
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
-- 11 -
(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
- 12 --
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~
- 13 -
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~
. . .
- 14 -
TABLE 2
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~
-- 15 --
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~
-- 16 --
- TABLE 3
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
., .
- 17 -
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
- 18 -
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
-- 20 -
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.
