Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
S3~7
Processes Utilizing a Polysaccharide Polymer
The present application has been divided out of
Canadian Patent Application Serial No. 513,834 filed July
15, 1986.
Xanthan gum is produced by bacteria of the genus
~anthomonas, such as the species campestris, albilineans,
fragaria, vesicatoria, and the like. Xanthan gum is a
widely used product due to its unusual physical
properties: extremely high specific viscosity and pseudo-
plasticity. It is commonly used in foods as a thickening
agent and in secondary oil recovery as mobility control
and profile modification agents and in petro]eum drilling
fluids.
Chemically, xanthan gum is an anionic heteropolY
saccharide. The repeating unit of the polymer is a
pentamer composed of five sugar moieties: two glucose,
one glucuronic acid and two mannose moieties. They are
arranged such that the glucose moieties form the backbone
of the polymer chain, and side chains of mannose-
glucuronic acid-mannose generally extend from alternate
glucose moieties. Often this basic structure is
specifically acetylated and/or pyruvylated. (Janson,
P.E., Kenne, L., and Lindberg, ~., Carbohydrate Research,
45, 275-282 (1975); Melton, L.D., Mindt, L., Rees, D.A.,
and Sanderson, G.R., Carboyhydrate Research, 46, 245-257
(1976~).
The s~ructure is depicte~ below:
;37
-- 2
I ~ ~n
~-o
t~ /~
t~
In spite of the broad utility of naturally
occurring xanthan gum, there are some situations where
its physical properties become limiting. In particular,
in secondary oil recovery it is not uncommon for the
temperature of the oil-bearing reservoir and salt
concentrations in the reservoir brine to be higher than
are optimal for xanthan solutions. When these conditions
occur, xanthan can precipitate, flocculate and/or lose
viscosity. Therefore there is a need for new
viscosifying products which perform well at high
temperature and high salt conditions.
It is an object of the present invention to
provide an improved process for increasing the viscosity
of an aqueous medium.
It is a further object of this invention to
provide an improved process for the recovery of oil from
an oil-bearing suhterranean formation.
6~
-- 3
In accordance with one aspect of this invention, there
is provided a process for increasing the viscosity of an aqueous
medium comprising dissolving a polysaccharide polymer containing
essentially no glucuronic acid moieties have a D-glucose:D-
mannose ratio of about 2:1, wherein the D-glucose moieties 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 in
an aqueous medium at a desirable concentration.
Other aspects of this invention are described in a
further divisional application.
The polysaccharide polymer is the subject of the parent
application, Canadian Patent Application Serial No. 513,834 which
issued as Canadian Patent 1,247,033 on December 20, 1988 and 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, th`e
polysaccharide polymer of this invention has a single sugar
moiety generally linked to alternate glucose moieties of the
backbone. The polysaccharide polymer is herein termed ~7poly-
trimer" 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.
~o /
~'~'
~ '~el
~6~S~7
As shown by the ~boYe, the polytrimer consists Or ~mennose linked
~lphe-[1,3] generslly to elternste moieties of bet~ [1,4] linked D-
glucose. As in xsnthan gum, an Acetic ~cid moiety can be, but is not
BIWByS, estelified Bt the 6-O position of m~nnose, BS described in
S Sutherl~nd, l.W., Csrbohydrste Polymers, ~? 107-115, (19B1). Although
the structure of the polys~cchsride polymer Is thought to be.~s shown,
it is possible th~t under certain conditions of synthesis, ~ m~nnose
moiety mQy not nlw~ys ~e linked ~t elternating glucose residues.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 depicts the sssumed p&thwQy of xanthAn gum biosynthe-
sis. It is bssed on the dstQ of severQI 1sbor~tories. See, lelpi, L.,
Couso, R.O., snd D~nkert, M.A., Biochem. Biophy. Res. Comm., 102,
1400-1408 (1981), FEBS Letters, 130, 253-256 (1981), Biochem. Intern.,
6, 323-333 ~1983); Osborn, M.J. and Weiner, I.M., J. Biol. Chem., 243,
2631-2639 (1967); Troy, F.A., Annusl Reviews of Microbiology, 33,
519-560 (1979). Abbrevistions used ~re: glu-glucose, gluA=glucuronic
Acid, msn=mQnnose, glu~lu=cellobiose, P=phosphate, PP=pyrophosph~te,
C55=isoprenoid lipid cflrrier, PEP=phosphoenolpyruvste, AcCoA=~cetyl
coenzyme A, I-V=glycosyltrsnsfer~ses, UDP=uridine 5'-diphosph~te,
2~ GDP=gusnosine 5'~iphosphste.
Figure 2 shows the viscosities of solutions of polytrimer ~nd
xAnthun gum, eQch st 1000 ppm in 10 weight percent NACI brine, ~s
function of sheflr r~te.
Figure 3 shows the rstio of viscosities of solutions of 1,000 ppm
polytrimer to xsnthfln gum ~s A function of brine s~linity.
Figure ~ shows the r~tio of viscosities of solutions of polytrimer
to xsnth~n gum AS ~I function of polymer concentrAtion in 10 weight
percent NACI brine.
Figure S shows the r~tio of viscosities of solutions Or 1,000 ppm
polytrimer to x~nthun gum as ~ function of temper~ture in brines of
v~rious s~linities.
~L~6f~37
~,
The polysaccharide polymer used in the processes
of this invention can be made with a cell-free enzyme
system or can be made by growing cells of an appropriate
mutant strain. Other means of preparing the polysac-
charide polymer are also described below.
The basic method relating to the use of acell-free system to make xanthan gum is described in
Ielpi, L., Couso, R.O., and Dankert, M.A. (FEBS Letters,
130, 253-256, (1981)) and can also be employed to make
the polysaccharide polymer. For example, wild-type
Xanthomonas cam~estris cells can he lysed hy a freeze-
thaw process and the suhstrates for polytrimer synthesis,
UDP-glucose and GDP-mannose, with or without acetyl-CoA,
can be added to the lysate. Alternate means of lysis may
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 path-
way. Since the enzymes have no UDP glucuronic acid to
add to the nascent chains, the pathway is blocked at
reaction IV (see pathway, Figure 1,) and the intermediate
isoprenoid lipid-pyrophosphate-glucose-glucose-mannose
accumulates. Surprisingly, the xanthan polymerase which
ordinarily acts on lipid-linked pentamer (glucose-
glucose-mannose-glucuronic acid-mannose) is able to
polymerize lipid-linked trimer, (glucose-glucose-
mannose.) Thus, the polytrimer of the present invention
can be synthesi%ed in vitro.
The cell-free synthesis of polytrimer described
above shows that Xanthomonas campestris cells have all
the enzymes necessary to synthesiæe polytrimer. However,
to use whole cells to synthesize polytrimer in vivo, a
means of blocking xanthan gum synthesis at reaction IV
(see Figure 1) is required. Mutagenesis can be employed
to block reaction IV.
~$4~i37
-- 6--
Transposons, including but not limited to l'nlO
and Tn903, can be used to mutagenize Xanthomonas. These
transposons confer resistance to tetracycline and
kanamycin, respectively. Transposons have the ability to
insert themselves into genes; when they do so, they cause
mutations by interrupting the coding sequence, (Kleckner,
N., Annual Reviews of Genetics, 15, 341 (1981)). The
transposons can be introduced into Xanthomonas on a
so-called suicide vector, such as pRK2013. This vector
10 has the ability to transfer itself into non-enteric
bacteria, such as 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
15 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. Survivors of such a challenge can
20 be screened for those which have lost the ability to make
xanthan gum. Such mutants appear less mucoid than wild-
type Xanthomonas.
Other means of mutagenesis can be employed togenerate mutants which have lost the ability to make
xanthan gum. Such means will readily occ~r to one skilled
in the art, and include, without limitation, irradiation,
recombinant DNA technology, and chemical mutagen treatment
(Miller, J.H., Experiments in Molecular Genetics (1972);
Davisr R.W., Bolstein, D., and Roth, J.R., Advanced
Bacterial Genetics (1980); Maniatis, T., Fritsch, E.F.,
Sambrook, J., Molecular Cloninq (1982), Cold Spring
. . ~
Harbor).
Although mutants can first he chosen which appear
less mucoid than wild-type, those desired retain the
ahility 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
~6~537
. 7
UDP-glucose, GDP-mannose, and UDP-glucuronic acid are
added as substeates, the product should be identical to
that produced when UDP-glucose and GDP-mannose are added.
Alternatively, appropriate mutants can be detected by
assaying the culture broth of each mutant for the presence
of polytrimer. Thus xanthan gum deficient mutants can be
found which appear to be blocked at reaction IV of the
xanthan gum pathway. A mutant of this description has
been placed on file at the American Type Culture
10 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, other
embodiments contemplate use of mutants in UDP-glucuronic
15 acid metabolism. Such a mutant has been isolated and
deposited at the American Type Culture Collection,
Rockville, Maryland, under the ATCC No. 531g6.
It is not beyond the scope of the invention
described in the parent application to employ an enzyme
20 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 degradation of natural xanthan gum as, for
25 example, by removing the terminal mannose and glucuronic
acid moieties from the side chains of xanthan gum.
Using similar schemes to mutagenize strains of
Xanthomonas, it is possible to obtain mutants which
produce other new polysaccharide polymers. For example, a
30 mutation in the acetylase gene yields completely
non-acetylated xanthan gum. When an acetylase mutation
and a glycosyltransferase IV mutation are put in the same
strain (a double mutant), a non-acetylated polytrimer is
produced. Other mutations and combinations of mutations
35 of the xantllan pathway are possible to yield new products.
~I Z~537
-- 8
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 assimilable carbon
sources such as glucose, sucrose, maltose, starch, invert
sugar, complex carbohydrates such as molasses or corn
syrup, various organic acids and the like. Mixtures oE
carbon sources can also be employed. The concentration of
carbon source supplied is o~ten between about 10 and 60
grams per liter. Also necessary for growth are an
10 assimilable sol~rce of organic or inorganic nitrogen,
generally between about 0.l and l.0 grams per liter, and
minerals, the choice of which are easily within the skill
of the art. Examples of suitable nitrogen sources are
ammonium salts, nitrate, urea, yeast extract, peptone, or
15 other hydrolyzed proteinaceous materials or mixtures
thereof. Examples of suitable minerals include
phosphorous, sulfur, potassium, sodium, iron, magnesium;
these are often added with a chelating agent such as EDTA
or citric acid.
Optimal temperatures for growth of Xanthomonas
generally are between about 18 and 35C, preferably
between about 28 and 32C. Xanthomonas cells are grown
aerobically by supplying air or oxyyen so that an adequate
level of dissolved oxygen is maintained, for example,
25 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, preferably at about 6.5 to 7.5.
The polysaccharide Polymer can be recovered from
fermentation hroths hy a suitahle means. Precipitation
30 with isopropanol, ethanol or other suitahle alcohol
readily yields the polytrimer gum. Generally, alcohols
are added to a concentration of about 50 to 75~, on the
basis of voluJne, preferably in the presence of potassium
chloride, sodium chloride or other salt. Alternatively,
35 the polymer can be recovered from the broth by ultra-
filtration.
~64~3~7
When chemical analyses are pre~ormed on
polytrimer gurn to determine the ratio of glucose:mannose,
a variation from the theoretical value of 2:1 is found.
The same type of variation is found when analyzing xanthan
gum. Measured ranges of the ratio of glucose:mannose will
generally be between about 1.4:1 and about 2.4:1.
Preferably the ratio will be hetween 1.7:] and 2.1:1.
Levels of acetylation of the mannose residues of
the polysaccharide polymer vary. In addition, it is not
beyond the scope of the inve~tion described in the parent
application to employ a microorganism to make 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 polysaccharide polymer.
Typically, concentrations of polytrimer in the
fermentation broth are about 0.1~ (w/w). Routine testing
of fermentation conditions 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 rnedium. The
viscosity of solutions of polytrimer is retained at
conditions of hgh temperatures and/or high salinity. Such
solutions can be prepared at any desirable concentrations,
preferably between about 0.01% and about 15% by dissolving
the polysaccharide polymer in an aqueous medium. The
polysaccharide polymer is ideally suited for use in
secondary oil recovery. The same techniques as are 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., ~.S. 3tl98,268.
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 polysaccharide polymer are appropriate for such
~ ~6~L~37
].o --
mobility control solutions. Other known additives may
also he used in, or in combination with, these solutions
to further enhance oil recovery. Such additives include,
for example, sur~actants and alkaline agents.
The polysaccharide polymer, like xanthan gum, can
also be used as a thickening agent in foods, cosmetics,
medicinal formulations, paper sizings, drillin~ muds,
printing inks, and the like. In addition it can be used
to reduce frictional drag of fluid flow in pipes.
The following examples are provided by way of
exemplification and are not intended to limit the scope of
the invention.
Example 1
This example shows how the polysaccharide polymer
can be prepared ln vitro, and identifies it as a truncated
product of the xanthan pathway.
Preparation of Lysates
Xanthomonas campestris B1459 S4-L was obtained
from Northern Regional Research Laboratories of the U.S.
Department of Agriculture. Bacteria were grown in YM
(yeast-malt medium) supplemented with 2% (w/v) glucose as
described by Jeanes, A., et al. (U.S. Department of
Agriculture, ARS-NC-51, 14 pp (1976)). Cultures were
grown to late log phase at 30C at 300 rpm. The cells
were titered on ~M 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, 70 mM~ pH 8.2 with 10 mM
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 evidenced 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 ~rozen in aliquots at -80C.
Protein concentration was determined with BIO RAD assay
i37
-- 11 --
Laboratories, Richmond, Cali~ornia) and was found to be 5
to 7 mg cell protein per ml of lysate.
Biosynthetic Assay Procedure
As described in Ie]pi, L., Couso, RØ, and
Dankert, M.A., FEBS Letters, 130, 253-256 (1981), an
aliquot of freeze-thawed lysate (equivalent to 300 to 400
ug protein), D~Aase I (10 ug/ml), and MgC]2 (8 mM) were
preincubated at 20C for twenty minutes. An equal
volume of 70 mM Tris-HC1, pH 8.2, with the desired
1~ radiolabeled sugar nucleotides (UDP-glucose and
GDP-mannose), with or without ~DP-glucuronic acid, was
added and incubated at 20C. ~t various times, the
reaction were stopped by the addition of EDTA to 4mM. The
samples were centrifuged; the pellets were washed two
times with bufEer. The supernatants were combined,
carrier xanthan (100 ug) was added, and the xanthan plus
synthesized polymer were precipitated with ethanol(60~)-KCI
(0.8%). The precipitated polymer was resuspended in water
and reprecipitated two more times to remove unincorporated
label. Radioactivity incorporated into the gum fraction
was determined in a liquid scintillation counter, and the
data were processed to obtain incorporation in terms of
pmoles.
;3~
-- 12 --
TABLE 1
Incorporstion Or labeled SUgQrS by freeze-th~w cell Iysalte
of X.. ~m~stris B1459 S4-L into gum
aum Fr sc tion (pmol)
I ncub~ t i on Mi x[ 3H]mQn r 14c~ gl c gl c/man
~UDPG , C;DPM 9 8 2 0 1 2 .1
+VDPG, GDPM, UDP-GA15 4 01 5 6 2 1 . O
dpm/pmol 3H = 442
1~C = 3~.5
UDPG = UDP-glucose glc - glucose
GDPM = GDP-m~nnose mAn = mQnnose
UDP-GA = UDP-gl ucuroni c AC i d
dpm = dislntegrstions per minute
pmo I = p i como I e
Cell Iys~tes of B1~59 S4-L were incubsted ~t 20C for 30 minutes and
processed to give the gum fractlons QS described in the text. The
molQr rstio of glucose to m~nnose is the r~tio of pmotes ot incorpor-
~ted carbon-14 to tritlum labeled sugars in the gum frQctions.
~2~ii37
- 13 _
~n the presence of sll three sugar constituents, the r~tio of
glucose: m~nnose was 1.0:1, QS expected for xQnthQn gum. When
UDP-glucuronic dcid w~s qbsent, the ratio W8S 2.1:1. See TAble 1.
This rutio is consistent with the hypothesis thQt tlle polysacchsride
polymer is formed of trimer units which are Intermediates in the
x~nth~n gum biosynthetic p~thwQy.
A pulse-ch~se in vitro experiment showed that lipid-linked
cellobiose (~ glucose dimer) w~s processed to lipid-linked trimer
~glucose-glucose-m~nnose) ~nd subsequently to polytrimer gum. A
freeze-th~w IysRte of str~in B1459 S~-L w~s prep~red ~s descri~ed
~bo~e. UDP-[14C~glucose wss Qdded to the Iys~te, comprising the
"pulsen~ ~nd r~diolQbeled cellobiose ~ccumulflted on the lipid c~rrier
during Qn incubQtion of 13 minutes. The "ch~se" consisted of ~ddition
of 100-fold excess unIabeled UDP~lucose ~s well ~s GDP{3H]m~nnose.
Aliquots of the incuba~ion mixture of Iys~te ~nd sugQr nucleotides were
removed ~t vQrious times ~nd processed to produce ~n org~nic extrsct
(lipid cQrrier-linked fraction) ~nd ~n ~queous fr~ction (COntQining gum).
The oligos~cch~rides o~ the or8anic extract were Qcid hydrolyzed from
the lipld carrier, dephosphorylQted, sep~r~ted by thin IQyer
chrom~tography, removed from the chromQtogrsms snd the rQdiolabel
qusntitQted. The results Rre shown in Table 2.
~L~69L5~37
TABLE 2
F~te ot tlDP-l14CJ glucose in pulse-chsse In Yi tro experiment
with ceal Iys~tes of B1459 S4-L
Pulse (12 min~ 9 pmol Llpid-l inked cel lobiose
ChRse (4 m;n~I pmol Lipid-llnked cellobiose
10 pmol Llpid-linked trimer
ChQse (16 min)0.5 pmol Llpld-ainked cellobiose
6 pmol Llpid-linked trimer
3 pmol Soluble polytrimer
Ch~se (48 min)0.2 pmol Lipld-linked cellobiose
0.~1 pmol Lipid-l{nked trimer
10 pmol Soluble polytrimer
The experiment~l conditions Rnd the processing of the orgQnic ~r~ction
snd the soluble gum fr~ction Qre described In the text of Ex~mple 1.
~L2~i37
- t5 -
The l~lbeled glucose ~rom UDP-~I~C]glucose, as c~n be seen in
T~ble 2, w~s irnmedi~tely ineorpor~ted into llpid-linked cellobiose in the
"pulsen. Upon ~ddition of GDP-mAnnose ~nd excess UDP~lucose (the
ch~se), the IQbeled cellobiose WQS conYerted rQpldly to lebeled lipid-
linked trimer, which w~s l~ter detected ~s polytrimer gum in the ~qu~
ous ~r~ction, at ~bout 16 mlnutes ~ter the ch~se beg~n. This
demonstr~tes the precursor-product relationshlps of UDP~lucose, lipi~
linked ce~lobiose, lipid-linked trimer, ~tnd polytrimer gum, ~nd their
relQtionships to the xsnthan biosynthetic p~thw~y,
Ex~mple 2
This exQmple demonstrstes the mol~r r~tio of glucose to
m~nnose in polytrimer gum synthesized in vltro by ~ glycosyltrsnsferase
IV-deficient mutant.
The method of prep~ring the Iysste is described above in Ex~m-
lS p2e 1. The strsfn used to prep~re the Iysete w~s th~t designQted
A~CC: No~ 53195. Added to the Iys~tte were either 1, 2 or 3
nucleotide-chsrged sug~rs, consisting of UDP-114C]glucose nlone,
UI)P-t1~C~glucose ~nd GDP-~3Hlm~nnose, or UDP-~14C]g' Icose,
GDP-f3H]m~nnose ~nd unlAbeled UDP~lucuronic Rcid. At 30 minutes
Qfter ~ddition of the sugQr substrQtes, the ~queous frQction WQS
processed ~nd sn~lyzed 8s described in ExQmple 1. Results ~re shown
in T~ble 3. When two sug~r substr~tes, UDP-glucose ~nd GDP-
mQnnose9 were present in the incub~tion mixture the molflr rQtio of
glucose to mQnnose found in the gum w8s 2.4:1. When 811 three sugflr
substrRtes were incubQted together with the Iysste, the resulting gum
hQd ~ ~3:1 molar r~tio of glucose to mannose.
Z6~L53
-- 16 --
TABLE 3
Incorporatlon of labeled sugars by freeze-th~w cell Iysate
of ATCC NQ. 53~95 into polytr Imer gum
_
Gum Fr~e t i on ( Pmol )
_ _ ~ _ __
Reac t i on Mi x E 3H]m~n r 1 4C~ gl e gl e/man
~ 2 UDPG, GDPM 71 17 4 2 . 4
+ 3 UDPG, Gl:)PM, UDP-GA 6 5 1 5 2 2 . 3
d pm/ pmo l 3~1 = 3 4 0
14C - 40
_________~ _________ _______ ___________~_____ _____
Abb r e v l ~ t i on s ~ r e e xp l a l n ed i n l egend t o T~ b l e 1 .
Cell lys~tes of ATCC No. 53195 were incubated at 20C for 30
minutes in the reaction mixes Indicsted ~nd processed to give the gum
fractlons as descrlbed in Example 1. The molar ratio of glucose to
m~nnose Indic~ted Is the ratio of pmoles of incorpor~ted csrbon-14 to
trltium labeled sug~rs in the processed frQctions.
~2~53~
- 17 --
The presence of UDP~lucuronlc ~cid has no effect on the r~tio
of glucose to mannose Incorpor~ted into A polysacchqride polymer when
the cell-free Iysste used is from a glycosyltransfer~se IV~eîicient
mut~nt. The biochemicAI phenotype of the mutant Iys~te when
incubAted with Qll three sugars is anslogous to thAt of the wil~type
Iysate when incubated with only two sug~r substrates, in th~t the in
~ritro produced ~ums both h~re ~ molar r~tio of ~pproximately 2:1 of
glucose to mannose moieties.
Exa m~e
This ex~mple demonstrQtes th~t the trimeric intermedi~te which
is polymerized to form polytrimer gum hss the s~me anomeric configu-
ration Or the sug~rs QS in x~nthan gum. In addition it demonstr~tes
th~t the mannose of the trimer is ~tt~ched to the non-reducing glucose
of cellobiose in the lipi~linked intermedi~te.
AlphA-mannosidQse (EC 3.2.1.24) and bet~-glucosidAse (EC 3.2~1.21)
were used to singly or sequentislly treQt the trimeric oligos~ccharide
which hQd been synthesized ~nd double l~beled in vitro ~s described in
Ex~mple 1. Alphs-mAnnosidRse will hydrolyze terminQI, unsubstituted
m~nnose residues ettAched through ~n alphA-1 linkQge. Bet~-glucosidase
2n will hydrolyze terminsll unsubstituted D~lucosyl residues ~ttQched in
betfl-1 link~ge.
The trimer W8S removed from the lipid snd dephosphorylAted.
This WQS then deAcetylsted by base trestment, such as pH12 for 2 to
3 hours, bec~use alphn-mannosidase cQnnot recognize acetylated mAnnose
2 5 moieties.
The results were As follows. Treatment of trimeric
oligosacch~ride with beta~lucosid~se left it unchsnged. When alphs-
manllOSidQSe WQS used to trest the trimeric oligos~ccharide, cellobiose
and mannose were formed. When the trimeric oiigomer wss treAted
with slphQ-m~nnosidase, rirst, Qnd beta-glucosidflse, second, glucose and
m~nnose were formed. The results confirm th~t m~nnose is ~ttllched
to the non-reducing glucose by ~n nlphQ-link~ge in the trimeric
53~
-- 18 --
intermedi~te, ~nd th~t the glucose moleties ~re beta-linked. This
confirms th~t trimer is An intermedi~te product of the norm~l xflnthfln
enzyme pAthwfly.
~e~
s This eXRmple shows the methods of mut~genesis And screening
which were employed to gener~te the mut~nt str~ins which *re x~nthfln
gu m deficient due to Q lesiosa in the gene for glycosyltrQns~erQse IV.
XQnthomones cflmpestris, geneticfllly mQrke~d with 8 chromosomal
resistflnce to streptomycin, was used QS ~9 recipient in ~ conjugfltion
with E. coli LE392 conteining plQsmid pP~K2013::Tn10. Plssmid
pRK2013 contflins Tn903 which encodes kan~mycin resistflnce, (Fi~urski,
D.H., ~nd Helinski, D.R., Proc. Natl. Acad. Sci., U.S.A., 76, 164B-1652
(1979),) and the plasmid csnnot replicate in XenthomonQs, (Dittfl, G., et
81., suprQ.) TrQnsposon TnlO encodes resist~nce to tetrQcycline.
Transconjugsnts were selected which were resistant to streptomycin And
kunQmycin, or streptomycin And tetracycline. The former occurred Qt
A frequency Or Qbout 4 X 10~6/recipient and presumAbly resulted from
Q trQnsposition of Tn903. The Iqtter occurred Qt ~ frequency or nbout
3 X 10~6/recipient And presum~bly resulted from Q trQnsposition of
TnlO into the genome Or XAnthomonas cempestris.
Auxotrophs were found Qmong these tr~nsconjugQnts Qt 8
frequency of ebout 2%; their needs were widely distributed Qmong the
verious nutritionfll requirements. This indicAtes thAt these transposons
do not have ~ perticulQrly preferred locus for inserti~n in
Xanthomonfls. Prototrophic revertants of the ~uxotrophs were selected,
nnd most were found to be drug-sensitive; this suggests th~t the
Auxotrophies were csused by tr~nsposon insertion.
To screen for x~nthsn gum rieficient mutAnts flmong the doubly
resistflnt tranScOnjUgQntS~ Congo P~ed dye, which enhQnces the
3~ morphologic~l distinction between xRnthQn gum produclng and non-
producing colonies, wQs Qdded to the solid mediA. ColoniQI morphology
was exQmined ~fter 7 to 12 days incubAtion Qt 30C. Xnnth~n gum
deficient mut~nts were found ~t A frequency of Approximately 10-4.
-- 19 --
To identify a glycosyltr~nsferase IY mut~nt rrom among the
xenthan gum dericient mutAnts, freez~thsw lysates ot each were pre-
p~red. RQdiolAbeled UDP-glucose and GDP-mannose were added with
or without UDP~lucuronic acid. The desired mutents mQde ~ gum
S h~ving R glucose:mannose r~tio of Qbout 2:1~ irre~pectiYe of the
- presence Or UDP~lucuronic acid. Sever~l mut~nts were fourid of this
description. They cont~in lesions due to TnlO insertion. Mut~nts
induced by Tn303 were also found h~ving this phenotype. In addition
mutflnts hflve been isolated heving this phenotype which were induced
by nitrosogu~nidine.
Ex~mple 5
This e~Rmple demonstr~tes the use oî e glycosy1transferase IV
deficient mutant to produce polytrimer gum in vivo.
To obt~in in YiVo synthesized gums, five liters e~ch of wild-type
NRRL B-1459 S4-L and the glycosyltr~nsferase IV deficient mutant of
Ex~mple 4 (ATCC No. 53195~ were ~erobic~lly grown in Q ~ermenter ~t
28C to 32C, with the pH controlled Qt pH 6.0 to 8Ø A minimnl
medium WQS used containing 10 g/l pot~ssium phosphQte, 1.43 g/l
flmmonium sult~te, 2 g/l citric ~cid, 30 g/l glucose, ~nd tr~ce ele-
20 ments. After 145 hours, the gums were recovered snd purified. The
cells were removed by centrifug~tion and the gums precipitated from
the broth by eddition of isopropanol (55% v/v) snd sodium chloride
(0.5% w/v). The precipit~tes were collected by filtr~tion snd
redissolved in weter. rhe gums were reprecipitated with isopropanol
25 (55% v/v) without s~lt ~nd redissolved in wster. The preparAtions
were dialyzed using 12,000 MW cutoff membrnne di~lysis tubing ag~inst
Wflter for three dAys.
The glucose:mannose ratios were determined by complete f~cid
hydrolysis of the polysnccharide polymers with subsequent analysis by
3l~ high performance liquid chromatogr~phy (HPLC), snd found to conform
to the r~tios found for the in vitro sy~sthesized polymers The
glycosyltr~nsfersse IV deficient mut~nt design~ted ATCC ~o. 53195
537
-- 20 --
m~de R gum with ~ glucose to m~nnose ratlo of ~bout 2.15:1, whereas
the wil~type m~de ~ gum of ratio flbout 0.96:1.
Other in vivo produced s~mples of polytrimer gum were ~ss~yed
by HPLC or by enzym~tic ~nalyses of the sug~rs sfter acid hydrolysis.
For the twenty-four ~n~lyses performed, the mol~r r~tios r~nge from
1.43:1 to 2.~:1 of glucose to m~nnose. The mean rRtio w~s 1.90 ~
0.15:1 for polytrimer m~de by the glycosyltr~nsrerase IV deficient
mutsnt strAin.
Also shown by the HPLC ~n~lysis ot the in vivo produced
polytrimer within detectsble limits were: 1-the ~bsence of glucuronic
acid; 2~the ~bsence of pyruvAte; 3-the presence of ~cetste; 4-the
sbsence of sugsrs other than glucose and mQnnose.
Ex~mple 6
This ex~mple shows th~t polytrimer provldes ~queous solutions
which exhibit improved rheologicAl properties compared to x~nthQn gum
over ~ r~nge of temperAtures And inorzanic sslt concentrQtions~
Solutions of polytrimer gum (synthesized ln vivo in ~ccordsnce
with Exmple 5) nnd x~nth~n gum (purified Pfizer Flocon 4800, were
prep~red ~t Q concentr~tion of 1,000 ppm in ~ w~ter cont~ining 10
weight percent sodium chloride.~ Polytrimer gum shows subst~ntislly
greQter viscosity thsn x~nthQn gum oYer Q wide rsnge of sheAr rates
(Figure 2).
The r~tio ot polytrimer to x~nth~n viscosity at room temper~ture
v~ries with w~ter s~linity Rnd is between 2 and 2.5 over ~ salinity
rQnge of O to 20 weight percent sodium chloride, flS shown in Figure
3. The r~tio of polytrimer viscosity to xQnthsn viscosity ~Iso Y~ries
with polymer concentr~tion ~Figure 43. Fin~lly, the improvement in
polytrimer viscosity over xsnth~n viscosity incresses with temper~ture
over ~ r~nge of 25 to 75C, for w~ter s~linities of 0 to 20 weight
3 percent sodium chlorlde ~Figure 5).
Since ~flri~tions of this invention will be ~ppnrent to those
skilled in the ~rt, it is Intended th~t this Invention be limited only by
the scope Or thc clflims.
