Biotechnol. Prog. 1990, 6, 182-187
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Genetic Engineering of Polysaccharide Structure: Production of Variants of Xanthan Gum in Xanthomonas campestrid Randal A. Hassler and Daniel H. Doherty* Synergen, Inc., 1885 33rd Street, Boulder, Colorado 80301
Xanthan gum is an extracellular heteropolysaccharide produced by the bacterium Xanthomonas campestris. Xanthan has wide commercial application as a viscosifier of aqueous solutions. Previously, through genetic engineering, a set of mutants defective in the xanthan biosynthetic pathway has been obtained. Certain mutants were shown to synthesize and polymerize structural variants of the xanthan repeating unit and thus produce “variant xanthans”. Initial studies of solution viscosities of these polymers, presented here, indicate that the variants have rheological properties similar to, but not identical with, xanthan. These results indicate that acetylation and pyruvylation can affect the viscometric properties of xanthan. Specifically, the presence of pyruvate increases viscosity, whereas acetate decreases viscosity. In addition, the elimination of sugar residues from xanthan side chains also has a major effect on viscosity. Compared to wild-type xanthan, polymer lacking the terminal mannose (polytetramer) is a poor viscosifier. In contrast, polymer lacking both the terminal mannose and glucuronic acid (polytrimer) is a superior viscosifier, on a weight basis. There is a negative effect of acetylation on the viscosity of polytetramer xanthan, but there is seemingly no effect of acetylation on polytrimer xanthan viscosity. The further study of these materials should provide insight into the relationship between xanthan structure and rheological behavior.
Introduction Xanthan gum is an extracellular heteropolysaccharide produced by the bacterium Xanthomonas campestris. This polymer enjoys wide commercial application as a viscosifier of aqueous solutions. The generally accepted structure (1,2) of the pentasaccharide repeating unit of xanthan is shown in Figure 1. Glucose residues are linked P-1,4 to form the backbone of the polymer. The side chain consists of mannose-glucuronic acid-mannose and occurs on alternating glucose residues of the backbone. The mannose residues are generally modified by the acetylation of the inner mannose and the pyruvylation of the outer mannose. The extent of these modifications is known to vary. Biosynthesis of xanthan gum in X . campestris has been extensively studied (3-7). The pentasaccharide repeating unit is assembled on an isoprenoid lipid carrier by sequential addition of individual sugar residues that are donated by sugar nucleotide diphosphate precursors. Each sugar addition is catalyzed by a specific glycosyltransferase enzyme. The mannose residues of the repeating unit are specifically acetylated and pyruvylated. The repeating unit is polymerized, and the polymer is subsequently secreted. Several groups (8-10) have reported the isolation of mutants of X . campestris that are defective in xanthan biosynthesis and the cloning of X . campestris DNA fragments, which complement these mutants. One report (7) describes a region of the X . campestris genome containing many clustered mutations. More detailed work (4; + Prepared for presentation at the American Institute of Chemical Engineers 1989 Annual Meeting, November 5-10, 1989; “Production of Specialty Chemicals, Polymers and Fibers via Recombinant DNA Methodology-ln/34f,
v
HO
Figure 1. Repeating structural unit of xanthan: G = glucose; M = mannose; GA = glucuronic acid; Ac = acetate; Pyr = pyru-
vate.
-
Doherty, Capage, and Vanderslice, unpublished) has identified a 15-kb region of DNA that contains a cluster of 12 genes required for xanthan biosynthesis. The DNA sequence of this region has been determined, and mutations in each of these genes have been obtained. The biochemical defects in xanthan biosynthesis that result from these mutations have been analyzed in vitro and in vivo. Five of these genes encode the glycosyltransferase enzymes, which direct synthesis of the lipid-linked pentasaccharide repeating unit. Four genes encode proteins involved either in polymerization of the repeating unit or possibly in subsequent events in xanthan biosynthesis. Three genes encode enzymes that catalyze modification of the mannose residues. Gene F encodes the enzyme acetylase I that modifies the inner mannose, and gene L encodes a ketalase that pyruvylates the outer mannose. Gene G was also found to encode an acetylase activ-
8756-7938/90/3006-0182$02.50/00 1990 American Chemical Society and American Institute of Chemical Engineers
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Table I. X. campestrjs Mutants Defective in Acetylation and/or Pyruvylation molar ratios of substituents gene F gene G gene L per glucoseu acetylase I acetylase I1 ketalase acetate* Dvruvatec + 0.57 0.33 0.00 1.15 + 0.34 0.61 + 0.00 0.47 0.32 0.09 0.00 0.44 0.35 0.00 0.00 0.00
X1396 Wild-type
X1398 Acy ll-
X1400
Acy I -
X1402 Acy I - A c y 1 1 -
genotype
strain X1396 X1397 X1398 X1399 X1400 X1401 X1402 X1403 Determined by HPLC as detailed in Methods. * Least significant difference at the 95% confidence level = 0.16. Least significant difference at the 95% confidence level = 0.04.
PYr
XI397 Ket
+
ity, termed acetylase 11. Surprisingly, it was found that this activity directs acetylation of the outer mannose. Little acetylation of the outer mannose occurs normally, but when pyruvylation of the outer mannose is blocked by mutations that inactivate the ketalase, high-level acetylation of the outer mannose results. Mutants were constructed that comprise all possible combinations of mutations in genes F, G, and L. The genotypes as well as the acetate and pyruvate contents of the variant xanthans produced by these mutants are summarized in Table I. Figure 2 depicts in schematic form the structures of the repeating unit(s) of each of these mutant gums. Analysis of the properties of this family of variant xanthans allows investigation of the effects of the individual modifications on polymer properties. It has also been found that mutations that inactivate transferase IV (3) and transferase V ( 4 ) result in biosynthesis of polymers with trisaccharide and tetrasaccharide repeating units, respectively. Double mutants defective in gene F (acetylase I) and gene K (transferase IV) or gene I (transferaseV) were constructed. These mutants produce non-acetylated polytrimer and polytetramer, respectively. These mutants and the resulting variant polymers are summarized in Figure 3. These variant xanthans provide an opportunity to assess the effects of the side-chain sugar residues on the properties of xanthan. This paper reports the preliminary analysis of the solution viscosities of these variant xanthans.
'
Methods Polymer Production and Purification. X . campestris strains were grown in baffled shake flasks in a reciprocating shaker in a medium consisting of 1.5 g/L yeast extract, 2.5 g/L Bacto-peptone, 2.0 g/L KHPPO~, 3.2 g/L K2HP04, 0.3 g/L MgS04, 0.2 g/L CaC12, 0.2 g/L NaC1, and 20 g/L glucose. Kanamycin sulfate (10 mg/L) was used as necessary to select for plasmid maintenance. After approximately 48 h of incubation at 30 "C, the raw culture broths were diluted (1/2 w/w to 1/9 w/w) with 0.1 M NaCl and centrifuged at approximately 14000g for 2 h at 10 "C in order to remove cells. The supernatants were then concentrated and diafiltered against three volumes of 0.1 M NaCl with Amicon hollow-fiber filter (10 OOO MW cutoff) and a peristaltic pump. Polymer Quantitation. The amount of polymer in each purified sample was determined by gravimetric analysis and by quantitation of glucose release following hydrolysis. In the gravimetric procedure, polymer was precipitated from a known quantity of sample by using two volumes of 2-propanol. The precipitates were then caught on tared 0.45-pm nylon filters (25-mm diameter), which
X1399 Acy II- Ket -
-
~
A
M
~ C
A
7 7 X1401 Acy 1- Ket -
C
M
Ac
X1403 Acy 1- Acy II- Ket -
Ac
Figure 2. Family of xanthan-based polymers varying in acetylation and/or pyruvylation. The repeating structural units of polymers from mutant strains of X. campestris with the designated mutations represented with AcyI- = defective acetylase I, AcyII- = defective acetylase 11, Ket- = defective ketalase, G = glucose, GA = glucuronic acid, M = mannose, Ac = acetate, and Pyr = pyruvate. These strains all carry a chromosomal deletion mutation that eliminates the entire gum gene cluster. The gum gene cluster is present in each strain on a recombinant plasmid. The indicated gum gene mutations are present in these cloned gum genes. X1361
V-
XI263
Iv-
X1419 V-Acy I -
X1454 IV-Acy I -
F i g u r e 3. Family of xanthan-based polymers with truncated side chains. The repeating structural units of polymers from mutant strains of X. campestris with the designated mutations represented with IV- and V- = defective glycosyltransferases IV and V, AcyI- = defective acetylase I, G = glucose, M = mannose, GA = glucuronic acid, and Ac = acetate. These strains all contain recombinant plasmids carrying mutated copies of the relevant gum genes. The chromosomal gum DNA corresponding to the cloned sequences has been deleted.
had been dried overnight at 95 OC. With a vacuum filtration apparatus, the precipitates were washed with three volumes of 70% 2-propanol. After drying for 16-24 h at 95 "C, the filters were reweighed and the polymer concentrations of the samples calculated. For the quantitation of xanthan by glucose, gums were hydrolyzed in 2 M trifluoroacetic acid at 120 "C for 2.5 h, cooled on ice, and then adjusted to pH 7.0 with 1.2 M Na2C03. The total glucose in each hydrolysate was measured enzymatically with a Sigma assay kit, which employs hexokinase and glucose-6-phosphate dehydrogenase (Sigma Procedure No. 16-UV). The amount of free, or non-xanthanassociated, glucose in each sample was determined directly without hydrolysis. The amount of xanthan-associated glucose was calculated by subtraction of the amount of free glucose from the total glucose amount. The concentration of xanthan in each sample was then calculated from the molecular weight of the corresponding repeating unit and the concentration of xanthan-associated glucose. All analyses were done in duplicate. Viscometry. For measurements of viscosity, samples were diluted with 0.1 M NaCl to concentrations of 0.05, 0.10, 0.15, and 0.2 wt %. The viscosities of all samples
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Table 11. Viscosities of Variant Xanthans (wt W ) Produced by X. campestris Mutants Defective in Acetylation and/or Pyruvylation viscosity,a CP strain 0.05 wt % 0.10 wt % 0.15 wt 5% 0.20 wt % XI396 2.5 7.5 13.0 23.0 X1397 2.0 1.0 2.5 4.5 7.5 X1398 2.5 13.0 26.0 3.5 8.0 13.5 XI399 1.5 12.0 XI400 4.5 16.5b 4.0 7.0 X1401 2.0 12.0 22.5 X1402 7.0 49.5 91.5 22.0 41.5 X1403 2.5 10.0 a
Measured in 0.1 M NaCl at 25 OC and 8 s-l, using a Brookfield
LVT viscometer. 0.12 wt %.
were measured with a Brookfield LVT viscometer equipped with a no. 18 spindle a t a shear rate of 8 s-l. The sample temperature was maintained a t 25 "C. Polymer Composition by HPLC. The molar ratios of the xanthan components were determined by HPLC analysis of samples hydrolyzed in 2 M trifluoroacetic acid as described above. The hydrolysates were analyzed with a Biorad HPX-87H carbohydrate/organic acids column a t 45 "C. At a flow rate of 0.6 mL/min, 0.01 N H2S04 was used as the solvent in a Beckman HPLC system. Detection was by a Biorad refractive index monitor and a Hewlett-Packard diode array detector at 214 nm.
Results Effects of Acetylation and Pyruvylation. In order to study the effects of changes in the acetylation and/or pyruvylation of the repeating unit of xanthan gum on the solution viscosity properties of the polymer, the mutant strains of X.campestris that produce the variant xanthans depicted in Figure 3 were cultured to produce samples of these polymers. The details of the growth of the bacteria and preparation of the polymers are given in Methods. Briefly, the microbes were cultured in shake flasks in rich medium a t 30 OC for approximately 48 h. The cultures were diluted in 0.1 M NaCl and centrifuged to remove cells. These supernatants were concentrated and diafiltered against 0.1 M NaC1. The polymer concentrations were determined as described in Methods, and samples were formulated to 0.05, 0.1, 0.15, and 0.2 wt ?6 in 0.1 M NaC1. Viscosities of these samples were measured with a Brookfield LVT viscometer with a no. 18 spindle a t a constant shear rate with the sample temperature maintained a t 25 "C. Thus, the viscosity measurements were made on polymers that had undergone minimal treatment and processing. The data obtained ought to closely reflect the viscosities of the native polymers produced in the culture broths. Table I1 shows the viscosities, as a function of concentration, for the eight polymers (depicted in Figure 2), which vary with respect to acetylation and pyruvylation. The viscosities, on a weight basis, of polymers with varying levels of acetate and pyruvate differed dramatically. All pyruvylated xanthan variants were of significantly higher viscosity than their non-pyruvylated counterparts (see X1396 vs X1397, X1398 vs X1399, X1400 vs X1401, and X1402 vs X1403). Elimination of acetate from the inner mannose, by genetic inactivation of acetylase I, resulted in increased viscosity (compare X1396 vs X1400, X1397 vs X1401, X1398 vs X1402, and X1399 vs X1403). Similarly, removal of acetate from the outer mannose, by genetic inactivation of acetylase 11, resulted in significantly increased viscosities for non-pyruvylated polymers (X1397 vs X1399 and X1401 vs X1403). For pyru-
'"7 /
11
x1
40
4 >;
30t
/
/
X1396
A
" 0.05
0 10
0 15
0.20
Gum, W t %
F i g u r e 4. Viscosities of wild-type (X1396, - -0-), pyruvylated but not acetylated (X1402, - -C -), and nonpyruvylated but fully acetylated (X1397, - -0-) xanthans in 0.1 M NaCl a t 25 "C as a function of concentration at a constant shear rate of 8 s-1. See Table 1 for the genotypes of these strains and Figure 3 for the repeating unit structures. Viscosities measured in centipoise (cP).
vylated polymers, inactivation of acetylase I1 has only a slight effect on acetate content and might therefore be expected to have an equivalently slight effect on viscosity. This was the case for the acetylase I+, ketalase+ strains, X1396, and X1398. However, in the strains mutated for acetylase I, but not ketalase, the inactivation of acetylase I1 did seem to significantly increase polymer viscosity (see X1400 vs X1402). The negative effect of acetate upon viscosity and the positive effect of pyruvate are strikingly contrasted in Figure 4, which presents viscosity curves for wild-type polymer (X1396), a non-pyruvylated but fully acetylated polymer (X1397), and a pyruvylated but non-acetylated polymer (X1402). The effects of acetylation and pyruvylation of the terminal mannose upon polymer viscosity are illustrated in Figures 5 and 6. Figure 5 presents viscosity curves for three variant xanthans, all of which are acetylated on the inner mannose but which differ with respect to modification of the outer mannose. The highest viscosity xanthan is pyruvylated on the terminal mannose (X1398). The nearly stoichiometric substitution of acetate for pyruvylate drastically reduced viscosity (X1397), while the polymer with an unmodified outer mannose (X1399) displayed an intermediate viscosity. Similar effects were seen by comparing an analogous set of three variant gums that are either pyruvylated, acetylated, or unmodified a t the terminal mannose, but which are all unmodified a t the inner mannose, as illustrated in Figure 6. Again, the highest viscosity was associated with the polymer that is pyruvylated on the outer mannose (X1402) and the lowest viscosity was associated with the polymer that is acetylated on the outer mannose (X1401), while the viscosity of the polymer with an unmodified outer mannose (X1403) was once more intermediate. These results show that pyruvylation of the outer mannose directly enhances viscosity but also does so indirectly by blocking acetylation of that residue. Moreover, similar effects of the modification of the outer mannose are observed for gums with either an unmodified or an acetylated inner mannose. Xanthan viscosity as a function of the degree of acetylation, in the absence of pyruvate, is illustrated in Figure 7. Reduced acetate levels resulted in higher viscosity. The viscosity of the polymer that is not modified on either mannose residue (X1403) was significantly higher than the viscosities of polymers acetylated on the inner
Biotechnol, hog., 1990,Vol. 6, No. 3
50
185
L
40 '
au CI
8
(1,
>
a0
201
t
X 1398
/
0 0.05
0.15
0.10
Gum, W t
0.20
YO
Figure 5. Viscosities of xanthans acetylated on the inner man-
nose but variably pyruvylated and acetylated on the terminal mannose in 0.1 M NaCl at 25 "C as a function of concentration -; X1398, -0-; at a constant shear rate of 8 s-1: X1397, - -0X1399, -0-. See Table 1for the genotypes of these strains and Figure 3 for the repeating unit structures. Viscosities measured in centipoise (cP).
0.05
X 1399
0.15
0.10
1
0.20
Gum. W t YO Figure 7. Viscosities of nonpyruvylated xanthans with varying levels of acetylation. Viscosities measured in 0.1 M NaCl
at 25 "C as a function of concentrationat a constant shear rate 8 s-~:X1397, - -0-;X1399, -O-; X1401, - -0- -;X1403, -0-. See Table 1 for the genotypes of these strains and Figure 3 for the repeating unit structures. Viscosities measured in centipoise (cP). Of
150
50
40
40
XI4031
-
100
t-
75
-
125
cn
"ACETYLATED PCCYTETRAMR
/
WLYTETMR
y o O*
"
0.05
0.10
0.15
0.20
Gum, W t %
Figure 6. Viscosities of xanthans not acetylated on the inner
mannose and variably pyruvylated and acetylated on the terminal mannose in 0.1 M NaCl at 25 "C as a function of concentration at a constant shear rate of 8 s-1: X1401, - -0--; X1402, 4; X1403, -e-. See Table 1for the genotypes of these strains and Figure 3 for the repeating unit structures. Viscosities measured in centipoise (cP). (X1399) or the outer (X1401) mannose. The viscosities of these two polymers were virtually equivalent and were greater than the viscosity of the polymer that is acetylated on both mannose residues (X1397). Effects of Side-Chain Truncation. In order to ascertain the effects of alterations in side-chain structure on the viscosity of xanthan, the mutant strains of X.campestris that produce variant gums with altered side chains, as shown in Figure 3, were grown in parallel along with an isogenic wild-type control. Polymers were prepared and analyzed as detailed in Methods and text above. The elimination of sugar residues from xanthan side chains also appears to have a major effect on xanthan viscosity, as shown in Figure 8. Polymer lacking the terminal mannose, termed polytetramer, had a much lower viscosity than did wild-type xanthan. It is not clear at this point to what extent the low viscosity of the polytetramer results, per se, from the elimination of the terminal mannose residue of the side chain as opposed to the concurrent loss of pyruvate. In contrast, the polytrimer polymer, which lacks both the terminal mannose
0.05
0 10
0.15
0.20
Gum, W t %
Figure 8. Viscosities of xanthan-based polymers with truncated side chains. Visccsities measured in 0.1 M NaCl at 25 "C as a function of concentrationat a constant shear rate of 8 s-1: polytrimer (X1263),-A-; polytetramer (X1361),-+; nonacetylated polytetramer (X1419), -+-; isogenic wild-type (X1431), -+-. See Figure 3 for repeating unit structures. Viscosities measured in centipoise (cP). and the glucuronic acid residues of the side chain, was more viscous on a weight basis than wild-type xanthan. The effects of acetylation on the viscosities of polymers with truncated side chains differed. A negative effect of acetylation on viscosity, similar to that observed for wild-type xanthan, was also seen for polytetramer (Figure 8). However, the viscosities of acetylated and nonacetylated polytrimer were very similar (data not shown).
Discussion The data presented here demonstrate that acetylation and pyruvylation can affect the viscometric properties of xanthan. In normal pentameric xanthan, a t least under the conditions of our measurements, the presence of pyruvate enhances viscosity, whereas acetate reduces viscosity. A negative effect of acetylation on viscosity is also observed with the polytetramer variant of xanthan but is seemingly absent for the polytrimer variant. The genetic manipulation of the genes encoding acetylase I and acetylase 11, respectively, indicates that the extent of acetylation affects viscosity but the position of the acetate does not.
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Mutations that result in the synthesis of gums with truncated side-chains also affect viscosity. Compared to wild-type xanthan, polytetramer, lacking the terminal mannose, is a very poor viscosifier. In contrast, polytrimer gum, which lacks both the terminal mannose and glucuronic acid, is a superior viscosifier on a weight basis. This result with the polytrimer has been reported previously (3), and Sutherland and Tait (11)have described a similar finding. They isolated a mutant of X . c a m p e s t r i s that produces a xanthan that contains predominantly disaccharide side chains which is a relatively poor viscosifier. This polymer is likely to be similar to our polytetramer variant. However, they showed that the viscosity of this material is vastly increased by treatment with p-glucuronidase, which removes the terminal glucuronic acid from the disaccharide side chain and thus produces a polytrimer-like polymer. As described above, our polytrimer variant is of far higher viscosity than the polytetramer variant. Other investigators have also studied the effects of pyruvylation and/or acetylation on xanthan viscosity under various conditions of salinity, polymer concentration, and shear rate and have reported results similar to ours (1220). However, other workers (21-23) have reported that pyruvylation and acetylation do not affect viscosity when associated differences in other polymer characteristics are taken into account. Some difficulties arise in attempting to compare directly the results from different investigators because of the use of polymer samples from different sources, the use of naturally produced polymers versus chemically deacetylated and depyruvylated polymers, and the use of a wide array of sample preparation protocols involving autoclaving, lyophilization, precipitation, filtration, and exposure to different solvents. Our approach to studying the effects of acetylation and pyruvylation on the viscosity of xanthan has been to compare polymers produced by isogenic mutants of X . campestris grown in parallel under identical culture conditions and to minimize the sample preparation steps. Thus, the introduction of other factors that may confuse the relationship of acetylation and pyruvylation to viscosity is reduced. In this study, we have reported the effects of acetylation and pyruvylation on polymer viscosities as measured under a limited set of experimental conditions, i.e., polymer concentrations between 0.05 and 0.20 wt % , in solution in 0.1 M NaCl, a t 25 "C, and at a shear rate of 8 s-1. Preliminary results have been obtained for different shear rates and temperatures; similar effects are observed between 8 and 80 s-l and a t temperatures of 25, 37, and 50 "C. It would be interesting to determine if the same effects are observed when various experimental parameters (polymer concentration, temperature, shear rate, ionic strength, divalent cation concentration, etc.) are systematically varied over wide ranges. Many factors, including molecular weight, polymer conformation, and intermolecular interactions, might affect the solution viscosity properties of xanthan. It is not known how the differences in the properties of the variant gums produced by these mutant strains of c a m p e s tris result from the mutations that affect acetylation, pyruvylation, and side-chain structure. These structural changes may alter the properties of the xanthan molecule directly. For instance, the structural changes might influence the polysaccharide's solution conformation. Previous studies have characterized different conformational states of xanthan in solution (24-28), and some results suggest that the pyruvylation and acetylation of
x.
the side chain can influence conformation. Our reported structural alterations could also affect intermolecular interactions of xanthan molecules in solution. It has been reported (29) that, under certain conditions, the extent of pyruvylation can affect intermolecular association of xanthan. Potentially, these structural changes could also affect some other unforeseen parameter(s) of polymer rheology. Alternatively, the observed differences in polymer viscosity could result from perturbations in xanthan biosynthesis caused by the alterations in the structure of the repeating unit. For example, the alteration of repeating-unit structure might result in synthesis of polymers of different molecular weights. Direct measurements of the molecular weights of these polymers would be informative but have not yet been made. Regardless of the mechanism by which these genetic changes in xanthan structure affect the viscosity of the polymer, a novel set of xanthan-like polymers has been produced by genetic engineering. Further characterization of these polymers may identify novel properties of these materials and could provide insight into the relationship between xanthan structure and function.
Acknowledgments We thank Anne Hill for preparation of this manuscript. This research was funded by a joint venture research agreement between Synergen and Getty Scientific Development Co., a wholly owned subsidiary of Texaco, Inc.
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