Variations in xanthan production by antibiotic-resistant mutants of Xanthomonas campestris

Mutants resistant to different antibiotics (streptomycin, tetracycline, ampicillin and penicillin) were obtained from several strains of Xanthomonas campestris and evaluated for xanthan production. Most of the mutants showed alterations in their polysaccharide production, either increasing, decreasing or totally losing their polymer-production capacity. The existence of two types of antibiotic-resistance mechanisms for the assayed drugs is suggested: one that affects xanthan production and another that does not. Differences in outer-membrane protein patterns of mutants that were simultaneously altered in antibiotic resistance and xanthan production were found, in comparison with their parental strains. These findings suggest the existence of a genetic relationship between antibiotic-resistance mechanisms and xanthan production. Some of the mutants obtained showed significant increases in broth viscosity and xanthan concentration. These results suggest that resistance to streptomycin and ampicillin can be used to obtain improved strains in plate screening assays. Received: 8 January 1997 / Received revision: 13 June 1997 / Accepted: 4 July 1997     

Effect of corn steep liquor on xanthan production by Xanthomonas campestris

Summary The addition of corn steep liquor (CSL) to batch cultures of Xanthomonas campestris using sucrose as carbon source stimulated cell growth rate, viscosity and xanthan production as compared to non-supplemented cultures. The addition of CSL to a basal medium at a dose of 1 g/l, increased xanthan production and viscosity by 22% and 44% respectively. CSL also shortened the cultivation time and promoted a more efficient sucrose utilization for polymer synthesis. After 72 h of incubation the xanthan yield per sucrose consumed in the CSL-amended culture was 0.63 g/g, this is, 15% higher than without CSL addition. At higher doses of CSL cell growth rate was also increased but not polymer production.     

Genetic engineering of polysaccharide structure: production of variants of xanthan gum in Xanthomonas campestris.

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.     

Use of agricultural wastes for xanthan production by Xanthomonas campestris

Four different acid-hydrolyzed wastes, from melon, watermelon, cucumber and tomato were compared for xanthan production. Growth of Xanthomonas campestris, xanthan biosynthesis, kinetics and chemical composition were investigated. Both growth and xanthan production were dependent on the acid hydrolysate concentrations and available nitrogen. Melon acid hydrolyzed waste was the best substrate for xanthan production. Exopolysaccharide obtained throughout this study was compared to commercial xanthan, showing a very similar chemical composition. Acid hydrolyzed wastes are proposed as a new carbon source for xanthan production. Received 16 July 1998/ Accepted in revised form 8 October 1998     

Xanthan is not essential for pathogenicity in citrus canker but contributes to<Emphasis Type="Italic"> Xanthomonas</Emphasis> epiphytic survival

Xanthan-deficient mutants of Xanthomonas axonopodis pv. citri, the bacterium responsible for citrus canker, were generated by deletion and marker exchange of the region encoding the carboxy-terminal end of the first glycosyltransferase, GumD. Mutants of gumD did not produce xanthan and remained pathogenic in citrus plants to the same extent as wild-type bacteria. The kinetics of appearance of initial symptoms, areas of plant material affected, and growth of bacteria inside plant tissue throughout the disease process were similar for both wild-type and mutant inoculations. Moreover, exopolysaccharide deficiency did not impair the ability of the bacteria to induce hypersensitive response on non-host plants. Apart from variations in phenotypic aspects, no differences in growth or survival under different stress conditions were observed between the xanthan-deficient mutant and wild-type bacteria. However, gumD mutants displayed impaired survival under oxidative stress during stationary phase as well as impaired epiphytic survival on citrus leaves. Our results suggest that xanthan does not play an essential role in citrus canker at the initial stages of infection or in the incompatible interactions between X. axonopodis pv. citri and non-host plants, but facilitates the maintenance of bacteria on the host plant, possibly improving the efficiency of colonization of distant tissue.     

Potential application of a low-viscosity and high-transparency xanthan gum produced from Xanthomonas campestris CCTCC M2015714 in foods

Xanthan gum is commonly used as a thickener in food industry, while the usage of xanthan gum as a dietary fiber is restricted for its low additive volume. Herein, the potential use of a low-viscosity and high-transparency xanthan gum as a dietary fiber was evaluated in vitro. This new xanthan shows better transparency and faster dissolution rate than most commercial products, and its viscosity increases along with the treatment of freeze–thaw cycles at ?20°C. Moreover, this new xanthan can absorb heavy metals (Pb, Cd, Cu) and retard starch digestion by glucoamylase. In summary, this new xanthan could be potentially used as a dietary fiber or fiber ingredient for preventing and treating diabetes, hyperlipemia, heavy metal poisoning, and cardiovascular diseases effectively.     

Stability of xanthan in aqueous sodium chloride at elevated temperature

Aqueous NaCl solutions of dimerized Na xanthan with salt concentrations of 0.005, 0.01 and 0.1 were exposed to 80°C for different time periods t, and their viscosities were determined as a function of t. The measured relative viscosities decreased markedly with t, suggesting that Na xanthan denatured at 80°C undergoes some conformation changes or degradation. The molecular weights of the test samples recovered at different t were estimated by viscometry in cadoxen, a single-coil solvent for xanthan, and were found to decrease monotonically with t. Thus, it was concluded that the observed decreases in relative viscosity are due primarily to degradation of Na xanthan.     

Anti-juvenile Hormone Effects of an Imidazole Compound (KK-42) in Different Larval Instars of Bombyx mori

The non-Newtonian behavior and dynamic viscoelasticity of solutions of the Ca salt of xanthan were measured with a rheogoniometer. The Ca salt of xanthan showed pseudoplastic behavior < 0.1 % but was plastic >0.3%. Compared with native, Na, and K salts of xanthan, the Ca salt had higher dynamic viscoelasticity at high concentrations. The apparent viscosity of Ca salt of xanthan was very large at low temperatures and decreased with increasing temperature. These suggest very strong intermolecular association of the xanthan (Ca salt) molecules, probably due to the formation of ionic force between adjacent charged trisaccharide side-chains via Ca²⁺ on different molecules. Possible structures for quaternary association including intramolecular association of xanthan molecules (Ca and K salts) in aqueous solution were proposed.     

Double-stranded helix of xanthan: Rigidity in 0.01M aqueous sodium chloride containing 0.01 N hydrochloric acid

Six samples of Na xanthan in 0.01M aqueous NaCl containing 0.01 N HCl (pH = 2) were studied by light scattering and viscosity. This study was motivated by the finding that the intrinsic viscosity [η] fairly sharply decreased when the pH of the solvent was lowered from about 6 to 2 by adding HCl to 0.01M aqueous NaCl in which Na xanthan dissolves as rigid dimers having a double-helical structure. The data for weight-average molecular weight, radius of gyration, and [η] showed that Na xanthan at pH = 2 remains a dimer behaving as a semiflexible chain. Data analysis in terms of known theories for unperturbed wormlike chains yielded 0.47 ± 0.02, 2.0 ± 0.6, and 68 ± 7 nm for the contour length h per main-chain residue, diameter d, and persistence length q of the dimer, respectively. these h and d values agreed with the pitch per main-chain residue and the diameter of the double helix of Na xanthan in 0.01 or 0.1M aqueous NaCl. However, the q value, which was close to the intrinsic persistence length q₀ ( = q in the absence of electrostatic interaction) of Na xanthan at pH = 2, was much smaller than the q₀ (106 nm) of this helix. We concluded that the xanthan dimer at pH = 2 assumes a double-helical structure, which is geometrically the same as, but is more flexible than, that at neutral pH.     

Production of xanthan gum and ice-nucleating material from whey by Xanthomonas campestris pv. translucens

Xanthomonas campestris pv. translucens IFO13599 could produce xanthan gum (18.5 mg/100 mg, lactose) with lactose as the growth substrate in spite of a low level of β-galactosidase. This productivity corresponded to one-fifth that with glucose. This strain could also produce ice-nucleating material having an ice-nucleating temperature, T ₅₀, of −2.8 °C with xanthan gum in the culture broth. We found that this strain produced both materials in whey medium from which the insoluble components had been removed. The production of xanthan with ice-nucleating material reached a maximum after cultivation for 168 h under optimum conditions. Furthermore, the xanthan obtained had a low viscosity because of its variant structure revealed, by TLC and HPLC analyses, to be lacking pyruvic acid. Furthermore, we concluded that this mixture had considerable potential as a regeneratic agent, when compared to other regeneratic agents such as carboxymethylcellulose. Received: 29 August 1997 / Received revision: 17 November 1997 / Accepted: 18 November 1997     

Improved strains for production of xanthan gum by fermentation ofXanthomonas campestris

Summary Two classes of mutants ofXanthomonas campestris B1459 were isolated that accumulate more xanthan gum than the parental wild-type in culture broths of shake flask cultures and both batch and fed-batch fermentations. The first mutant class was resistant to the antibiotic rifampicin and accumulated, on average, about 20% more xanthan gum than wild-type. The second mutant class, a derivative of the first, was resistant to both bacitracin and rifampicin, and accumulated about 10% more xanthan than its parent. On a weight basis, the viscosities of the polysaccharides made by each strain were not distinguishable. Only a subset of the drug-resistant mutants were overproducers of xanthan. The biochemical basis for the overproduction of xanthan by the mutant strains has not been determined. Both new strains served as recipients for recombinant plasmids bearing xanthan genes and further augmented the effects of multiple copies of those genes on xanthan productivity.     

Mutants ofXanthomonas campestris defective in secretion of extracellular enzymes

Summary Mutants ofXanthomonas campestris B 1459 were isolated that are defective in secretion of both cellulase and amylase. Both enzymes accumulated in the periplasmic space. The defects in secretion of cellulase or amylase were partly overcome by introducing into the mutants specific multiple copies of DNA cloned fromX. campestris, and presumed to code for cellulase or amylase enzymes. The mutant strains also showed reduced amounts of extracellular pectinase and protease activities, as if the mutants were generally defective for secretion of extracellular enzymes. The mutants showed reduced pathogenesis for turnip seedlings. The secretion-defective mutants may allow production of xanthan gum with reduced cellulose, pectin, protein and starch-degrading enzyme activities, thereby allowing more widespread mixing of microbially produced xanthan gum with these commercially important water-soluble polymers.     

Biodegradation of xanthan by salt-tolerant aerobic microorganisms

Summary Three salt-tolerant bacteria which degraded xanthan were isolated from various water and soil samples collected from New Jersey, Illinois, and Louisiana. The mixed culture, HD1, contained aBacillus sp. which produced an inducible enzyme(s) having the highest extracellular xanthan-degrading activity found. Xanthan alone induced the observed xanthan-degrading activity. The optimum pH and temperature for cell growth were 5–7 and 30–35°C, respectively. The optimum temperature for activity of the xanthan-degrading enzyme(s) was 35–45°C, slightly higher than the optimum growth temperature. With a cell-free enzyme preparation, the optimum pH for the reduction of solution viscosity and for the release of reducing sugar groups were different (5 and 6, respectively), suggesting the involvement of more than one enzyme for these two reactions. Products of enzymatic xanthan degradation were identified as glucose, glucuronic acid, mannose, pyruvated mannose, acetylated mannose and unidentified oligo- and polysaccharides. The weight average molecular weight of xanthan samples shifted from 6.5·10⁶ down to 6.0·10⁴ during 18 h of incubation with the cell-free crude enzymes. The activity of the xanthan-degrading enzyme(s) was not influenced by the presence or absence of air or by the presence of Na₂S₂O₄ and low levels of biocides such as formaldehyde (25 ppm) and 2,2-dibromo-3-nitrilopropionamide (10 ppm). Formaldehyde at 50 ppm effectively inhibited growth of the xanthan degraders.     

Modified xanthan—its preparation and viscosity

Xanthan with various pyruvic acid and acetate contents has been prepared from a single commercial polysaccharide sample using optimised chemical conditions (acid and alkali hydrolysis, respectively) for removal of acetal and acyl groups. The only significant change found on analysis of the modified xanthans was loss of pyruvic acid and/or acetate; no low moleculur weight carbohydrate-containing material was released. Contrary to some previous reports, evidence is presented to show that the pyruvic acid acetal and o-acetyl contents of xanthan do not affect solution viscosity. The viscosities of native, pyruvate-free and pyruvate/acetate-free xanthan solutions (0·3% w/v) were similar at shear rates 8·8–88·3 s^?1 in both distilled water and 1% KCl. Over the concentration range 0·2-1·5%, the viscosities of native and pyruvate-free xanthan at 10 s^?1 were similar. The viscosity increase on addition of 1% KCl to salt-free xanthan solutions was independent of pyruvic acid acetal substitution. Our results suggest that xanthan samples with various pyruvic acid acetal and o-acetal contents, prepared under different fermentation conditions of Xanthomonas campestri should not normally be used for assessing the contribution of these groups to solution viscosity.     

Effects of yeast extract and glucose on xanthan production and cell growth in batch culture of Xanthomonas campestris

Although available kinetic data provide a useful insight into the effects of medium composition on xanthan production by Xanthomonas campestris, they cannot account for the synergetic effects of carbon (glucose) and nitrogen (yeast extract) substrates on cell growth and xanthan production. In this work, we studied the effects of the glucose/yeast-extract ratio (G/YE) in the medium on cell growth and xanthan production in various operating modes, including batch, two-stage batch, and fed-batch fermentations. In general, both the xanthan yield and specific production rate increased with increasing G/YE in the medium, but the cell yield and specific growth rate decreased as G/YE increased. A two-stage batch fermentation with a G/YE shift from an initial low level (2.5% glucose/0.3% yeast extract) to a high level (5.0% glucose/0.3% yeast extract) at the end of the exponential growth phase was found to be preferable for xanthan production. This two-stage fermentation design both provided fast cell growth and gave a high xanthan yield and xanthan production rate. In contrast, fed-batch fermentation with intermittent additions of glucose to the fermentor during the stationary phase was not favorable for xanthan production because of the relatively low G/YE resulting in low xanthan production rate and yield. It is also important to use a moderately high yeast extract concentration in the medium in order to reach a high cell density before the culture enters the stationary phase. A high cell density is also important to the overall xanthan production rate. Received: 30 September 1996 / Received revision: 21 January 1997 / Accepted: 10 February 1997     

Relationship between the Rheological Properties of Thickener Solutions and Their Velocity through the Pharynx as Measured by the Ultrasonic Pulse Doppler Method

The dependence of the dynamic viscoelastic parameters of carboxymethylcellulose (CMC), xanthan gum, and guar gum solutions on the angular frequency (ω) was compared with that of their viscosity (μ) on the shear rate (γ). In addition, the effect of these rheological properties on the maximum velocity through the pharynx, V _max, as measured by the ultrasonic pulse Doppler method, was investigated. The CMC and guar gum solutions examined were taken as a dilute solution and a true polymer solution, respectively. The xanthan gum solution was taken as a weak gel above 0.5% and a true polymer solution below 0.2%. The maximum velocity, V _max, of the thickener solutions correlated well with μ, the dynamic viscosity η′, and the complex viscosity η^*, especially those measured at γ or ω of 20–30 s^?1 (or rad/s) and above, suggesting that μ, η′, and η^* are suitable indexes for care foods of the liquid type for dysphagic patients.     

Kinesin velocity increases with the number of motors pulling against viscoelastic drag

Although the properties of single kinesin molecular motors are well understood, it is not clear whether multiple motors pulling a single vesicle in a cell cooperate or interfere with one another. To learn how small numbers of motors interact, microtubule gliding assays were carried out with full-length Drosophila kinesin in a novel motility medium containing xanthan, a stiff, water-soluble polysaccharide. At 2 mg/ml xanthan, the zero-shear viscosity of this medium is 1,000 times the viscosity of water, similar to cellular viscosity. To mimic the rheological drag force on the motors when attached to a vesicle in a cell, we attached a 2 μm bead to one end of the microtubule (MT). During gliding assays in our novel medium, the moving bead exerted a drag force of 4–15 pN on the kinesins pulling the MT. The velocity of MTs with an attached bead increased with MT length and with kinesin concentration. The increase with MT length arose because the number of motors is directly proportional to MT length. Our results show that small numbers of kinesins cooperate constructively when pulling against a viscoelastic drag. In the absence of a bead but still in the viscous medium, MT velocity was independent of MT length and kinesin concentration because the thin MT, like a snake moving through grass, was able to move between xanthan molecules with little resistance. A minimal shared-load model in which the number of motors is proportional to MT length fits the observed dependence of gliding velocity on MT length and kinesin concentration.     

Inter- and Intra-Manufacturer Variability in Pharmaceutical Grades and Lots of Xanthan Gum

A pharmaceutical formulation typically contains one or more excipients in addition to the active pharmaceutical ingredient(s). Though excipients have been considered inert components of a formulation, variability in their properties has been shown to affect the performance of drug dosage forms and delivery systems. This study investigates the inter- and intra-manufacturer variability among different NF grades and lots of xanthan gum made by two manufacturers. As many formulators rely on compendial standards to monitor and control the variability of excipients, this study focuses on the adequacy of the NF specifications, in particular the viscosity specification, to discern the variability in solution properties of different pharmaceutical grades and lots of xanthan gum. All the grades and lots in this study were NF grade materials. Xanthan gum solutions were prepared in accordance with NF test methodology and were rheologically evaluated using a rotational rheometer. Both steady shear measurements and small amplitude oscillatory measurements were carried out on 1% w/w xanthan gum solutions. Results showed significant inter- and intra-manufacturer variability among the NF grades and lots of xanthan gum that was not reflected in the NF viscosity test specifications.     

Response time and electrorheology of semidiluted gellan, xanthan and cellulose suspensions

Response times with electrical fields of gellan and xanthan dry powder suspensions of 25, 32 and 53 μm average diameter and concentrations of 1.0, 1.5 and 2.0% (w/w) dispersed in commercial corn oil were optically measured through a specifically designed set up. In all cases, the delay time was proportional to 1/E^a, where E is the applied field and a is an adjustable parameter. The values of parameter a were very different from the typical value of some known electrorheolgical fluids. Response time of gellan suspensions was shorter than the one obtained for xanthan and it is comparable to the time found by using silica particles in silicon oil. Response times for cellulose were very large and the fibrillation phenomenon was negligible for E<1.0 kv/mm.

Viscosity measurements of semidiluted xanthan, gellan and cellulose suspensions (1.0 and 1.5% w/w) under the influence of electrical fields, were performed in a parallel plates rheometer. Results in the range of stress <70 Pa showed that viscosity values of gellan suspensions were larger than those obtained with xanthan or cellulose under the same applied electric field at shear rates higher than 10 s⁻¹. However, cellulose suspensions showed larger viscosity values compared with the ones measured with xanthan and gellan suspensions at very low shear rates. Dielectric measurements of cellulose, xanthan and gellan 1.5% w/w suspensions were performed in the range 10⁰–8×10⁴ Hz. Results agree with a Maxwell–Wagner type relaxation model.     

Xanthan Lyase of Bacillus sp. Strain GL1 Liberates Pyruvylated Mannose from Xanthan Side Chains

When the bacterium Bacillus sp. strain GL1 was grown in a medium containing xanthan as the carbon source, the viscosity of the medium decreased in association with growth, showing that the bacterium had xanthan-depolymerizing enzymes. One of the xanthan-depolymerizing enzymes (xanthan lyase) was present in the medium and was found to be induced by xanthan. The xanthan lyase purified from the culture fluid was a monomer with a molecular mass of 75 kDa, and was most active at pH 5.5 and 50°C. The enzyme was highly specific for xanthan and produced pyruvylated mannose. The result indicates that the enzyme cleaved the linkage between the terminal pyruvylated mannosyl and glucuronyl residues in the side chain of xanthan.