The structure of a novel polysaccharide isolated from Zymomonas mobilis determined by nuclear magnetic resonance spectroscopy

Abstract

A novel polysaccharide has been observed in uivo by 13C nuclear magnetic resonance (NMR) spectroscopy of the gram-negative bacterium, Zymomonas mobilis. The polysaccharide, which was not removed from the Zmobilis cell by washing with 0.86 % saline, was extracted by mild acid treatment. The structure was elucidated by a combination of NMR techniques, including proton and proton-carbon two-dimensional methods. The structure was determined to be -a-fructofuranosyl-(2-1)-~-fructofuranosyl-(2-6)-. The polysaccharide is unlike levan, a p-(2-6)-fructose polymer which is produced by many bacteria, including Z.mobilis, when grown on sucrose. The polysaccharide isolated here is produced by cells cultured on glucose, fructose or sucrose.Polyfructosides are generally subdivided into two main classes : levans, in which the main interglycoside linkage is p(2 -6), and inulin, in which the main linkage is p(2 -1) [1]. Both inulin and levans are widely distributed throughout the plant kingdom, where they occur as both linear and branched polymers of comparatively low molecular mass and usually contain a small porportion of glucose in addition to fructose.Microbial polyfructosides are generally of the levan type. Levan is produced by both gram-positive and gram-negative bacteria [2] including Zymomonas mobilis. Z . mobilis is a gramnegative bacterium which can ferment only glucose, fructose and sucrose to ethanol anaerobically via the EntnerDoudoroff pathway. Growth on sucrose results in the synthesis of levan while in the absence of sucrose no levan production occurs. The levan produced by Z . mobilis has been shown to be typical of the bacterial levans containing only fructose in the furanose ring form, linked p(2 -6) with p(2 -1) branch points and a very high molecular mass (approximately 10 MDa) [3].The novel polyfructoside described here has been isolated from Z . mobilis cells grown on glucose and has been found to have a different structure to that of levan and a lower molecular mass. MATERIALS AND METHODS Ma terialsSephacryl 200 was obtained from Pharmacia ; the anionexchange resin, AG3-X4A, was obtained from Bio-Rad. D 2 0 ( 2 99.75 % D) was obtained from the Australian Atomic Energy Commission, Lucas Heights. All other reagents were analytical reagent grade.

Respiratory behaviour of a Zymomonas mobilis adhB::kanr mutant supports the hypothesis of two alcohol dehydrogenase isoenzymes catalysing opposite reactions

“…It is well established that the maximum value of Y ETOH in Z. mobilis catabolism under anaerobic conditions is close to 0.51 g of ethanol per gram of consumed glucose [18,21], and that under aerobic growth conditions Y ETOH is lower due to competition for NADH between the respiratory chain and ethanol synthesis [15]. In the aerobic chemostat culture of the mutant strain, we found Y ETOH to be 0.22 g g À1 , which constitutes 43% of the maximum value.…”

“…It must be emphasized that the measured bulk reactant ratios for the ADH reaction in both strains were closely similar, generally exceeding K eq by 3 orders of magnitude, and hence could not explain the extremely different responses of the strains to the ethanol pulse. Taking the measured values for ethanol, acetaldehyde and intracellular NADH concentrations (Table 2), 6.5 for intracellular pH [18], 3.3 ll per mg of dry weight for the intracellular volume [19], and (for the lowest estimate of the reactant ratios) assuming that NAD + makes up most of the 5 mM intracellular NAD(P)(H) pool, detected in Z. mobilis by NMR [20], we come to reactant ratios of 2.6 · 10 À9 M for ZM6 and 12.6 · 10 À9 M for the ADH II-deficient strain. These are indeed the lowest estimates of the reactant ratios, because: (i) by assuming the intracellular acetaldehyde/ethanol ratio equal to that of the outer medium, we are neglecting the fact that acetaldehyde is a more volatile compound than ethanol, and hence, the true intracellular acetaldehyde/ethanol ratio might be higher; (ii) a lower value for the intracellular volume of Z. mobilis has been reported by Osman et al [21], which would result in a higher measured intracellular NADH concentrations.…”

Purification of a novel fructosyltransferase from Lactobacillus reuteri strain 121 and characterization of the levan produced

“…Comparison with the published 13 C chemical shifts of fructan produced by Lb. reuteri strain 121 grown on MRSr media [8] and Z. mobilis levan [14] indicated that the FTF fructan had a [C6)-L-D-Fruf-(2C] n structure, a levan. Further con¢r-mation was obtained from the identical 1 H NMR spectra of the FTF fructan (results not shown) and the fructan produced by MRSr grown Lb.…”

Cloning and sequencing of the sacA gene: characterization of a sucrase from Zymomonas mobilis

“…Inocula (5% in volume) were grown for 18 h in stationary flasks. Cultures were run at 30°C in a 2-liter fermentor containing 1.5 liters of medium with mild agitation (200 rpm), and pH was controlled at 5.5 by addition of 2 subtilis levans (average molecular mass, 15,000 daltons) per ml and the enzyme solution. Aliquots of 10 Al were taken every 30 min (for 2 h) and treated as above for the exchange reaction.…”