Agric. Biol Chem., 49 (4), 959~966, 1985 959
Structure of an Acidic Polysaccharide from Acetobacter sp. NBI 1022f Kenji Tayama, Hiroyuki Minakami, Etsuzo Entani, Seiichi Fujiyama and Hiroshi Masai Nakano Biochemical Research Institute, Nakano Vinegar Co., Ltd., Honda, Aichi 475, Japan Received July ll, 1984
Anacetic acid bacterium which produces a new type of extracellular soluble polysaccharide was isolated from vinegar mash. The isolated strain, NBI 1022, was tentatively identified as Acetobacter aceti subsp. xylinum. The polysaccharide, named AM-2, was composed ofD-glucose, l- rhamnose, D-mannose, D-glucuronic acid, and 0-acetyl in a molar ratio of approximately 4 : 1: 1 : 1 : 1. From the results ofmethylation, Smith degradation, and partial acid hydrolysis of the polysaccharide and its derivatives, the polysaccharide AM-2 may have a branched structure containing a backbone chain of /?-(l->4)-linked D-glucose residues and a side chain shown as l- rhamnosyl-(l ->6)-/?-D-glucosyl-(l ->6)-D-glucosyl-(l ->4)-D-glucuronosyl-(l ->2)-d-
The acetic acid bacteria assigned to the MATERIALS AND METHODS genera Acetobacter and Gluconobacter pro- duce several polysaccharides such as cel- Materials. A /M,4-D-glucanase (cellulase) from Aspergillus niger was purchased from the Sigma Chem. lulose,1* levan,2) dextran,3) soluble glucan,4) Co., St. Louis, Missouri, U.S.A. Glycogen, dextran B- and acidic polysaccharide.5) In previous pa- 1 355-S from Leuconostoc mesenteroides,9) and the polysac- pers,6'7* wehave reported that isolated strains charide from Xanthomonasoryzael0) were used as ref- belonging to Acetobacter species produce a erence standard in analysis of methylated sugars. newtype of extracellular polysaccharide des- Isolation and characterization of polysaccharide- ignated AM-1. In the course of studies on producing acetic acid bacterium. The methods for isolation acetic acid bacteria producing much acetic of polysaccharide-producing acetic acid bacteria, mainten- acid,8) we have found that a newly isolated ance of culture,, cultural and biochemical tests, identifi- acetic acid bacterium produces another type of cation of ubiquinone type, and DNAbase composition polysaccharide, a highly viscous heteropoly- were described previously.6) saccharide. This polysaccharide, tentatively Purification of polysaccharide and preparation of car- named AM-2, has been found to be composed boxyl-reduced polysaccharide. Purification of polysac- of D-glucose, L-rhamnose, D-mannose, d- charide and preparation of carboxyl-reduced polysaccha- glucuronic acid, and 0-acetyl in a molar ratio ride were performed by the methods described previous- of approximately 4: 1 : 1 : 1 : 1, differing in its ly.6) componentsfromthe polysaccharides report- General analytical methods for polysaccharide and its ed before. hydrolysis products. Reducing sugars were chromato- In this paper, we describe characteristics of graphed on Toyoroshi No. 51A paper by descending the newly isolated strain NBI 1022 and the method using a solvent system (v/v) of n- structure of the highly viscous heteropolysac- butanol-pyridine-water (6 : 4 : 3), and detected by spray- charide AM-2. ing with alkaline silver nitrate reagent. For identification
Extracellular Polysaccharides Produced by Acetic Acid Bacteria. Part II. 960 K. Tayama et al. of disaccharides, thin-layer chromatographywasused by sample (20mg) in 0.05m acetate buffer (3ml) at pH 5.0 the method of Hansen.n) For analysis of enzymatic and 30°C for 48 hr. The incubation mixture was heated in digestion products, thin-layer chromatography was done a boiling-water bath, and desalted with ion-exchange on pre-coated silica gel glass plates (Merck art. No. 5715) resin. The product was examined by thin-layer using a solvent system (v/v) of isopropanol-acetone-0. 1 m chromatography. lactic acid (4:2:2). Polysaccharide and oligosaccharide were hydrolyzed Methylation, periodate oxidation, and Smith degradation with 2m trifluoroacetic acid at 100°C for 18hr and 4hr, of polysaccharide and oligosaccharide. Methylation of respectively. Gas-liquid chromatography (GLC) was done polysaccharide and oligosaccharide was performed by the with a Hitachi model 124 gas chromatograph with a flame- method of Hakomori.17) Polysaccharide (20mg) was dis- ionization detector. Methylated sugars were converted to solved in dimethyl sulfoxide (2ml) and stirred for 20min their corresponding alditol acetates, and separated at at 50°Cin nitrogen atmosphere. The solution was treated 180°C on a column packed with 3% ECNSS-MGaschrom with 2m methylsulflnyl carbanion (0.5 ml) for 3 hr at room Q (0.4x200cm) or at 190°C on a surface-coated open- temperature and then with methyl iodide (1.5 ml) for 1.5 hr tubular glass capillary column coated with Silar-10C12) at 20cC. The mixture was dialyzed against water and dried (G-SCOT, 0.28mmx30m). GLC of neutral monosac- in vacuo. The methylation was repeated twice, and the fully charides was done after conversion to their corresponding methylated polysaccharide was hydrolyzed with 90% alditol acetates with a 3% ECNSS-Mcolumn at 190°C. formic acid (lml) for 16hr at 100°C, followed with 2m GLC of neutral disaccharides was done with a 3% SE-30 trifluoroacetic acid (lml) for 5hr at 100°C. The hy- column at 260°C after trimethyl-silylation.13) For sepa- drolyzed products of the methylated polysaccharide were ration by GLC of the products obtained by complete reduced with sodium borohydride and the corresponding Smith degradation, the hydrolysis products were reduced alditols were acetylated by heating with pyridine-acetic with sodium borohydride and then acetylated. anhydride (1 : 1) for 2hr at 100°C for analysis by GLC. Colorimetric determination of uronic acid was done by a Oligosaccharides (3mg) were methylated by the same modified carbazole method.14) procedure. The methylated oligosaccharide was extracted 13C-Nuclear magnetic resonance (13C-NMR) spectra with dichloromethane after evaporation of methyl iodide, were obtained using sample in D2O in 10mm diameter and the extract was washed with distilled water and then tubes at 90°C with a JEOL FX-100 spectrometer operating dehydrated with MgSO4.The extract was applied to a at 25MHz, and chemical shifts were recorded in ppm Sephadex LH-20 column (1 x60cm) which was fully down field from internal dioxane (67.40ppm). Molecular washed with dichloromethane. Fractions containing weight determination of polysaccharide was done by the methylated oligosaccharide were collected and evaporated meniscus depletion method15) using a partial specific vol- to dryness. The methylated oligosaccharide was hydro- umeof 0.59ml/g. Ultracentrifugal analysis of polysac- lyzed, reduced, and acetylated by the procedure described charide wasconductedusing an analytical ultracentrifuge, above for analysis by GLC. Spinco Model E. Optical rotation was measured with a For periodate oxidation and Smith degradation, the Union PM-101 automatic digital polarimeter. polysaccharide (lOOmg) was oxidized with 0.05 m sodium The acetyl content of polysaccharide was determined by metaperiodate (100 ml).at 4°.C in the dark. After complete two methods. First, the polysaccharide was deacetylated oxidation (6 days), the oxidation was stopped by the with 0.01 m potassium hydroxide in the presence of po- addition of ethylene glycol (10ml). The mixture was tassium chloride (1% w/v) at room temperature, and an dialyzed and converted to the corresponding polysac- aliquot of the solution was titrated with 0.01 n sulfuric charide' polyalcohol by the addition of sodium borohy- acid using a pHmeter. The second method was a col- dride. A portion of the polysaccharide polyalcohol was orimetric procedure based on the color complex formation hydrolyzed with 2n sulfuric acid for 6hr at 100°C (com- of an acyl hydroxamic acid and ferric ions.16) plete Smith degradation), and another portion was hy- The pyruvic acid content of polysaccharide was de- drolyzed with 0.1n sulfuric acid for 20hr at 21°C (mild termined by an enzyme assay method as follows. The Smith degradation). pyruvic acid was freed from the polysaccharide by acid treatment (0.1 mHC1 or 0.1 mtrifluoroacetic acid at 100°C Partial acid hydrolysis and fractionation of polysac- for 3hr) and then the solution was neutralized with 2m charide. The purified polysaccharide (9g) was heated at sodiumcarbonate. To an aliquot of the neutralized so- 100°C for 3hr in 0.25m trifluoroacetic acid (900ml), and lution (2 ml), the mixture of lactate dehydrogenase (20 un- then the hydrolyzate was evaporated, dissolved in water its), 1 m triethanolamine (l ml) and 1% NADHsolution (400ml) and added to 95% ethanol (1.2liter). The result- (50/d) was added, and absorbance at 340nm was mea- ing precipitate (I) was collected by centrifugation and the sured at 5 min intervals until stable. resulting supernatant (I) was evaporated to a syrup. The The enzymatic hydrolysis of the polysaccharide fraction precipitate (I) was dissolved in water and half of it was obtained by partial acid hydrolysis ofpolysaccharide AM- heated at 100°C for 3 hr in 0.25 Mtrifluoroacetic acid. The 2 was done by incubation of /M,4-D-glucanase (5 mg) and hydrolyzate was evaporated, dissolved in water, and added Structure of Polysaccharide from Acetobacter sp. 961 to 95%ethanol. The resulting precipitate (II) was collected Table I. Physiological and Biochemical by centrifugation and the resulting supernatant (II) was Characteristics of Isolated evaporated to a syrup. Precipitates I and II were separately Strain NBI 1022 dissolved in water, dialyzed exhaustively, and lyophilized. Supernatants I and II were combined and applied to a F eature Dowex 1 x 4 (5 x 20cm, formate form) column, which was P igm entation then washed with water to collect neutral oligosaccharides. Production of acetic acid + Acidic oligosaccharide was eluted with 1 mformic acid and Overoxidation of ethanol (Frateur) + then purified to a homogeneousstate by preparative paper (C arr) + chromatography on Whatman3MMpaper with a solvent Oxidation of acetate + system (v/v) of «-butanol-pyridine-water (6:4:3). lactate + Neutral oligosaccharides were fractionated on a column Decomposition of lactate to carbonate + of charcoal (5x25cm, Activated Charcoal for Dihydroxyacetone from glycerol + Chromatography, WakoPure Chemical Industries, Ltd., FeCL reaction on glucose broth Japan). After washing with distilled water, oligosac- fructose broth charides were eluted stepwise with 4% (800ml), 7% Growth on Frateur's Hover medium-ethanol (800ml), and 25% aqueous ethanol (800ml). Each fraction Frateur's Hoyer medium-glucose + was evaporated to dryness and neutral oligosaccharides as Growth on glutamate agar + fl well as acidic oligosaccharide were purified to a homo- m an nitol agar + geneous state by preparative paper chromatography. Formation of gluconate + 5-keto gluconate + 2-k etog luconate + 2, 5-diketogluconate RESULTS Acidic heteropolysaccharide production + Cellulose production Characteristics of isolated strain V itam in req uirem ent + The isolated strain NBI 1022 was non- C atalase + motile, gram-negative, rod-shaped, 0.6 ~0.7 x O xidase 1.0~ 1.8 jLim in cell size and obligately aerobic. G elatin ase Formation of indole Nospores and no flagella were observed. The Formation of H2S isolated strain grew at pH 3.5-7.5 (30°C) and Reduction of nitrates at 17~37°C (pH 6.5). The optimum tempera- Reaction in milk ture for growth was around 30°C. Acid for- V P -test M R -test + mation was observed strikingly from L-arabi- nose, D-xylose, D-glucose, D-mannose, D-galac- Very weak. tose, trehalose, melibiose, glycerol, ethanol, n- propanol, and ft-butanol, and weakly from sucrose, D-mannitol, and inositol, but not physicochemical characteristics of the purified from D-fructose, maltose, lactose, D-sorbitol, polysaccharide AM-2. No nitrogen was de- raffinose, L-sorbose, isopropanol, methanol, or tected in the purified preparation. In 13C- starch. Table I shows other physiological and NMRspectroscopy, the presence of both de- biochemical characteristics of the isolated oxy sugars and acetyl groups was suggested strain. The ubiquinone type of the isolated because signals were observed at 17.6, 21.2, strain was found to be Q10, differing from that and 174.2ppm, and the dominant glycosidic of A. aceti subsp. orleanensis IFO 13752 (type, linkages of the polysaccharide were assumed Q9).6) The DNA base composition of the to be ^-configuration because of the chemical isolated strain was calculated to be 59.5 mol% shift of anomeric carbon at 103.5 and guanine plus cytosine. 101.5ppm and of [a]D+18° (c=0.2, water). The acid hydrolyzates of the native polysac- Characteristics ofpolysaccharide charide gave four spots corresponding to d- The purified higly viscous polysaccharide glucose, L-rhamnose, D-mannose, and d- AM-2gave a sharp, single peak on analytical glucuronic acid on both paper and thin-layer ultracentrifugation (Fig. 1). Table II shows the chromatograms, and the gas-liquid chroma- 962 K. Tayama et al. tographic analyses of alditol acetates derived ratios for D-glucose, L-rhamnose, and d- from the native polysaccharide and its mannose of 4.1:0.98:1.0 and 5.1:0.95:1.0, carboxyl-reduced derivative showed molar respectively. It is apparent that the increase in glucose in the carboxyl-reduced derivative cor- responds to the amount of glucuronic acid in the native polysaccharide. The content of d- glucuronic acid coincided with the result of colorimetric determination (uronic acid, 1 3%). The acetyl content was determined to be 5.4%, but pyruvic acid was not detected in the poly- saccharide. Therefore, polysaccharide AM-2 was composed of D-glucose, L-rhamnose, d- mannose, D-glucuronic acid, and O-acetyl in themolarratioof4:1:1:1:1.
Methylation analysis and Smith degradation of Fig. 1. Sedimentation Diagram of Purified Polysac- polysaccharide charide AM-2.The results of methylation analysis of the Measurementwas done at a concentration of2.5 mg/mlof native polysaccharide AM-2 and its carboxyl- polysaccharide AM-2 in 0.1 m NaCl. The photograph was reduced derivative are shown in Table III. The taken at 54min after attaining top speed (40,000rpm) at increase in the relative amount of 2,3,6-tri-<9- 20°C. Direction of sedimentation was left to right. methyl-D-glucose in the carboxyl-reduced de- rivative compared with that in the native Table II. Physicochemical Characteristics of Purified Polysaccharide AM-2 polysaccharide should correspond to the pres- ence of (l ->4)-linked D-glucuronic acid. The Molecular weight 2.1 x lO6 polysaccharide had a branched structure with Sedimentation coefficient 5.35 (0.25% solution) teo,w) a repeating unit in an average of seven sugar O p tical rotation + 18- (c = 0.2, w ater) residues and had L-rhamnose as terminal res- V iscosity 1200cps (1% solution) idues and D-glucose residues as branch points. Elementary analysis 39.91% There were two (l-^6)-linked D-glucose res- 6.27% N O% idues, one (l ->2)-linked D-mannose residue, Ash 3.05 -/ one (l ->4)-linked D-glucose residue, and one (1 -^-linked D-glucuronic acid residue. The
Table III. Molar Ratios of Methylated Sugar Fragments from Hydrolyzates of Methylated Native Polysaccharide AM-2 and Its Carboxyl-reduced Derivative
M o la r ra tio O -M eth y la te d su g a r M o d e o f (a s a ld ito l a ce ta te ) lin k a g e N a tiv e O R e d u c ed O
2 ,3 , 4- T ri - O- m et h yl -L - r ha mn o se R h a -(1 - 0 . 8 2 0 . 7 9 3 , 4, 6 -T r i -O - me t h yl - D- m an n o se 蠎2 )-M a n -(l - 1. 0 1. 0 2, 3, 4 -T ri -O -m et hy l- D- gl uc os e -6 )-G lc -( l - 1. 9 1. 9 2, 3, 6 -T ri -O -m et hy l- D- gl uc os e 4 )-G lc -( l - 1. 1 2 .1 2 ,6 -D i -O - me th y l- D -g lu c os e 蠎4 )-G lc -( l - 1. 2 1. 1 3 T
Native polysaccharide AM-2and its carboxyl-reduced derivative are abbreviated as Native and Reduced. Structure of Polysaccharide from Acetobacter sp. 963 paper chromatographic analysis indicated the and 2,3,6-tri-(9-methyl-D-glucose in a molar presence of glycerol, erythritol, and glucose as ratio of 1.0: 1.0. The oligosaccharide had the degradation products by complete Smith deg- same retention time as authentic cellobiose on radation and of glycerol and a small amount a 3% SE-30 column after trimethylsilylation.13) of glucosyl erythritol as degradation products Additonally, the oligosaccharide had the same by mild Smith degradation. The molar ratio of mobility (7?/ 0.35) as authentic cellobiose on glycerol, erythritol, and glucose was 2.5: thin-layer chromatography,n) so neutral oli- 1.0 : 1.1, respectively, in complete Smith degra- gosaccharide II was identified as cellobiose. dation. This value agreed well with the val- Acidic oligosaccharide I. The purified oligo- ue expected from the methylation analysis saccharide (15mg) showing RGlc 0.22 on pa- mentioned above. respondingper chromatogramto D-glucuronicgave two spotsacid(1 : and1) cor-d- Fragmentation ofpolysaccharide by partial acid mannose on acid hydrolysis. The GLCanal- hydro lys is ysis of hydrolyzates of the methylated acidic The purified polysaccharide AM-2(9 g) was oligosaccharide showed the presence of 3,4,6- partially hydrolyzed for investigation of de- tri-O-methyl-D-mannose, so acidic oligosac- tailed structure. The acid hydrolyzates were charide I was identified as D-glucuronosyl- purified to a homogeneous state by ion- ( l ->2)-D-mannose. exchange- and charcoal-column chromatog- raphies, followed by preparative paper chro- Analysis ofpolysaccharide residues obtained by matography, as described above. partial acid hydrolysis Neutral oligosaccharide I. The purified oli- Precipitates I and II obtained by partial acid gosaccharide (432mg) eluted from the char- hydrolysis of the polysaccharide AM-2were coal column with 7%aqueous ethanol showed composed of D-glucose, D-mannose, d- RGlc 0.58 on a paper chromatogram with the glucuronic acid, and L-rhamnose in molar solvent system (v/v) of «-butanol-pyridine- ratios of3.7:1.0:1.0:0.2 and 2.6:1.0:1.0:0, water (6:4:3) and [
Table IV. Molar Ratios of Methylated Sugar Fragments from Hydrolyzates of Methylated Native Precipitates I and II and Methylated Carboxyl-reduced Precipitates I and II Obtained FROM POLYSACCHARIDE AM-2
M o lar ratio
O -M ethylated sugar M od e of Nat ive pp tfl R ed uce d p pta (as ald itol acetate) link age
I II I II
2, 3,4-Tri-O-methyl-L-rhamnose R h a-(1 - 0. 3 1 0 . 2 8 2,3,4,6-Tetra-O-methyl-D-glucose G lc-(1 - 0.6 3 0 .2 2 0 .6 2 1 .1 3,4,6-Tri-O-methyl-D-mannose 蠎2)-M an -(l - 1.0 1.0 1.0 1.0 2,3,4-Tri-O-methyl-D-glucose �6)-G lc-(l - 0. 9 1 0 . 9 3 2, 3,6-Tri-O-methyl-D-glucose >4)-G lc-(l - 1 . 1 1 . 2 2 .0 1 . 2 2,6-Di-O-methyl-D-glucose 4 )-G lc-(l - 1.2 1 2 1.1 1.2 3 T
Native precipitate and carboxyl-reduced precipitate are abbreviated as Native ppt and Reduced ppt. changed. the ubiquinone type of A. aceti subsp. or- D-Glucose, D-glucuronosyl-(l ->2)-D-man- leanensis was Q96) and that of the isolated nose, and cellobiose were obtained by fur- strain was Q10. Yamadaet al.20'21) reported ther partial acid hydrolysis (0.1 mtrifluoro- that Acetobacter xylinus (sic) (A. aceti subsp. acetic acid, 100°C, 3hr) of precipitate II, but xylinum) and the peritrichously flagellated in- laminaribiose and nigerose were not obtained. termediate strains {A. aceti subsp. liquefaciens) Moreover, the action of /M,4-D-glucanase on are exceptional in having Q10 among the carboxyl-reduced precipitate II resulted in Acetobacter strains. The isolated strain was the release of an oligosaccharide having RGlc apparently defferent from A. aceti subsp. liq- 0.20 on thin-layer chromatogram. uefaciens because the isolated strain did not produce y-pyrones, brown pigment, or 2,5- DISCUSSION diketogluconate but did produce a large amount of polysaccharide AM-2.From these The isolated strain was a gram-negative, observations, we judged, according to Bergey's obligately aerobic rod that grew at pH 3.5 and Manual 8th Ed. and other papers,20'21] that the oxidized ethanol to acetic acid. Additionally, it isolated strain maybe A. aceti subsp. xylinum, oxidized lactate and acetate to carbon dioxide although it produced a large amount of poly- and water. Therefore, the isolated strain was saccharide AM-2. Recently, Gossele et al.22) classified into the genus 'Acetobacter according have proposed a new species, Acetobacter han- to Bergey's Manual 8th Ed.18) and a recent senii, which includes some strains of A. aceti report of Gossele et all9) The isolated strain subsp. xylinum, A. aceti subsp. orleanensis and was positive for catalase, ketogenesis in glyc- A. pasteurianus subsp. pasteurianus, and erol, and formation of gluconate, 2-ketoglu- Acetobacter pasteurianus emend, which in- conate, and 5-ketogluconate from glucose, but cludes other strains ofA. aceti subsp. xylinum, negative for growth on ethanol and produc- A. aceti subsp. orleanensis, A. pasteurianus tion of cellulose, brown pigment, and y-py- subsp. pasteurianus and so on. Yamada23)have rones (FeCl3 reaction in glucose broth was postulated a revived name, Acetobacter xylinus positive). According to Bergey's Manual 8th (sic), which includes Q10-having A. aceti subsp. Ed., the isolated strain seemed to be xylinum strains. Concerning the systematic Acetobacter aceti subsp. orleanensis, however, position of our strain, more detailed studies Structure of Polysaccharide from Acetobacter sp. 965
are necessary. åº4)-/8-D-Glc-(l-M)-/0-D-Glc-(l- Valuable evidence was obtained not only by 3 analysis of oligosaccharides but also by anal- t 1 ysis of polysaccharide residues after partial D-Man 2 acid hydrolysis of polysaccharide AM-2. We t found the following indications, comparing 1 D-GlcA the results of methylation analysis of the poly- 4 saccharide and precipitates I and II (Table III t 1 and IV): 1) by the single partial acid hydrolysis D-Glc of the pofysaccharide, D-glucose residues ap- 6 /3 t +OAc peared as newterminal residues corresponding 1 D-Glc to the release of L-rhamnosyl terminal residues 6 and the decrease in (l->6)-linked D-glucose t residues; 2) by the double partial acid hy- 1 drolysis of the polysaccharide, D-glucuronic L-Rha acid residues appeared as new terminal res- Fig. 2. Possible Structure of a Repeating Unit ofPoly- idues corresponding to the release of both l- saccharide AM-2. rhamnosyl and D-glucosyl terminal residues Glucose, rhamnose, mannose, glucuronic acid, and O- and the decreases in (l->6)-linked D-glucose acetyl are indicated as Glc, Rha, Man, GlcA, and OAc, and (l -»4)-linked D-glucuronic acid residues. respectively. Also, it was shown that further partial acid hydrolysis of precipitate II yielded cello- peating heptasaccharide unit as shown in Fig. 2. biose and D-glucuronosyl-(l ->2)-D-mannose. Polysaccharide AM-2is similar to an extra- Precipitate II is composed of D-glucose, d- cellular polysaccharide, which is composed of mannose, and D-glucuronic acid in a molar glucose (3mol), rhamnose (l mol), mannose ratio of 2: 1 : 1 and has glucuronosyl residues (1mol) and glucuronic acid (lmol), from as non-reducing terminals and glucosyl resi- Acetobacter xylinum reported by Valla and dues as branch points, indicating that the Kjosbakken.5) However, there are some differ- backbone chain of polysaccharide AM-2 is ences in components and molar ratio of sugar D-glucan and that a side chain, shown as l- components between the two polysaccharides. rhamnosyl-(l ->6)-/?-D-glucosyl-(l ->6)-d- Moreover, an important difference is that the glucosyl-(l ->4)-D-glucuronosyl-(l ->2)-d- polysaccharide AM-2 contains cellobiose mannose, is linked at alternate D-glucose units as structural parts which is not present residues of the backbone. The products ob- in their polysaccharide. tained by partial acid hydrolysis of precipi- It is interesting that Acetobacter xylinum tate II were D-glucose, D-glucuronosyl-(l ->2)- strains produce several polysaccharides4~7) in D-mannose, and cellobiose, but laminari- addition to cellulose. Couso et al.24) succeeded biose and nigerose were not detected. Addi- in the sequential in vitro synthesis of a he- tionally, the action of cellulase on the car- terooligosaccharide diphosphate prenol using boxyl-reduced precipitate II resulted in the an EDTA-treated Acetobacter xylinum strain. release of an oligosaccharide. Therefore, it It is expected that the detailed structural fea- seems that polysaccharide AM-2is formed of tures of polysaccharides AM-1and AM-2will a linear backbone chain of (l -»4)-linked /?-d- yield newdevelopments in the studies on bio- glucosyl units to which side chains are linked synthesis of several extracellular heteropoly- at 0-3 positions of D-glucose residues of saccharides in Acetobacter xylinum. the backbone. Although the position of O- acetyl substituents has not been establish- Acknowledgment. We thank Dr. A. Misaki, Osaka ed, polysaccharide AM-2possibly has a re- City University, for his valuable advice in the structural 966 K. Tayama et al. analysis of polysaccharides. S. A. Hansen, /. Chromatogr., 107, 224 (1975). N. Shibuya, J. Chromatogr., 208, 96 (1981). REFERENCES T. Bhatti, R. E. Chambers and J. R. Clamp, Biochim. Biophys. Acta, 222, 339 (1970). 1) S. Hestrin, M. Aschner and J. Mager, Nature J. T. Galambos, Anal. Biochem., 19, 119 (1967). 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