Agric. Biol. Chem., 50 (5), 1271 ~1278, 1986 1271

Structure of an Acidic Elaborated by Acetobacter sp. NBI 10051"

Kenji Tayama, Hiroyuki Minakami, Seiichi Fujiyama, Hiroshi Masai and Akira Misaki* NakanoBiochemical Research Institute, NakanoVinegar Co., Ltd., Handa, Aichi 475, Japan * Faculty of Science of Living, Osaka City University, Sugimoto-cho, Sumiyoshi, Osaka 558, Japan Received November 19, 1985

An extracellular acidic polysaccharide elaborated by Acetobacter sp. NBI1005 was composed of D-, D-, D-, and D-glucuronic acid (approximate molar ratio, 6 : 2 : 1 : 1). Methylation and fragmentation analysis by partial acid hydrolysis indicated that the polysaccharide has a branched structure containing a backbone chain of /?-(l ->4)-linked D-glucose residues, two out of every four glucose residues being substituted at the 0-3 positions to form two kinds of branches, one consisting of D-mannose and D-glucuronic acid residues and the other of (l ->6)-linked D-galactose and D-glucose residues.

Some microorganisms belonging to Ace- cosyl-(l ->4)-D-glucuronosyl-(l ->2)-D-man- tobacter species have been knownto produce nose.9) This paper reports the structural fea- extracellular , such as cellu- ture of polysaccharide AM-1, as revealed by lose,1* ,2) levan,3) and an acidic poly- methylation, fragmentation analysis, and en- saccharide,4* and also soluble, /?-(l-»2)- zymatic degradation. branched, £-(1 ^4)-D-glucan5) and )8-(l -»2)- D-.6) In the course of study on acetic MATERIALS AND METHODS acid bacteria (genera Acetobacter and Glu- conobacter) having a high productivity of Materials. Polysaccharide AM-1of Acetobacter sp. NBI 1005, used in this study, was prepared and purified by the acetic acid from ethanol,7) we found that some methods reported in our previous paper.8) A /?-1,4-d- strains of Acetobacter sp. are capable of pro- glucanase from Aspergillus niger was purchased from the ducing new types of water-soluble acidic Sigma Chem. Co. heteropolysaccharides, tentatively designated polysaccharide AM-1 and AM-2. Polysac- General methods. All evaporations were conducted be- charide AM-1 is composed of D-glucose, d- low 40°C. Paper chromatography was performed on Toyoroshi No. 51A paper by the descending method using galactose, D-mannose, and D-glucuronic acid, the solvent system (v/v): 1-butanol-pyridine-water (6; while polysaccharide AM-2is composed of d- glucose, L-, D-mannose, and D-glu- 4 : 3). Preparative paper chromatography was done with Whatman3MMpaper. Reducing sugars on paper chro- curonic acid.8'9) matograms were detected with alkaline silver nitrate Our previous study showed that polysac- reagent. Thin-layer chromatography was carried out with charide AM-2 has a branched structure con- the solvent 1-propanol-nitromethane-water (10 : 2 : 3) and sugars were detected by spraying with 50%sulfuric acid in taining a backbone chain of /?-(l -»4)-linked d- ethanol. The polysaccharide was hydrolyzed with 2m glucose residues and a side chain shownas l- trifluoroacetic acid for 18 hr at 1OO.°C. For hydrolysis of rhamnosyl-(l ->6)-D-glucosyl-(l ->6)-D-glu- with the acid, samples were heated at

Extracellular Polysaccharides Produced by Acetic Acid Bacteria. Part III. 1272 K. Tayama et ah

100°C for 4hr. After the acid was distilled off in vacuo, the perature and then with methyl iodide (1.5ml) for 1.5hr at hydrolysate was analyzed by paper chromatography or 20°C. The mixture was dialyzed against water and dried in gas-liquid chromatography (GLC). GLCwas conducted vacuo. The methylation was repeated twice, and the fully with a Hitachi gas chromatograph model 124 equipped methylated polysaccharide was hydrolyzed with 90% with a flame-ionization detector. Neutral sugars and their formic acid (1 ml) for 16hr at 100°C, followed by heating methyl derivatives were converted into their correspond- with 2m trifluoroacetic acid (1 ml) for 5hr at 100°C. The ing alditol acetates, and were analyzed using a column hydrolyzed products of the methylated polysaccharide packed with 3% ECNSS-M Gaschrom Q (0.4 x 200cm) were reduced with sodium borohydride and the resulted at 190°C, and 180°C, respectively. The alditol acetates of alditols were acetylated by heating with pyridine-acetic methylated sugars were also analyzed' on a capillary anhydride (1 : 1) for 2hr at 100°C for analysis by GLC. glass column coated with Silar-10C1O) (G-SCOT, Oligosaccharides (3 mg each) were methylated by the same 0.28mmx30m) at 190°C. 13C-NMRspectra were ob- procedure. The methylated was extracted tained using samples in D2Oin 10mmdiameter tubes at with dichloromethane after evaporation of methyl iodide, 90°C with a JEOL FX-100 spectrometer operating at and the extract was washed with distilled water and then 25MHz. Optical rotation was measured with a Union dried on MgSO4.The extract was applied on a Sephadex automatic digital polarimeter, model PM-101. The LH-20 column (1 x 60cm) which was throughly washed enzymatic hydrolysis of the high molecular weight frac- with dichloromethane. Fractions containing the methyl- tion (5 mg), obtained by a mild acid hydrolysis of poly- ated oligosaccharide were collected and evaporated to saccharide AM-1, was carried out by incubation with dryness. The methylated oligosaccharide was hydrolyzed, /M,4-D-glucanase (1 mg) in 0.05m acetate buffer (2.5 ml) reduced, and acetylated by the procedure described above, at pH 5.0 and 37°C for 24hr. The incubation mixture and analyzed by GLC. was heated in a boiling water bath and desalted with ion-exchange resin (Amberlite IR-120), and the Periodate oxidation and Smith degradation of polysac- products were examinedby paper chromatography. charide. The polysaccharide (100mg) was oxidized with 0.05m sodium metaperiodate (100ml) at 4°C in the dark. Carboxyl-reduction of acidic polysaccharide. Carboxyl After complete oxidation (6 days), the oxidation was groups of the acidic polysaccharide were reduced by the stopped by the addition of ethylene glycol (10ml). The method of Taylor and Conrad.11] Anaqueous solution mixture was dialyzed and converted into the correspond- of the native polysaccharide (50mg in 100ml of water) ing polysaccharide-polyalcohol by the addition of sodi- was adjusted to pH 4.7 with 0.1m hydrochloric acid, um borohydride. A portion of the polysaccharide-poly- followed by the addition of l-ethyl-3-(3-dimethylamino- alcohol was hydrolyzed with 1 m sulfuric acid for 6hr at propyl) carbodiimide (500mg), and the stirred reaction 100°C (complete Smith degradation), and another por- mixture was maintained at pH 4.7 for 3 hr by the addition tion was hydrolyzed with 0.05m sulfuric acid for 20hr of 0.02m hydrochloric acid. After addition of sodium at 25°C (mild Smith degradation). borohydride (500mg) and one drop of «-octanol, the mixture was stirred overnight and then dialyzed exhaus- Partial acid hydrolysis ofpolysaccharide. The polysac- tively against water. The reduced polysaccharide (45 mg) charide (25g) was heated with 0.25 m trifluoroacetic acid was obtained by lyophilization. (2.5 liters) for 3 hr at 100°C. After evaporation of the acid, the hydrolysate was dissolved in water (800 ml), and etha- Methylation analysis. Methylation of polysaccharide nol (2.4 liters) was added to the solution, and then the and oligosaccharide was performed by the method of resulting precipitate was collected by centrifugation to give Hakomori.12) Polysaccharide (20mg) was dissolved in a degraded polysaccharide. The supernatant was evapo- dimethyl sulfoxide (2ml) and stirred for 20min at 50°C at rated to a small volume, passed through a column of nitrogen atmosphere. The solution was treated with 2m Dowex 1 x4 (5 x20cm, formate form), and washed with methylsulfinyl carbanion (0.5ml) for 3hr at room tern- water to give a mixture of neutral oligosaccharides. Acidic

Table I. Yields of Neutral Oligosaccharide Fractions from Partial Acid Hydrolysate F raction E lu en t W eight (m g) C o m p onents

1 Water (3000 ml) 15258 Glucose, galactose 2 4% Ethanol (1500ml) 1898 D isacch aride 3 7% Ethanol (1000ml) 530 D i- a nd tri-sacch aride 4 12% Ethanol (1100ml) 1099 Tetra- and pentasaccharide 5 25 % E tha noH 850 ml) 987 H igher oligo saccharid es 6 War m 50% ethanol ( 800ml) 1090 Unkn own brown materials Structure of Polysaccharide from Acetobacter sp. 1273

Table II. Molar Ratio of Methylated Sugar Fragments from Hydrolyzates of Methylated Native and Carboxyl-reduced Polysaccharide AM- 1 M o la r ra tio O -M e th y la ted su g a r M o d e o f (a ld ito l ac eta te) lin k a g e N a tive C ar b o x y l-re d u c e d

2, 3 ,4 ,6- Te tra -O -m eth yl -D- gl uc ose G lc -( 1 - 1. 0 1. 8 3 , 4, 6 - Tr i -O - m et h yl - D- m a nn o se 蠎2 )-M a n -( l - 1. 2 1. 1 2 ,3 ,4 -T ri -O -m et hy l- D- g lu co se 蠎6 )-G lc -( l - 2 .0 2,3, 6-T ri-O -m e th y l-D -g lu c o se 4 )-G lc -(l - 2 . 0 2 . 2 2, 3, 4- Tri -O- me thy l-D -g ala cto se 蠎6 )-G a l-( l - 2 . 0 2 . 0 2 ,6 -D i -O - me th y l- D -g l uc os e 蠎4 )-G lc-(l - 2 . 0 2 . 0 3 T

oligosaccharides adsorbed on the column were eluted with methylated, and the partially methylated 1 m formic acid. The neutral oligosaccharides were frac- sugars in the acid hydrolysate were analyzed tionated by a column (5 x 25cm) of charcoal (Activated by GLC. The results are shown in Table II. Charcoal, WakoPure Chemical Ind.). After washing the columnwith water, oligosaccharideswerestepwiseeluted Identities and proportions of the cleavage with 4%, 7%, 12%, and 25%aqueous ethanol, and finally fragments of the native and carboxyl-reduced with warm 50% ethanol. The yields of oligosaccharides polysaccharides indicate that polysaccharide from the partial acid hydrolysate of the polysaccharide are AM-1 has a highly branched structure with a shown in Table I. Each fraction was evaporated to dryness and further purified by preparative paper chromatography repeating unit of eleven sugar residues. It may using Whatman 3MMpaper. The precipitate (degraded contain a backbone chain of /?-(l ->4)-linked polysaccharide) obtained by addition of ethanol to the D-glucose residues, like polysaccharide AM- mild acid hydrolysate was dissolved in water, dialyzed 2,9) and two out of the four D-glucose residues exhaustively, freeze-dried, and subjected to structural are branched at the 0-3 positions. There are analysis. two kinds of side chains, one is terminated RESULTS with D-glucose residues and the other is with d- glucuronic acid residues, as indicated by the Polysaccharide AM-1, isolated from the increase in approximately one mole of the culture filtrate of Acetobacter sp. NBI 1005, tetra-O-methyl-D-glucose in the methylated, was shown to be homogeneous by ultracen- carboxyl-reduced polysaccharide. In addition, trifugal analysis.8) This polysaccharide was the polysaccharide contains (1 -^-linked d- composed of D-glucose, D-galactose,. D-man- glucose, (l ->6)-linkded D-galactose, and (l -> nose, and D-glucuronic acid in the molar ratio 2)-linked D-mannoseresidues. They are most of 6.3:2.1 : 1.0: 1.2, as reported in our previ- probably located in the side chains, as revealed ous paper.8) It contained neither deoxy sugars by the fragmentation analysis (partial acid nor acetyl groups, as revealed by the 13C- hydrolysis). NMR spectroscopy. Its optical rotation [a]D The results of methylation analysis were + 12° (c=0.2, water) and the chemical shift of supported by Smith degradation. When the the anomeric carbon at 103.67ppm in 13C- polysaccharide-polyalcohol derived by per- NMR spectrum indicated that the dominant iodate oxidation and subsequent borohydride D-glycosidic linkages must be /^-configuration. reduction was completely hydrolyzed with acid (complete Smith degradation), glycerol, Methylation analysis and Smith degradation erythritol, and glucose were yielded in the In order to obtain an information on the molar ratio of 3.1:1.1:1.0. When the poly- mode of glycosidic linkages, both the native saccharide-polyalcohol was subjected to a and carboxyl-reduced polysaccharide were mild acid hydrolysis with diluted acid for 20 hr 1274 K. Tayama et al. at 25°C (mild Smith degradation), a small gave only D-glucose. This oligosaccharide amount of glucosyl erythritol was obtained in gave, on methylation and subsequent acid addition to glycerol. The former should have hydrolysis, 2,3,4,6-tetra- and 2,3,4-tri-O- arisen from such a sequence: -^4 or 6)-Glc- methyl-D-glucose in the molar ratio of 1.0 : 1.9. (1 ->4)-Glc-(l ->4)-Glc-(l ->. From these results and the susceptivility to- t ward jS-D-glucosidase, the oligosaccharide B 3 is characterized as gentiotriose. Fragmentation ofpolysaccharide by partial acid hydro lys is Te trasaccharide In order to know the sequential arrange- Oligosaccharide C (20mg) had RGlc 0.19 ment of the sugar residues, the polysaccharide and [a]D -7° (c=0.1, water). Acid hydrolysis (25 g) was subjected to partial acid hydrolysis. gave D-glucose and D-galactose (1.0 : 3.3), in- After the acid was distilled off, the high- dicating that this oligosaccharide is a tetra- molecular-weight fraction (degraded poly- saccharide. Whenthe was saccharide) was separated by precipitation methylated and hydrolyzed with acid, 2,3,4,6- with ethanol (3 volumes). The oligosaccharides tetra-O-methyl-D-glucose, 2,3,4-tri-(9-methyl- in the supernatant solution were fractionated D-glucose, 2,3,5-tri-(9-methyl-D-galactose and by a column ofDowex1 x 4 and charcoal, and 2,3,4-tri-(9-methyl-D-galactose were yielded in were respectively isolated by preparative paper the molar ratio of 1.0:2.1 :0.67:0.34. From chromatography. These procedures afforded its low optical rotation, most sugar residues four kinds of neutral oligosaccharides (A -D) in this oligosaccharide may have ^-configu- and an aldobiouronic acid. They were char- rations. On partial acid hydrolysis, it gave acterized mainly by methylation analysis as gentiobiose and gentiotriose in addition to shown below. galactose. Thus, the tetrasaccharide was ten- tatively characterized as (9-/?-D-grucosyl-(l -> D isaccharide 6)-0-/?-D-glucosyl-(l ->6)-O-jS-D-glucosyl- Oligosaccharide A (358 mg), which had RGlc (l->6)-D-galactose, although a part of the 0.48 (1-butanol-pyridine-water, 6 : 4 : 3) and D-galactose residue at the reducing end ap- [a]D + 10° (c=0.1, water), was composed ofd- peared to have a form. glucose and D-galactose in the molar ratio of 1.0: 1.1. Acid hydro-lysis of the methylated Higher oligosaccharide yielded 2,3,4,6-tetra-O-methyl-D- Oligosaccharide D (38mg) had RGlc 0.12 glucose, 2,3,5-tri-O-methyl-D-galactose, and and [a]D -13° (c=0.1, water). It was com- 2,3,4-tri-O-methyl-D-galactose in the molar posed of D-glucose and D-galactose (1.6 : 1.0), ratio of 1.0 : 0.66 : 0.33. When treated with j8-d- and acid hydrolysis of the methylated deriva- glucosidase, this oligosaccharide released d- tive yielded 2,3,4,6-tetra- and 2,3,4-tri-O- glucose, indicating that the glucose residue at methyl-D-glucose, 2,3,5-tri- and 2,3,4-tri- the non-reducing terminal is in the j8-type. The O-methyl-D-galactose, in the molar ratio of quantitation of these methylated sugar frag- 1.0:2.3:0.3:2.0. These results indicate that ments indicates that oligosaccharide A is 0-/?- the oligosaccharide is probably a pentasac- D-glucosyl-(l->6)-D-galactose. It should be charide, consisting of two (l->6)-linked d- noted that D-galactose residues are present glucose residues and two (l-»6)-linked d- both in the furanose (two-third) and the galactose residues, and one non-reducing (one-third) forms. terminal of D-glucose residue. Tr isaccharide Aldobiouronic acid Oligosaccharide B (120mg) had RGlc 0.54 An aldobiouronic acid (76 mg), adsorbed on and [a]D +8° (c=0.1, water). Acid hydrolysis a Dowex 1 x4 (formate form) column and Structure of Polysaccharide from Acetobacter sp. 1275 eluted with 1 m formic acid, was purified by 3,4,6-tri-(9-methyl-D-mannose, 2,3,6-tri-O- paper chromatography. It had RGlc 0.21 and methyl-D-glucose, and 2,6-di-O-methyl-D- Md ~34° (c=0.1, water). The acid hydroly- glucose in the molar ratio of0.1 : 1.0:2.6: 1.1. sate gave two spots on the paper chromato- In another experiment, the degraded poly- gram, corresponding to D-glucuronic acid and saccharide was treated with carbodiimide, D-mannose, respectively. GLCanalysis of the followed by sodium borohydride reduction, hydrolysate of the methylated aldobiouronic and then methylated. On acid hydrolysis, it acid gave 3,4,6-tri-O-methyl-D-mannose, gave 2,3,4,6-tetra-O-methyl-D-glucose, 3,4,6- as the neutral sugar. Thus, the aldobiouronic tri-O-methyl-D-mannose, 2,3,6-tri-O-methyl- acid was identified as (9-/?-D-glucuronosyl- D-glucose, and 2,6-di-O-methyl-D-glucose in (l ->2)-D-mannose. the molar ratio of 1.1: 1.0:2.6: 1.1. When the degraded polysaccharide was subjected to Analysis of acid-degraded polysaccharide further mild acid hydrolysis (0.25 M trifluoro- The degraded polysaccharide obtained by »4)-/9-D-Glc-(1-»4)-/?-D-Glc-(1- ethanol precipitation from the mild acid hy- t drolysate was purified by dialysis, followed by 1 ethanol precipitation. This degraded poly- D-Man 2 saccharide, composed of D-glucose, D-man- t 1 nose, and D-glucuronic acid in the molar ratio D-GlcA of 3.5: 1.0: 1.0, was methylated. The methyl- Fig. 1. Structure of Repeating Unit of Acid-degraded ated, degraded polysaccharide gave on acid Polysaccharide, Obtained by Mild Aid Hydrolysis of hydrolysis 2,3,4,6-tetra-O-methyl-D-glucose, Polysaccharide AM-2.9)

100 70 40 i~00 70" 40 P PTT1 P PTT1 Fig. 2. 13C-NMR Spectra of Acid Degraded Polysaccharides of AM-1 (A) and AM-2 (B). 1276 K. Tayama et al.

acetic acid at 100°C for 3 hr), an aldobiouronic DISCUSSION acid, identified as O-/^D-glucuronosyl-(l ->2)- D-mannose, and were released, but Some of acetic acid bacteria (genera Ace- laminaribiose and nigerose were not detected. tobacter and Gluconobacter) elaborate cellu- These results suggest that the degraded poly- lose,1* dextran,2) levan,3) /Kl->2)-branched saccharide from AM-1resembles the acid- £-(l -+4)-D-glucan5) and £-(l ->2)-D-glucan.6) degraded polysaccharide from AM-2, whose In addition, extracellular productions of repeating unit is shown in Fig. 1.9) To con- other types of polysaccharides were report- firm this, 13C-NMR spectra of the present ed, an -like polysaccharide by A. degraded polysaccharide and that from poly- pasteurianus,13) a -like polysaccharide saccharide AM-1were compared. As shown in by A. acidum-mucosum1^ (A. pasteurianus),15) Fig. 2, the two spectra are very similar, in- and polysaccharide-like substances by Bacte- cluding the chemical shifts of C-l (105.25 and rium aceti viscosum16) (G. oxydans subsp. 102.79ppm), substituted C-4 (81.14ppm), industrius),15) and by A. capsulation11} (G. C-2, C-3, C-5 (74.30, 75.29 and 78.04ppm), oxydans subsp. industrius) 15) although their and C-6 (62.95ppm). chemical natures have not yet been elucidated. The above-mentioned structural data in- As reported in our previous paper,8) the dicate that polysaccharide AM-1has a back- extracellular polysaccharides produced by two bone chain of /?-(l ->4)-linked D-glucose res- strains of acetic acid bacteria belonging to A. idue, and that two kinds of side chains, one pasteurianus and G. oxydans subsp. industrius consisting of (l ->6)-linked D-glucose and d- are essentially same polysaccharide as poly- galactose residues and the other consisting of saccharide AM-1in terms of their sugar com- the jS-D-glucuronosyl-(l ->2)-D-mannose unit, positions. In order to know the chemical na- are attached at the 0-3 positions of the /?- ture of these similar polysaccharides, com- (l ->4)-linked D-glucose residues of the back- posed of D-glucose, D-galactose, D-mannose, bone chain, respectively. It appeared that side and D-glucuronic acid (approximate molar chains of the neutral oligosaccharide units ratio, 6:2: 1:1), the structural analysis of were preferentially split during the mild acid polysaccharide AM-1was necessitated. hydrolysis to give the degraded polysaccharide Polysaccharide AM-1, reported here, was having branches of the aldobiouronic acid homogeneous in the ultracentrifugal analysis, unit, whosestructure is very similar to that and had a mol. wt. 1 x 106 daltons. The results from polysaccharide AM-2 (see, Fig. 1). It of methylation analyses of the native and is interesting that D-galactose residues locat- carboxy-reduced polysaccharide indicated that ed in the side chains maybe present in both polysaccharide AM-1, like polysaccharide pyranose and furanose forms, as indicated by AM-2, contains the backbone of /?- methylation analyses of the oligosaccharides (l ->4)-linked D-glucose residues. However, released by the mild acid hydrolysis of the polysaccharide AM-1 contains two different polysaccharide. As regards the arrangement of types of side chains attached at the 0-3 posi- sugar residues in the neutral side chains, the tions of the /?-(l -»4)-linked D-glucose residues, most probable sequence may be /?-D-glucosyl- one terminated with j8-D-glucose residues and (1 ->6)-/?-D-glucosyl-(l ->6)-/?-D-glucosyl- the other with /?-D-glucuronic acid residues, (l ->6)-D-galactosyl-(l -->6)-D-galactose, as re- respectively. Informations on the precise vealed by methylation analysis, Smith deg- structural feature of the polysaccharide were radation, and isolation of the acidic and provided by the data of fragmentation analy- neutral oligosaccharides. sis. Whenpolysaccharide AM-1was partially hydrolyzed with dilute acid, several neutral and acidic oligosaccharides were released. They were purified and characterized: O-jS-d- Structure of Polysaccharide from Acetobacter sp. 1277

/9-D-Glc-(1-*6)-D-Gal

A /3-D-G1c-(1-»6)-/3-D-Glc-(l->6)-D-Glc B /S-D-Glc-(1->6)-/S-D-Glc-(1->6)-/9-D-Glc-(1-»6)-D-Gal /3-D-G1cA-(1-»2)-D-Man

C E

>4)_/3_D-Glc-(1-»4)-/?-D-Glc-(1-*4)-/^-D-Glc-(1->4)-/?-D-Glc-(1-

t 1 D-Man 2 t 1 ^-D-GlcA- F Fig. 3. Structures of the Oligosaccharides. The oligosaccharide A, B, and C in the text are shown as A, B, and C in this figure, respectively. Aldobiouronic acid and a high molecular weight fraction left after mild acid hydrolysis of the polysaccharide were indicated as E and F, respectively.

>4)-/?-D-Glc-(l->4)-/?-D-Glc-(1-4)-/?-D-G1c-(1->4)-/?-D-G1c-(1- 3 3

t1 t1 D-Gal D-Man 6 2 t1 t1 D-Gal /3-D-GlcA 6 t 1 /3-D-Glc 6 t 1 /tf-D-Glc 6 t ] /9-D-Glc Fig. 4. Proposed Structure of the Repeating Unit of Polysaccharide AM-1. glucosyl-(l ->6)-D-glucose (Fig. 3 (A)), <9-/?-d- AM-1represents a core portion of the poly- glucosyl-(l ^6)-O-j8-D-glucosyl-(l ->6)-D-glu- saccharide, although it still contained the acid- cose (Fig. 3 (B)), <9-jtf-D-glucuronosyl-(l ->2)- resistant glucuronosyl-mannosyl side chains, D-mannose (Fig. 3 (E)), and O-jS-D-glucosyl- neutral oligosaccharides released by mild acid (1 ->6)-O-j3-D-glucosyl-(l -+6)-O-jS-D-glucosyl- hydrolysis might have arisen from neutral side (1 ^6)-D-galactose (Fig. 3 (Q). chains having the sequence: /?-d-G1c-(1 ->6)-/?- The degraded polysaccharide, which was d-G1c-(1 -+6)-j8-d-G1c-(1 -^-D-Gal-O ->6)- relatively acid-resistant and left after the mild D-Gal. Thus, a possible structure of the re- acid hydrolysis, was shown by methylation peating unit of polysaccharide AM-1can be analysis to have an essentially samestructure depicted, as shown in Fig. 4. The most sugar to that obtained from polysaccharide AM-2 residues building up the polysaccharide may (Fig. 1). This was confirmed by 13C-NMR have ^-configurations, as indicated by the analysis, which showed that both the degraded chemical shift of C-l (103.67ppm) in its 13C- polysaccharides from AM-1and AM-2gave NMRspectrum, a low optical rotation, and almost identical 13C-NMR spectra. Assum- also the isolation of jS-(l-^6)-linked gluco- ing that the degraded polysaccharide from oligosaccharides. Thus, the structural feature 1278 K. Tayama et al

åº4)-/3-D-Glc-Cl->4)-/3-D-Glc-Cl- REFERENCES 3 t 1 1) S. Hestrin, M. Aschner and J. Mager, Nature D-Man 2 (London), 159, 64 (1947). t 2) E. J. HehreandD. M. Hamilton, J. Biol. Chem., 192, 1 161 (1951). D-GlcA 4 3) M. S. Loitsyanskaya, Tr. Petergof. Biol. Inst. T Leningrad. Gos. Univ., 19, 20 (1965). i D-Glc +OAc 4) S. Valla and J. Kjosbakken, Can. J. Microbiol., 27, 6 599 (1981). T i 5) J. R. Colvin, L. Chene, L. C. Sowden and M. Takai, D-Glc 6 Can. J. Biochem., 55, 1057 (1977). t 6) A. Amemura, T. Hashimoto, K. Koizumi and T. 1 L-Rha Utamura, J. Gen. Microbiol., 131, 301 (1985). 7) S. Ohmori, H. Masai, K. Arima and T. Beppu, Agric. Fig. 5. Structure of a Repeating Unit ofPolysaccharide Biol. Chem., 44, 2901 (1980). AM-2.9) 8) H. Minakami, E. Entani, K. Tayama, S. Fujiyama and H. Masai, Agric. Biol. Chem., 48, 2405 (1984). 9) K. Tayama, H. Minakami, E. Entani, S. Fujiyama of polysaccharide AM-1somewhat resembles and H. Masai, Agric. Biol. Chem., 49, 959 (1985). that of polysaccharide AM-2 (see, Fig. 5), 10) N. Shibuya, J. Chromatogr., 208, 96 (1981). ll) R. L. Taylor and H. E. Conrad, Biochemistry, ll, except the sugar arrangement in the side 1383 (1972). chains. Xanthan produced by Xanthomonas 12) S. Hakomori, J. Biochem., 55, 205 (1964). campestris contains the backbone chain of /?- 13) J. Frateur, P. Simonart and T. Coulon, Antonie v. (l-»4)-linked D-glucose residues,18'19* but Leeuwenhoek, 20, 111 (1954). polysaccharide AM-1 is apparently differ- 14) J. Tosic and T. K. Walker, J. Gen. Microbiol., 4, 192 ent from the hitherto known polysaccha- (1950). 15) J. De Ley and J. Frateur, "Bergey's Manual of rides in respect to the sequential arrange- Determinative Bacteriology," 8th Ed., ed. by R. E. ments of sugar residues. Buchanan and N. E. Gibbons, Williams and Wilkins, As elucidated by our studies, it is interest- Baltimore, 1974, pp. 251 and 276. 16) J. L. Baker, F. E. Dayand H. F. E. Hulton, J. Inst. ing that somestrains of Acetobacter species Brew., 18, 651 (1912). produce soluble branched polysaccharides 17) J. L. Shimwell, J. Inst. Brew., 42, 585 (1936). having cellulose backbone. Structures of these 18) P.-E. Jansson, L. Kenne and B. Lindberg, branched polysaccharides may receive atten- Carbohydr. Res., 45, 275 (1975). tion in view of taxonomy of acetic acid bac- 19) L. D. Melton, L. Mindt, D. A. Rees and G. R. teria and also their biosynthetic mechanisms in Sanderson, Carbohydr. Res., 46, 245 (1976). relation to cellulose synthesis.