Agric. Biol. Chem., 50 (5), 1271 ~1278, 1986 1271 Structure of an Acidic Polysaccharide 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-glucose, D-galactose, D-mannose, 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 polysaccharides, such as cellu- ture of polysaccharide AM-1, as revealed by lose,1* dextran,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-glucan.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-rhamnose, 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- oligosaccharides 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 oligosaccharide 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.