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Purification of cyclic β-1,2-glucan synthetase from Agrobacterium radiobacter
JOURNALOF FERMENTATIONANDBIOENGINEERING Vol. 72, NO. 6, 416-421. 1991
Purification of Cyclic fl-l,2-Glucan Synthetase from Agrobacterium radiobacter SHINICHI KINOSHITA, l* MOTOMI NAKATA, 1 AKINORI AMEMURA, z AND HISAHARU TAGUCHP
Department of Fermentation Technology, Faculty of Engineering, 1 and Inst. Sci. Ind. Res., 2 Osaka University, Yamadaoka, Suita-shi, Osaka 565, Japan Received 17 June 1991/Accepted 13 September 1991 A cyclic ~ l , 2 - g l u c a n synthetase was solubilized by sonicating 100,000 × g pellets of the crude extract of
Agrobacterium radiobacter IFO 12665 A1-5 in the presence of ethylenediaminetetraacetic acid. The enzyme was separated by DEAE-Sephadex column chromatography into adsorbed and nonadsorbed fractions (cyclic ~ l , 2 - g l u c a n synthetases I and II, respectively). Both enzymes were further purified by Toyo-pearl HW55 gel filtration and high performance liquid chromatography on Shodex WS-803. Neither enzyme could penetrate into 4% polyacrylamide gel in sodium dodecyl sulfate polyacrylamide gel electrophoresis. The specific activity of synthetase I was 9.5 x 10 -4 units/rag and it was purified 26-fold from the crude extract with a yield of 1.9%. Synthetase ! was optimal at 33°C and pH 8-9 for the enzyme reaction. The activity was almost stable up to 40°C for 30 rain, and mostly inactivated at 50°C for 30 rain. The enzyme activity was stimulated about three times with the addition of 10-20 mM FeCI3. The enzyme produced the same cyclic fl-l,2-glucan with a polymerization degree of 17-24 from UDP-glucose that the cell produced. The specific activity of synthetase II was 23.6 x 10 -4 units/rag and it was purified 60-fold from the crude extract with a yield of 2.3%.
national Plc., Backinghamshire), 1/21 of 10 mM ATP, 2 al of 20 mM NAD, 2/21 of 0.1 M ferric chloride, 5/21 of 0.1 M magnesium chloride, 12/A of 0.1 M glycine-NaOH buffer, pH 8 (10). After incubation for 30 min at 30°C, the reaction was terminated by adding 5/21 of 25% trichloroacetic acid (TCA), followed by centrifugation at 12,000 rpm for 3 rain. Ten /A of the supernatant was spotted on filter paper (Toyo no. 50, 40×40cm), which was developed descendingly by the solvent of 1-butanol: pyridine: water ( 6 : 4 : 3 ) . After drying, the paper was cut into 3mm lengths and the radioactivity determined by a liquid scintillation counter (LS-7500, Beckman Instruments Inc., CA, USA). One unit of enzyme activity was defined as the amount of enzyme needed to incorporate 1/~mol of glucose into the glucan fractions in 1 min. Enzyme activity was also determined using cold UDP-Dglucose in the following reaction mixture: 1.0 ml of the enzyme solution, 0.5 ml of 1 mg/ml UDP-D-glucose, 0.1 ml of 10mM ATP, 0.4ml of 20mM NAD, 0.2ml of 0.1M ferric chloride, 0.5ml of 0.1 M magnesium chloride, 0.8ml of 0.1 M glycine-NaOH buffer. After incubation for 30 rain at 30°C, the reaction was terminated by adding 0.5 ml of 2 5 ~ TCA, followed by centrifugation at 5,000 rpm for 5 rain. To the supernatant, 7 ml of ethanol was added and centrifuged. To this supernatant a further 40 ml of ethanol was added, and it was again centrifuged. The precipitate was dissolved in 100/tl of water and 10/~1 of it was analyzed by high performance liquid chromatography (HPLC, 880-PU, Jasco, Tokyo) with a column of Shodex KS-2 (0.8 × 30cm) and a refractometer (830 RI, Jasco, Tokyo) using water as a solvent. The authentic cyclic fl-l,2-glucan was kindly donated by Mr. Higasiura, Daikin Co. Ltd., Osaka. Other determinations Protein concentration was determined from the adsorption at 280 nm, and one adsorption unit was assumed to be 0.5 mg/ml protein. Chromatography Paper chromatography was carried out using filter paper, Toyo no. 50 (40 × 40 cm) with a
Agrobacterium tumefaciens infects the dicotyledon, forms nodules in the root, and produces glucans of low molecular weight (1), which are mainly composed offl-l,2glucans (2). This kind of glucan was also produced in the liquid cultures of some species of Agrobacterium (3) and Rhizobium (4). Agrobacterium (5) and Rhizobium (6-8) produced cyclic fl-l,2-glucans, whose polymerization degrees were in the range of 17-24 (9). Cell-free synthesis of this glucan has previously been carried out with 100,000 × g pellets of A. radiobacter IFO 12665 A1-5 (10). Here we succeeded in solubilizing cyclic glucan synthetase I and II from the 100,000×g pellets, purified them, and determined some of their properties. MATERIALS AND METHODS Strain and cultivation Agrobacterium radiobacter IFO12665 A1-5 used in this experiment was obtained by treating strain IFO12665 (11) with N-methyl-N'-nitro-N"nitroso guanidine, producing only cyclic fl-1,2-glucans as a sole extracellular polysaccharide. The bacterium was inoculated from the slant culture into a 100-ml conical flask containing 20 ml of a medium comprising glucose 40g, (NH4)2SO4 1.5 g, KH2PO4 1 g, MgSO4.7H20 0.5 g, Na2CO3 10g, NaC1 10mg, CaC12. 2H20 10 mg, MnC12.4HzO 10 mg, FeC13-6H20 10 mg, ZnC12 0.07 mg, CuSO4.SH20 0.05 mg, and H3BO3 0.01 mg in 1 l (pH 7) and it was incubated on a rotary shaker at 30°C for 24 h. This culture was transferred to a 2-l (Sakaguchi-flask containing 500ml of the medium and incubated for another 24 h. Determination of cyclic ~-l,2-glucan synthetase activity The enzyme activity was determined in the follow-
ing reaction mixture: 5/A of the enzyme solution, 3 ~1 of UDP-(U-14C)-D-glucose (278 mCi/mmol, Amersham Inter* Corresponding author. 416
VOL. 72, 1991
CYCLIC fl-I,2-GLUCAN SYNTHETASE
solvent system of 1-butanol-pyridine-water (6 : 4 : 3). Sugars were detected by the alkaline silver nitrate method (12) and the cyclic glucan was usually detected by radioautography. T h i n layer chromatography was carried out using a Kieselgel plate 60 F254 (20 x 20 cm, E. Merck) with a solvent system of 1-butanol-ethanol-water (5 : 5 : 4). Sugars were detected by spraying 5%0 sulfuric acid in methanol, and cyclic glucan was usually detected by radioautography. R E S U L T S AND D I S C U S S I O N
Localization of cyclic ~-l,2-glucan synlhetase and its solubilization The cells were harvested from 5 l of the culture, washed with 5 0 m M Tris-HC1 buffer, pH 7.5, suspended in the buffer, and disrupted by French Pressure Cell (Ohtake Mfg., Tokyo). After centrifuging at 10,000 x g for 30 min, 80% of the enzyme activity in the cell homogenate was recovered in the 10,000 x g supernatant fraction as described before (10). This was again centrifuged at 100,000x g for 3 0 m i n by ultracentrifugation (55P-7, Hitachi Mfg., Tokyo). Only 7% of the activity was obtained in the supernatant, and 61% of the activity was recovered in the 100,000xg pellet fraction, which meant that the enzyme in this preparation was still insoluble. To solubilize the enzyme, the crude enzyme was treated with various kinds of detergents and the activity in the 100,000xg supernatant was determined (Table 1). None of the detergents was satisfactory for enzyme solubilization and Triton X-100 was rather inhibitory. Next, we sonicated the enzyme with ethylenediaminetetraacetic acid (EDTA). By this treatment, two-thirds of the activity was solubilized. In further experiments this method was employed. Purification of cyclic ~-l,2-glucan synthetases The solubilized enzyme was purified by DEAE-Sephadex column chromatography (Fig. 1). The activity appeared ill tWO peaks (nonadsorbed and adsorbed fractions eluted by
417
TABLE 1. Effect of various treatments on the solubilization of cyclic fl-1,2-glucan synthetase Enzyme activity (10 4units/ml) Supernatant Pellet 1.9 12.0 3.6 11.1 2.5 1.0 2.8 7.7 1.3 10.1 2.5 9.0 5.7 5.5 3.2 7.1 7.6 3.6
Treatment a None Tween 80 Triton x 100 Briji 35 Deoxycholic acid Emalugen 905 EDTA Sonication Sonication with EDTA
a The concentration of detergents and EDTA was 1% and 10 mM, respectively, and the enzyme was incubated for 30 rain at 30°C. Sonication (Kaijo-Denki Co., Kobe) was carried out at 20 kc for 5 min. After the treatment the enzyme solution was centrifuged at 100,000 z g for 30 rain and the activity of the supernatant was determined.
0.3 M NaCI). The activity recovery was less than 50%0 in this chromatography. We then atempted to elute the activity from the column with another 500 ml of 10% NaC1, but no activity was eluted. We started to purify the enzyme from the second peak (absorbed fraction), which was designated as cyclic fl-l,2-glucan synthetase I and the enzyme in the first peak as cyclic fl-l,2-glucan synthetase II. The active fractions of the second peak (synthetase I) were pooled, and concentrated to 10 ml by ultrafiltration with a m e m b r a n e of 50,000 molecular cut-off (UHP-76 and UK-50, Toyo Filter-paper Co. Ltd., Tokyo). It was charged on a Toyo-pearl H W 5 5 column (2.5 x 9 0 c m ) preequilibrated with 50 m M Tris buffer, pH 7.5. The protein was eluted in the first small peak and the second big peak, and the activity was detected in the first peak only (data not shown). The active fractions were pooled and concentrated to
Synthetase II Activity 5
E
4 ,t
x
b
g 3 o
>
t~
2 o
hi
n
0 0
20
40
60 Fraction
80
I00
120
0 40
number { 2 0 ml each)
FIG. 1. Purification of cyclic fl-l,2-glucan synthetase by DEAE-Sephadex column chromatography. Crude enzyme solution (200 ml) was charged on a DEAE-Sephadex column (4 x 55 cm) preequilibrated with 50 mM Tris. HCI buffer, pH 7.5, and eluted with 400 ml of the buffer and 21 of linear gradient of NaC1 (0-1 M) in the buffer.
418
K I N O S H I T A ET AL.
J. FERMENT. BIOENG.,
3.0 ml; the concentrate was then purified by H P L C on a gel filtration column (data not shown). The protein was eluted in a big peak near the void volume at 11 min and several small peaks at 14-18 min. The activity was present in the first peak. A f t e r repeating H P L C , the active fractions were p o o l e d and concentrated to 1.0 ml. F r o m the elution volume, the molecular weight o f this enzyme seemed very high, and in fact it could not penetrate even into 4%0 polyacrylamide gel in sodium dodecyl sulfate polyacrylamide electrophoresis ( S D S - P A G E , 13). Thus we treated the enzyme p r e p a r a t i o n with 10 m M E D T A , 0.1% SDS, and 0.01% dithiothreitol (DTT) at 30°C for 30 min, purified it again by the H P L C , and the protein was eluted in several small peaks at 11-15 min. The activity was observed in the 11.5 min-eluate. A f t e r repeating H P L C , the active fractions were pooled and concentrated, and the concentrate was used as purified cyclic fl-l,2-glucan synthetase I in further experiments. The purification is summarized in Table 2. The specific activity o f synthetase I was 0.5 x 10 4 u n i t s / m g and it was purified 26-fold from the crude extract with a yield o f 1.9°//00. The first peak fractions (synthetase II) o f the D E A E Sephadex c h r o m a t o g r a p h y were pooled, concentrated, and sonicated for 5 min in the presence o f 10 m M E D T A , 0.1% SDS, and 0.01% DTT. The concentrate was then purified again by D E A E - S e p h a d e x column c h r o m a t o g r a phy. Most o f the activity was a d s o r b e d and eluted at 0.4 M NaC1. The active fractions were purified by Toyo-pearl HW55 gel filtration and H P L C as described above. Synthetase II was purified 60-fold from the crude extract with a yield o f 2.3%0, and the specific activity was 23.6 u n i t s / m g , which was m o r e than 2 times higher than that o f synthetase I. The UV spectrum o f synthetase II showed an a d s o r p t i o n peak at 260 nm with a shoulder at 290 nm, whereas that of synthetase I showed a peak at 280 nm with a small shoulder at 290 nm. Synthetase II might contain a nucleotide, but since we could not confirm it. We did not conduct further study of the properties o f this synthetase II. Properties of cyclic ~l,2-glucan synthetase I The purified synthetase I was analyzed by S D S - P A G E , but
T A B L E 2.
al
0
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4
8
/
12 Time
I
16
I
20
:>4
I
28
(rain)
FIG. 2. Elution profile of the purified cyclic fl-l,2-glucan synthetase I in H P L C on WS-803. Enzyme (100/zl) was injected onto a H P L C gel filtration column (0.8x 5 0 c m , Shodex WS-803, ShowaDenko Co., Tokyo) with a UV detector (880-PU, Jasco, Tokyo) at a flow rate of 1.0 m l / m i n using 50 m M Tris. HC1 buffer, p H 7.5. The enzyme was eluted by a linear gradient of NaCl (0-1.0 M in 30 ml of the buffer).
even in 4%0 polyacrylamide gel the protein showed a faint staining at the t o p o f the gel, which means that it could not penetrate well into the gel. The H P L C elution pattern in gel filtration on Shodex WS-803 gave a symmetrical single peak at 11.5 min, as shown in Fig. 2 and we estimated the molecular weight to be about 350,000, though it was just a limit o f the reference curve o f this column. This enzyme m a y not be a simple protein. We measured the sugar content in the enzyme preparation by a phenol sulfuric acid m e t h o d (14) and found a b o u t 10% o f sugar in the enzyme. It was unusual that the bacterial enzyme was a glycoprotein. The sugar o f the enzyme was analyzed. The enzyme was treated with 0.1 N HC1 at 100°C for 60 min, and then the protein was precipitated with ethanol, the supernatant was dried, and the released sugars were analyzed by paper c h r o m a t o g r a p h y . Two sugar spots were observed; one was found to be mannose, but the other spot did not coincide with ordinary sugars and it was negative in the Elson-Morgan reaction (15), which means it was neither hexosamine nor N-acetylhexosamine. The ethanol precipitate was dissolved in a small a m o u n t o f water, which was analyzed again by SDS-
Purification o f cyclic fl-l,2-glucan synthetase I and 11
Purification step
Total protein (mg)
Crude enzyme" 8,550 Sonic and E D T A b 2,700 Synthetase I DEAE-Sephadex (I) 85.0 Ultrafiltration 78.5 Toyo-pearl HW55 22.0 Ultrafiltration 20.0 Showdex WS-803 (I) 13.5 E D T A , SDS, D T T c 13.0 Shodex WS-802 (II) 6.6 Synthetase II DEAE-Sephadex (I) 54.0 E D T A , SDS, D T T ~ 33.0 DEAE-Sephadex (1I) 30.0 Ultrafiltration 27.0 Toyo-pearl HW55 9.2 Ultra filtration 9.0 Shodex WS-803 (I) 3.3
Total Specific Yield activity activity (10 3units) (10 4units/mg) ( ~ ) 331 230
0.4 1.1
100 69
17.0 15.0 12.0 11.0 9.0 6.8 6.2
2.0 1.9 5.5 5.5 6.7 5.2 9.5
5. l 4.5 3.6 3.3 2.7 2.1 1.9
48.0 17.4 15.0 13.2 9.0 8.4 7.8
8.9 5.3 5.0 4.9 9.8 9.3 23.6
14.4 5.2 4.5 4.0 2.7 2.5 2.3
a 10,000 × g s u p e r n a t a n t . b 100,000 X g supernatant. T h e a c t i v i t y w a s d e t e r m i n e d a f t e r dialysis.
o
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pH
FIG. 3. Effect of pH on cyclic fl-l,2-glucan synthetase l activity. The enzyme activity was determined by using substrates of the following pHs at 30°C in 30-min reactions: p H 7.0-9.5, 50 m M T r i s . H C l buffer (solid symbols); p H 8 - 1 0 . 5 , 5 0 m M glycine.NaOH buffer (open symbols).
VOL. 72, 1991
CYCLIC fl-I,2-GLUCAN SYNTHETASE
TABLE 3. Effect of the reaction components on cyclic fl-l,2-glucan synthetase activity
P A G E . It gave a dense band near the top and a few faint bands at less moved positions. The mobility o f this main band was almost same as that o f ferritin (molecular weight 440,000); we estimated that the molecular weight o f the enzyme would be a b o u t this value if it was a simple pro-tein, and the few m i n o r bands would be due to partiallyb r o k e n peptides formed during the hydrolysis. The enzyme was treated with 1 N N a O H at 100°C for 60 min, but no sugar was released, which meant that the enzyme did not have any O-glycoside linkage via serine or threonine. The enzyme was partially hydrolyzed with trifluoroacetic acid, but no oligosaccharide was released. This meant that any nascent cyclic fl-l,2-glucan was not included in the enzyme protein.
Components NAD MnCIz (0.9 mM) (lOmM)
ATP (0.3 mM) + + + +
+ ~ ~
Relative activity (O//oo)
FeC13 (lOmM)
+ +
+ +
+
+
+ {-
+ + +
4-
+
100 a 48 98 100 114 28 85 27 29 43 52 18 18b
+ + +
The enzymatic properties of cyclic ~-l,2-glucan synthetase I The optimal p H of the enzyme reaction was
+
f o u n d to be at p H 8-9 and the enzyme was rather stable in an alkaline side, as shown in Fig. 3. The optimal t e m p e r a t u r e o f the enzyme reaction was found to be 33°C as shown in Fig. 4A, and activity was scarcely detected at temperature higher than 50°C. The enzyme was roughly stable up to 40°C and was almost negligible after incubation at 50°C (Fig. 4B). The effect o f c o m p o n e n t s in the enzyme reaction on the activity was determined. In omission tests, FeCI3 was most effective as shown in Table 3, while in a d d i t i o n tests, A T P and N A D did not have any effect on the activity. FeCI3 and MnC12 were effective, and especially when both metals were present. A T P or N A D had a small effect only together with FeC13 and MnCI2. W e a d d e d A T P and N A D in the enzyme reaction mixture according to the previous m e t h o d (10), but they might not be absolutely necessary. The effect o f metal ions (each 10 mM) on the enzyme activity in the presence o f 10 m M MnCIE was studied. FeCI:~ stimulated the activity very much (276% o f the control), CaCIE, MnC12 (total 20 raM) and MgCI2 were a little effective (133, 125 and 110%, respectively), but ZnC12, NiC12, COC12, CuC12, and CrCI3 were inhibitory (43, 44, 62, 67, and 69%, respectively). The effect o f FeC13 concentrations
+
~
+
a 8.0 x 10 4 units/ml. b With 100 mM EDTA. on the activity was also studied. The activity increased with an increase o f FeC13 concentration up to 1 0 m M , where it was 280% o f the control. This stimulation was maintained at 10-20 mM, but at more than 20 m M it decreased (100% at 30 mM). In the enzyme reaction, UDP-glucose was used as a substrate. We tested other sugars and derivatives such as gluc o s e - l - p h o s p h a t e , glucose-6-phosphate, glucose, sophorose, and sophorotriose together with U T P (or UDP), A T P , and N A D instead o f UDP-glucose, but none o f them showed any sugar polymerization. Sophorose and sophorotriose did not inhibit the cyclic glucan synthesis from UDP-glucose.
Analysis of the products obtained by the enzyme reaction We determined the enzyme activity by the polymerization of glucose from UDP-glucose and carried out the following experiments to confirm that the products formed by the enzyme reaction were surely cyclic /?-l,2glucans. The reaction products were prepared from non-
(A)
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419
(B)
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o
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,
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,
20
30
40
Temperature
i 50
(=C)
,
60
20
0
'
O
2'0
~
'
~
40
Temperature
h
L
60
(*C)
FIG. 4. Effect of temperature on cyclic fl-l,2-glucan synthetase I activity (A) and stability (B). (A) The enzyme activity was determined at different temperatures (20-60°C) at pH 8.0 in 30-min reaction; (B) The enzyme was incubated at different temperatures (0-70°C) at pH 8.0, and then the residual enzyme activity was determined under the standard conditions.
420
KINOSHITA ET AL.
J. FERMENT.BIOENG.,
=
~,.,~oProduct ._~ U
L~4,6 -Tri-O - methyl-D-glueitol
o
i'o
z0
3'0 Time
5'0
6'o
I
0
(min]
FIG. 5. Analysis of the methylated hydrolysate of the enzyme reaction products by gas chromatography. Gas chromatograph (GC-7A, Shimadzu Mfg, Kyoto) with an FID detector; column (0.3 × 200 cm), 0.3% OV-275 and 0.4% GEXF-1150; temperature program; 140°C for 4 min, temperature gradient from 140 to 180°C, and 180°C for 16 min.
radioactive UDP-glucose as described in Methods, methylated and hydrolyzed as described before (10). In gas chromatography analysis, and the hydrolysate was eluted in a single peak at 21.5 m i n (Fig. 5), which coincided with an authentic 3,4,6-tri-O-methyl-D-glucitol. 2,3,4,6-Tetra-Omethyl-D-glucitol, 2,4,6-tri-O-methyl-D-glucitol, 2,3,6-triO-methyl-D-glucitol, 2,3,4-tri-O-methyl-D-glucitol, 2,3di-O-methyl-D-glucitol were not detected. F r o m these resuits, the products were f o u n d to be cyclic fl-l,2-glucans. This result indicated that the enzyme (a single component) could carry out the initiation, elongation, and cyclization of cyclic fl-l,2-glucan synthesis. The reaction products were analyzed by thin layer chromatography. The spots were similar to the authentic cyclic fl-l,2-glucans obtained by fermentation. The size of the cyclic oligomers produced by the enzyme reaction was analyzed by H P L C (9). The elution pattern showed one u n k n o w n peak at 13 min and several peaks comprised of cyclic glucan oligomers of 17-24 glucose units at 31-59 min, similar to those of authentic cyclic fl1,2-glucans obtained by fermentation (Fig. 6). The peak eluate at 13 min was collected and analyzed by the following two methods; (i) after hydrolysis by 1 N HC1, glucose was recognized by paper chromatography (10); (ii) after methylation and hydrolysis, no distinct peak was obtained in gas chromatography (10). Hence the linkage pattern of glucose could not be determined. ACKNOWLEDGMENT We thank Mr. H. Higashiura, Daikin Co. Ltd. for the gift of cyclic fl-1,2-glucan. REFERENCES 1. Melntire, M.C., Peterson, W.H., and Rikor, A.J.: A poly-
Ill
I
20
I
I
|
40 Time
I
60
|
i
80
( men }
FIG. 6. Analysis of sizes of the cyclic glucan produced by the enzyme reaction by HPLC. The product (100pl) was injected onto a column (ERC-NH-II71, Elmer Opt. Corp.) and eluted with 50% acetonitrile at a flow rate of 0.5 ml/min.
2.
3. 4. 5.
6.
7. 8.
9.
10.
11. 12.
saccharide produced by the crown-gall organism. J. Biol. Chem., 143, 491-496 (1942). Putman, E. W., Potter, A. L., Hodgson, R., and Hassifl, W. Z.: The structure of crown gall polysaccharide I. J. Am. Chem. Soc., 72, 5024-5026 (1950). Gorin, P. A. J., Spencer, J. F. T., and Westlake, D. W. S.: The structure and resistance to methylation of 1,2-fl-glucanfrom species of Agrobacteria. Can. J. Chem., 39, 1067-1073 (1961). Dendonder, R. A. and Hassid, W. Z.: The enzymatic synthesis of a (fl-l,2)-linked glucan by an extract of Rhizobium japonicum. Biochim. Biophys. Acta, 90, 239-248 (1964). Hisamatsu, M., Amemura, A., Matsuo, H., and Harada, T.: Cyclic (1 ÷2)-fl-D-glucanand the octasaccharide repeating-unit of succinoglycan produced by Agrobacterium. J. Gen. Microbiol., 128, 1873-1879 (1982). Zevenhvizen, L. P. T. M.: Cellular glycogen, fl-l,2-glucan, polyfl-hydroxybutyric acid and extracellular polysaccharides in fastgrowing species of Rhizobium I. Antonie van Leewenhoek, 47, 481-497 (1981). Abe, M., Amemura, A., and Higashi, S.: Studies on cyclicfl-l,2glucan obtained from periplasmic space of Rhizobium trifolii cells. Plant Soil, 64, 315-324 (1982). Amemura, A., Hisamatsu, M., Mitani, H., and Harada, T.: Cyclic (1 ~2)-fl-D-glucan and the octasaccharide repeating-unit of extracellular acidic polysaccharide produced by Rhizobium. Carbohydr. Res., 114, 277-285 (1983). Koizumi, K., Okada, Y., Horitama, S., Utamura, T., Hisamatsu, M., and Amemura, A.: Separation of cyclic (1 *2)-pD-glucan (cyclosophoraoses) produced by Agrobacterium and Rhizobium, and determination of their degree of polymerization by high performance liquid chromatography. J. Chromatogr., 265, 89-96 (1983). Amemura, A.: Synthesis of cyclic (1 *2)-fl-o-glucanby cell-free extracts of Agrobacterium radiobacter IFO 12665 bl and Rhizobium phaseoli AHU 1133. Agric. Biol. Chem., 48, 1809-1817 (1984). Hisamatsu, M., Ott, S., Amemura, A., and Harada, T.: Changes in ability of Agrobacterium to produce water-soluble and waterinsoluble fl-glucan. J. Gen. Microbiol., 103, 375-379 (1975). Aano, K. and Seno, N.: Isolation and detection of sugars by paper chromatography (in Japanese), p. 376-395. In Kotake, M.,
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CYCLIC fl-I,2-GLUCAN SYNTHETASE
421
et al. (ed.), Jikken kagaku koza, vol. 23 (part 1), Maruzen,
al. (ed.), Jikken kagaku koza, vol. 23 (part 1), Maruzen, Tokyo
Tokyo (1957). 13. Davis, B. J.: Disc electrophoresis. Anals. New York Acad. Sci., 121,407-427 (1964). 14. Anno, K. and Seno, N.: Isolation and analysis of sugars; phenolsulfuric acid method (in Japanese), p. 421-422, In Kotake, M. el
(1957). 15. Anno, K. and Seno, N.: Isolation and analysis of sugars; color reaction of amino-sugar (in Japanese), p. 370-376. In Kotake, M. et al. (ed.), Jikken kagaku koza, vol. 23 (part 1), Maruzen, Tokyo (1957).
Purification of Cyclic fl-l,2-Glucan Synthetase from Agrobacterium radiobacter SHINICHI KINOSHITA, l* MOTOMI NAKATA, 1 AKINORI AMEMURA, z AND HISAHARU TAGUCHP
Department of Fermentation Technology, Faculty of Engineering, 1 and Inst. Sci. Ind. Res., 2 Osaka University, Yamadaoka, Suita-shi, Osaka 565, Japan Received 17 June 1991/Accepted 13 September 1991 A cyclic ~ l , 2 - g l u c a n synthetase was solubilized by sonicating 100,000 × g pellets of the crude extract of
Agrobacterium radiobacter IFO 12665 A1-5 in the presence of ethylenediaminetetraacetic acid. The enzyme was separated by DEAE-Sephadex column chromatography into adsorbed and nonadsorbed fractions (cyclic ~ l , 2 - g l u c a n synthetases I and II, respectively). Both enzymes were further purified by Toyo-pearl HW55 gel filtration and high performance liquid chromatography on Shodex WS-803. Neither enzyme could penetrate into 4% polyacrylamide gel in sodium dodecyl sulfate polyacrylamide gel electrophoresis. The specific activity of synthetase I was 9.5 x 10 -4 units/rag and it was purified 26-fold from the crude extract with a yield of 1.9%. Synthetase ! was optimal at 33°C and pH 8-9 for the enzyme reaction. The activity was almost stable up to 40°C for 30 rain, and mostly inactivated at 50°C for 30 rain. The enzyme activity was stimulated about three times with the addition of 10-20 mM FeCI3. The enzyme produced the same cyclic fl-l,2-glucan with a polymerization degree of 17-24 from UDP-glucose that the cell produced. The specific activity of synthetase II was 23.6 x 10 -4 units/rag and it was purified 60-fold from the crude extract with a yield of 2.3%.
national Plc., Backinghamshire), 1/21 of 10 mM ATP, 2 al of 20 mM NAD, 2/21 of 0.1 M ferric chloride, 5/21 of 0.1 M magnesium chloride, 12/A of 0.1 M glycine-NaOH buffer, pH 8 (10). After incubation for 30 min at 30°C, the reaction was terminated by adding 5/21 of 25% trichloroacetic acid (TCA), followed by centrifugation at 12,000 rpm for 3 rain. Ten /A of the supernatant was spotted on filter paper (Toyo no. 50, 40×40cm), which was developed descendingly by the solvent of 1-butanol: pyridine: water ( 6 : 4 : 3 ) . After drying, the paper was cut into 3mm lengths and the radioactivity determined by a liquid scintillation counter (LS-7500, Beckman Instruments Inc., CA, USA). One unit of enzyme activity was defined as the amount of enzyme needed to incorporate 1/~mol of glucose into the glucan fractions in 1 min. Enzyme activity was also determined using cold UDP-Dglucose in the following reaction mixture: 1.0 ml of the enzyme solution, 0.5 ml of 1 mg/ml UDP-D-glucose, 0.1 ml of 10mM ATP, 0.4ml of 20mM NAD, 0.2ml of 0.1M ferric chloride, 0.5ml of 0.1 M magnesium chloride, 0.8ml of 0.1 M glycine-NaOH buffer. After incubation for 30 rain at 30°C, the reaction was terminated by adding 0.5 ml of 2 5 ~ TCA, followed by centrifugation at 5,000 rpm for 5 rain. To the supernatant, 7 ml of ethanol was added and centrifuged. To this supernatant a further 40 ml of ethanol was added, and it was again centrifuged. The precipitate was dissolved in 100/tl of water and 10/~1 of it was analyzed by high performance liquid chromatography (HPLC, 880-PU, Jasco, Tokyo) with a column of Shodex KS-2 (0.8 × 30cm) and a refractometer (830 RI, Jasco, Tokyo) using water as a solvent. The authentic cyclic fl-l,2-glucan was kindly donated by Mr. Higasiura, Daikin Co. Ltd., Osaka. Other determinations Protein concentration was determined from the adsorption at 280 nm, and one adsorption unit was assumed to be 0.5 mg/ml protein. Chromatography Paper chromatography was carried out using filter paper, Toyo no. 50 (40 × 40 cm) with a
Agrobacterium tumefaciens infects the dicotyledon, forms nodules in the root, and produces glucans of low molecular weight (1), which are mainly composed offl-l,2glucans (2). This kind of glucan was also produced in the liquid cultures of some species of Agrobacterium (3) and Rhizobium (4). Agrobacterium (5) and Rhizobium (6-8) produced cyclic fl-l,2-glucans, whose polymerization degrees were in the range of 17-24 (9). Cell-free synthesis of this glucan has previously been carried out with 100,000 × g pellets of A. radiobacter IFO 12665 A1-5 (10). Here we succeeded in solubilizing cyclic glucan synthetase I and II from the 100,000×g pellets, purified them, and determined some of their properties. MATERIALS AND METHODS Strain and cultivation Agrobacterium radiobacter IFO12665 A1-5 used in this experiment was obtained by treating strain IFO12665 (11) with N-methyl-N'-nitro-N"nitroso guanidine, producing only cyclic fl-1,2-glucans as a sole extracellular polysaccharide. The bacterium was inoculated from the slant culture into a 100-ml conical flask containing 20 ml of a medium comprising glucose 40g, (NH4)2SO4 1.5 g, KH2PO4 1 g, MgSO4.7H20 0.5 g, Na2CO3 10g, NaC1 10mg, CaC12. 2H20 10 mg, MnC12.4HzO 10 mg, FeC13-6H20 10 mg, ZnC12 0.07 mg, CuSO4.SH20 0.05 mg, and H3BO3 0.01 mg in 1 l (pH 7) and it was incubated on a rotary shaker at 30°C for 24 h. This culture was transferred to a 2-l (Sakaguchi-flask containing 500ml of the medium and incubated for another 24 h. Determination of cyclic ~-l,2-glucan synthetase activity The enzyme activity was determined in the follow-
ing reaction mixture: 5/A of the enzyme solution, 3 ~1 of UDP-(U-14C)-D-glucose (278 mCi/mmol, Amersham Inter* Corresponding author. 416
VOL. 72, 1991
CYCLIC fl-I,2-GLUCAN SYNTHETASE
solvent system of 1-butanol-pyridine-water (6 : 4 : 3). Sugars were detected by the alkaline silver nitrate method (12) and the cyclic glucan was usually detected by radioautography. T h i n layer chromatography was carried out using a Kieselgel plate 60 F254 (20 x 20 cm, E. Merck) with a solvent system of 1-butanol-ethanol-water (5 : 5 : 4). Sugars were detected by spraying 5%0 sulfuric acid in methanol, and cyclic glucan was usually detected by radioautography. R E S U L T S AND D I S C U S S I O N
Localization of cyclic ~-l,2-glucan synlhetase and its solubilization The cells were harvested from 5 l of the culture, washed with 5 0 m M Tris-HC1 buffer, pH 7.5, suspended in the buffer, and disrupted by French Pressure Cell (Ohtake Mfg., Tokyo). After centrifuging at 10,000 x g for 30 min, 80% of the enzyme activity in the cell homogenate was recovered in the 10,000 x g supernatant fraction as described before (10). This was again centrifuged at 100,000x g for 3 0 m i n by ultracentrifugation (55P-7, Hitachi Mfg., Tokyo). Only 7% of the activity was obtained in the supernatant, and 61% of the activity was recovered in the 100,000xg pellet fraction, which meant that the enzyme in this preparation was still insoluble. To solubilize the enzyme, the crude enzyme was treated with various kinds of detergents and the activity in the 100,000xg supernatant was determined (Table 1). None of the detergents was satisfactory for enzyme solubilization and Triton X-100 was rather inhibitory. Next, we sonicated the enzyme with ethylenediaminetetraacetic acid (EDTA). By this treatment, two-thirds of the activity was solubilized. In further experiments this method was employed. Purification of cyclic ~-l,2-glucan synthetases The solubilized enzyme was purified by DEAE-Sephadex column chromatography (Fig. 1). The activity appeared ill tWO peaks (nonadsorbed and adsorbed fractions eluted by
417
TABLE 1. Effect of various treatments on the solubilization of cyclic fl-1,2-glucan synthetase Enzyme activity (10 4units/ml) Supernatant Pellet 1.9 12.0 3.6 11.1 2.5 1.0 2.8 7.7 1.3 10.1 2.5 9.0 5.7 5.5 3.2 7.1 7.6 3.6
Treatment a None Tween 80 Triton x 100 Briji 35 Deoxycholic acid Emalugen 905 EDTA Sonication Sonication with EDTA
a The concentration of detergents and EDTA was 1% and 10 mM, respectively, and the enzyme was incubated for 30 rain at 30°C. Sonication (Kaijo-Denki Co., Kobe) was carried out at 20 kc for 5 min. After the treatment the enzyme solution was centrifuged at 100,000 z g for 30 rain and the activity of the supernatant was determined.
0.3 M NaCI). The activity recovery was less than 50%0 in this chromatography. We then atempted to elute the activity from the column with another 500 ml of 10% NaC1, but no activity was eluted. We started to purify the enzyme from the second peak (absorbed fraction), which was designated as cyclic fl-l,2-glucan synthetase I and the enzyme in the first peak as cyclic fl-l,2-glucan synthetase II. The active fractions of the second peak (synthetase I) were pooled, and concentrated to 10 ml by ultrafiltration with a m e m b r a n e of 50,000 molecular cut-off (UHP-76 and UK-50, Toyo Filter-paper Co. Ltd., Tokyo). It was charged on a Toyo-pearl H W 5 5 column (2.5 x 9 0 c m ) preequilibrated with 50 m M Tris buffer, pH 7.5. The protein was eluted in the first small peak and the second big peak, and the activity was detected in the first peak only (data not shown). The active fractions were pooled and concentrated to
Synthetase II Activity 5
E
4 ,t
x
b
g 3 o
>
t~
2 o
hi
n
0 0
20
40
60 Fraction
80
I00
120
0 40
number { 2 0 ml each)
FIG. 1. Purification of cyclic fl-l,2-glucan synthetase by DEAE-Sephadex column chromatography. Crude enzyme solution (200 ml) was charged on a DEAE-Sephadex column (4 x 55 cm) preequilibrated with 50 mM Tris. HCI buffer, pH 7.5, and eluted with 400 ml of the buffer and 21 of linear gradient of NaC1 (0-1 M) in the buffer.
418
K I N O S H I T A ET AL.
J. FERMENT. BIOENG.,
3.0 ml; the concentrate was then purified by H P L C on a gel filtration column (data not shown). The protein was eluted in a big peak near the void volume at 11 min and several small peaks at 14-18 min. The activity was present in the first peak. A f t e r repeating H P L C , the active fractions were p o o l e d and concentrated to 1.0 ml. F r o m the elution volume, the molecular weight o f this enzyme seemed very high, and in fact it could not penetrate even into 4%0 polyacrylamide gel in sodium dodecyl sulfate polyacrylamide electrophoresis ( S D S - P A G E , 13). Thus we treated the enzyme p r e p a r a t i o n with 10 m M E D T A , 0.1% SDS, and 0.01% dithiothreitol (DTT) at 30°C for 30 min, purified it again by the H P L C , and the protein was eluted in several small peaks at 11-15 min. The activity was observed in the 11.5 min-eluate. A f t e r repeating H P L C , the active fractions were pooled and concentrated, and the concentrate was used as purified cyclic fl-l,2-glucan synthetase I in further experiments. The purification is summarized in Table 2. The specific activity o f synthetase I was 0.5 x 10 4 u n i t s / m g and it was purified 26-fold from the crude extract with a yield o f 1.9°//00. The first peak fractions (synthetase II) o f the D E A E Sephadex c h r o m a t o g r a p h y were pooled, concentrated, and sonicated for 5 min in the presence o f 10 m M E D T A , 0.1% SDS, and 0.01% DTT. The concentrate was then purified again by D E A E - S e p h a d e x column c h r o m a t o g r a phy. Most o f the activity was a d s o r b e d and eluted at 0.4 M NaC1. The active fractions were purified by Toyo-pearl HW55 gel filtration and H P L C as described above. Synthetase II was purified 60-fold from the crude extract with a yield o f 2.3%0, and the specific activity was 23.6 u n i t s / m g , which was m o r e than 2 times higher than that o f synthetase I. The UV spectrum o f synthetase II showed an a d s o r p t i o n peak at 260 nm with a shoulder at 290 nm, whereas that of synthetase I showed a peak at 280 nm with a small shoulder at 290 nm. Synthetase II might contain a nucleotide, but since we could not confirm it. We did not conduct further study of the properties o f this synthetase II. Properties of cyclic ~l,2-glucan synthetase I The purified synthetase I was analyzed by S D S - P A G E , but
T A B L E 2.
al
0
I
I
4
8
/
12 Time
I
16
I
20
:>4
I
28
(rain)
FIG. 2. Elution profile of the purified cyclic fl-l,2-glucan synthetase I in H P L C on WS-803. Enzyme (100/zl) was injected onto a H P L C gel filtration column (0.8x 5 0 c m , Shodex WS-803, ShowaDenko Co., Tokyo) with a UV detector (880-PU, Jasco, Tokyo) at a flow rate of 1.0 m l / m i n using 50 m M Tris. HC1 buffer, p H 7.5. The enzyme was eluted by a linear gradient of NaCl (0-1.0 M in 30 ml of the buffer).
even in 4%0 polyacrylamide gel the protein showed a faint staining at the t o p o f the gel, which means that it could not penetrate well into the gel. The H P L C elution pattern in gel filtration on Shodex WS-803 gave a symmetrical single peak at 11.5 min, as shown in Fig. 2 and we estimated the molecular weight to be about 350,000, though it was just a limit o f the reference curve o f this column. This enzyme m a y not be a simple protein. We measured the sugar content in the enzyme preparation by a phenol sulfuric acid m e t h o d (14) and found a b o u t 10% o f sugar in the enzyme. It was unusual that the bacterial enzyme was a glycoprotein. The sugar o f the enzyme was analyzed. The enzyme was treated with 0.1 N HC1 at 100°C for 60 min, and then the protein was precipitated with ethanol, the supernatant was dried, and the released sugars were analyzed by paper c h r o m a t o g r a p h y . Two sugar spots were observed; one was found to be mannose, but the other spot did not coincide with ordinary sugars and it was negative in the Elson-Morgan reaction (15), which means it was neither hexosamine nor N-acetylhexosamine. The ethanol precipitate was dissolved in a small a m o u n t o f water, which was analyzed again by SDS-
Purification o f cyclic fl-l,2-glucan synthetase I and 11
Purification step
Total protein (mg)
Crude enzyme" 8,550 Sonic and E D T A b 2,700 Synthetase I DEAE-Sephadex (I) 85.0 Ultrafiltration 78.5 Toyo-pearl HW55 22.0 Ultrafiltration 20.0 Showdex WS-803 (I) 13.5 E D T A , SDS, D T T c 13.0 Shodex WS-802 (II) 6.6 Synthetase II DEAE-Sephadex (I) 54.0 E D T A , SDS, D T T ~ 33.0 DEAE-Sephadex (1I) 30.0 Ultrafiltration 27.0 Toyo-pearl HW55 9.2 Ultra filtration 9.0 Shodex WS-803 (I) 3.3
Total Specific Yield activity activity (10 3units) (10 4units/mg) ( ~ ) 331 230
0.4 1.1
100 69
17.0 15.0 12.0 11.0 9.0 6.8 6.2
2.0 1.9 5.5 5.5 6.7 5.2 9.5
5. l 4.5 3.6 3.3 2.7 2.1 1.9
48.0 17.4 15.0 13.2 9.0 8.4 7.8
8.9 5.3 5.0 4.9 9.8 9.3 23.6
14.4 5.2 4.5 4.0 2.7 2.5 2.3
a 10,000 × g s u p e r n a t a n t . b 100,000 X g supernatant. T h e a c t i v i t y w a s d e t e r m i n e d a f t e r dialysis.
o
•
o
•
i 0 •
o
>
g
c w
I
7
I
I
8
9
I0
I
I
II
pH
FIG. 3. Effect of pH on cyclic fl-l,2-glucan synthetase l activity. The enzyme activity was determined by using substrates of the following pHs at 30°C in 30-min reactions: p H 7.0-9.5, 50 m M T r i s . H C l buffer (solid symbols); p H 8 - 1 0 . 5 , 5 0 m M glycine.NaOH buffer (open symbols).
VOL. 72, 1991
CYCLIC fl-I,2-GLUCAN SYNTHETASE
TABLE 3. Effect of the reaction components on cyclic fl-l,2-glucan synthetase activity
P A G E . It gave a dense band near the top and a few faint bands at less moved positions. The mobility o f this main band was almost same as that o f ferritin (molecular weight 440,000); we estimated that the molecular weight o f the enzyme would be a b o u t this value if it was a simple pro-tein, and the few m i n o r bands would be due to partiallyb r o k e n peptides formed during the hydrolysis. The enzyme was treated with 1 N N a O H at 100°C for 60 min, but no sugar was released, which meant that the enzyme did not have any O-glycoside linkage via serine or threonine. The enzyme was partially hydrolyzed with trifluoroacetic acid, but no oligosaccharide was released. This meant that any nascent cyclic fl-l,2-glucan was not included in the enzyme protein.
Components NAD MnCIz (0.9 mM) (lOmM)
ATP (0.3 mM) + + + +
+ ~ ~
Relative activity (O//oo)
FeC13 (lOmM)
+ +
+ +
+
+
+ {-
+ + +
4-
+
100 a 48 98 100 114 28 85 27 29 43 52 18 18b
+ + +
The enzymatic properties of cyclic ~-l,2-glucan synthetase I The optimal p H of the enzyme reaction was
+
f o u n d to be at p H 8-9 and the enzyme was rather stable in an alkaline side, as shown in Fig. 3. The optimal t e m p e r a t u r e o f the enzyme reaction was found to be 33°C as shown in Fig. 4A, and activity was scarcely detected at temperature higher than 50°C. The enzyme was roughly stable up to 40°C and was almost negligible after incubation at 50°C (Fig. 4B). The effect o f c o m p o n e n t s in the enzyme reaction on the activity was determined. In omission tests, FeCI3 was most effective as shown in Table 3, while in a d d i t i o n tests, A T P and N A D did not have any effect on the activity. FeCI3 and MnC12 were effective, and especially when both metals were present. A T P or N A D had a small effect only together with FeC13 and MnCI2. W e a d d e d A T P and N A D in the enzyme reaction mixture according to the previous m e t h o d (10), but they might not be absolutely necessary. The effect o f metal ions (each 10 mM) on the enzyme activity in the presence o f 10 m M MnCIE was studied. FeCI:~ stimulated the activity very much (276% o f the control), CaCIE, MnC12 (total 20 raM) and MgCI2 were a little effective (133, 125 and 110%, respectively), but ZnC12, NiC12, COC12, CuC12, and CrCI3 were inhibitory (43, 44, 62, 67, and 69%, respectively). The effect o f FeC13 concentrations
+
~
+
a 8.0 x 10 4 units/ml. b With 100 mM EDTA. on the activity was also studied. The activity increased with an increase o f FeC13 concentration up to 1 0 m M , where it was 280% o f the control. This stimulation was maintained at 10-20 mM, but at more than 20 m M it decreased (100% at 30 mM). In the enzyme reaction, UDP-glucose was used as a substrate. We tested other sugars and derivatives such as gluc o s e - l - p h o s p h a t e , glucose-6-phosphate, glucose, sophorose, and sophorotriose together with U T P (or UDP), A T P , and N A D instead o f UDP-glucose, but none o f them showed any sugar polymerization. Sophorose and sophorotriose did not inhibit the cyclic glucan synthesis from UDP-glucose.
Analysis of the products obtained by the enzyme reaction We determined the enzyme activity by the polymerization of glucose from UDP-glucose and carried out the following experiments to confirm that the products formed by the enzyme reaction were surely cyclic /?-l,2glucans. The reaction products were prepared from non-
(A)
E
419
(B)
5
ioo
4-
80
{:
___3
-
60
.-> • --
o
2
4O
.=-I
0
IO
~
,
~
,
20
30
40
Temperature
i 50
(=C)
,
60
20
0
'
O
2'0
~
'
~
40
Temperature
h
L
60
(*C)
FIG. 4. Effect of temperature on cyclic fl-l,2-glucan synthetase I activity (A) and stability (B). (A) The enzyme activity was determined at different temperatures (20-60°C) at pH 8.0 in 30-min reaction; (B) The enzyme was incubated at different temperatures (0-70°C) at pH 8.0, and then the residual enzyme activity was determined under the standard conditions.
420
KINOSHITA ET AL.
J. FERMENT.BIOENG.,
=
~,.,~oProduct ._~ U
L~4,6 -Tri-O - methyl-D-glueitol
o
i'o
z0
3'0 Time
5'0
6'o
I
0
(min]
FIG. 5. Analysis of the methylated hydrolysate of the enzyme reaction products by gas chromatography. Gas chromatograph (GC-7A, Shimadzu Mfg, Kyoto) with an FID detector; column (0.3 × 200 cm), 0.3% OV-275 and 0.4% GEXF-1150; temperature program; 140°C for 4 min, temperature gradient from 140 to 180°C, and 180°C for 16 min.
radioactive UDP-glucose as described in Methods, methylated and hydrolyzed as described before (10). In gas chromatography analysis, and the hydrolysate was eluted in a single peak at 21.5 m i n (Fig. 5), which coincided with an authentic 3,4,6-tri-O-methyl-D-glucitol. 2,3,4,6-Tetra-Omethyl-D-glucitol, 2,4,6-tri-O-methyl-D-glucitol, 2,3,6-triO-methyl-D-glucitol, 2,3,4-tri-O-methyl-D-glucitol, 2,3di-O-methyl-D-glucitol were not detected. F r o m these resuits, the products were f o u n d to be cyclic fl-l,2-glucans. This result indicated that the enzyme (a single component) could carry out the initiation, elongation, and cyclization of cyclic fl-l,2-glucan synthesis. The reaction products were analyzed by thin layer chromatography. The spots were similar to the authentic cyclic fl-l,2-glucans obtained by fermentation. The size of the cyclic oligomers produced by the enzyme reaction was analyzed by H P L C (9). The elution pattern showed one u n k n o w n peak at 13 min and several peaks comprised of cyclic glucan oligomers of 17-24 glucose units at 31-59 min, similar to those of authentic cyclic fl1,2-glucans obtained by fermentation (Fig. 6). The peak eluate at 13 min was collected and analyzed by the following two methods; (i) after hydrolysis by 1 N HC1, glucose was recognized by paper chromatography (10); (ii) after methylation and hydrolysis, no distinct peak was obtained in gas chromatography (10). Hence the linkage pattern of glucose could not be determined. ACKNOWLEDGMENT We thank Mr. H. Higashiura, Daikin Co. Ltd. for the gift of cyclic fl-1,2-glucan. REFERENCES 1. Melntire, M.C., Peterson, W.H., and Rikor, A.J.: A poly-
Ill
I
20
I
I
|
40 Time
I
60
|
i
80
( men }
FIG. 6. Analysis of sizes of the cyclic glucan produced by the enzyme reaction by HPLC. The product (100pl) was injected onto a column (ERC-NH-II71, Elmer Opt. Corp.) and eluted with 50% acetonitrile at a flow rate of 0.5 ml/min.
2.
3. 4. 5.
6.
7. 8.
9.
10.
11. 12.
saccharide produced by the crown-gall organism. J. Biol. Chem., 143, 491-496 (1942). Putman, E. W., Potter, A. L., Hodgson, R., and Hassifl, W. Z.: The structure of crown gall polysaccharide I. J. Am. Chem. Soc., 72, 5024-5026 (1950). Gorin, P. A. J., Spencer, J. F. T., and Westlake, D. W. S.: The structure and resistance to methylation of 1,2-fl-glucanfrom species of Agrobacteria. Can. J. Chem., 39, 1067-1073 (1961). Dendonder, R. A. and Hassid, W. Z.: The enzymatic synthesis of a (fl-l,2)-linked glucan by an extract of Rhizobium japonicum. Biochim. Biophys. Acta, 90, 239-248 (1964). Hisamatsu, M., Amemura, A., Matsuo, H., and Harada, T.: Cyclic (1 ÷2)-fl-D-glucanand the octasaccharide repeating-unit of succinoglycan produced by Agrobacterium. J. Gen. Microbiol., 128, 1873-1879 (1982). Zevenhvizen, L. P. T. M.: Cellular glycogen, fl-l,2-glucan, polyfl-hydroxybutyric acid and extracellular polysaccharides in fastgrowing species of Rhizobium I. Antonie van Leewenhoek, 47, 481-497 (1981). Abe, M., Amemura, A., and Higashi, S.: Studies on cyclicfl-l,2glucan obtained from periplasmic space of Rhizobium trifolii cells. Plant Soil, 64, 315-324 (1982). Amemura, A., Hisamatsu, M., Mitani, H., and Harada, T.: Cyclic (1 ~2)-fl-D-glucan and the octasaccharide repeating-unit of extracellular acidic polysaccharide produced by Rhizobium. Carbohydr. Res., 114, 277-285 (1983). Koizumi, K., Okada, Y., Horitama, S., Utamura, T., Hisamatsu, M., and Amemura, A.: Separation of cyclic (1 *2)-pD-glucan (cyclosophoraoses) produced by Agrobacterium and Rhizobium, and determination of their degree of polymerization by high performance liquid chromatography. J. Chromatogr., 265, 89-96 (1983). Amemura, A.: Synthesis of cyclic (1 *2)-fl-o-glucanby cell-free extracts of Agrobacterium radiobacter IFO 12665 bl and Rhizobium phaseoli AHU 1133. Agric. Biol. Chem., 48, 1809-1817 (1984). Hisamatsu, M., Ott, S., Amemura, A., and Harada, T.: Changes in ability of Agrobacterium to produce water-soluble and waterinsoluble fl-glucan. J. Gen. Microbiol., 103, 375-379 (1975). Aano, K. and Seno, N.: Isolation and detection of sugars by paper chromatography (in Japanese), p. 376-395. In Kotake, M.,
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CYCLIC fl-I,2-GLUCAN SYNTHETASE
421
et al. (ed.), Jikken kagaku koza, vol. 23 (part 1), Maruzen,
al. (ed.), Jikken kagaku koza, vol. 23 (part 1), Maruzen, Tokyo
Tokyo (1957). 13. Davis, B. J.: Disc electrophoresis. Anals. New York Acad. Sci., 121,407-427 (1964). 14. Anno, K. and Seno, N.: Isolation and analysis of sugars; phenolsulfuric acid method (in Japanese), p. 421-422, In Kotake, M. el
(1957). 15. Anno, K. and Seno, N.: Isolation and analysis of sugars; color reaction of amino-sugar (in Japanese), p. 370-376. In Kotake, M. et al. (ed.), Jikken kagaku koza, vol. 23 (part 1), Maruzen, Tokyo (1957).