Agric. Biol. Chern., 53 (10), 2661 -2666, 1989 2661

The Acceptor Specificity of Amylomaltase from Escherichia coli IFO 3806

Sumio Kitahata, Hiromi Murakami, Yoshiaki Sone* and Akira Misaki* Osaka Municipal Technical Research Institute, 6-50 Morinomiya 1-chome, Jyoto-ku, Osaka 536, Japan * Department of Food and Nutrition, Faculty of Science of Living, Osaka City University, Sugimoto-cho, Sumiyoshi, Osaka 558, Japan Received April 10, 1989

The acceptor specificity of amylomaltase from Escherichia coli IFO 3806 was investigated using various and alcohols. D-, D-glucosamine, TV-acetyl-D-glucosamine, D-, D- , isomaltose, and were efficientacceptors in the transglycosylation reaction of this enzyme. It was shownby chemical and enzymic methods that this enzymecould transfer glycosyl residues only to the C4-hydroxyl groups of D-mannose, TV-acetyl-D-glucosamine, D-allose, and D- xylose, producing terminated by 4-O-a-D-glucopyranosyl-D-mannose, 4-O-a-D- glucopyranosyl-A^-acetyl-D-glucosamine, 4-O-a-D-glucopyranosyl-D-allose, and 4-O-a-D-gluco- pyranosyl-D-xylose at the reducing ends, respectively.

Amylomaltase (1,4-a-D- : 1,4-a-D- pared in a pure form free from a-amylase (EC 3.2.1.1). glucan 4-a-glycosyl-transferase, EC 2.4. 1.25) Other materials. D-Mannose, D-glucosamine, TV-acetyl- is an enzyme which catalyzes both glucosyl- D-glucosamine, and D-xylose were purchased from transfer and 4-a-glucanosyl-transfer reactions. Nakarai Chemical Co. and 2-deoxy-D- was pur- This enzyme has been isolated and purified chased from Sigma Chemicals Co. Isomaltose was sup- from the Escherichia coli strain ML30,1} E. coli plied by Hayashibara Co., Ltd. These saccharides were strain ML308,2) and Pseudomonas stutzeri used after purification by paper chromatography. The other saccharides and their derivatives were of the highest NRRL 3389.3) grade commercially available. In our previous paper,4) we reported the purification of the amylomaltase from E. coli General methods. The amylomaltase activity was as- IFO 3806 to homogeneity by SDS-polyac- sayed using as a substrate by measuring the rylamide gel electrophoresis. amount of glucose released, as described previously.40 The amount of glucose was measured using the HK, G6P-DH, This paper describes the acceptor specific- NADPsystem. The total sugar and were ities of the amylomaltase from E. coli IFO measured colorimetrically by the phenol-sulfuric acid 3806 and the structure of the transglycosyla- method6) and the Somogyi-Nelson method,7' respective- ly. Glucosamine was measured by the Elson-Morgan tion products. method.8) Paper chromatography was done on Toyo No. 50 filter Materials and Methods paper (40 x 40cm) for qualitative and preparative chro- matography, using 1-butanol, pyridine, and water (6 : 4 : 3, Enzyme preparation. The amylomaltase from Esch- by volume) as a solvent system with multiple ascents. The erichia coli IFO 3806 was purified to a homogeneous sugars on a paper chromatogramwere detected with silver state, as described previously.40 A crystalline preparation nitrate reagent after the glucoamylase treatment by the of glucoamylase (EC 3.2.1.3) was supplied by Ueda method of Kainuma and French.9' Thin layer chromatog- Chemical Co., Ltd. a-Glucosidase (EC 3.2.1.20) from raphy (TLC) of the reaction products was done on Saccharomyces sp. was supplied by Toyobo Co., Ltd. /?- Kieselgel 60 plates (Merck Co., Ltd.) using ethylacetate, Amylase (EC 3.2.1.2) from defatted soybeans5) was pre- acetic acid, and water (3: 1 : 1, by volume) as a solvent 2662 S. Kitahata et al. system, with two developments. The on 13C-NMR spectra. Natural-abundance proton-de- TLCplates were shown by heating at (110~ 120)°C after coupled 13C-NMR spectra were recorded at 40°C with a spraying with sulfuric acid-methanol. JEOL-FMNGX-400 spectrometer operating at 100 MHz in the pulsed F.t. mode on solution in D2O(20~60mg Characterization of oligosaccharides. To identify the in 0.5ml of D2O). Chemical shifts (S, ppm) constituent sugars of oligosaccharides, a sampleof oligo- were measured with an internal standard of acetone-d6 saccharides (1 mg) was heated with 1 m hydrochloric acid whose chemical shift was set to (330.40 relative to the (1 ml) at 100°C for 1 hr, and the hydrolyzate was deionized signal of tetramethylsilane. by an anion exchange resin (Diaion SA 10A, OH~form). The hydrolysis products were identified by paper chroma- tography, by comparing their Rf values with those of Results authentic sugars. For the reduction of oligosaccharides, a sample of oligosaccharides (5mg, 1 ml) was reduced with Acceptor specificity sodium borohydride (5 mg) at 40°C for 20 hr. The reaction The acceptor specificity of amylomaltase mixture was treated with a cation exchange resin (Diaion from E. coli IFO 3806 was examined using SK1B, H+ form) to remove sodium ion, and the solution various and their derivatives. wasevaporated to dryness and resolved by methanol. The enzyme (663u/ml, lOjul) was incubated Boric acid in the residue was removed by repetitive evaporations with methanol under reduced pressure. The with 120//I of 1.0% soluble containing degree of polymerization (DP) of the oligosaccharides was an acceptor at 0.1 m at 40°C. After 20hr, 20/d estimated essentially by the method of Timmel10) and was taken for paper chromatography. The calculated from the following equation. DP=A/(A -B): amounts of transfer products were estimated A, amount of total sugar of the parent (before reduction) estimated by the phenol-sulfuric acid from the intensities of the corresponding spots method; B, amount of total sugar of the oligosaccharide after coloration with the silver nitrate reagent. after reduction with sodium borohydride. The results are summarized in Fig. 1 and Table I. The effective acceptors were D-glucose, Estimation of specific optical rotation ofoligosaccharides. methyl-a- and jS-D-glucoside, phenyl-a- and /?- The specific optical rotation of the oligosaccharides was D-glucoside, L-, D-xylose, D-mannose, measured in 5%aqueous solution at 20°C with an auto- A^-acetyl-D-glucosamine, D-glucosamine, 2- matic polarimeter (SEPA-200 high sensitive polarimeter, Horiba). deoxy-D-glucose, D-allose, , and iso- . On the other hand, D-, sugar Methylation analysis of oligosaccharides. Adry sample alcohols (such as sorbitol and xylitol) and (10mg) was dissolved in dry dimethylsulfoxide (2ml) and glycerol were not effective. converted into the sugar alkoxide with fresh methylsulfinyl carbanion (0.5 ml), prepared by the method of Sand ford and Conrad,11} at room temperature for 2hr with stirring Structure of the oligosaccharides synthesized by under a nitrogen atmosphere. The resulting sugar alkoxide transglycosylation to D-glucose, methyl-oc- was then methylated by the addition of methyl iodide and fi-D-glucoside, and phenyl-cn- and /?-d- (1.5ml) at 20°C for 1hr. The methylated product was glucoside applied on to Sep-Pak C18 Cartridges (Waters Associates The transglycosylation products with d- Inc.) and eluted with methanol. Complete methylation of glucose, methyl-a- and jS-D-glucoside, and the resulting methylated product was confirmed by the infrared spectrum. The completely methylated oligosac- phenyl-a- and jS-D-glucoside as acceptors were charide was hydrolyzed with formic acid (90%, v/v) at enzymically hydrolyzed with glucoamylase 100°C for 3hr, then with 2m trifluoroacetic acid at 100°C and /?-amylase. The hydrolysis products were for 5 hr. The methylated monosaccharides were reduced examined by paper chromatography or thin with sodium borohydride and then acetylated by heating with pyridine and acetic anhydride (1 : 1) at 100°C for 2hr. layer chromatography. The transglycosylation The mixture of alditol acetates so obtained was gas-liquid products to D-glucose gave only glucose by chromatographed (GLC) using a column (2 mlong) of 3% ECNSS-M on Gas Chrom Q at a gas flow rate of glucoamylase action, and maltose and malto- 30ml/min at 180°C12) or a Silicone OV-225 column at by /?-amylase action. The transglycosyl- 170°C for 40min and then raised to 210°C at a rate of ation products to methyl-a-D-glucoside gave 3°C/min. glucose and methyl-a-glucoside on hydrolysis with glucoamylase, and gave maltose, methyl- Acceptor Specificity of Amylomaltase 2663

Fig. 1. Paper Chromatogram of Transfer Products to Various Acceptors by Amylomaltase from E. coli IFO 3806. The reaction mixture containing 1%soluble starch, acceptor (0.1 m), and amylomaltase (6.6unit) was incubated at 40°C for 20hr. TwentyjA of the reaction mixture was spotted on filter paper and chromato- graphed as described in Materials and Methods. M, reference maltooligosaccharides; B, blank (no enzyme); T, test; (1), control (reaction mixture without acceptor); (2), (3), (4), (5), (6), (7), (8), (9), (10), and (1 1), trans- fer products to methyl a-glucoside, methyl /?-glucoside, phenyl a-glucoside, phenyl /?-glucoside, D-mannose, 2-deoxy-D-glucose, N-acetyl-D-glucosamine, D-xylose, isomaltose, and D-allose, respectively. Gl, glucose; G2, maltose; G3, maltotriose; G4, maltotetraose; G5, maltopentaose.

Table I. The Acceptor Specificity of clear that the amylomaltase from E. coli IFO Amylomaltase from E. coli IFO 3806 3806 transfers glycosyl residues only to the C4- Acceptor Transfer products hydroxyl group of D-glucose, methyl-a- and /?- D-glucoside, and phenyl-a- and /?-D-glucoside, D-Glucose + producing oligosaccharides terminated by ac- Methyl-a-, ^-D-glucoside + Phenyl-a-, ^-D-glucoside + ceptors at the reducing ends. D-Mannose + 2-Deoxy-D-glucose + Isolation of the transfer products to D-mannose, D-Glucosamine + N-acetyl-D-glucosamine, D-allose, and D- 7V-Acetyl-D-glucosamine + D-Allose + xy lose D-Galactose - The reaction mixture (30ml) containing 1 g D-Xylose + of soluble starch, 2g of acceptor, and the Isomaltose + enzyme (7500u) was incubated at 40°C. After +, transfer products formation; -, no transfer 48hr, the reaction mixture was heated at product. 100°C for 10 min to inactivate the enzyme, and then diluted to 0.5% concentration with 200 ml of 20mMacetate buffer (pH 4.5). To increase a-glucoside and methyl-a-maltoside on hy- the yield of the resulting from drolysis with jS-amylase. In the case of transglycosylation to acceptor, and hydrolyze transglycosylation products with methyl-jS-D- the degradation products from soluble starch glucoside and phenyl-a- and /?-D-glucoside, to glucose, the reaction mixture was hydro- similar results were obtained. Considering the lyzed by glucoamylase. The treated solution known action patterns of glucoamylase and /?- was put on to the active carbon column. The amylase, and the hydrolysis products obtained column was thoroughly washed with distilled from the transglycosylation products, it is water, and then with 10% ethanol solution. 2664 S. Kitahata et al.

The eluate with 10% ethanol solution which lose, and xylose, respectively (Fig. 2). When contains the smallest transglycosylation prod- the glucose and TV-acetyl-glucosamine in the uct in molecular weight, but still contains acid hydrolyzates of the original and reduced small amounts of other saccharides, was fur- oligosaccharide GGNAcwere measured, the ther purified by preparative paper chromatog- ratio of glucose to N-acetyl-glucosamine were raphy, and then lyophilized. Each lyophilized 1.1 : 1.0 and 1.0:0, respectively. The degree of sample gave a single spot on the paper polymerization of the oligosaccharides GM, chromatogram. GA, and GXwere estimated to be 2.0 by the method described in Materials and Methods. Identification of the constituent sugars, reducing Bythe action of a-glucosidase from brewer's ends, and degree of polymerization of the yeast, oligosaccharides GM,GGNAc,GA, and transfer products GXwere completely hydrolyzed to glucose The constituent sugars and the reducing end and corresponding monosaccharides (Fig. 3), group of the transfer products to D-mannose suggesting that the glucosidic linkage is the a- (GM), jV-acetyl-D-glucosamine (GGNAc), d- type. These results indicate that the oligosac- allose (GA), and D-xylose (GX) were examined charide GM is a-glucosyl-D-mannose, GG- by acid hydrolysis of the oligosaccharides and NAc is a-glucosyl-N-acetyl-D-glucosamine, its reduced products. On acid hydrolysis, the GAis a-glucosyl-D-allose, and GXis a-gluco- oligosaccharide GM, GGNAc, GA, and GX syl-D-xylose. gave glucose and mannose, glucose and N- acetyl-glucosamine, glucose and allose, and Methylation analysis glucose and xylose, respectively. On acid hy- To examine the glucosidic linkages of these drolysis of the reduced oligosaccharides, the oligosaccharides, these samples were used for reduced GM, GGNAc,GA, and GXgave only methylation analysis. GLCof the alditol ace- glucose with concomitant disappearance of the tates of the methylated sugar fragments from spots of mannose, A^-acetyl-glucosamine, al- GM, GX, GGNAc, and GA revealed the presence of 2,3,4,6-tetra-O-methyl-D-glucose, 2,3,6-tri-O-methyl-D-mannose, 2,3-di-O-meth- yl-D-xylose (identified by comparison of their retention times with those of authentic speci-

Fig. 2. Paper Chromatogram of Acid Hydrolyzates of Fig. 3. Paper Chromatogram of Hydrolyzates by the Oligosaccharides GXand Reduced GX. Brewer's Yeast a-Glucosidase of the Oligosaccharides X, D-xylose; R, reduced GX; A and RA, acid hydrol- GM, GX and GGNAc. yzates of GXand reduced GX, respectively. Other ab- A, acceptor; B, transfer products; T, a-glucosidase di- breviations are as in Fig. 1. gestion. Other abbreviations are as in Fig. 1. Acceptor Specificity of Amylomaltase 2665

Table II. 13C-NMRChemical Shift" Data for Disaccharides

Residue C- l C-2 C-3 C-4

GX a-Glc (l -*4) 72.94 73.51 70.26 73.51 a-Xyl 72.25 72.94 78.70 60.76 jS-Xyl 74.52 75.46 78.70 64.87 GM a-Glc (l -+4) 72.70 73.82 70.30 73.56 61.90 a-Man 71.59 70.30 76.42 71.94 61.47 jg-Man 71.94 72.70 76.36 75.60 61.47 GGNAc a-Glc (l->4) 72.67 73.62 70.30 73.62 61.58 a-GlcNAc 54.79 70.30 78.75 71.ll 61.50 )5-GlcNAc 57.59 75.19 78.05 75.56 61.50

Chemical shift (S) in ppm downfield from tetramethylsilane.

mens), and 3,6-di-<9-methyl-Af-acetyl-D-gluco- Table III. The Specific Optical Rotation samine and 2,3,6-tri-(9-methyl-D-allose (tenta- of the Prepared Oligosaccharides and tively identified by comparison of their reten- Related Compounds tion times with those in references).13'14) These Compound [a] n° results suggest that the oligosaccharides GM, 3-O-a-D-Glucopyranosyl-D-mannose16) + 27.9° GX, GGNAc, and GAare 4-0-a-glucosyl-D- 4-O-a-D-Glucopyranosyl-D-mannose1 7) +115.0° mannose, 4-Oa-glucosyl-D-xylose, 4-0-a-glu- + 105.6° cosyl-7V-acetyl-D-glucosamine, and 4-O-a-glu- 4-O-a-D-Glucopyranosyl-A^-acetyl-Prepared glucosylmannose +110.0° cosyl-D-allose, respectively. D-glucosamine1 8) + 96.9° 3-O-a-D-Glucopyranosyl-D-xylose19)Prepared glucosyl-iV-acetyl-D-glucosamine +101.6° 13 C-NMR analysis 4-O-a-D-Glucopyranosyl-D-xylose1 9) + 95.0° The 13C-NMRdata for solution of GX, + 95.8° GM, and GGNAcin D2Oare shown in Table Prepared glucosylxylose II. The resonances were assigned on the basis of comparison with the data in the literature15) 4-0-a-D-glucosylmannose ([a]D + 1 15.0°), and and the relative intensities. The a-configu- that of the glucosylxylose was +95.8°, close rations of the D-glucosyl residues in these to the value for 4-0-a-D-glucosylxylose ([a]D disaccharides are confirmed by the C-l res- +95.0°). onances at 3 100.57 (GX), 3 100.75 (GM), and S 100.73 (GGNAc). The data in Table II also Discussion confirm the linkages at position 4 of the accep- tor residues on the basis of the downfield Several reports have so far been published displacement of C-4 resonances by about 7 ~9 on the acceptor specificity of D-enzymefrom ppmcomparedwith those of the unsubstituted potato, and amylomaltase from E. coli monosaccharides. ML308. Peat et al studied the acceptor speci- ficity of potato D-enzymeand pointed out that Specific optical rotation of the glucosyl- the ideal acceptor was a-D-glucopyranose, and mannose, glucosyl-xylose, and glucosyl-N- any departure from this structure was accom- ace tyl-glucosam ine panied by a diminution, or by a complete loss The structures of the saccharides GM, GX, of acceptor activity.20) Saccharides such as and GGNAc were confirmed by comparing D-glucosamine, 7V-acetyl-D-glucosamine, and the specific optical rotation with those in re- isomaltose have no activity as acceptors of ferences (Table III). The glucosylmannose the transglycosylation by potato D-enzyme. was +105.6°, the value being close to that of Wiesmeyer and Cohn have reported that d- 2666 S. Kitahata et al. glucose, D-mannose, and methyl-a-D-malto- glucose, D-glucose-1-phosphate, and maltose side have activity as the acceptor of trans- in the reaction mixture. Thus, amylomaltase glycosylation by amylomaltase from E. coli from E. coli IFO 3806 may be a unique enzyme ML308, but D-glucose and D-mannose deriv- capable of synthesizing 4-0-a-D-glucopy- atives at the Cl position, such as methyl-a-D- ranosyl-D-glucosamine and 4-0-a-D-glucopy- glucoside, phenyl-a-D-glucoside, methyl-a-D- ranosyl-7V-acetyl-D-glucosamine as single di- mannoside, and cellobiose have no activity saccharides, from starch in the presence of d- as acceptors.21) glucosamine and N-acetyl-D-glucosamine as Wehave investigated the acceptor specificity the acceptor, respectively. of cyclodextrin glucanotransferase (CGTase) and found that saccharides which are epimers References or have substitutions of the D-glucopyranose at C2 position, such as D-mannose, d- 1) W. J. Whelan, FEESLett., 1, 1 (1968). 2) H. Wiesmeyer and M. Cohn, Biochim. Biophys. glucosamine, and N-acetyl-D-glucosamine had Acta, 39, 417 (1960). no activity as acceptors of the intermolecular 3) J. Schmidt and M. John, Biochim. Biophys. Acta, transglycosylation by CGTase. 566, 100 (1979). In this paper, we have shownthat the amylo- 4) S. Kitahata, H. Murakami and S. Okada, Agric. BioI. maltase from E. coli IFO 3806 has a broader Chem., 53, 2653 (1989). 5) J. Fukumoto and Y. Tsujisaka, Kagaku to Kogyo, 28, acceptor specificity than potato D-enzymeand 282 (1954). the amylomaltase from E. coli ML308,and the 6) M. Dubois, K. A. Gilles, J. K. Hamilton, P. A. various derivatives of D-glucopyranose, such Rebers and F. Smith, Anal. Chem., 28, 350 (1956). as methyl- and phenyl-D-glucoside, isomaltose, 7) M. Somogyi, J. Biol. Chem., 195, 19 (1952). cellobiose, D-glucosamine, and A^-acetyl-D- 8) S. Gardell, Acta Chem. Scand., 7, 207 (1953). 9) K. Kainuma and D. French, FEBS Lett., 5, 257 glucosamine were also efficient acceptors of (1969). the transglycosylation by this enzyme. Several 10) T. E. Timmel, Svensk Papperstid., 63, 668 (1960). reports have been presented dealing with en- ll) A. Stand ford and H. E. Conrad, Biochemistry, 5, zymes which transfer glycosyl groups to d- 1508 (1966). mannose, D-glucosamine, and Af-acetyl-D- 12) H. Bjorndal, B. Lindberg and S. Svenssan, Acta glucosamine. a-Glucosidase from brewer's Chem. Scand., 21, 1801 (1967). 13) K. Ohtani and A. Misaki, Carbohydr. Res., 87, 275 yeast transfers the glucosyl groups from (1980). phenyl-a-D-glucoside to C3- (mainly) and C6 14) Y. Tsuburaya, personal communication. (minor)-hydroxyl groups of D-mannoseas ac- 15) K. Bock, C. Pedersen and H. Pedersen, Adv. Carbohydr. Chem. Biochem., 42, 193 (1984). ceptors, producing two kinds of a-D-glucosyl- 16) A. M. Gakhokidze and I. A. Gvelukashvil, /. Gen. D-mannose.22) When amylomaltase from E. Chem., 22, 143 (1952). coli ML308is incubated with soluble starch 17) W. N. Haworth, E. L. Hirstand R. J. W. Reynolds, in the presence of D-mannose as acceptor, /. Chem. Soc, 1934, 302. glucosyl-D-mannose is synthesized. (The struc- 18) Z. Selinger and M. Schramm, J. Biol. Chem., 236, ture is not definite.) Maltose phosphorylase 2183 (1961). from Neiseria perflava has been reported to 19) S. Chiba, N. Takahashi and T. Shimomura, Agric. produce glucosyl-7V-acetyl-D-glucosamine and Biol. Chem., 33, 813 (1969). 20) S. Peat, W. J. Whelan and G. Jones, /. Chem. Soc, glucosyl-D-glucosamine from maltose and N- acetyl-D-glucosamine and maltose and d- 21) H. Wiesmeyer1957,and M. Cohn, Biochim.2490.Biophys. Acta, glucosamine in the presence of phosphate, 39, 427 (1960). respectively.17) However, in addition to the 22) S. Chiba and T. Shimomura, Agric. Biol. Chem., 33, 807 (1969). transglycosylation product, there are d-