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The Acceptor Specificity of Amylomaltase from Escherichia Coli IFO 3806

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The Acceptor Specificity of Amylomaltase from Escherichia Coli IFO 3806 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 sugars and sugar alcohols. D-Mannose, D-glucosamine, TV-acetyl-D-glucosamine, D-xylose, D- allose, isomaltose, and cellobiose 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 oligosaccharides 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-glucan : 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-glucose 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 maltotriose 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 reducing sugar 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 carbohydrates 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 disaccharide 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 monosaccharides 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 starch 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 oligosaccharide (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-sorbose, 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, sucrose, and iso- maltose. On the other hand, D-galactose, 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 triose 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.
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