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In water; at 40℃;pH 6.0;Acetate buffer; Enzymatic reaction;Reactivity; |
Experiment 12-1Product from Maltose by the Enzyme ReactionAqueous maltose solution and acetate buffer (pH 6.0) were mixed to give final concentrations of 1percent (w/v) and 10 mM, respectively, to make into a substrate solution. The substrate solution was admixed with 10 units/g-solid substrate of alpha-glucosyltransferase, obtained by the method in Experiment 6, and followed by the enzyme reaction at 40° C. and pH 6.0. Aliquots were sampled from the reaction mixture with time and the reaction was stopped by keeping at 100° C. for 10 min. The saccharide compositions of the samples were measured by HPLC and GC. HPLC and GC were carried out under the conditions described in Experiment 4-2. The results are in Table 10. TABLE 10 Reaction Saccharide Composition (percent, w/w) Time DP1 DP2 DP3 (hr) Glucose Maltose Isomaltose Neotrehalose Maltotriose Panose Isopanose Isomaltotriose DP4 DP5 DP6 0 0.0 99.2 0.0 0.0 0.8 0.0 0.0 0.0 0.0 0.0 0.0 1 12.1 55.0 0.7 0.0 13.2 16.1 0.0 0.0 2.9 0.0 0.0 2 18.4 33.5 1.0 0.0 14.4 24.1 0.0 0.0 7.6 1.0 0.0 4 23.7 17.3 2.5 0.0 10.0 29.3 0.0 0.0 13.0 4.2 0.0 8 27.0 10.4 4.7 0.0 4.9 27.8 0.0 0.0 15.9 5.3 4.0 24 30.4 6.0 10.0 2.6 1.4 11.0 1.0 3.6 15.3 10.2 8.5 48 31.5 6.2 13.8 3.9 0.7 4.1 1.5 4.2 12.4 10.3 11.4 DP: Glucose polymerization degreeAs is evident from the results in Table 10, at initial stage (after one hour) of the reaction, glucose, maltotriose, and panose were formed as major reaction products from the substrate, maltose, by the action of the alpha-glucosyltransferase of the present invention. Also, oligosaccharides with a glucose polymerization degree of 4 and 5 were formed when the reaction time was elapsed 2 to 4 hours. Accompanying with the progress of the reaction, the content of maltotriose reached a maximum, 14.4percent, at 2 hours and then decreased; and the content of panose reached a maximum, 29.3percent at 4 hours and then decreased; and the content of isomaltose was increased with the decrease of the contents of maltotriose and panose. Further, the contents of isomaltose and oligosaccharides with a glucose polymerization degree of 4 or higher were increased until 48 hours.From these results, it was revealed that the alpha-glucosyltransferase of the present invention acts on maltose and forms glucose, maltotriose, and panose by catalyzing both alpha-1,4 and alpha-1,6 glucosyl transfer at initial stage of the reaction; and forms isomaltose, which is formed by alpha-1,6 glucosyl transfer to glucose, and isopanose and isomaltotriose, which are formed by alpha-1,4 and alpha-1,6 glucosyl transfer to isomaltose accompanied with the progress of the reaction. Since the identification of many kinds of oligosaccharide with glucose polymerization degree of 4 or higher is difficult in this experiment, the reaction mechanism of the enzyme was investigated in the following Experiment 12-2 using maltopentaose, whose glucose polymerization degree is higher than maltose, as substrate. |