We have studied the anomeric configuration of the saccharides produced by rice-debranching enzyme, pullulanase, and isoamylase with 1 H-NMR spectrometer. 1) Some workers described quantitative measurement of the anomers produced by amylases, glucosidases, and other glycosidases by polarimetric 2 -8 ) and gas chromatographic 9 ) techniques.However, the anomeric form of D-fructofuranose or the terminal reducing residue of fructose oligomers produced by j3-D-fructofuranoside hydrolases (invertases, inulinases, and levanases) have not been studied because it is difficult to measure the anomeric form of these saccharides in which the methine proton ofC-2 is lacking using 1H-NMR spectrometry. Therefore, we intend to apply 13C-NMR spectroscopy for clarification of the anomeric configuration of such saccharides although this method is not always quantitative. This paper deals with identification of the anomeric form of D-fructofuranose released from sucrose by invertases.Baker's yeast invertase (800 U/mg protein) and Candida uti/is invertase (365 U/mg protein) were purchased from Sigma Chemical Co. A solution of the enzyme protei:ll in°2°w as dialyzed against 0 2 0. A solution of sucrose (Wako Junyaku Co., Ltd.) in 0 2 0 was lyophilized.The baker's yeast or C. utilis invertase (3000 U in 0.2 ml of 020) and sucrose (200 mg in 0.5 ml of 0.005 M sodium acetate buffer in 020' pH 5.0) were incubated in an NMR spectrometer tube at 20°C and 13C-NMR spectra were taken at intervals of 0,5,8, 15, and 60 min; 1,4 dioxane was used as the internal standard. Chemical shifts of carbons of sucrose 10 ) and fructose 11 ) (a,j3-D-fructofuranose and j3-D-fructopyranose) reported by H. C. Jarrell et al. and S. J. Angyal et al.. were referred to in this experiment. The spectrum of sucrose at zero time is shown in Fig. 1. The signals for Cl, C2, C3, C4, C5, and C6 of the j3-Dfructofuranosyl residue of sucrose occurred at b 62 23 104. 50, 77.32, 74.86, 82.19, and 63.23, respectively,~nd were separated from those for C 1, C2, .. " C6 of the a-D-2347 glucopyranosyl residue.The signals for C l-C6 of D-fructose or D-glucose released from sucrose by the ac:tion of baker's yeast invertase are shown in Fig. 1. After 5 min, six strong signals (b 63.61, 102.26,76.27,75.32,81.46, and 62.23) du~to Cl, C2, C3, C4, C5, and C6 of j3-D-fructofuranose and the same number of signals as the D-fructofuranose based on C l-C6 of a-D-glucopyranose were observed accompanied by disappearance of the carbon signals of sucrose. The (2~1) 13-linkage in sucrose had hydrolyzed completely in this time and the fructose released from sucrose was composed of almost only j3-D-fructofuranose.After 8 min the carbon signals associated with [3-Dfructopyranose were detected clearly at b 64.78 (C 1), 98.83 (C2), 68.40 (C3), 70.51 (C4), 70.01 (C5), and 64.19 (C6), and intensities of their signals gradually increased thereafter. Also, weak carbon signals based on a-Dfructofuranose appeared at b 63.80 (Cl), 105.16 (C2), 82.63 (C3), 76.90 (C4), 81.93 (C5), and 62.01 (C6) at this phase, ...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.