Melibiose from raffinose

Fletcher and Diehl, in studying the preparation of melibiose from raffinose by the fermentative hydrolysis of the trisaccharide, noticed a new form of the disaccharide. By observing the mutarotation of the new form, and by comparing the infrared spectra with that of an authentic... 

Melibiose (6-0-(3-D-Gal-D-G1c) (disaccharide) Widespread in plant exudates from Raffinose hydrolysis Sweet [[Pg.404]

An a-D-galactosidase from a strain of E. coli, which was adapted to synthesize the enzyme, was stable in concentrated, but not in dilute, solution. The enzyme (pH optimum 6.8, temperature optimum 37 °C, Km for 4-nitrophenyl a-D-galacto-pyranoside, melibiose, and raffinose 1.07 x 10, 2.33 x 10 , and 3.65 x 10 moll S respectively) also possesses weak glycosyltransferase activity. 

Melibiose is a reducing disaccharide [a-D-gaiactopy ranosy i-(1,6)-o-gi ucopy ranose] obtained from raffinose by yeast fermentation. 

That melibiose-l-phosphate cannot react with fructofuranose is inferred from the inability of the enzyme to phosphorolyze raffinose. 

Melibiose occurs in Nature primarily as a constituent of the trisaccharide raffinose, although it has been identified in such plant exudates as ash manna and an exudate from mallow. Historical details of the discovery and preparation of melibiose have been reviewed by Harding68 and will not be reiterated here. Modern methods for the preparation of melibiose have been recorded by Hudson and Harding60 and by Bates and associates.66 Melibiose can be purchased from a number of chemical-supply firms. 

The exact structure of raffinose follows most simply from the fact that it is cleaved by mild acid hydrolysis, or by invertase action, to give D-fructose and melibiose,7 and by almond-emulsin a-D-galactopyrano-sidase to give D-galactose and sucrose.16... 

De Whalley and his coworkers have made extensive studies on the application of paper chromatography to raffinose. To separate raffinose from large proportions of sucrose, a 1-butanol-benzene-pyridine-water solvent was used.140 81 To avoid pyridine, a 1-propanol-ethyl acetate-water mixture was recommended.141 As a color reagent, a-naph-thol-phosphoric acid was used. In this manner, less than 1 % of raffinose in cane sugar could be separated and determined. Partial hydrolysis of the raffinose, using invertase directly on the filter paper, proved useful in some instances.93 142 Melibiose was then detected with 3,5-dinitrosali-cylate142 or with diethyl-p-phenylenediamine sulfite.93... 

Fig. 1. Electrophoresis of sugars (A) sucrose, raffinose, and fructose (B) sucrose, raffinose, melibiose, and glucose (C) sucrose, raffiinose, stachyose, and fructose 40 v./em., 8-12 milliamperes, 1.5 hours, 1.5 X 22.5 inch strip, Whatman No. 1, 0.05 M borate buffer solution, pH — 9.2 (B) sprayed with benzidine + acetic acid, (A), (0), and (D) with 1-naphthol 4- phosphoric acid (D) kestose, sucrose, raffinose, and stachyose same conditions except for 3 hour run and origin line in center of strip instead of 9 inches from cathode end 5-20 ig. of each sugar applied.

Using chromatographic and heat inactivation procedures, Pazur succeeded in separating transfructosidase from Aspergillus oryzae cultures, containing a mixture of transglucosidase, invertase, and maltase. In tracer experiments the C -fructosyl units in radioactive sucrose were transferred to nonradioactive raffinose. Hydrolysis of the radioactive fructosylraffinose into fructose and melibiose, followed by a determination... 

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