Dialysis of Oligosaccharides

Abstract

IDENTIFICATION of oligosaccharide intermediates liberated during the enzymatic hydrolysis of a polysaccharide provides information on both the structure and mode of hydrolysis of the polymer. Isolation of these intermediates in good yield is facilitated if they can be removed from the enzyme site before further hydrolysis occurs. Perila and Bishop¹ accomplished this removal by hydrolysing a glucomannan in a dialysis sac to give a good yield of disaccharides (28 per cent) and lesser yields of tri- and tetra-saccharides (8 per cent each), and Timell² likewise hydrolysed a glucuronoxylan to moderate yields of oligosaccharides (none of these authors specified the grade of dialysis tubing used). Similarly, with a protozoal xylanase, we found that a convincing demonstration of the liberation of xylodextrins of a degree of polymerization (DP) > 3 was only obtained when the xylan was hydrolysed by the enzyme with concurrent dialysis in 20/32 ‘Visking’ tubing (Union Carbide Corp, N.Y.). Oligosaccharides of DP 3–5 are of relatively low molecular weight, but in our experience they dialysed at far slower rates than those reported for comparable peptides (see Table 2, Craig and King³). The dialysis of various oligosaccharides was therefore investigated in a simple dialysis chamber consisting of a piece of glass tubing (7 × 3.5 cm diam.) closed at one end with a sheet of opened-out dialysis tubing. The closed end dipped just below the external solution (10 ml.), the oligosaccharide solution (10 mg in 2 ml.; 3.3 cm² of membrane per ml.) was placed inside the chamber and both solutions were gently stirred with magnetic stirrers. At intervals, portions (100 µl.) of external solution were removed for the measurement of dialysed oligosaccharide so that the time for the diffusion of half the initial oligosaccharide (half-escape time) could be calculated. Three grades of ‘Visking’ seamless cellulose tubing were used; 20/32, 36/36 and C-75 heavy wall.