Chromatographic separation of nucleosides using a cross-linked xylose isomerase crystal stationary phase

Cross-linked xylose isomerase (EC 5.3.1.5., from Streptomyces rubiginosus) crystals (CLXIC) packed into a 7.8 x 300 mm steel column showed specific affinity towards uridine (Urd), cytidine (Cyd), adenosine (Ado), guanosine (Guo), and thymidine. These nucleosides eluted out of the CLXIC column in the same order as the corresponding nucleoside bases, indicating that the retention depends mainly on the base component of the molecule. The interaction of nucleosides with the CLXIC material was not based merely on ion exchange or hydrophobic interactions but also on the unique properties of the CLXIC column. Decrease in temperature increased the retention but not the resolution factors of the adjacent nucleosides. The CLXIC column maintained its separation capacity even when 100 mg of ribonucleosides in equimass amounts were injected into the column in a volume of 1 mL corresponding to 10% of the total column volume. Analysis of sugar beet molasses, a side stream from sucrose production, showed it to contain 1-2.5 mg mL(-1) of Urd, Cyd, Ado, and Guo. The CLXIC column was able to separate and enrich these nucleosides also from highly viscous sugar beet molasses. The CLXIC column was especially efficient in the purification of guanosine. Other commercially interesting sugar beet molasses components such as the acidic compounds betaine, gamma-amino butyric acid, and D- and L-pyroglutamic acids or neutral sucrose did not interact with the CLXIC material.