Preparation of Affinity Columns of α-Mannose-binding Bulb Lectins and Resolution of Mannose-containing Glycans : Microanalysis of Brewing Products

Abstract

Affinity columns of two unique α-mannose-binding lectins, isolated from bulbs of Zephyranthes carinata (ZCA) and Crocus vernus (CVA), the latter highly specific to Man (α-1,3) Man, were used as probes of biochemical fractionation of glycans and glycoproteins. Thus, the α-mannan of yeast cell-wall was selectively separated from glycogen, either by CVA or ZCA column. The horse radish peroxidase glycoprotein (PDG), which contains biantennary mannose-terminated carbohydrate moiety, was purified by ZCA-column. When the purified PDG was further applied onto CVA column, two fractions were separated, one (65 %) not retained on the column, and the other (35 %) retained was eluted with 20mM diaminopropane (DAP), suggesting structural heterogeneity of PDG, with regard to the carbohydrate sequence.

The ZCA-column, strongly binds to terminal a-mannose residues (Man), was used for fractionation of brewing liquor products, including several kinds of commercial beer and "happou-shu", wine and also sake, and to provide a one-step microanalysis of content of yeast mannan. A small aliquot of each sample was dialyzed, and the non-dialyzable fraction (Mw. > 6,000) was applied onto the affinity column, from which starch and other non-retaining polymers were removed. The column retained α-mannan, which should be liberated from yeast during fermentation process was eluted with 20-40mM DAP. Thus, most of commercial beer (either domestic or imported) showed similar elution profiles and mannan contents, 20-25mg / 100ml of beer, whereas all "happou-shu" samples showed lower mannan contents, approximately 10mg/100ml, suggesting different brewing process. On the other hand, the sake samples were shown to contain 60mg/100ml of mannan, their elution profiles were not same from each other, probably due to a variety of fermentation process.