Purification of alternanase by affinity chromatography.

The enzyme alternanase, produced by Bacillus sp. NRRL B-21195, hydrolyzes alternan, a polysaccharide produced by certain strains of Leuconostoc mesenteroides that consists of glucose linked by alternating alpha(1-->6), alpha(1-->3) linkages. The main product of enzymatic hydrolysis by alternanase is a novel cyclic tetrasaccharide of glucose that also has alternating linkages between the glucose moieties. An improved purification scheme for alternanase has been developed that incorporates the use of isomaltosyl units linked to agarose for selectively binding the alternanase enzyme. Bound enzyme was eluted with 0.5 M sodium chloride and was nearly pure after this procedure. When followed by preparative isoelectric focusing, a single band of 117 kDa was measured when the purified protein was analyzed by HPLC size-exclusion chromatography/multiangle light scattering. The purification procedure can be scaled to permit large quantities of enzyme to be purified in high (36%) yield.