Debranching enzymes of potato tubers (Solanum tuberosum L.). II. Purification of a pullulanase (R-enzyme) from potato tubers and comparison of its properties with those of the potato isoamylase.

A pullulanase (R-enzyme) was purified from potato tubers by fractional precipitation at pH 5, 20% ethanol precipitation, 40% saturated (NH4)4SO4 precipitation, ion exchange chromatography on Whatman DE 32 and finally by affinity chromatography on Sepharose 6B-αCD. The final preparation having a specific activity of 11.4 U/mg was obtained with a yield of 35.6%. The purified enzyme separated on gel filtration on Sephadex G-150 into 2 forms, the apparent molecular weight of which were calculated to be 220, 000 and 87, 000. Two forms converted spontaneously to each other. The pullulanase hydrolyzed pullulan most rapidly, β-LDs more rapidly than their original polysaccharidesbut could not act on oyster glycogen. It released maltosyl and maltotriosyl residues from poly-and oligosaccharides at approximately the same rate. All these catalytic properties are characteristic to cereal pullulanases and close to those of Aerobacter aerogenes (Klebsiella pneumoniae) pullulanase. The potato isoamylase purified in the previous paper could not hydrolyze pullulan at all but instead hydrolyzed oyster glycogen quite well. It favoured polysaccharides with longer branch chains than those with maltose and maltotriosyl stubs. Maltosyl stubs are debranched much more slowly than maltotriosyl stubs by this enzyme. All these properties are identical to those of Pseudomonas isoamylase. Data presented in this paper indicated clearly that potato tubers have a pullulanase and an isoamylase.